Wilson Lab Wiki

Using the lab's AI skills

Mon, 06 Jul 2026 00:00:00 +0000

The lab keeps its AI tooling in one place: wilson-lab-ai-skills. If you use Claude to help with lab work, start there.

What it is#

A shared repo of things that make an AI agent genuinely useful for our work:

skills/ — reusable “skills” that teach Claude a lab workflow once, so nobody has to re-explain it. Examples: reading long PDFs, putting files on the lab server, keeping this wiki tidy.
tutorials/ — slides from lab-meeting intros to agentic AI.
mcp/ — notes on connectors (MCP servers) worth plugging in.
CLAUDE.md — standing instructions agents follow when working in the repo.

How to use a skill#

You don’t “run” a skill. Open the repo in Claude Code or Cowork, and skills load themselves when your request matches — you just ask for the thing. For example, “read this thesis and summarise it” pulls in the PDF skill; “put these slides on the lab server” pulls in the server skill. If nothing matches, Claude just works normally.

PCR for confirming transgenes

Fri, 26 Jun 2026 17:02:53 +0000

Protocol by Helen Yang, transcribed by Janki Patel.

Drosophila genetics allows us to phenotypically score for the presence of transgenes in our fly stocks. There may come a time when you make a mistake (just how I did which lead me to write this page) and you need to check for the transgene using PCR. Alternatively, you may also need to run PCR when you are trying to recombine two transgenes onto the same chromosome.

MedTech Gas Ordering

Mon, 06 Apr 2026 17:20:25 +0000

The current gas czar orders our general use lab gas from MedTech (not Airgas) using their website.

Login

https://medtech.synergyomni.net/User/Login

user: 485196

pass: 485196

Notes

As of 04/2026 we pay for this using the HHMI procurement card (not an RBA/standing PO).
We order “H” cylinders typically, but the tank farms will fit the larger “J” cylinders as well.
Using “QuickEntry” is probably the easiest way to, order. Just use the codes below:

CO2

Lab Website

Thu, 05 Mar 2026 16:07:43 +0000

As of 2026, Harvard Medical School uses Drupal for managing websites. The official guide can be found here: https://drupal.harvardsites.harvard.edu/site-management

To edit the website, Rachel (or the current Website Czar/lab admin) must add you as a New User. Note that only Editors and Administrators can “Publish” edits, Authors can only draft changes that must be approved by another user: https://drupal.harvardsites.harvard.edu/managing-users

Then login via the “Admin Login” at the bottom right of the page using your Harvard Key credentials. From there, editing should be relatively straightforward!

Lab Calendars

Fri, 13 Feb 2026 15:39:53 +0000

All shared calendars should be run through the flypokers (wilson lab) account Lab Gmail (flypokers)

Currently, the lab calendars include:

Rachel’s Calendar: for scheduling meetings
Lab Away: for noting when you are out of lab for >1 day (I.e. vacation, doctor appointments, etc)
Birthdays: for past and present member birthdays
Berg 1-4 Schedules: for keeping track of day-to-day use of 2-photon rigs

To add yourself to any of these calendars:

Log on to the flypokers gmail account
Navigate to Google Calendar
Find your calendar(s) of interest and select “Add people” and share with full changes and managing permissions

Writing a Thesis in the Program in Neuroscience (PiN) – Harvard GSAS

Thu, 08 Jan 2026 15:25:13 +0000

GSAS provides requirements and formatting guidelines for PhD dissertations. Currently, PiN does not impose additional requirements beyond GSAS standards, though there is general consensus that dissertations should contain necessary components and follow formal formatting.

Word#

Harvard offers suggested formatting through LaTeX, but LaTeX can be challenging to learn and use. To address this, I’ve created a Word document that replicates Harvard’s recommended dissertation formatting. While there is no single “required” format, this template has been shared with several students who found it helpful, and it has been approved by multiple committees.

Illustrator Tips

Wed, 07 Jan 2026 16:34:16 +0000

Philosophy & Getting Started#

Start Early, Practice Often

It’s never a bad time to generate clean, pretty figures
Start learning early—it will pay off tremendously during manuscript preparation
Sketch your figure layout on paper or digitally first
Create a Sandbox file to experiment with new designs and variants
- This can also be helpful when making multiple variants of a schematic for a talk or poster
Typically easiest to import data as SVGs (vector format)

Manuscript submissions

Tue, 06 Jan 2026 18:08:28 +0000

HHMI rules#

New manuscript submissions to a journal must include a “standard notice” from HHMI (see attached doc).
Ask Rachel to use this tool to determine how HHMI policies apply: https://workspace.hhmi.org/app/task/compliance-wizard and to use this tool to determine if our HHMI budget can be used to pay journal fees: https://workspace.hhmi.org/app/task/journal-search
if we upload our final draft, HHMI will submit it to PubMed Central on our behalf: https://workspace.hhmi.org/app/dashboard

NIH rules#

The NIH Public Access Policy was revised last year to implement a zero-embargo requirement, requiring that federally-sponsored manuscripts accepted for publication on or after 7/1/25 must be deposited in PubMed Central and made publicly available immediately upon the official publication date with no embargo period. However, some journals are fighting this: e.g., Springer Nature titles now require authors to buy “gold” full OA (the Published Version of Record) directly from the publisher at substantial costs (e.g., 9.500 EURO for Nature) if they need to deposit an unformatted author-manuscript version of a paper in PubMed Central to meet NIH requirements. The following resources can help you identify publishers with green OA policies that comply with NIH requirements:

Behavioral Genetics Experiments

Wed, 05 Nov 2025 20:24:37 +0000

Behavioral genetics typically includes experiments to optogenetically or chemogenetically activate or inhibit neuron(s) of interest. Here is a general list of best practices when approaching these experiments.

Behavioral Genetics Strategies#

Activation Strategies#

CsChrimson: red-shifted channelrhodopsin best targeted with 590-700nm light between 20-90 μW/mm². Can be tagged with various fluorophores, we have CyRFP3 and mVenus in house

Inhibition Strategies#

GTACR: anion channelrhodopsin best targeted with ~520nm light with ~2 mW/mm². Can be tagged with various fluorophores, we have EYFP in house.
- caveats: all experiments done in darkness (panels activate ACR). Required light intensity often prompts significant control effects. Dependent on local chloride dynamics as well
HfACR/ RubyACR: red-shifted anion channelrhodopsin best targeted with ~660nm light with ~20 μW/mm². Can be tagged with various fluorophores, we have EYFP in house.
- caveats: newer reagent, Sophia/Victoria beginning to use
Shibire: temperature-sensitive gene that, when heated, the gene’s product (dynamin orthologue) is disrupted, leading to a halt in synaptic vescle recycling and inhibiting synaptic transmission. Permissive temperature is 19-22 deg C, and restrictive temperature is 29-31 deg C.
- caveats: temperature can be challenging to control perfectly accurately
Kir: inwardly rectifying potassium channel.
- caveats: permanently silences neurons, potential developmental effects/compensation
Tetanus toxin light chain (TeTxLC): permanently blocks synaptic transmission, silencing neurons.
- caveats: permanently silences neurons, potential developmental effects/compensation

As a reminder, there are specific versions of each of these genetic strategies, be conscientious when selecting a variant. For example, different variants of GTACR have different properties.

Flystocks database

Sat, 30 Aug 2025 20:22:56 +0000

We maintain a list of “key shareable stocks” in our flystocks database.

Key shareable stocks are stocks that other lab members might be interested in using.

These include:

balancer stocks
effectors (UAS-GFP, LexAOp-Chrimson, etc.)
wild type stocks (these are often used on constructing genetic contol genotypes)
useful mutations (e.g., norpA mutants, which are blind)
especially common or useful Gal4 or LexA lines (e.g., for driving expression panneuronally, or in P1 neurons, or EPG neurons, etc.)

Please keep your list of key shareable stocks up to date.

LED Rings

Wed, 04 Jun 2025 15:29:16 +0000

LED Ring hardware / software setup description.

UPDATE 20260206, this information is being migrated to a github repository https://github.com/wilson-lab/led_ring_arena

Hardware

For 40 LED ring:

cut filters / diffuser paper to 143 mm x 39 mm, then trim to size once in holder

There is a small channel in the base of the ring that will hold the filter paper roughly in place as you tape it. You can use transparent tape first to hold everything in place. Then trim off excess with a sharp blade.

WLU: Signal processing

Wed, 16 Apr 2025 15:14:01 +0000

Slides and references from April 16, 2025 WLU on signal processing by Helen Yang

Topics#

Measurements = signal + noise
Analog vs. digital signals
- Sampling and quantization
- Analog-to-digital conversion
- Aliasing
Filtering
- As convolution
- Causal vs. acausal filters
- Types of filters: low-pass, high-pass, band-pass, notch
- Frequency domain vs. time domain
- Applications of filtering
Circular data

Slides#

📎 250416_WLU_signalProcessing.pptx

References#

Smith, Steven. The Scientist and Engineer’s Guide to Digital Signal Processing. California Technical Publishing. 1997

Unexpected visitor protocol

Wed, 19 Mar 2025 20:36:23 +0000

If visitors appears on campus claiming to be a government official or a law enforcement official, protocol is as follows:

HUPD should be called immediately (617-495-1215).
Harvard Office of General Counsel should be called immediately if a search warrant is presented (617-495-1280); they have the role of evaluating the legal grounds for the warrant.
Visitors should be escorted to a conference room if they are already inside a building.
HUPD should mediate all interactions with the visitors.
If the visit is after hours, HUPD can contact OGC directly.
General guidance on responding to law enforcement requests for access to university spaces (Harvard OGC)

Safety planning:

"Quick Fix" Meetings

Fri, 04 Oct 2024 15:09:25 +0000

“Quick Fix” meetings are generally held on Fridays at 11am, in our “journal club” time slot. The goal of “quick fix” or “10 min brainstorm” meetings is for each person in lab to have the opportunity to crowdsource a problem that they are facing to the lab. Anyone can ask a question on a single topic. Attendance is optional. Please feel free to attend and ask, or attend without asking. This is a great way to learn about the nitty gritty issues underlying the techniques we use in the lab.

Scalebars with connectome volumes

Wed, 21 Aug 2024 15:44:33 +0000

How to fetch volume dimensions (in nm)#

To fetch the dimensions of the entire brain neuropil (in nm):

boundingbox(FAFB14NP.surf)

Note - Once you know the x dimension of the full brain neuropil in nm, you can then generate a scale bar in um over a length that makes sense for your plot (e.g., 50 or 100 um)

Note - the brain used for the connectome is dehydrated, meaning that it is slightly smaller than what you would expect with a hydrated brain (e.g., confocal stack).

SendCutSend

Wed, 14 Aug 2024 21:20:10 +0000

SendCutSend is a service for cutting and processing 2d materials, including a range of plastics and sheet metal. They can perform a range of post processing steps like bending, tapping, countersinking, anodizing, and powder coating. It is also fast and cheap.

Lab account:

Username: flypokers@gmail.com Pw: OregonR?

You can organize your files into folders based on project/device/category.

When ordering, please make sure you select tax exempt during checkout. The tax exempt status on the account was registered using an HHMI ST-5 certificate, so I believe one should use HHMI funds (Card, PO).

Data Acquisition WLU 2024

Wed, 15 May 2024 14:52:41 +0000

2-Photon Data Acquisition Wilson Lab University 2024

Google Slides from presentation that Carl and Stephen made… please edit and improve this as a resource.

https://docs.google.com/presentation/d/1VHHnvDD-fCE0xmLnoHl_uX-K9oXo-RBOMWNeGEMO3oI/edit?usp=sharing

Planning notes:

https://docs.google.com/document/d/1YrA8rXMlU15x0A-EBOeNKDhFaCZS_9coCPl-o5mUOgg/edit?usp=sharing

210922 WLU on 2P imaging

Mon, 29 Apr 2024 13:36:12 +0000

WLU presented by Stephen Holtz and Helen Yang on September 22, 2021

Topics covered:

Fluorescence imaging: theory and approaches
Parts and function of a two-photon microscope
Safety
Equipment use and care

📎 WLU_2PLSM_09_22_2021.pptx

2-photon microscope configurations and information

Thu, 28 Mar 2024 17:22:08 +0000

This page is meant to keep the current configurations of all the 2-photon microscopes we have in an easy to access way. Information useful for maintenance or troubleshooting, like serial numbers, who used them in the past, RMA numbers, and when components were modified. Try to maintain all sections as you make changes, add in notes if something was repaired or is not functioning properly. Try not to add sections that are under flux unless necessary.

Lab Birthdays

Wed, 06 Mar 2024 17:34:19 +0000

Jingxuan: Feb 14

Elena: Feb 21

Noah: March 26

Janki: April 11

Matt: April 13

Fernanda: April 15

Helen: June 14

Allie: Aug 5

Carl: Aug 12

Stephen: Sept 10

Sophia: Sept 15

Alex: Sept 23

Diego: Sept 23

Pablo: Sept 24

Rachel: Oct 23

TTX (tetrodotoxin) SOP/SDS

Tue, 13 Feb 2024 20:25:12 +0000

TTX IS A FEDERALLY REGULATED “SELECT AGENT” AND REQUIRES TRAINING PRIOR TO USE

Standard Operating Procedure (SOP)#

We are required to keep a SOP by EH&S, failure to follow the regulations in it may result in us no longer being able to use TTX or (worse) we may be subject to random inspections.

We keep the SOP as a google doc so it can be easily printed and sent:

https://docs.google.com/document/d/1XUTlD5VZ9RBVUSnxm2s8P6kcZLQbBFMHAn1k1jkMw5I/edit

240207 WLU: Making transgenic flies

Wed, 07 Feb 2024 19:29:16 +0000

Slides from WLU

📎 240207WLU_MakingTransgenicFlies_v2.pptx

Spreadsheet of attP sites and their genomic locations

📎 attP landing sites.xlsx

Fluorescence in situ hybridization (FISH)

Wed, 03 Jan 2024 21:50:00 +0000

Our protocol is a concise 2-day RNA FISH protocol. The goal of FISH is to detect the existence and/or quantify the abundance of an RNA of interest in the whole-mount adult fly brain. Our protocol is adapted from the one originally reported in https://doi.org/10.1016/j.ymeth.2017.06.025.

Probe design#

Our probes come from a company named Biosearch Technology. The principle of their probes is: a library of 50 oligos will be used to hybridize onto your RNA. Each oligo is 18-22nt long. Only when most of the 50 oligos bind to the RNA molecule will the fluorescence shine. This technology has greatly improved the SNR.

BANC

Fri, 15 Dec 2023 13:24:59 +0000

BANC is the first full female Brain-And-Nerve-Cord volumetric EM data set. It was generated by Wei-Chung Lee’s lab, and segmented by Zetta AI. The FlyWire team at Princeton is responsible for systematic proofreading, beginning with DNs and ANs.

To get access to the BANC, please ask Rachel to contact Wei or follow this link to request access through Flywire. Note a few restrictions:

We cannot publish reconstructions from the dataset until Wei and his lab do so, or until December 31, 2024, whichever comes first.
Anyone using the BANC data prior to the first publication from WCL also must agree to include WCL and WCL lab members that generated the dataset (Jasper Phelps and Minsu Kim) as authors on their publication.
To publish neurons from FlyWire/BANC, a member lab must submit a list by emailing flywire@princeton.edu or through a planned online submission system, and then must make that list available in the paper.

Please see the attached document for full terms of use.

Annual safety (EHS) inspection

Mon, 27 Nov 2023 20:02:22 +0000

Recommendations#

Keep personal care products outside the lab/bench area (store them in your locker or in personal bags).
If keeping flies close to personal desks, make a clear divide between these biological materials and personal stuff.

Preparation for inspection checklist:#

Read and fill out the Lab safety Orientation Checklist (it has useful links to Safety resources).
Make sure there is no food or food-related trash in the lab or lab trash bins.
Make sure “fly morgues are capped”.
Make sure that biowaste boxes/bins are not overfilled.
Make sure that the emergency eyewashes are unobstructed.
Make sure paper towels and soap are provided at the sinks.
Make sure all gas tanks that are not in use are capped.
Make sure people do not have flies on their personal desks.
Make sure you move your plants somewhere outside the lab area (for example, you can put them in your locker).
Make sure apple cider vinegar, nail polisher, and other potentially edible reagents are labeled ‘for lab use only’.

Staying connected (seminars etc.,)

Fri, 21 Jul 2023 19:52:54 +0000

HMS Neurobiology keeps a master calendar of department events.

The Harvard Brain Initiative keeps a master list of all neuroscience seminars in the Boston area.

Neuronet is the internal repository for resources and information about the HMS Department of Neurobiology.

“Who Do I Contact About…” is the NeuroNet page that routes you to the person in HMS Neurobio who can best answer your question.

World Wide Neuro hosts links to neuroscience seminars presented anywhere in the world, available online.

Fly Making Overview (BestGene, Twist)

Thu, 13 Jul 2023 16:11:15 +0000

BestGene can do injection services quickly and return balanced flies in a pretty painless way.

Account#

Our lab has an account to help keep track of past orders.

Username: flypokers@gmail.com Password: wilsonlab

Overview of making a fly:#

To generate flies with a transgene in a specific landing site, you need to first pick one they support: https://www.thebestgene.com/PhiC31InfoPage.do. Note: you can use other sites (but it might be odd given the large selection), but you will need to supply them with the fly and use additional services not mentioned below.
Next, you will need to ship them the plasmids for doing the injection. It is easiest to ship a small amount and have them amplify it.
To ship a small amount of DNA and have BestGene generate flies with your transgenes in a specific landing site, you would use:
- Service I: PhiC31 Premium - Plan H plus balancing (ship balanced line)
  - This means the only flies you will get are balanced, there are other options which are faster but will require more work on your end (e.g. plan H). Make sure you understand what they’ll be sending you.
- Service Z1: Regular plasmid DNA Midiprep. One midiprep is enough for 3 “Service Quantity”
  - This allows you to send a smaller amount of DNA they can use for multiple injections.
Fill out their order form (link at the top of their page), and attach a Purchase Order for the amount of money the order will cost. Alternatively, you can add a credit card to the account and have them charge that (but be careful! this can get expensive and you don’t want to hit the limit).
Note they will want you to tell them the restriction enzyme sites to use for their quality checks - you will want to check that they work ahead of time. For the pJFRC7 and pJFRC19 uploaded to Twist, it appears that NotI and Xba1 work well.
Ship them the DNA and plasmids, and wait patiently for the flies to trickle in.

Other notes#

Their purchasing page has a list of other services and pricing here.
Also see the page on WellGenetics /fly-stocks-and-crosses/wellgenetics-services/

Spreadsheet of attP landing sites and their locations

Room scheduling

Wed, 12 Jul 2023 15:10:22 +0000

Instructions for scheduling rooms can be found here https://neuro.hms.harvard.edu/resources/room-scheduling. You need access to “neuronet” to see this page, and should be prompted to request access when you login to the page.

However, it just redirects to https://calendars.med.harvard.edu/ - which you can login to with Username: neuro Password: harvard.

Note to book Arm 108 you need to email neuro_ops@hms.harvard.edu (Steve and Josh).

The door to each room now has a QR code that will take you to the reservation calendar for so that you can book a room in real time. Note that reservations for Goldenson 122 and Warren Alpert 236 will require approval before they’re considered final.

Benchling (storing biological sequences)

Wed, 12 Jul 2023 12:46:30 +0000

The lab has a “benchling” account for storing (in silico) your genetic sequences like transgenes, plasmids, primers, and so forth. This is very useful as a centralized reference and location to start building from.

https://benchling.com/organizations/wilson_lab_hms/

For generating transgenes our current best practice is to upload an annotated transgene to a nicely named folder in “Wilson Lab Projects (Main)” project. When you put this into a plasmid, it would be good to also add that plasmid to the site in a corresponding folder, and even better to re-annotate it if the annotations have been lost.

Plasmid Generation

Wed, 12 Jul 2023 12:38:06 +0000

Note: for having transgenic flies made see:

/fly-stocks-and-crosses/wellgenetics-services/
or, consider making a BestGene page to document that service (preferred by some).

Genscript#

Genscript will synthesize and clone plasmids for generating flies. Compared with Twist Bioscience, they are somewhat more expensive and slower, but they will synthesize sequences that Twist will not. They are probably your best bet if you’re trying to synthesize enhancer elements (to make driver lines). Genscript also does custom cloning if you already have your DNA of interest and just want to move it to another vector.

Controlling enclosure temperature and humidity

Wed, 14 Jun 2023 19:16:11 +0000

Work in progress - NP 2023-06-14

It is sometimes advantageous for fly-on-ball behavior to have the behavior enclosure warmer and more humid than room temp. It is also useful to be able to log ambient temperature and humidity in the enclosure as an experimental variable.

There are a lot of way to achieve control and measurement, but here is a system that has worked for me:

Heaters and humidifiers#

Get a heater and a humidifier. These would ideally be fairly bare-bones as we’re going to switch them on and off based on the measured temp/humidity. Built in thermostats may be factory tuned with some fixed hysteresis that might be larger than the narrow range of temp that we want to keep the enclosure at.

Image analysis resources

Wed, 24 May 2023 20:20:27 +0000

Pete Bankhead’s Introduction to Bioimage Analysis is a good place for beginners to start.

The Harvard Image Analysis Collaboratory in Armenise 531D is available for in-person consultations on image analysis problems.

Image.sc is an online discussion forum for image analysis problems.

Personnel-funding assignments

Sun, 12 Mar 2023 14:20:33 +0000

When making purchases for the group, please use HHMI funds when possible.

When making purchases specific to your project, please use the following table:

Open embedded document

Fly Food Supplies

Tue, 07 Mar 2023 16:57:40 +0000

1. Vials#

We get empty wide vials from:

LabExpress - https://www.lab-express.com/flyfoodsupplies.htm

Catalog No. 8008-10: Tray-packed wide vials, polystyrene, 100 vials/tray, 10 trays/case.
5 tray option also available

Genesee Scientific - (Cat #: 32-110), material “Polystyrene”, packaging “Tray Packed”, size “Wide”.

If backordered, try other material options “polypropylene” or “K-resin”. The material does not matter that much.
polypropylene is what our fly-food comes in, and it is a bit more durable for large containers.

Protolabs

Mon, 23 Jan 2023 03:37:59 +0000

For 3D Printing and CNC Machining common lab supplies.

Please keep projects organized and separate different design files.

https://buildit.protolabs.com/projects

Email: flypokers@gmail.com

Password: OregonR?

Please note: As of 3/1/2023, HHMI requests that all Protolabs requests be processed through the P2P purchasing system and not paid for with the ProCard. To do this you need to get a quote for your order and send it to someone with access to the P2P system (e.g. Stephen). There should be a “download PDF” button somewhere on the page after you’ve finished configuring the part.

Notes on setting up and de-noising an imaging rig

Mon, 16 Jan 2023 18:24:49 +0000

Most of these tips are based on advice from Stephen Holtz when Habiba and Stephen revamped Berg4, so a big thank you to Stephen!

General principles/tips#

When setting up a rack (and this isn’t specific to imaging), it is worth the extra time to consider ergonomics, placing equipment in convenient locations (ex: at eye level), and practicing good cable management. The rig is something you will likely be using daily so putting in the extra time in the beginning can save a lot of time in the future.
This might sound obvious, but try to have all equipments/DAQs on the same side of the rack so you can see everything and where all the connections are going. This will also allow you to have shorter cables.
It is useful to have the bottom portion of the side of the blackout box closest to the rack covered with a curtain that you can flexibly snake wires through. You can then velcro the curtain to the air table. To make sure the side curtain is completely blacked out, you can make the curtain extra long and use magnets to drape over the side of the air table.
If possible, have the imaging-related cables exit the box separately from the other cables (such as behavior cables). For example, on Berg4, all the imaging cables exist from the top of the box and all the behavior cables from the side of the box. This is helpful for cable management and it also decreases the likelihood that you will pull on an imaging cable when moving behavior cables around.
The air table is designed to reduce motion, but if you have anything coupled to the air table that isn’t on the air table, this can introduce motion. So to reduce motion, make sure nothing rigid is coupled to air table. This also means that you shouldn’t to make the cables going to/from the rack and air table too short.
It is really useful to set up your rack so that you have shelves right under power strips. This will allow you to place power blocks on the shelf since they shouldn’t be on the floor. This also helps with organization and preventing the cables from dangling precariously and potentially getting unplugged.
You can use 1ft power extension cables to connect power blocks to the power strips without having them take up extra room and block other power inlets on the power strip.
Make cables as short as possible. This is especially important for data cables such as the PMTs. But make sure the cables coming to/from the rack aren’t too short that they would pull on the air table and produce motion.
Separate the main bundles of power strip and data cables along different sides of the rack. Since it is usually easy to get extension cables for the power strips, consider bundling them along the further end of the rack (from the air table). You can then bundle any data cables on the opposite side of the rack (closer to the air tables so that the data cables don’t have to be too long)
BNC paththroughs (ThorLabs part BNCB2) are very nice for mounting the PMT cables to the rack so that they are held securely in place and to reduce the amount of cable that is precariously dangling.

3D Printing (in house)

Wed, 11 Jan 2023 16:01:21 +0000

Overview#

How to use our resin 3D printer, wash and cure station in the WLI TOBIN.

Hardware#

We currently have an ELEGOO Mars 5 printer and an ELEGOO Mercury Plus wash and cure station.

Preparing your design#

What you will need:

.stl 3d file(s) of your part(s). Can be exported from many programs: e.g., Onshape, Autodesk Inventor) but should be exported as a format in mm (the default for the ‘slicing software’).
.goo print file to upload to the printer. This is the configured print as it will happen on the printer, and can have many different parts with different orientations. Currently the best software to use is Lychee Slicer https://doc.mango3d.link/
- NOTE: you can use CHITUBOX Basic for the Mars 5, but newer versions have removed the option to use anti-aliasing from the basic edition. This is quite useful for most of the things we print.

Mars 5 + Lychee Slicer Tips#

This software operates very similarly to CHITUBOX a screenshot of the very conservative settings for the resin/print when using the ABS-like V3 (grey, not green as it says in the screenshot)

Loading dock

Thu, 01 Dec 2022 18:17:41 +0000

The Warren Alpert loading dock’s address is 200 Longwood Ave.

The loading dock hours of operation are from 7:30am-4:00pm.

You may arrange to have deliveries made to the dock before 9:00am and after 11:00am by calling 617-432-1718. If you are coordinating a move or have a delivery that will require the use of the dock for more than an hour, it is necessary that you give as much advance notice as possible. If there are any issues please call Devonne Countryman at 617-432-7909.

Planning fly work around time away from the lab

Wed, 16 Nov 2022 13:53:29 +0000

Here is how to minimize the disruption to your fly work (and therefore your experiments) when you plan to be away from the lab for many days.

Crosses for Building New Stocks#

For any crossing scheme, you should have the entire plan written out before you start, so you should know when you start that you won’t have a stable stock by the time you’ll be away. Assuming you’re diligent about collecting flies and have enough copies of the cross at each step, two weeks per generation is a good estimate for the timeline.
Once you’re about one to two generations out from the time you’ll be away, it should be clear when you’ll have the parents for the cross that will overlap with the time you’ll be away. That is, if you were to continue on your current trajectory, will they eclose while you’re away? Or will you have them collected 3 days before you leave?
The ideal situation is to have the cross set up before you leave and to have enough vials to collect from when you get back. There are a couple ways to do this:
- Make sure you have enough parents in hand to set up the desired number of copies of the cross about a week before you have to leave. Set up the crosses at 25 deg, and flip the crosses after 2-3 days, as usual. After at least 2 days at 25 deg for any given vial, move the vial to 18 deg. (Setting up a cross at 18 deg will work, but crosses do much better when the parents are given a few days at 25 deg to mate and lay eggs.) Flies take 2X as long to develop at 18 deg compared with 25 deg. Importantly, this applies even if you move the flies from 25 deg to 18 deg; the remaining development time is approximately doubled. For example, a cross started at 25 deg for 3 days and then moved to 18 deg will take about 15-17 days total to eclose; a cross started at 25 deg for 1 week and then moved to 18 deg will take about 12-13 days total to eclose. In other words, by setting up the cross early and then moving various flips to 18 deg after an appropriate amount of time, you can make sure the flies don’t eclose until you get back while ensuring you have enough vials to collect from. With this approach, it is better to delay the crossing scheme by one generation to ensure that you have enough parents several days before you leave than to push through one more generation.
- Set up the crosses and ask a labmate to flip them for you every few days while you’re away. Depending on how long you’ll be gone, asking them to move earlier flips to 18 deg can ensure you won’t miss any virgins.
- If you’ll be gone longer than about 2 weeks, the first approach doesn’t work very well, as the flies will eclose before you get back, and the second approach may be less than ideal as the earlier flips will eclose before you get back and the later flips may not produce as many progeny as you need. You can instead collect the parents in separate vials and keep them at 18 deg, and then ask a labmate to put them together and flip them every few days on a timeline that means you’ll be back before the flies eclose.
- Of course, the last option is to ask a labmate to continue the crossing scheme as if they were you, collecting virgins and genotyping and such as needed. However, as elaborated on below, this would be a very big favor you’re asking for.
If you need virgins from a stock for future crosses, remember to expand those while you’re away as well. The first 2 approaches above work just as well for stocks in vials or bottles.

Experimental Crosses#

You can get clever with temperature for your crosses for building stocks, but I don’t recommend doing that with your experimental crosses, as that will introduce a variable you really don’t want to have to worry about.
Instead, ask a labmate to flip your experimental crosses for you.
Set up new copies of the crosses right before you leave, so they won’t die out before you get back.
If you’ll be away for long enough that you’re concerned the cross will die out before you get back even if you set it up right before you leave, collect the parents, keep them at 18 deg, and ask a labmate to set up the cross some time into your time away such that they eclose right when you get back.

Stock collection#

Be aware of when your stock collection is due to be flipped, so that you know in advance that it’s due when you’ll be away
If that’s the case, you should probably flip your collection early, before you leave. If it’s due only a few days before you get back and your usual flipping schedule incorporates that amount of leeway, then flipping late is okay.
Flipping so early that the first progeny haven’t yet eclosed (less than 12-14 days from the date of last flip for stocks on white food at room temp) is not a great idea, as you’ll be relying on old flies to propagate the stock instead of new ones. Being in this situation (i.e. flipping when you get back is too late) means you’re probably away for >3 weeks, so hopefully, you know about that well in advance. I recommend shifting the previous flip early as well as the current one so that each flip has enough time.
If you shift the flipping schedule for one copy of your stock collection, you should shift the flipping schedule of the other copy correspondingly so that they stay optimally staggered.

Asking Your Labmates to Do Your Fly Work#

Asking your labmates to help you out with your fly work when you’re away can prevent you from waiting for experimental flies or from failing a multi-generational crossing scheme. You should definitely ask them for help. That being said, some types of fly work are small favors and some types of fly work are big favors, and you should be cognizant of what you’re asking.

Notes on known issues with Berg-2

Fri, 21 Oct 2022 19:48:27 +0000

I. COM port errors from scanimage – AKH, August 2022

Problem description: scanimage throws an error at start-up that one of the COM ports for the mirrors is not available, despite the fact that the COM port in question shows up in the windows device manager.

Troubleshooting notes: SH and I first changed the COM port numbers in the machine data file from 5–>81 and 5–>80. That didn’t hold upon restarting, but we then set them to 020 (Port_#0006.Hub_#0001) and 021 (Port_#0014.Hub_#0001) and that ultimately worked. We made this change in both the windows device manager and the machine data file (see the code snippet below). We also had to reverse the direction of one of the mirrors (MAIN CONTROLS > File > Machine Configuration > ThorLabs BScope1 Set up). After making this change, we restarted the computer several times and it eventually “stuck.”

Labeling with neurobiotin and biocytin

Mon, 22 Aug 2022 23:01:22 +0000

There are two compounds commonly used to dye fill cells during whole-cell patch-clamp recordings: neurobiotin and biocytin (although many alternatives exist, such as lucifer yellow, horseradish peroxidase, and Alexa 488). Conventional wisdom holds that neurobiotin and biocytin are the smallest and least toxic. Here is a dump of what I currently know about each based on personal experience and talking with lab members:

Neurobiotin (from Vector Labs)

Fills cells far more completely, and faster, at lower concentrations.
Possibly some toxicity, although this seems to be cell-type, and concentration specific.

Biocytin (from Fisher)

Spectra-Physics Laser

Mon, 08 Aug 2022 19:21:04 +0000

This is the laser that is split between Berg 3 and Berg 4. It is controlled by a laptop on the Berg 4 rack as of 03/2024.

General#

Model: Mai Tai HP 2040S with DeepSee

Serial: 7056-EV

Service#

Chiller#

The chiller should be set to 21C and maintain this temperature. **If the chiller is not maintaining this temperature then try to 1) bring the room temperature down, and/or 2) clean the chiller using fluids under the sink in WAB 305. The chiller fluid must not be allowed to get too low, and the fluid should be routinely drained and and cleaned (~every 6/months). During draining and cleaning the laser should be off.

Replacing halogen lamps on dissection scopes

Wed, 13 Jul 2022 15:33:37 +0000

This is relevant to dissection scopes that use Halogen light sources (not the newer ones that use LEDs), specifically KL 1500 LCD light sources (manual).

You might need to replace the lamp if the light stops working and you get an Err1 (see page 18 of the manual for more details).

These are the compatible halogen lamps for this model (page 20 of the manual). We currently use the Osram 64634 HLX 15V 150W lamps.

Registration using CMTK plugin in FIJI

Thu, 30 Jun 2022 13:14:11 +0000

by Isabel D’Alessandro and Emily Kellogg

Registering a Light-Level Stack to a Standard Template Brain #

You can use CMTK to register your light-level confocal stack to a standard Drosophila template brain. Follow the instructions here to install the CMTK Registration GUI Fiji plugin. The Fiji GUI seems only to work on Linux (and not on Windows), so you can use a Linux virtual machine to run this GUI. Alternatively, if you install CMTK on your Windows system (see forum discussions here and here for how to do this), you can run the same programs from the command line. There is a guide on Google Drive that is linked here.

Offboarding

Tue, 28 Jun 2022 17:54:28 +0000

This page is meant to contain instructions/guidelines for what to do as you leave the lab. Please add more sections as needed.

Fly Stocks#

Please discuss your fly stocks with Rachel, to determine which stocks (if any) should be kept in the lab, and how to distribute them accordingly.

Slack#

Under our current slack plan (Pro), workspace administrators (Karen, Stephen, many others) can export a copy of all Public channels, but cannot for private channels, or DMs. If you would like a copy of the current history please ask, it may take a day or so for slack to finish exporting to a zipped filestructure full of .json files for each day of active messages within each channel. You can import this into another slack workspace you make yourself if you want to browse it more easily.

Berg status

Fri, 29 Apr 2022 15:44:13 +0000

Here, we can keep track of key measures from our two-photon microscope systems, in order to monitor for problems and understand the past history of the microscopes:

date	berg	power at sample at 100% (milli Watts)	Extinction ratio (power out of Pockel cell at 100% / power in at 0%)	Pockel cell holding voltage	Alignment assessment
04/27/2022	III	115	537 (537/1)	-0.52	Workable. Re-alignment needed at low priority
12/16/2022	IV	117		-0.29	Quick alignment starting at the periscope because power at sample was 70mW
1/26/2023	IV	~150	~800 (goes up to 1000)	-0.31	Much improved beam shape and power! Full realignment starting at the laser aperture, trhough half wave plate, P cell and allignment inside the scope. Fixed scanimage calibration issues to improved read out extinction ratio.	Will add picture later, pretty circular

Notes on getting robust behavior

Fri, 15 Apr 2022 15:25:58 +0000

Suggestions on how to improve behavior at each step of the dissection/preparation process.

A healthy, intact, freely walking fly will walk at 10 mm/s or more, with rest periods clearly delineated from walking periods, so that the histogram of forward velocities is clearly bimodal (see e.g. DeAngelis et al. 2019, Figure 1). On a spherical treadmill setup, this should also be visible, as in these data from Gaudry et al. 2013, Supplementary Figure 1; note also that the distribution of lateral (sideways) velocities is fairly symmetric around zero:

Notes on Saline

Wed, 13 Apr 2022 21:52:11 +0000

This page is meant to document the results from a series of experiments under different saline conditions, as performed by Matt Collie in the spring of 2022. Behavior was assessed based on 1) the fly’s directional velocity distribution and 2) the fly’s engagement with the stimulus (in this case, chasing an oscillating dark bar).

Quick disclaimer - many of these experiment conditions have a sample size of 1 or 2, so please take findings with a grain of salt.

Gas Flow (in 2p and main lab)

Thu, 10 Feb 2022 22:32:43 +0000

This page is meant to explain exactly how gas gets from your tank to your rig. For the most part, we all have a functional understanding of what knobs we can turn to change the pressure at our rig. However, with a stronger understanding of how gas actually flows from tank to rig, hopefully this page allows you to:

a) hook things up properly

b) troubleshoot when something feels awry

G4 Arenas: Set up notes

Fri, 22 Oct 2021 17:52:46 +0000

Introduction#

In general, the Reiser Lab Github has thorough documentation for assembling and configuring the G4 arena. This guide is meant to help with supplementary information and tips. The section headers and organization of this guide match the section headers and organization of the github page, so that both can be used simultaneously. If you publish expleriments using G4 Arenas, please cite Isaacson et al. (2022) and link to the Reiser Github page.

Applying Dispase

Mon, 30 Aug 2021 17:53:13 +0000

Purpose#

Technique used when one needs to patch a cell body that is typically occluded by a tightly-associated glial sheath. This type of sheath cannot be removed by traditional de-sheathing or cleaning techniques, and is more common in cells that are more superficial in the brain. You can identify whether your cell is sheathed based on the appearance (cell body appears textured rather than smooth, with small dark puncta on the surface) and whether it is even possible to break in to the cell even with a good seal and large pipette tip.

Laser permit

Wed, 21 Jul 2021 14:31:04 +0000

📎 Laser permit.pdf

Laser Permit

Wed, 21 Jul 2021 14:30:26 +0000

📎 Laser permit.pdf

neuprint-python

Tue, 20 Jul 2021 10:35:01 +0000

contributed by Habiba Noamany & Rachel Wilson

You can query the neuprint hemibrain dataset programatically using Python.

If you’re starting from scratch, begin by installing miniconda from here.

For Windows, open Anaconda Prompt. For Mac, open the terminal.

Enter:

conda create -n neuprint
conda activate neuprint

Next, install neuprint python using conda (documentation here):

conda install -c flyem-forge neuprint-python

Then install Jupyter notebook, matplotlib for plotting, pandas for interacting with the hemibrain data, numpy for matrix manipulations and some other plotting software used in the neuprint-python tutorial:

Balls for behavior

Sat, 26 Jun 2021 16:06:08 +0000

All of the current lab members use 9 mm diameter foam balls for behavior, although in the past some people have used 6 mm balls, and also used different plastic materials.

We now make the foam balls ourselves. We obtain the foam from General Plastics, by requesting a “sample kit” of blocks of various foams. We usually use FR-4615 foam (although we have tried FR-4510 in the past - this is a more porous foam than FR-4615). Foam samples are stored in the behavior hardware drawer in the main lab (under the soldering station).

Berg-2 operation

Fri, 25 Jun 2021 20:27:03 +0000

Berg-2 has two computers, Wombat, which operates fictrac and MATLAB scripts, and Cassowary, that operate ScanImage. Typically, Cassowary awaits a trigger from Wombat in order to start an imaging experiment. Wombat will extract experimental variables (e.g. ball movements via fictrac) and send commands to deliver, for example, optogenetic stimulation. Cassowary only handles the imaging. Berg-2 has two mirror paths to the sample, one for the laser and one for its LED illumination. The code that Michael uses for Wombat can be found here.

Fly Incubators

Wed, 23 Jun 2021 19:23:15 +0000

The following jobs fall under the purview of the current Incubator Czar.

Current Incubators#

Each incubator was named after a character from the 1989 cult classic Bill and Ted’s Excellent Adventure.

Bill#

25*C, 8am-8pm
Temp/Humidity Control - house air/water

Ted#

25*C, 4am-4pm
Temp/Humidity Control - house air/water

Joan of Arc [primary]#

25*C, 8am-8pm
Temp/Humidity Control - house air/water

So-Crates#

18*C, 8am-8pm
Temp/Humidity Control - water pan

📎 Incubator Names.pptx

Maintenance#

The incubators should be cleaned every 6-12 months.
The transducer used by the humidifiers is a consumable, and should usually be replaced every 1-2 years.

Cleaning#

Darwin Chambers suggests using white vinegar for cleaning. Gloves should be worn at all times.

Iontophoresis

Mon, 31 May 2021 20:36:04 +0000

Iontophoresis can be used to expel or hold charged particles. Using a glass pipette, we can use iontophoresis to emit a solution into the brain. Because it does not rely on mechanical force, this can be used in conjunction with smaller diameter pipettes than mechanical air pump methods.

The solution#

When preparing a solution to be used with iontophoresis, you must be aware that the particle you wish to emit must be charged, and all particles you wish to emit must thave the same charge. For example, ATP in solution is a weak acid with a pea value of ~6.5, meaning that half of the ATP molecules are de-protonated, and therefore negatively charged, at a pH of 6.5. We therefore also need an acidic fluorophore. Alexa hydrazides, dextrans, and Lucifer yellow are, like de-protonate ATP, anions, so you need to pass a negative current to emit them from the pipette. See here for how to make an ATP solution with an Alex Fluor. Remember to test the pH of the solution. For more information see here.

Lab photos

Mon, 17 May 2021 18:24:19 +0000

At the esplanade for Yvette’s farewell

Celebrating in WAB 326 after Paola’s defense

Sasha’s post-defense celebration

Party train at Barcelona

Wilson lab tattoo artwork (art by Alexandra Moore, arm model Michael Marquis)

Laser Cutter

Sun, 16 May 2021 23:11:48 +0000

The laser cutter can be used to cut mainly 2D designs into acrylic, wood, card and other materails. We have a Universal Laser Systems machine.

Access#

Email Ofer/Pavel (the Instrumentation Core) requesting laser cutter training. They will provide you with a time.
Make an account here: ppms.us/hms-ric
After the training, email Ofer/Pavel your Harvard ID number so you can be given card access to the room.

Making a design file#

You can use many softwares for making a vector drawing (AutoCAD or Inventor) that can be used to direct the laser cutter. For example: Inventor -> .dxf file export -> illustrator to check design is good.
However, it is perhaps most simply done in Adobe Illustrator. Make a vector drawing of the design you wish to cut in a sensibly scaled space, e.g. inches or millimeters.
To make interlocking tabs for 3d builds, you can take tab designs from here: makercase.com.
Note the ‘kerf’. You need to adjust for our 200-micron laser beam. Kerf should be beam diameter divided by two (i.e. 100).
Colors are used to set different defaults of the laser cutter’s laser power and speed:
- Color mode needs to be RGB
- By convention, Red - full on RGB red (R, full, G is0 and B is 0) - full power, cut through.
- Black - etch in a raster, can be used for text.
Lines need to be 1/1000 of an inch or less, otherwise the machine will attempt to raster rather than perform a clean cut.
Multiple lines = multiple cuts, so make sure your lines in your vector drawing do not overlap otherwise they will be widened because the laser will run over the same position multiple times.
Open your file on the computer that is connected to the laser cutter. It should have the most up-to-date Illustrator.
Illustrator -> ctrl+p -> ‘print’ the file, opens in the laser cutter’s software, ready to be sent to the machine (if not just double click on the UCP shortcut in the PC desktop).

The Machine#

The largest 2D material size it can fit is 32’’ by 18’’, the cutter’s max size. Often, the biggest commercial is 2 feet by one foot.
Check plastic thickness with calipers. Remove any top surface paper. Keep on paper at the bottom, which protects from dirty honeycomb.
Turn the machine on by hitting the switch on the bottom right side. Turn on the airflow to the beam head by turning the tap at the back of the machine by 90 degrees.
The software that controls and sends designs to the machine is Universal Laser Systems control panel UCP.
A design printed from Illustrator will appear as the design you see in UCP. You can drag it around the cutting surface, but you are best served by having it near the origin (the leftmost corner) as this is where the laser power will be strongest.
Within UCP you can set colour meanings. Colour modes run in the order shown on panel. You can load saved settings (already good saved settings for most materials) by selecting ‘load’.
Position design to have a 0.2, 0.2 origin (top left corner), rather than 0,0 to buffer yourself from potentially hitting the edge of the arena, off of your plastic.
Auto Z should be enabled - for planar cutting.
You may need to calibrate the Z position of the cutting plane. For thick plastic, shift focus to be in the middle.
Cutting is not uniform. The closer to 0,0, the stronger the laser will be. Up the power if doing large designs.
Power and speed are inversely linked. decreasing speed by 2 would also increase power by ~2.
If you see small flames that disappear in a few seconds, this is fine. A fire that sustains itself for 10s of seconds can be extinguished using the pressurized air nozzle on a yellow cord, to the right of the room (as you face the laser cutter).
If lines are not cutting through fully, try running a second time after adjusting speed and/or z-axis, for example, increase speed from 3% to 3.5%, and move z from 0.15 to 0.02 (or 0).

Shop rules

Laser permit

Thu, 13 May 2021 17:50:33 +0000

We have three lasers in the lab at the moment (one Excimer, two multiphoton lasers). They get inspected every two years (most recent renewal: May, 2021). Laser Safety Officer is in charge of assembling the relevant documents and communicating with the EHS Radiation Safety.

All the users need to take the laser training online, and renew every two years (including lab members who use the shared confocal facility).

Laser Inspection 2023#

📎 SOP_Class4_Laser_rev2023.docx

Coherent Vision-S laser

Tue, 11 May 2021 15:52:51 +0000

Please log error messages and problems in this log.

11/2025 service updates

Serial for unit: GDP.1180646.10060

Serial for “head” GDP.1001H.1311

Service contract renewals at coherent are all handled by annette.davis@coherent.com

10/21/2022 service note

We identified the issue of causing the error. It is due to motor homing problem every time when we turn the laser from active to standby. The motor sometimes doesn’t home correctly so the output wavelength can be off from the required wavelength quite a bit (usually more than +/- 5nm is problematic).

Microscope components and alignment

Mon, 10 May 2021 21:48:37 +0000

Background Resources#

General principles of microscopy are covered in an excellent book “Fundamentals of Biomedical Optics” sitting in the Wilson Lab library. Nikon Microscopy U by Michael Davidson is also a great resource.
Laser scanning basics are covered in this Thorlabs Laser Scanning Microscopy Tutorial.
Svoboda and Yasuda 2006 has a nice summary of the principles behind two-photon microscopy.

Components#

There are several optics components that make up our two photon systems. Some differences exist between the systems, but the following core components are shared (and differences noted). A convenient way to break them down is into 1) laser and ancillary components, 2) table optics, and 3) the microscope proper.

Mass flow controller (MFC)

Fri, 07 May 2021 14:59:00 +0000

Mass flow controllers allows us to precisely control the flow of gas, and is especially useful for experiments on olfaction and wind. This page contains way more information that you might need, but it maybe helpful if you see an unexpected behavior when using a MFC.

Choosing a model#

You can choose a max flow rate from a wide range of values. Note that for the Aalborg MFC, the accuracy is ~1% of the max flow rate.

ScanImage Setup (Download + Installation)

Fri, 07 May 2021 14:20:18 +0000

We use ScanImage software to acquire our imaging data (documentation).

Support#

Tickets can be submitted here: https://www.mbfbioscience.com/advanced-support

Drivers and new versions (2023+) downloaded here: https://support.mbfbioscience.com/downloads

user: Rachel.Wilson

pass: flypokers

Email Contact: rachel.irene.wilson@gmail.com

Licensing#

If you are starting on a rig without really ‘dug in’ users please upgrade to use bugfixes and keep use unified.

We need to renew our a license whenever we want to upgrade to a new release. We get a discount if we upgrade with every new release, but we are not obliged to upgrade with every new release. Note that transitioning to 2023 requires contacting ScanImage. To see the licenses go to https://support.mbfbioscience.com/my_subscriptions

Dissection hooks

Thu, 06 May 2021 16:53:38 +0000

Dissection ‘hooks’ and ‘sticks’ are useful for removing fat bodies and other detritus when dissecting, as well as ‘hooking out’ muscle 16, which otherwise causes the brain to lightly pulse. These hooks and sticks are custom-made and electrolytically sharpened. This process involves using a 10 Amp transformer and is one of the most dangerous processes in the lab. Take great care and ask a current lab member for help the first time you attempt this.

MP-225

Tue, 04 May 2021 19:47:39 +0000

The MP-225 Micromanipulator is designed for the specific use of moving micropipettes in three-dimensional space in increments of micrometers into and out of an optical pathway of a microscope. The system provides for a synthetic 4th axis for diagonal advancement of the pipette; and up to 16 different angles can be configured. Its manual can be found here. Please refer there esp. for construction and setting up the angle of your diagonal mode through the DIP switches on the back of the human-interface device.

Berg-1 two-photon microscope

Tue, 04 May 2021 16:39:29 +0000

Computers#

~~Condor (ScanImage) condor.med.harvard.edu (10.119.96.25)~~ currently on leave
Crane (ScanImage) crane.med.harvard.edu (10.119.97.143)
Puma (behavior): puma.med.harvard.edu (10.119.96.44)
Stardock Multiplicity connects the mouse and keyboard between the two computers

Software#

MATLAB 2018b (with Instrument Control Toolbox)
ScanImage 2020 Premium (documentation)
NI Max 20.0
FlexRio 20.1

Excitation pathway#

Detection pathway #

Hamamatsu GaAsP PMT (H7422): amplify electrons generated by the detection of photons, the output is current.
Thorlabs transimpedance amplifier (TIA60): converts current to voltage

Pico Pump

Tue, 04 May 2021 15:04:04 +0000

Designed to simplify intracellular injection and a variety of other microinjection tasks, PicoPumps use carefully regulated air pressures for securing cells and injecting them with fluid. Injected volumes range from picoliters to nanoliters. Separate ports supply positive and negative pressure—positive pressure for high-pressure ejection, and suction for supporting the cell or for filling the pipette from the tip. A second pressure port maintains a low positive “holding” pressure to the injecting pipette between injection pulses, to prevent fluid uptake through capillary action. A manual for PicoPumps (we have PV800 PicoPumps in the lab) can be found here. Newer models are available:

Lab Away Dates

Tue, 04 May 2021 13:15:01 +0000

Logging Away Dates#

When you take a day off and/or will be working remotely for an extended period of time, please log those dates on our shared “Lab Away” calendar so all lab members are aware.

Importantly, please remove any regularly scheduled meetings that you will be missing from Rachel’s calendar. Please also update any rig-sharing partners or close collaborators of your plans.

Thank you!

Adding Yourself#

Any lab member with current access to the “Lab Away” calendar can grant new users access.

FANC

Fri, 16 Apr 2021 11:56:41 +0000

FANC is a complete EM dataset of the female fly ventral nerve cord (VNC).
It is documented in Phelps et al. 2020.
We have access to this dataset by agreement with the consortium of researchers who are reconstructing it.
To access the data, please ask Rachel to obtain a login from Wei.
Users must agree to the community guidelines before starting to work in FANC.
Raw data can be browsed via the FANC CATMAID platform.
The Slack channel for this group is http://fanc-reconstruction.slack.com/. For access to autosegmentation and proofreading, consult the instructions on the FANC Slack #proofreading-access channel.

Overview: closed-loop virtual reality for flies

Wed, 14 Apr 2021 14:55:41 +0000

This document gives you an overview of how to use our G3 VR set ups with awake behaving flies on a ball, in concert with FicTrac and custom code for delivering your visual stimulus.

Hardware Assembly#

There are various hardware set ups that could work. Here we give an example of a simple set up, with common equipment within the Wilson lab.

Construct or otherwise acquire a G3 arena
Construct or otherwise acquire a controller box for the arena.
- Load it with the appropriate C code.
Inline heater (e.g. TC-324C, Warner Instruments)
FLIR camera (e.g. Chameleon3)
IR light source
Fly ball (machined from foam, painted with black dots)
Fly ball holder (3D printed)
Fly mount (3D printed)

Software installation#

Install Spinnaker
Install the latest MATLAB, Visual Studio and PyCharm
Install FicTrac
- Download our starting config .txt file to C:/dev/fictrac/sample/ and .py Phidget helper file to C:/dev/fictrac/scripts/ respectively.
Clone matlab-panels to your computer and add this to your Matlab path
- Create a panels pattern, possibly by using the example Matlab code provided
- Burn your pattern to an SD card as instructed here using an SD card reader and your computer.
In order to use the serial port on the controller box you need to install the FDI driver.

Software testing#

We will need to test FicTrac, your pane code for the G3 arena and both together in closed-loop.

G3 Arenas Part 3: pattern and function guide

Mon, 12 Apr 2021 21:51:17 +0000

Patterns are created in MATLAB using matlab-panels and saved as .mat files locally. They are then burned to an SD card using matlab-panels, and this card is inserted into the controller box. Patterns can then be deployed to the LED panels of the G3 arena very fast and their position updated by reading variables from FicTrac, using low level C code on the controller box. The Reiser lab guide can be found here, code was written mainly by Reiser lab members. Multiple authors (inc. Mark Frye, Michael Reiser, Michael Dickinson, Stephen Holtz, Chuntao Dan, Sung Soo Kim, John Tuthill) have build a guide for panel use available here. The following is a slightly simplified version.

Amazon.com Purchases

Mon, 12 Apr 2021 13:43:14 +0000

If you want to order from Amazon, send your request to Rees. Please don’t log into the lab Amazon account. If the purchase can be charged to a particular grant, let Rees know when you Slack him your request.

G3 Arenas Part 2: software and general usage

Fri, 09 Apr 2021 21:17:01 +0000

G3 panels along with FicTrac are used in the lab for fly virtual reality experiments. Multiple authors (inc. Mark Frye, Michael Reiser, Michael Dickinson, Stephen Holtz, Chuntao Dan, Sung Soo Kim, John Tuthill) have build a guide for panel use available here. The guide is actually written for tethered, flying flies where wing-beats are analysed rather than walking flies on a ball. The Reiser lab guide can be found online at their satellite site here.

Pressurised ATP delivery

Wed, 07 Apr 2021 23:50:32 +0000

Flys do not naturally express ATP receptors, they are not encoded in the D. melanogaster genome. Neurons that have been made to express the ionotropic purinoceptor P2X2 (Valera et al. 1994), i.e. under the control of GAL4, can be stimulated by ATP. ATP can be delivered locally using a micropipette loaded with ATP in solution, which it inserted into the brain to target a loci of interest.

The Miesenböck group first did this using caged ATP that was photo-released by widefield UV illumination (Lima et al.) . The Wilson group used P2X2 and pressurized local ATP delivery to get specific responses from neurons (Liu et al. 2012). ATP has also been delivered using electrophoresis (Handler et al. 2019), which we have yet to try.

Filter cubes

Wed, 07 Apr 2021 22:36:22 +0000

Filter cubes take an input light, e.g. white light from a mecury lamp or white LED, and filter it, so that only light of allowed wavelengths hit your sample. The filtered light is directed to your sample by bouncing off the dichroic mirror in the filter cube. Emitted light from your sample of a certain set of different wavelengths are permitted to pass up, through the dichroic mirror and to your camera. There are many filter types. Here are some we use in the lab:

Pipettes

Wed, 07 Apr 2021 21:27:03 +0000

Pipettes start life as glass tubes that need to be pulled by our micropipette puller so that they taper to a fine point.

Major glass tubes in use#

item	specs	uses	useful puller programs
Sutter Instrument BF150-110-7.5	‘Thinwall’ Heavy Polish Borosilicate glass 1.5 OD 1.1 ID 7.5 cm Long filament: yes	electrophysiology, mostly ‘cleaning’ use instead of un-polished WPI glass to avoid re-chloriding wire after scraping
Sutter Instrument BF150-86-7.5HP	‘Thickwall’ Heavy Polish Borosilicate glass 1.5 mm OD 0.86 mm ID 7.5 cm Long filament: yes	electrophysiology
Warner G100-F		Pressurised ATP delivery Pressurised KCl delivery	56 [ATP delivery - Alex Bates]

Filling a pipette#

You will need to fill your pipette with fluid. To do this:

Pipette puller

Wed, 07 Apr 2021 20:44:39 +0000

Micropipettes are used for electrophysiology and fluid delivery. The pipette puller we use in the lab is a P-97 Flaming/Brown Micropipette Puller from Sutter. The manual is available here. Users need to load a glass pipette and run a program, which may have one or more pull cycles, stored on the device. Each program has its own number and will produce pipettes of different tapers, resistances, tip diameters, etc. Pipettes are then automatically pulled. Briefly:

Dissection Adhesives

Tue, 30 Mar 2021 02:42:46 +0000

Under construction!

For physiology dissections we typically use some kind of adhesive to restrain the fly. UV-cured adhesives are very convenient because they tend to cure very quickly.

WARNINGS:

UV light is dangerous! It can lead to short-term blindness and long-term damage to the skin and eyes.
Do not expose your skin to the LED, wear gloves or move your hands.
Do not look directly at the LED or at light emitted by the LED.

Commonly used adhesives:

Paying for WellGenetics order

Fri, 26 Mar 2021 15:54:55 +0000

Once WellGenetics has generated a quote, please send it to Karen Harmin, who will pay for it using HHMI funds. Note that WellGenetics services are quite difficult to pay for using Harvard (NIH) funds. WellGenetics will want half the price paid up front, with the other half paid later. A discount may be applied to the last payment, which makes the payment more complicated to process.

Basics#

The payment is split into two parts:

Forcep sharpening with Corte Instruments

Fri, 26 Mar 2021 11:27:30 +0000

How to place orders using HHMI’s procurement system - AKH, 3/11/24

1. Pack up the forceps and complete the Corte order form

Gather up the forceps that you’d like to have sharpened and check each pair quickly to make sure that they’re not super gnarled and completely beyond repair. They should all have protective plastic tips. Separate out the number of forceps that you want to be sharpened into A tips and D tips and put them in separate baggies with tape labels, e.g., “5 pairs, type A”. Pack them up in a small, well-padded box. Type A tips are longer and finer/sharper – good for desheathing. Type Ds have slightly shorter shanks and are a bit bigger and bitey-er at the very tip – good for removing little bits of airsac and fat without puncturing the sheath. People seem to like both, so I try to make sure we have a 50:50 stash of As and Ds in the “freshly sharpened forceps” box at all times.

Giving a talk

Thu, 25 Mar 2021 23:58:42 +0000

Lab meeting presentations#

Goals: Start by thinking about your goals for the presentation. What do you need from the group? Is there a decision you’re facing where you need to think through the options? Is there something that’s confusing you or defeating you? Don’t give us a blow-by-blow account of everything you’ve done. Tell us what we need to know to help you achieve your goal for this meeting.
Limit your content. Lab meeting should be a discussion, not a monologue. If you cram in too much material, you shut out discussion, which is a waste of an opportunity. Aim for 15 slides or fewer.
Describe your data: Whenever you show a data plot, you should explain every aspect of that plot. Read us the axis labels. Tell us what different colors or symbols represent. Then point out the feature(s) of the plot that you want us to notice. Give us time to see what you want us to see. Do this for every single plot. If you skip any step, your audience will be lost. If you feel self-conscious doing this, then you need to get over that self-consciousness. If you feel you don’t have time for this, then you need to be more selective in limiting your content. When you show a schematic or a model, you should also walk us through it carefully, just as if it were data.
Embrace messiness: Don’t discard loose ends and contradictory data. What doesn’t fit? What might lead to a distinct or contradictory story? The group might be able to help you see these things in a new light. If you hide your puzzles, you miss an opportunity to benefit from group puzzle-solving.
Tell a story: Whenever possible, try to tell the story you might tell in a manuscript, or at least a chapter in that story. This doesn’t mean showing all your data. What is means is that you try to sketch the arc of a story. Often you will want to skip some past “chapters” in a given lab meeting in order to focus on recent material: just devote a quick high-level slide to relevant past chapters, to remind us how those chapters advance the story. If your story ends abruptly (because you don’t know the ending), then that’s fine - just get us started.
Help the team: Consider points that might be useful to other projects. What will particularly interest your audience? Is there any news that they can use?
Tell us what to expect: Give us a quick visual roadmap at the beginning. If we know the sections of your talk in advance, we can pace our questions accordingly. If you tell us where you particularly want feedback, we can refrain from trivial questions in the rest of the presentation.
Go big: The presentation screen in WAB236 is small, and many folks are sitting far from the screen. So you need to make your display items big, with big fonts. Consider limiting each slide to just 1 or 2 large display items. Otherwise we can’t see your data and it’s hard to avoid tuning out.
General advice on research seminars (see below) applies to lab meeting presentations also.

Research seminars#

Opportunities to give talks
- HMS Neurobio SysClub (see https://neuro.hms.harvard.edu/events and the SysClub calendar)
- CBS Neurolunch
- HMS Neurobio Pizza talks - ask Rachel!
- conferences
- others? add them here…
How to give a talk - by David Stern
MCB 208. Talking about Science
some suggestions from Rachel
- Before your start outlining your talk, identify a specific person you want to imagine in the audience. Ideally, this person is a nonexpert, somebody you really respect, but also someone who is somewhat sympathetic. Try developing the entire talk with that person in mind. With each slide or major point, ask yourself, “What does this person know? What don’t they know? What will interest them? What will confuse them?” The more you can put yourself in their shoes, the better your talk will be.
- Plan on 1 slide per ~2 minutes. If you have 20 minutes and 20 slides, you have far too many slides; 10 would be a better number.
- Don’t write an elaborate written script for yourself before practicing your talk out loud. You should develop your spoken delivery by speaking, not writing. Speaking and writing are different. If you read a script you have written without much oral practice, you will find yourself stumbling over it, and your audience will stumble also. Oral practice will keep your delivery spare and powerful.
- Stand up for your oral practice. It makes it feel more real. You should be on your feet as much as possible – not sitting at your desk fussing with slides and typing your script.
- Aim to show relatively few data plots (often just 1 or 2 plots per slide), but the plots you show should be rich, and you should spend plenty of time on each plot.
- Whenever you show a data plot, you should explain every aspect of that plot – including the axes, the symbols, the colors, etc. Walk the audience through the plot methodically. If you feel you don’t have time for this, then it’s a sign that the data plot is not all that essential, or you are trying to pack in too much material. If you walk them through the plot, then the audience has time to actually comprehend it. But if you just flash the plot up without methodically explaining it, there is really no time to extract any meaning from it.
- Establish a clear, strong visual style, and stick to that style in the talk. For example, try to employ schematics that you can re-use at several points in the talk. When possible, try to build up your schematics throughout the talk. Try to use a consistent color-code for different types of data.
- If you have text on your slide, you should speak that text aloud. For example, if you have a phrase on the slide, then you should speak that exact phrase. If you have a sentence, you should speak that exact sentence. Don’t paraphrase the text, because this forces your audience to read one text while listening to an alternative (similar but competing) phrasing. This is simply confusing.
- Even if you are clear, some audience members will get lost. You can get them back on track by starting with a Roadmap/Outline/Agenda slide, and then revisiting that slide when you transition to a new major section of the talk.
- Some points will be relevant for everybody, whereas a few slides will be mainly for experts in the field. It’s OK to have some points “for experts only”, but in this case, be sure to signal this to the audience. For example, you might say, “Now, if you’ve actually done these sorts of experiments, you might know that it’s critical to control for X, and so I just want to show you how we control for that….” Or: “This slide is mainly for the handful of experts in the room, but I need to show it, so please bear with me….”.
- Some of your results will be unsurprising, whereas other results may be surprising. Your audience will be more interested – and better able to follow the talk – if you make a clear distinction between surprising and unsurprising results. For example, you can say some results are “as expected”, or “as you might have predicted”, or some experiments were “confirming”. When you talk about something being surprising, you can say “We were surprised”, or “We didn’t expect”, etc.
- The Introduction and Conclusion sections are arguably the most important sections. The job of the Introduction section is to tell the audience what gap you are going to fill, and why they should care. The job of the Conclusion section is to summarize the high-level take-home messages and why they should care. If you are tempted to skimp on these sections, or if you feel nervous about them, it’s a sign that you should spend some time brainstorming about your Introduction and Conclusion with a colleague or mentor, to try to refine your big-picture pitch. It’s common to feel nervous about these sections, but the solution is to spend time thinking about them, and get help early and often.

Chalk talks#

How to design a chalk talk- the million dollar sales pitch - by Erik Lee Snapp
How to give a chalk talk - by Leslie Vosshall
Q&A on chalk talks - a panel discussion
some suggestions from Rachel (specifically re: “job interview chalk talks”)
- Your goal in the chalk talk is to communicate your future research Aims and to foster discussion/excitement in the audience. The audience will not get excited unless you let them talk and interact with you. So you must regard this as a managed conversation and not a monologue.
- If you have a 1 hr time slot, then you need to plan for no more than 20 minutes of “presenter content”. In other words, when you practice in a room alone, your content should not last >20 min.
- Start with a statement of your overarching question, problem, or hypothesis. Try to deliver a bold and confident statement that they will remember. Good eye contact and body language are crucial at this particular moment.
- Then do a quick summary of your Aims (1-2 minutes of presenter-content per Aim, ~5 min presenter content total). If you are lucky, nobody will interrupt you during this quick summary. You can try to forestall interruptions by saying at the outset that you are going to begin with a quick summary of each Aim that will last ~5 min. “After I finish that quick summary would be a great time to start asking big-picture questions. At that point I’ll loop back around and discuss each Aim in more detail.”
- After you finish your summary, say “This is a great time to ask big-picture questions, before I go into detail.” The questions that will make you shine are the “big-picture” questions… assuming you can answer them. So encouraging big-picture questions is a strategic move. Piddly little questions are dangerous because they tend to pull you down into tiny details that will be boring to non-experts. (Here are some good big-picture questions: “These seem like fundamental questions - can you educate us about why they are still open questions in your field?” “Can you tell us about your long-term vision?” “How might your work change paradigms in your field?” “What is the most exciting thing you think you might discover in the next 5 or 10 years?” “Can you draw our attention to the most innovative part of your research program?” “What’s your approach to balancing risk-taking and exploration with productivity?” “What would you say is the most innovative part of your research program?”)
- After your quick summary, plan to go through each Aim again in more detail (~5 min per Aim, no more than 15 min of presenter-content in total). You will be interrupted constantly, so this will last forever.
- Try to respond to each question as if you really wanted the feedback – i.e., you must suppress your irritation and impatience. On the other hand, fakeness is easy to detect. So the challenge is to get yourself into a mindset where the valuable thing is the discussion, not the content you have planned. Remember that each audience member will want to talk, and letting them talk makes them happy.
- If you are getting dragged down with piddly little questions too early, it’s OK to try to defer one of them, in order to send a message to the audience and to pick up your pace. E.g., “If it’s OK with you, I’d like to defer that question for a few minutes until we get to the part of the talk where we discuss Aim 2 in more detail.” But then you must remember to go back to that question… and that imposes a cognitive load on you.
- As you go, it’s extremely helpful to telegraph which Aims or sub-Aims are the “safer” ones, and which are the “riskier” ones. A good research program has a balanced portfolio. Don’t make them guess.
- If it looks like you’re not going to make it through your Aims, you can ask them to let you jump ahead. At this point, feel free to break the rules if you need to. E.g., if Aim 2 is sort of standard stuff (slightly ho-hum) and you really want to get to Aim 3, then it would be OK to skip to Aim 3. That said, it’s fine if you never get to Aim 3 if it’s not your most exciting Aim. Do whatever you need to do in order to get them excited about your research. Your goal is not to get through your content. Your goal is to get them excited. Use a time-crisis as an opportunity to focus on your best stuff.
- If at all possible, prepare the whiteboard / zoom-board ahead of time. You will want to establish a space for each Aim. Draw in any relevant circuit diagrams or other crucial visuals. But there is a chance you won’t get any time in advance to prepare the board (and you will be pretty rattled anyhow), so practice your quick draw skills in advance. If color is important to you, then bring your own whiteboard markers; there is also a slight chance you will be given a blackboard, so make sure you have a plan in mind for monochrome.
- Adjust your plans for writing/drawing to your skill level. Don’t make them watch you slowly write/draw for a long time. But don’t write/draw so fast that it’s illegible. If you are not good at writing/drawing on a whiteboard, then plan to use the marker very sparingly, and then do it carefully. It’s better to have a few neatly written things, versus many sloppily written things.
- Sometimes people will ask how your Aims might map onto your first R01 application. There is no right or wrong answer here, as long as your answer is coherent. Just be ready for it.

2P photostimulation + imaging

Thu, 25 Mar 2021 22:16:07 +0000

NOTE IN 2026: Some of the limitations mentioned here are no longer applicable, this needs to be updated by current users.

Berg2 has a Galvo-Galvo-Res scan system (see this page in the ScanImage documentation for an explanation of the concept behind this system) that allows us to conduct near-simultaneous 2P photostimulation and imaging despite the lack of a dedicated photostimulation laser and optical path (with some important limitations). If you have questions about this process you can ask Michael about it if he’s still in the lab (or if he’s no longer in the lab but you ask very nicely).

De-noising a rig

Thu, 25 Mar 2021 17:11:21 +0000

While all of the rigs in the lab are built on an antivibration table and placed within a Faraday cage in order to dramatically reduce movement and external electrical noise, respectively, there are many electrical devices within the proximity of the rig that can act as potential sources of noise if not grounded/shielded properly. Your electrode is highly sensitive to sources of noise, as it acts as an antenna by design, picking up any stray electrical radiation and converting such into time-varying currents. These currents will generate observable voltages as they flow through resistance in the circuit, such as the high-impedance junction between your pipette and the cell.

Making internal

Wed, 24 Mar 2021 20:05:09 +0000

K+ aspartate internal solution (w/ biocytin)

Based on the standard recipe for internal from the Wilson lab, updated by Yvette 10/2020

Note: to make Cs aspartate internal substitute KOH for CsOH

	Final Conc [mM]	Mol. Mass [g/mol]	grams in 5 mL	uL in 5 mL	grams in 20 mL	uL in 20 mL
KOH (1M stock)	140	-	-	700	-	2800
aspartic acid	140	133.1	0.093	-	0.372	-
HEPES	10	238.3	0.012	-	0.048	-
EGTA	1	380.4	0.002	-	0.008	-
KCl (50 mM stock)	1	-	-	100	-	400
MgATP*	4	507.2	0.010	-	0.040	-
Na3GTP*	0.4	523.2	0.001	-	0.004	-
biocytin hydrazide	13	386.5	0.025	-	0.100	-
**ALTERNATIVE:neurobiotin citrate	15	478.5	0.036	-	0.1440

ATP, GTP and Neurobiotin should be stored in the freezer (-20C) in a container w/ drierite.

** alternatively you can use neurobiotin instead of biocytin. Do not use both! It seems that neurobiotin can provide better fills in certain cell types, however there are also anecdotal observations of it making certain cell types unhealthy.

Making saline

Wed, 24 Mar 2021 20:04:57 +0000

We perfuse a saline solution over the fly’s brain, on the posterior part of the holder, whenever we’re doing physiology, whether it is electrophysiology or calcium imaging.

To make it, you first need to fill a large flask with about 5 L of miliq water.
Add a magnetic stir to it, place the flask on top of the stirring plate, and turn it on, making sure that a vortex is created.

O2

Wed, 24 Mar 2021 18:08:44 +0000

O2 is a Linux-based platform for high performance computer that we can use at Harvard. This should be used for things that require a lot of computing power and therefore are slow to run locally. An example is registration of imaging sessions. Quarterly computation and storage charges apply to non-Quad groups, but not us.

Usage#

To start using it, you need to create an account.

If you use Linux or Mac, you can use the native terminal application to interact with the O2 server. If you use Windows, you will need to install a program like MobaXterm to interact with it.

Dissection guide

Tue, 23 Mar 2021 19:51:30 +0000

Equipment#

Dissection-suitable microscope
Forceps:
- Fine, sharp
- Thicker, blunt
- ‘Grippy’, fine but blunted
Small electric heater
Hypodermic needle
UV glue + gun
Optional: custom hook
Fly holder
- Recommended: cone-shaped holders for walking behavior and visual field-of-view
  - The design files for the standard, T shaped, and circular (C) cone-holders are attached below
  - Depending on the average size of your fly, you can adjust the slot size by adjusting the thickness parameters
  - Ordering: CNC Machined from Protolabs out of black delrin
  - Note: the very original cone-shaped holder design was provided by the Maimon Lab. They’ve been acknowledged in Fisher et al. If you revert to the original Maimon lab holder design, they should be acknowledged.

Design Files:

FLIR + Spinnaker + SpinView

Tue, 23 Mar 2021 17:10:25 +0000

Spinnaker SDK is FLIR’s GenICam3 API library. SpinView is the GUI for Spinnaker. The FLIR Spinnaker toolbox enables supported FLIR cameras play with the Image Acquisition Toolbox for MATLAB. The Spinnaker SDK supports FLIR USB3, 10GigE, and most GigE area scan cameras. Supported platforms: Windows 7 (32- and 64-bit) / Windows 10 (32- and 64-bit) / Desktop Ubuntu 18.04 (64-bit) / Desktop Ubuntu 16.04 (32-bit) / Ubuntu 18.04 (ARM64) / Ubuntu (16.04 ARMHF & ARM64) / MacOS (Mojave & High Sierra). Btw, a real spinnaker is actually a special kind of sail, which fills with wind and balloons out at the front of a sailboat.

FicTrac Part 3: closed-loop setup

Mon, 22 Mar 2021 15:55:15 +0000

To use FicTrac with a closed-loop setup (to provide real time feedback), you need to configure FicTrac to output data via a socket port. To do so, you will need to (1) set a valid sock_port in your configuration text file and (2) write a python script in the fictrac/script directory for receiving data via said socket. The python script can also be used to output variables of interest as voltages via a Phidget device, a 4 channel analog output device (+/- 10V) that can be connected via USB cable.

Postdoc onboarding

Mon, 22 Mar 2021 14:13:47 +0000

So, you’ve arrived! Now what? A fair bit actually. The following guide is written mainly for non-US citizens who are coming to the lab for a postdoc, but some of it could be useful for international graduate students and US postdocs. It can be helpful to attend the new postdoc orientation. In sum, you need to do the following:

HMS Onboarding Paperwork
Obtain your Harvard University ID card
Obtain appropriate log-ins. Your Harvard Key is a Harvard-wide log-in; your HMS Account is for HMS-specific services. You need both. When you’ve completed the onboarding paperwork, you’ll be sent your HUID number (needed to obtain a Harvard Key login) and your HMS login (needed to create your HMS account)
Enroll in Harvard Benefits, including medical insurance
Make sure your myHMS works and download appropriate software
Obtain appropriate building/room accesses
Obtain access to the Lab Safety Training course, as well as other Harvard on-line training courses, at the Harvard Training Portal. To do this, either Rachel or Diego can add you to the lab roster on PeopleSoft.
Other items for aliens:
- Get a bike
- Get a phone contract
- Register with the Harvard International Office
- Open a US bank account
- Get a social security number

HMS Onboarding Paperwork#

For HR appointment-related matters - including Visas, Intern, LHT, and Visiting Student onboardings, your point of contact is neuro_appointments@hms.harvard.edu.

Mental health

Sat, 20 Mar 2021 21:07:37 +0000

contributors: Melanie Basnak, Rachel Wilson

If you are a graduate student, you can see a therapist for free at Harvard University Counseling and Mental Health Services for short term treatment, and/or you can see an outside professional for longer periods.

If you a postdoc or a research assistant,

The mental health resources you have access to will depend on your health insurance plan.
You are eligible to participate in a variety of classes and workshops offered by the Harvard Longwood Office of Employee Development and Wellness.
Postdocs can participate in theregular workshops about mental health and wellness offered by the Office for Postdoctoral Fellows.

Some recommended therapists are listed here. For PiN students, Susan Jackson can also recommend therapists who have helped PiN students in the past.

FlyWire

Sat, 20 Mar 2021 10:20:59 +0000

Overview#

FlyWire is a platform for interacting with the FAFB dataset. There are some key differences relative to v14/CATMAID/walled garden:

The FlyWire team realigned the raw EM data, and this alignment is better than the v14 alignment.
As a result, the autosegmentation is better, with many fewer small fragments.
FlyWire uses the Neuroglancer interface rather than CATMAID.
FlyWire is open to everybody.

Dorkenwald et al. 2022 provides an overview of FlyWire.

Neuron activation and inhibition

Fri, 19 Mar 2021 14:44:12 +0000

Drosophila neurons can be activated or inhibited using different methods, and some are more appropriate under specific conditions.

Neuron activation#

The activation of neurons can be achieved through the following methods:

1) Using an LED of appropriate wavelength to drive activation of Chrimson in the neurons of interest.#

Chrimson is a red shifted opsin that can be expressed in neurons of interest. When shining a light of appropriate wavelength, this depolarizes the neurons.

ScanImage Operation (Bergamo-2P)

Fri, 19 Mar 2021 14:31:30 +0000

See ScanImage Setup for installation etc.,

Triggering ScanImage from another computer#

(As of 2023 this note does not currently outline the newest way to remotely configure scanimage, but should still be functional).

If you’re triggering ScanImage acquisition from a different computer (e.g. a computer that monitors and controls behavior), you’ll need two things:

TCP/IP connection between the two computers.
The computer that’s sending the trigger will need to specify the correct IP address of the scan image computer in connect_to_scanimage.m. Make sure to request a static IP from HMS IT (and name your computer) for the ScanImage computer if you haven’t done so already.
ScanImage computer will need to be running fly_tracker_server_v2.m so that it’s port is ready to receive the message. The file name name will be send from the behavior computer to the scanimage controller, and will extract the trial duration from the file name, and set the number of volumes to be acquired.
Triggering through the NI system.
Make sure to generate imaging trigger (HIGH during the entire duration of the trial) in your behavior program and send this trigger through a digital output channel.
This imaging trigger needs to be fed into one of the PFI channels in a NI-DAQ breakout panel (e.g. one associated with Pockels cell control). Make sure to specify this panel as digitalIODeviceName in the Machine Data File of ScanImage.
Specify the PFI channel in the Trigger Controls in ScanImage. Select the appropriate PFI in the “Terminal” and choose “rising” Edge.

Acquire imaging data#

Setting up the scope#

To acquire imaging data, open MATLAB and type ‘scanimage’. This should open a small prompt asking you for a ‘machine data file’ and a ‘user settings file’.

G3 Arenas Part 1: assembly

Thu, 18 Mar 2021 17:18:48 +0000

Parts List:

A Printed Circuit Board (PCB)
- Note: This is available as both 270 and 360 degree variants
Small circuit elements to solder onto the PCB
- Power Connector (CP-2350-ND)
- Power Switch Toggle (CKN1513-ND)
- I2C Port (609-1478-ND)
- Connection Headers (A26513-40-ND)
- Connection Jumpers (S9001-ND)
- 10uF Capacitors (478-4170-ND)
- 8-Position Receptacle Connectors (SAM1222-08-ND)
- Note: There is a model PCB with components already soldered onto it as an example
LED Panels
- Green panels are better for behavioral experiments. However, they are less compatible with imaging experiments. Therefore, blue panels should be used from the get-go if you plan on doing any imaging for consistency
- Most arenas consist of 2 rows of panels, this means you will need between 18-24 panels total depending on your configuration. It is good practice to save extra LED panels from the same lot should any fail later.
Panel Microchips
- Interface between your code and the LED panels. Microchips in the designated panel drawer have already been programmed.
White Light Diffuser Film
- 1 layer, cut out squares and adhere directly to each individual LED panel
Blue/Green Gel Filters
- 3-4 layers, taped to form an even cylinder
- note: if you are using the panels for imaging, you might also want to add a layer of Marine blue filter, which blocks some of the bluer components that can generate autofluorescence coming from the fly, and decrease the SNR.
Laser Cut Parts
- Square platform base with screw holes - for easier mounting
- Circular top - for securing top row LED panels in place
3D Printed Ball Holder (6 or 9 mm)

FicTrac Part 1: initial installation

Thu, 18 Mar 2021 17:07:48 +0000

IMPORTANT NOTE: There seems now to be a FicTrac installer, you could give it a try or else follow the instructions below. Note that the installer is pre-built to work with point grey USB3 cameras, so if you plan on using GigE, a different camera brand, or need to customize aspects of the installation (for example, changing the config window size), follow the instructions to build from source, below.

FicTrac is a software than enables you to track the fly’s projected motion as it ’navigates’ while tethered to a ball. The software has a relatively active reddit forum with some questions and answers that may assist debugging: https://www.reddit.com/r/fictrac/

Searching for Genetic Lines

Thu, 18 Mar 2021 17:00:22 +0000

This page is a list of online tools to search for Gen1, VT, and Split-GAL4 driver lines that label a cell of interest. Usually, the neuron(s) are found in one of the connectome datasets, and their EM skeleton can be compared to existing genetic lines. This can be done through two main web pages, NeuronBridge and PatchPerPix. These two sites will output lines that MAY contain your neuron of interest. From there, these lines should be validated using FlyLight.

Electrophysiology for Drosophilists

Tue, 16 Mar 2021 13:54:30 +0000

Google Slides - create and edit presentations online, for free. — Create a new presentation and edit with others at the same time. Get stuff done with or without an internet connection. Use Slides to edit PowerPoint files. Free from Google.

#

Supplementary Material

Fundamental circuit properties :

https://www.scientifica.uk.com/learning-zone/understanding-the-cell-as-an-electrical-circuit

Learning coding, data science, & stats

Tue, 16 Mar 2021 13:39:25 +0000

There are SO many ways to learn now it can be overwhelming. Here are some suggestions from lab members about how to go about learning to code and get into data analysis. This page from the Pearson Lab at Duke has (an opinionated, but very) useful set of suggestions: https://pearsonlab.github.io/learning.html.

Online Courses#

Software Carpentry and Data Carpentry have many short open-source courses with all the code and documentation available. They are short, well made, and to the point (also many are in Spanish). Here are some of note:
- Plotting and Programming in Python a very nice introduction that highlights modern tools for data science and visualization.
- Programming in MATLAB just a simple set of examples and quick start-up :)
- Programming in R Basically the same scope as the MATLAB course but in R
- The Unix Shell highly recommended if this has always seemed like a morass!
- There are many other short courses on domain specific analysis too, have a look!
CS50: Introduction to Computer Science, suggested by Jenny Lu and Helen Yang
Lab in Cognition and Perception a really nice introduction to statistics and python for analysis in the guise of a cognitive science course
Harvard’s online R courses, suggested by Rachel Wilson
edX: Using python for research, suggested by Stephen Holtz
coursera’s Machine Learning, suggested by Gu lab members

O’Reilly Learning#

Technical books are available through O’Reilly Learning Platform to which Harvard gives access. Of particular interest:

Immuno reagents log

Mon, 15 Mar 2021 18:22:25 +0000

Open embedded document

Supply database

Mon, 15 Mar 2021 18:20:36 +0000

Open embedded document

Vendor database

Mon, 15 Mar 2021 18:19:42 +0000

Open embedded document

Contact numbers

Mon, 15 Mar 2021 18:18:24 +0000

Open embedded document

Patching guide

Mon, 15 Mar 2021 13:01:25 +0000

Cleaning#

Using a large pipette tip, carefully blow or suction (applying positive pressure to blow away debris can be useful for initial cleaning of a larger area while suctioning off debris tends to work better for more targeted/careful cleaning of a specific target) off any cells or debris occluding your cell of interest
- It can be helpful to swipe the cleaning pipette either to the side or up while suctioning in order to pull away unwanted tissue
If your cell is “clean”, you should see it move freely, and/or “dimple” when positive pressure is applied

Patching#

Set up the amplifier

Posters

Mon, 15 Mar 2021 11:41:18 +0000

PhD Posters has a great tool for printing out posters, and is local to the Longwood Medical Area.

We pay with the HHMI P-card and use the tax exemption code 94808064

Molecular biology equipment

Mon, 15 Mar 2021 01:34:10 +0000

PCR Machines#

We can use, after asking permission, PCR machines in the Yellen lab (next door, ask permission from Tanya) or in the Weitz lab (upstairs by the elevators, ask permission from Darko).

Gel doc#

The gel doc system is in Goldenson 523, down the hall from the HCNR office. It is the first piece of equipment in the room, on the right. Do not touch the computer or the outside of the gel doc system with gloves on: follow the one-glove policy to avoid EtBr contamination in this room and elsewhere.

Two-photon analysis pipeline

Sun, 14 Mar 2021 17:16:56 +0000

by Tatsuo Okubo, Stephen Holtz, Helen Yang, Jenny Lu, Michael Marquis and Melanie Basnak

We all use different custom code to analyze our calcium imaging data, but there are some steps that most of us need to take. Next is a description of these steps, along with a list of the different methods used in the lab:

Removing flyback frames#

This step is required if you are doing volumetric imaging. When you acquire a volume, a piezo platform moves the objective, so that you can acquire slices that are further down in z as the volume progresses. When you reach your final slice, the platform needs to move for the scope to start acquiring at z=0 again. This takes a few frames, and you will need to discard them. You can see what this motion should ideally be and what it actually is in scanimage, in the ‘Fast Z controls’ window (making sure ‘Enable’ is checked), clicking the ‘actuator tuning’ button. It gives you a curve like the following:

Water purifier

Fri, 12 Mar 2021 19:36:57 +0000

When we need a replacement cartridge, the Water Purifier Czar will order directly from Millipore (https://www.emdmillipore.com/Web-US-Site/en_CA/-/USD/QuickPurchase-Start, user name: flypokers@gmail.com , password: wilsonlab320).
The filter cartridge is part number SIMPAKKD2, and the final bacterial filter (the bell-shaped filter at the outlet point) is part number MPGP02001. Always replace these at the same time.
For servicing, contact our Millipore rep Ray Reilly, 800-645-5476 ext. 6795, raymond_reilly@millipore.com. The SIMPAKKD2 cartridges are specifically for a system with input water that has already been partially purified by reverse osmosis (“RO water”). Our water purifier is a Millipore “Synergy” system (part # SYNS60001).

UPS

Fri, 12 Mar 2021 19:36:26 +0000

You might specifically need to send a package via UPS if, for example, another party is paying for shipping and requests that you use their UPS account number. To generate a shipping label, navigate to the UPS website and login using the username WilsonLab and password flypokers. Even if you are billing the charges to the recipient, you need to provide a UPS account number corresponding to the sender (i.e., us). Our account number is 23V173, and this is associated with our P-card. After you enter the information for the shipment, the browser should prompt you to print the shipping label. You should tape this to the outside of the box with clear plastic tape. The box should be placed in the Department of Neurobiology mailroom. The HMS mail staff will take the box down to their office, and UPS will pick it up from there.

2P troubleshooting (hardware/software)

Fri, 12 Mar 2021 19:35:38 +0000

FastZ error that involved improper syncing with the NI PXIe and a possible “Machine Data File” error:

Make sure the connection to the NI PXIe is intact, and powercycle the NI PXIe and the computer.

If both the “GR mirror” and “Flipper mirror” controls are greyed out in ScanImage, it’s possible that Windows has assigned them the same COM port. To check, open the Windows device manager and look for the two entries called “Thorlabs flipper mirror” in the COM ports section. If they are both assigned to the same COM port, try the following: unplug the USB cables from both mirror control units, then unplug both power cables. After 10 sec, plug the power cables back in. Plug one of the USB cables back in and wait for the device to show up in the Windows device manager. Then, plug the other cable back in.

Tracing from confocal images

Fri, 12 Mar 2021 19:34:13 +0000

To trace the morphology of a neuron from a confocal z-stack, open the stack in Fiji. Launch the Simple Neurite Tracer plugin (Plugins -> Segmentation -> Simple Neurite Tracer). Instructions for using the Plugin are posted here. When tracing, it’s helpful to selecting the viewing option “View Paths (2D): parts in nearby slices”. Draw paths through all the neurites of the neuron. The paths do not need to join up if you goal is simply to create a visual representation of the fill (and not to do any quantitative analysis on the fill) – the tracing does not need to represent a single unbroken skeleton, and so it’s adequate for the endpoints of two paths to be extremely close. Save the skeletonized path file (File -> Save traces file). Then use the Fill Out command to automatically generate a 3D volume around the skeleton. Select all the paths from the list (in the All Paths window), and select the “Fill Out” button. Fiji will search for the boundaries of the fill, and you can set the threshold for the boundary as a free parameter (with the Set button). The algorithm does not need to converge – you can pause the process if it stalls and still potentially recover a satisfactory fill. It’s useful to try a few boundary settings to determine what result best resembles the original fill. Save the fill for further processing. The fill will render as a white neuron on a black background; you can then open the z-projection in Photoshop and invert the colors in the image to obtain a black neuron on a white background.

Purchasing tools

Fri, 12 Mar 2021 19:33:35 +0000

We have an online account with True Value Hardware (http://www.truevalue.com, use account e-mail riwilson@caltech.edu, password amsterdam).

It is not possible to get tax-exempt ordering from Home Depot, so just pay tax if needed. The Assad lab and the Regehr lab both have extensive collections of exotic tools that we may borrow if we do so politely. The thin cutting wheels for the Dremel tool are Dremel part # 409 or 425. Dremel 425 corresponds to Home Depot #162935. Dremel #409 is available on Amazon.com.

Temperature (in lab)

Fri, 12 Mar 2021 19:30:55 +0000

The temperature in all the lab rooms is “controlled” by the Facilities Department. To change the temperature, call facilities at 617-43(2-1901).

Telephone

Fri, 12 Mar 2021 19:30:08 +0000

The lab phone number is: 617-432-5569. To get an outside line, dial 9. Then dial as you would any other phone.

Domestic calls: 9 -1 - Area Code - Number.

International calls: 9 - 011 - Country Code - Number.

Lab Supplies

Fri, 12 Mar 2021 19:09:34 +0000

Always go through the HHMI P2P or the HMS B2P system for purchasing from these suppliers.

McMaster-Carr has a very large catalogue of raw materials (acrilic sheets, metal, etc.,), electrical components (wires, plugs, etc.,), building supplies (screws, bolts, velcro, etc.,), as well as various curated products.
- They have extremely fast shipping and helpful human-on-phone support.
Another source for electrical and mechanical supplies is MSC Industrial Supply (http://www.mscdirect.com).
- Use customer number 2060124 for tax-exempt status and faster processing.
A good source for many “standard lab” supplies and equipment is VWR (http://www.vwr.com), since Harvard gets special pricing and free shipping from VWR. Our customer # is 2076338.
For cheaper prices on glassware and other Corning brand products, a good source is Westnet.
Cole Parmer is a good source for valves, fittings, specialized tubing, etc. Use customer number 019180-15.

NeuroOps

Fri, 12 Mar 2021 19:07:49 +0000

The NeuroOps department comprises Joshua Lefurge and Steve Sleboda.
Josh and Steve can be reached by emailing Neuro_Ops@hms.harvard.edu, or by dropping by their office (Goldenson 206), or by phoning their office at 617-432-2251.
Joshua Lefurge is the Assistant Research Operations Manager (Joshua_Lefurge@hms.harvard.edu). He should be the primary point of contact for most of your research operations issues, including
- getting a locker
- key access
- obtaining/removing surplus furniture
- small construction or repair jobs
- moving heavy items between our lab and the loading dock
- removing old equipment or furniture to be donated or trashed
- monitoring the quality of lab water and air
- leaks, drips, and condensation
- pest control
- strange odors
- problems with lab lighting, temperature control, trash removal, biohazard bag removal, or ventilation that HMS facilities is not solving
Steve Sleboda is the Department of Neurobiology Manager of Research Operations (steven_sleboda@hms.harvard.edu, for urgent issues you can call his cell at 617-831-6432). He is the person to contact if Josh Lefurge is unavailable. Steve mainly manages departmental operations policy, space issues, renovation issues, etc., but he can pinch-hit for Josh as needed.

Solenoid valves

Fri, 12 Mar 2021 19:06:02 +0000

Our preferred isolation solenoid valves are from the Lee company (LHD series, www.theleeco.com). Note that they often have a 6-8 week lead time, so if you see that we are running low (say <4 valves remaining) it’s a good idea to place a preemptive order. We typically use either the 12V or 24V valves.

For the LHD series the distal port is normally open, the proximal port is normally closed, and the center port is common (you can seal off the NC or NO port to use it as a 2-way valve).

Software - Gene Construction Kit

Fri, 12 Mar 2021 19:05:00 +0000

This is a DNA construct management tool that is very useful when building new DNA plasmids. It keeps DNA sequence information together with user- and software-defined annotations in a user-friendly graphical format.

In June 2013, we purchased a non-expiring Windows license for this software that can be used on ONE computer at a time. The software can be installed in demo version on multiple computers, but full functionality (saving and exporting) is available for only a single machine. Please check with other lab members before converting your copy of the software to full functionality.

Server

Fri, 12 Mar 2021 18:45:56 +0000

We have access to the Wilson Lab folder on the NB1 server. Access NB1 by name or as \134.174.164.101 . login with firstinitial,lastname; password is firstinitial,lastinitial,roomnumber.

We also have Wilson Lab file shares consisting of HMS “Active collaborations” storage and “Standby” storage (see a description of these types of storage here).

The “Active collaborations” directory is our main lab shared server for everyday use, and has a storage limit of 79 TB (as of April 2021). It’s located here:
- Mac/Linux: smb://files.med.harvard.edu/neurobio/wilsonlab/
- Windows: \files.med.harvard.edu\neurobio\wilsonlab\
The “Standby” directory is intended for data that is rarely accessed (e.g. backup copies of 2P imaging data or archived data from lab alumni), and has a storage limit of 36 TB (as of Apr 2021). It’s located here:
- Mac/Linux: smb://standby.files.med.harvard.edu/hms/neurobio/wilson/collaborations
- Windows: \standby.files.med.harvard.edu\hms\neurobio\wilson\collaborations

Until further notice, please don’t move new data onto the standby server, as we are getting close to exceeding its storage capacity.

Optomechanical components

Fri, 12 Mar 2021 18:27:11 +0000

Optomechanical components are stored in WAB305 in the blue bins on the rack next to the sink.

Additional components are stored in the drawers under the sink in WAB305.

Thorlabs posts are tapped for 8-32 threads (the little hole) and 1/4-20 threads (the big hole). Some posts are metric, indicated by a thin etching by the “top” smaller holes.

Narishige manipulator threads are M2.5 (2.5 mm diameter) x .45 mm thread pitch.

Copier - scanner

Fri, 12 Mar 2021 18:25:41 +0000

The copier on the 4th floor of Goldenson is also a scanner, and it has been set up to scan to a folder within the Neurobiology Shared directory on the server. Our lab’s code is 499.

To scan rather than copying:

press the physical “Scanning” button on the left
on the LCD screen, press the “Department” button
press the physical green “Start” button on the right
once it completes, go to your computer and find the newly scanned documents under the “Neurobiology Shared” share, in a folder named “DEPT_SCANS”. Be aware that everyone in Neurobiology can access this folder, so use caution and plan accordingly

There is also a scanner in the Neurobiology Graphics Lab (Goldenson 158). Our general access Goldenson key permits us access to this facility even on evenings and weekends, if needed. See also: Neurobio Dept graphics lab.

Electrophysiology rig configuration

Fri, 12 Mar 2021 17:57:00 +0000

Generic useful oscilloscope settings#

trigger: type: edge, source: AC line, slope: rising, mode:auto, coupling: noise reject
all channels: probe gain = 1×
no external trigger

Whole-cell patch-clamp recordings#

40×WI objective
variable output on A-M Systems Model 2400 amplifier to A-to-D board; using Model 2400 filter this output at 5kHz, gain=50×, probe gain set to low
Im output on on A-M Systems Model 2400 amplifier → to input channel 1 on LPF202A auxillary amplifier; using the LPF202A amplify this 100× (filter on “bypass”) → to oscilloscope channel 2 (“current”) and A-to-D board
×10Vm output on A-M Systems Model 2400 amplifier → to input channel 2 on LPF202A auxillary amplifier; using the LPF202A amplify this 1× and low-pass filter at 2kHz → to oscilloscope channel 1 (“voltage”)
Oscilloscope channel 1 (“voltage”) set to DC coupled, 88mV/div vertical scale, 100ms/div horizontal scale; channel 2 (“current”) set to DC coupled, 2V/div
amplifier in Iclamp or Vclamp mode
electrode angle ~30°

Single-sensillum recordings#

50× air objective
put the condenser diaphragm completely open, and switch from the polarizer filter to the heat-shielding filter
×10Vm output on A-M Systems Model 2400 amplifier → to input channel 2 on LPF202A auxillary amplifier; using the LPF202A amplify this 20× and low-pass filter at 2kHz → to oscilloscope channel 1 (“voltage”) and A-to-D boardoscilloscope channel 1 set to AC coupled, 88mV/div vertical scale, 100ms/div horizontal scale all other oscilloscope channels off amplifier in I=0 mode
electrode angle ~35°
antenna is stabilized by two hooks: one coming from the top and right (this should be on a Sutter manipulator) and the other from the left and bottom (a coarse manipulator will work); both the hooks should come in horizontally for best stability.
The method for stabilizing the palp is a little bit different than that for the antennae. The palp sits on a coverslip and it is anchored from the top by a cleaning pipette. The cleaning pipette should come at ~30°. The recording pipette should also come in at a steeper angle than for the antennae. 45° should work fine but there does not seem to be any reason not to go as high as the air-objective will allow.

Radio Shack power cords

Fri, 12 Mar 2021 17:54:45 +0000

The color code for Radio Shack power cords is nonstandard and funky. The code is given in the table below. These assignments were determined empirically:

	Radio Shack	Standard U.S.	Other
Ground	Green	Green	Green
Neutral	Red	White	Blue
Live	Black	Black	Green with yellow lining

Printers

Fri, 12 Mar 2021 17:53:48 +0000

Toner for the printer can be purchased through the OfficeMax punchout or Amazon. Note: The HP version is worth the extra money, other printer-compatible brands have leaked or lasted for a small fraction of the time when tried in the past. Update here if one is found to be reliable.

HP - 508X High Yield Black Toner Cartridge (CF360X) - CF360X, Black
HP - 508X High Yield Cyan Original LaserJet Toner Cartridge (CF361X) - CF361X, Cyan
HP - 508X High-Yield Yellow Toner Cartridge (CF362X) - CF362X, Yellow
HP - 508X High-Yield Magenta Toner Cartridge (CF363X) - CF363X, Magenta

The Wilson Lab printer IP address is 10.11.176.93.

Piezoelectric actuators

Fri, 12 Mar 2021 17:50:46 +0000

Here are some guidelines and information on the piezoelectric actuators from Physik Instrumente (PI). Users should also read the following manuals:

E-802 Servo-Controller Submodule (PZ 150E User Manual),
E-509 Position Servo-Control Module (specifically section 4 E-509 Calibration Routines),
E-801 Sensor Submodule (PZ 117E User Manual, specifically the section on E801B1008)

General terminology#

The terms “piezo,” “actuator,” “piezoelectric actuator,” and “piezo stack,” all refer to a stack of piezoelectric elements encased in a metal tube. The term “actuator” is preferred by the PI engineers.
All of the piezoelectric actuators we have are “preloaded” meaning there is a static load applied such that they are able to be bidirectionally moved.
The “probe” is anything that we move with an actuator.
The piezoelectric actuators are either open or closed loop. All of the open loop piezos have just one thin grey cable that is for charging the stack to drive motion. The closed loop piezos have two cables, where the second is part of a feedback system.
All piezos, both closed and open loop require an amplifier to convert our command voltages (e.g. generated in matlab) to a high voltage / current signal that will charge the piezoelectric stack and thereby drive the actuator. The amplifiers are all identical.
Importantly, each closed loop piezo is paired with a “controller”. These controllers contain servos that allow for both precise positional control and a readout of current position. Controllers are factory calibrated (in a way that we cannot recreate in lab) to ensure linearity in control and accurate positional information. See below for more guidelines / rules about these.
PI sells both large and small chassis that house several controllers and amplifiers. These distribute power to the amplifier and controller, and have some logic for additional, optional boards. Some amplifiers and controllers have multiple channels. It is not always straightforward which amplifier slot links to which controller channel.

Rules for using PI piezos#

When adding or removing a probe (small, front adapter): apply even pressure and use a small, 5mm on the flat to prevent movement. Failure to do so may cause a crack in the ceramic element and require repair and re-calibration. Alternatively, the stack may become misaligned and the feedback signal will be misleading.
Be mindful when mounting actuators. Do so using either the rear threads or with an even clamp along the body length; do not over tighten and do not bend the cable exit. For example, do not use a mount that will apply focal pressure and cause damage to the housing. This may compromise the calibration and introduce artifacts not seen in the sensor signal.
Never swap a controller and actuator, keep them paired. They are paired when factory calibrated. For multi-channel servos, the correct channel is indicated. Swapping can result in feedback signals off by large margins, or ringing in step responses that may not be visible in the feedback signal. In the worst case it will irreversibly damage a piezo and require factory repair (e.g. if over-voltages are constantly applied).
Always use extension cables with the closed-loop actuators. Do not EVER cut the cables. Extension cables are only strictly required for the short travel actuator, it hasbeen calibrated with the cable by our request. Cutting cables results in a need to both re-calibrate and re-connectorize.
Be careful when calibrating actuator step responses. Do not EVER change the potentiometers associated with static calibration when changing the step response (see below). This will result in a need to factory re-calibrate.

Delivering mechanical stimuli#

Displacement (voltage charging) is bandlimited by the capacitive load of the stack and the current delivery capabilities of the amplifier. Charts by load are in PI documentation. This is responsible for hard limits on different frequency amplities.
When changing the step response, familiarize yourself with all the potentiometers, and take measures prevent yourself from touching those that influence the static calibration in particular. An excellent practice is placing a piece of clean room tape over top of all the potentiometers except those used in the step response.
Servo feedback is bandlimited, and so linearity is only guaranteed below this point. Check the model for specifications.
Resonance listed in PI documentation is only axial resonance for an unloaded piezo. The cantilever resonance on an attached probe will be much lower and can only be measured with a separate sensor (e.g. a laser Doppler vibrometer positioned at ~90 degrees to the displacement). Also, if adding a load this will modestly reduce the axial resonance.
Splitting an analog signal going to both the piezo amplifier and a DAQ board will typically result in “sampling noise” that exists in the piezo displacement. This is sometimes hard to detect but can be bypassed by sending a separate copy on a different analog output channel.
There is a small voltage offset on all analog output channels on DAQ boards. Not accounting for this will result in “jumps” typically at physiological scale at the beginning and end of stimuli. An initial calibration routine using the servo feedback (or direct measurement of the channel) can null out this effect.

Good habits#

To avoid temperature fluctuations, it is OK to leave amplifiers on, however it is not required.
Before use, “exercise” the piezo with the servo off by turning the DC-OFFSET knob all the way in each direction a few times.
Perform a “zero-point adjustment” (See E-509 Position Servo-Control Module User Manual PZ 77E) every week or so, or write a matlab routine that helps check if it is required.

Neurobio Dept photocopier

Fri, 12 Mar 2021 17:48:35 +0000

The Neurobio Department photocopier is outside Goldenson 420 (next to the fax machine). For “departmental ID”, punch in 499. (This charges copies to our lab.) Leave “password” blank. Press the “ID” key, and you’re ready to copy. After you’re done, press the “ID” key again to clear our code from the memory. Press the yellow “clear mode” and the red “clear/stop” buttons at the same time to logout.

pH Meter

Fri, 12 Mar 2021 17:47:14 +0000

Use: To easily move the probe between solutions, tighten or loosen the red knob on the articulated arm holding the probe, it’s pretty slick.

Calibration:

To calibrate, follow the instructions on the page sticky-noted on the manual on the shelf above the meter with the buffer aliquots. For a 3 point calibration using the 4, 7, 10 buffers, you may need to manually enter the values, it shouldn’t need to be done again, but instructions for how to do so are in the manual.

Parking at HMS

Fri, 12 Mar 2021 17:45:31 +0000

Occasionally another vendor or visitor will request a short-term parking pass for the garage under the Alpert building. You’ll need to find out how large their car is. The Quad garage uses vehicle lifts and there are height restrictions. SUVs, trucks, vans and larger passenger vehicles will not fit in the Quad garage and will have to use the NRB garage. (Note that parkers in the Quad garage generally have to leave their keys with the valet; parking in NRB is one-level parking and parkers can take their keys with them.) (This matters to some people.)

Paraformaldehyde

Fri, 12 Mar 2021 17:41:10 +0000

Paraformaldehyde solution (32%) is kept in the fume hood in sealed 10-ml glass ampules. When you break open a new ampule, please transfer the contents to a clean 10-ml scintillation vial, and clearly label and date the vial. If the date on the current vial indicates that it is >2 months old, the contents should be disposed of in the paraformaldehyde waste container (see Hazardous Waste below). New ampules can be ordered from Electron Microscopy Sciences (www.emsdiasum.com/formaldehyde), product # 15714; be sure to order the 10-ml ampules and not the large bottles.

Ordering through HMS (NIH projects)

Fri, 12 Mar 2021 17:39:15 +0000

First-time users need to take ROPPA training to get access to the B2P system.
When you’ve completed your training, contact Mary Kate Galusha in the Neurobio finance office and ask to be given access to B2P.
When you’re able, log into B2P and set up your account by following the steps on the quick-start guide on the first page: Under User Profile (upper right-hand corner), update your phone number, email, etc. Under Default User Settings, click Default Addresses and select the shipping address for Warren Alpert. Under Default User Settings, update Chart of Account Favorites to reflect the billing codes you’ll most frequently use.
Harvard’s B2P system is very similar to HHMI’s P2P system; if you’ve used P2P, you’ll have no trouble with B2P. If you’ve used neither, then you need someone to show you how to do it.

Ordering -- HHMI ProCard

Fri, 12 Mar 2021 17:31:12 +0000

Can you use the Procard? Yes, IF:

You intend to charge HHMI funds;
Your order is under $1,000 (conference registrations can go up to $1,500);
Your order is NOT on this list:

Anything that can be purchased through the HHMI P2P system (this includes anything from Amazon – do not use the ProCard for Amazon purchases.)
Restricted items such as hazardous and controlled substances
Travel, including Ubers and Lyfts
Payment of an outstanding invoice
Gift cards
In-restaurant dining
Setting up a Paypal account
Purchases from foreign suppliers

How to use the Procard:

Ordering -- HMS PCard

Fri, 12 Mar 2021 17:28:54 +0000

You can use the P-card if

you intend to charge NIH funds or grants/fellowships administered by the Neurobio finance office (i.e., which have a 33-digit billing number)
your order total is $500 or less
your order is not from an HCOM Marketplace Vendor (HCOM Marketplace Vendors applicable to us would be Thorlabs, VWR, Invitrogen, Sigma-Aldrich, McMaster-Carr, Digi-Key, Newark, Biorad, Thermofisher, Qiagen, Grainger, W.B. Mason, MSC Industrial Supply, or Creative Office Pavilion)

To use the Harvard P-card,

Ordering through HHMI

Fri, 12 Mar 2021 17:26:04 +0000

Orders charged to HHMI funds are placed via the HHMI P2P system. Please consult an authorized HHMI purchaser within the lab for this information. Currently, these are: Rachel Wilson, Stephen Holtz, and Allie Moore.

Large purchases require a quote, and “HHMI” must be somewhere on the quote. Have the quote formatted so that it’s addressed to: “Rachel Wilson Lab, HHMI/Harvard Medical School.”
When you’re in P2P, the title must be formatted as: CC30620 [vendor] mm/dd/yy [your initials]; if the item is a rush order, type RUSH after your initials.
If you are placing an HHMI order and have questions, please contact Rees Collier, or Adam Hepp (heppa@hhmi.org) or Cindy Truong (truongc@hhmi.org). Adam is located in Maryland and Cindy at HMS; both are available to help.
If you have questions about HHMI payments that are not P2P-related (e.g., questions about blanket POs, invoices from Harvard core facilities, etc.), please write to bwhsoo@hhmi.org; this email inbox is monitored by the HHMI staff that work in Enders 661 (Mary Ellen Morency, Nick DeSimone?, and Cindy Truong).

Odor delivery devices

Fri, 12 Mar 2021 17:13:38 +0000

A printed circuit board is available for adapting 0-5V control signals to drive 12V or 24V solenoid valves in olfactometers. The details on the most recent version (4.1) of Joe’s olfactometer can be found at https://github.com/joebell/valveDriver. The components for assembling the driver boards are kept in a box labeled “olfactometer driver parts” in the back of the lab.

Office Supplies

Fri, 12 Mar 2021 17:12:49 +0000

OfficeMax is the “preferred vendor” for HHMI labs. Because we would like to save Startup grant as much as possible and try to use the HHMI grant first, it is highly recommended to purchase from Purchases have to be made via the HHMI website. Please contact StephenHoltz to place an office supplies order.

If office supplies are being purchased through any of the HMS funding sources, W.B. Mason is the preferred vendor. Please contact Stephen Holtz to place an office supplies order.

Needles/syringes

Fri, 12 Mar 2021 17:10:02 +0000

Disposable syringe needles are available from the Neurobiology Supply Cabinet on the the 1st floor of Goldenson (across from room 159). See Steve Sleboda for the key. You must sign out any needles.

Molecular biology supplies

Fri, 12 Mar 2021 17:05:43 +0000

Autoclaved glassware#

Autoclaved glassware is found in the lower cabinet next under the chemicals bench. As it gets depleted, you’ll need to replenish this yourself. The first thing to do is to get training from Steve Sleboda in the operation of the glasswashers and autoclaves on the second floor. To do a glassware run, borrow a trolley from Tanya in the Yellen lab. Take a couple of autoclavable trays from the lower cabinet opposite the glassware (these are the larger trays on the bottom). Autoclave tape is in the “molecular biology drawer”and aluminum foil is on the shelf above. First run thing through the washers, then the autoclave. For smaller items like lids, there is heavy netting to put above the tray inserts, however, this doesn’t work well for stir bars, which end up at the bottom.

Software Installations

Fri, 12 Mar 2021 17:01:54 +0000

Download Sources#

FAS#

If you are an FAS affiliate (via status as graduate student, professor, or teaching assistant) you can download software from the expansive list on the FAS IT website downloads.fas.harvard.edu/download.

HMS#

If you are an HMS affiliate (postdoc or technician) you likely do not have access to the larger catalogue of FAS downloads, but can use those listed here https://software.hms.harvard.edu/all-hms-it-software-z. Note if you are transitioning from graduate student to some other role, your access to FAS content may terminate before other IT things (email, etc.,).

USPS

Fri, 12 Mar 2021 16:59:14 +0000

You can charge USPS postage on work-related letters and packages to our Startup account. To do this, fill out a Outbound Mail Account form (available in the mailroom) with the following billing code:

520-45318-8700-025881-730001-0000-66271

Or, pick up a pre-filled version of this form from Rachel’s office.

This is what the bottom of the form should look like (fill out the top with your package specification):

Note that the edge has been cut off, but it should end in 66271 (I will upload a better picture later)

Lunch Train

Fri, 12 Mar 2021 16:57:16 +0000

Lunch train generally leaves between 11:30am and 12:00pm. In honor of Emre, we try to eat early. The departure of lunch train can be signaled by pulling twice on an imaginary “train whistle” with your right hand.

Lockers

Fri, 12 Mar 2021 16:56:37 +0000

A new lab member can obtain a locker in the hallway by contacting Neuro Ops (neuro_ops@hms.harvard.edu) and asking for an available locker near the lab. Steve or Josh will give you the locker number together with a three-digit combination for the lock.

Turn right three times. Stop at First Digit.
Turn left one full turn passing 1st number and stop at Second Digit.
Turn right and stop at Third Digit and open. Might need to rotate the dial to do so.

Library (Countway)

Fri, 12 Mar 2021 16:55:38 +0000

Countway library at HMS can help you:

get a book from another Harvard library
get a book from a non-Harvard library
get a pdf of an article or book chapter at another Harvard library or non-Harvard library

GetIt! is the Harvard Library portal for accessing these services.

You’ll have to register the first time, and provide our 33-digit billing code. The “item number” is 8540 (meaning library photocopying), so the 33-digit code is: 520453188540025881730001000066271

Maintenance of multiphoton lasers

Fri, 12 Mar 2021 16:54:18 +0000

SpectraPhysics MaiTai laser#

Support engineer is Jian Lu (Jian.Liu@newport.com).

Coherent Vision-S laser#

Service contract sales and administration: Annette Davis (p. 800-367-7890 x4 | f. 408-404-0898, Annette.Davis@coherent.com).

Technical Support (+1 800 367 7890, customer.support@coherent.com).

A field engineer that covers the Boston area is Falk Preller (Falk.Preller@coherent.com).

Laser monitoring (all users)#

Keep an eye on the laser power, the output stabiliy, the time it takes to mode-lock, and the shape of the spectrum. Below is an example of laser showing signs of continuous-wave (CW) breakthrough, something that we don’t want to see! If you notice something strange, try 800 nm and see if the laser can mode-lock. Since more pump laser power is required for this wavelength, you might be able to detect something abnormal.

Lab laptop

Fri, 12 Mar 2021 16:34:13 +0000

The Wilson Lab laptop is an Asus. The password is “flypoker”. It is intended primarily for lab meeting presentations. It is sometimes locked to the desk in the rear corner of the lab, and the code for the lock is 0420.

HMS IT

Fri, 12 Mar 2021 16:32:06 +0000

All HMS IT support requests need to be initiated by sending an email to itsupport_EQ@hms.harvard.edu or by calling 617-432-2000 (press option 7). Phone support is available during business hours as well as after hours.

Computers needing hands-on IT support can be dropped off and picked up outside the Goldenson 540 IT Office.

Tony Luong Tony_Luong@hms.harvard.edu (617-432-1759) is one member of the East Quad HMS IT team who often supports users in the Department of Neurobiology.

Imaging core facilities

Fri, 12 Mar 2021 16:13:19 +0000

When using these core facilities:

If you have any questions, especially pertaining to operations which might damage the microscope, please ask a staff member. After hours, don’t hesitate to contact Rachel (home: 617-487-5301).
If you remove or install objectives on the microscope, do so very carefully. Dropping one of these can destroy it.
We are charged for every minute you’re logged in (even if you didn’t reserve that time), so don’t forget to log out when you are done.
We are charged the full time reserved if the reservation is not removed from the calendar 24 hrs. in advance.
If you are the last user of the day, please shut off the laser according to the facility’s procedures. We will be charged for the whole night if you don’t.
Do not attempt to use this equipment without registering and getting training by the staff.

Neurobiology Imaging Facility#

The Neurobiology Imaging Facility is located in Goldenson 242.

Hazardous Wastes

Fri, 12 Mar 2021 16:11:14 +0000

Diego is the current Safety Czar. Please direct questions to him.

General guidelines for waste disposal can be found on the flip-chart posted by the entrance to the 2P room. Please look here first for answers to questions about waste disposal.

If you want general guidance on what you can put down the sink, consult this.

Hazardous chemical waste should not go down the sink. If you are unsure whether something is hazardous chemical waste, please consult this or this. Note that hazardous chemical wastes include:

Halo Tag Ligands

Fri, 12 Mar 2021 16:10:10 +0000

The Janelia Fluor Halo Tag ligands are now commercially available from Promega.

📎 JF_Dye_HaloTag_Snaptag_Product Info.pdf

📎 Grimm2017_Protocol_SynthesisOfJaneliaFluorHaloTag.pdf

Green Lab Practices

Fri, 12 Mar 2021 16:09:29 +0000

Harvard Green Labs initiative provides tips for conserving energy and reducing waste.

Items we won’t use can be made available to other labs via the Labs Reuse List.

Used Millipore water purifier filters should be placed in the lab’s Satellite Accumulation Area for waste (i.e., in the fume hood) with a tag on them. EH&S should then be contacted to request pickup for recycling the filters.

Key tips:

Shut the fume hood when not in use.
Turn off equipment at the end of the work day, when possible.
Use your own coffee cup and water bottle whenever possible.
Put recyclable items in the blue bins (paper, small cardboard, coffee cups, milk cartons, juice boxes, plastics #1-7, unbroken glass, aluminum cans, alumminum foil and trays).

The Longwood Campus “Reuse Room” (a.k.a. the “Freecycle Room”) is open weekly on Wednesdays from 12:30-2pm and is staffed by volunteers. It’s located on the HSPH loading dock, which can be accessed either by crossing the loading dock area from the HMS side to the HSPH, or from the Kresge building into Building 1 down to the basement level. From HSPH there are signs on the walls showing the way to the Reuse Room. The room accepts:

Gas tanks

Fri, 12 Mar 2021 16:02:29 +0000

Tank farm management#

At any given time, at least two tanks should be hooked up to a tank farm, with green stickers designating them as in-use (i.e., with “Full” stickers removed).
The valve should be pointing toward the in-use tanks.
If the in-use tanks have low pressure (<1000 psi), then two back-up tanks should be attached and turned on.
When a tank is empty, remove the “in-use” sticker, disconnect it, and replace the valve cap.

Suppliers#

We purchase our main gas supply from MedTech (see below), but occasionally need to purchase equipment or specialty gasses from AirGas. Our AirGas account number is 3076751 regardless of the purchase method (HHMI funds or Harvard funds). We purchase our premix gas for the excimer laser directly from NovaGas, who is the vendor that Airgas actually subcontracts to.

Driver line collections

Fri, 12 Mar 2021 15:58:48 +0000

GMR Gal4 lines#

GMR lines Gal4 lines need two citations, one for the overall methodology (Pfeiffer et al. 2008) and one for the generation of the complete set with accompanying images (Jenett et al. 2012).
All GMR Gal4 lines are inserted into the attP2 site on 3L.
GMR Gal4 lines available from BDSC are listed here.
You can search the FlyLight site for GMR lines having expression in particular regions. This site shows low-resolution movies.
An alternative interface is Virtual Fly Brain.
NBLAST can be used to search the GMR Gal4 line collection using morphometric criteria.
We have archived higher-resolution images for 3,505 selected GMR Gal4 lines. These images are kept on our server. These images are kept as read-only for most lab members to discourage deletion.
Note that a small fraction of GMR lines at Bloomington are actually incorrect, due to stock contamination. (The same is probably true of the GMR LexA lines and the VT lines.)

GMR LexA lines#

GMR LexA lines available from BDSC are listed here.
All GMR LexA lines are inserted into the attP40 site on 2L.
Projected stack images of brains for all 1450 GMR LexA lines that are available to us through BDSC are in the following directory on Wilson lab server (Wilson Lab\Flylight browser\rubin_lexA_line_imgs). You can load them all at once and then quickly screen through lines for further follow up with imageJ.

VT lines Gal4 and LexA lines#

Vienna Tiles (VT) lines need one citation (Tirian et al. 2017). Table 2 of this paper lists all VT Gal4 lines and their status.
All VT Gal4 lines are inserted into the attP2 site on 3L.
VT LexA lines are inserted in either attP40 (2L) or attP2 (3L).
You can search the FlyLight site for VT Gal4 lines having expression in particular regions. This site shows low-resolution movies.
Images of VT Gal4 lines and VT LexA lines be viewed at Virtual Fly Brain.
Some VT Gal4 lines are obtainable from the VDRC VT Collection.
Note that many VT Gal4 lines have been culled from the VDRC VT Collection; culled stocks are listed here. If one of these stocks is important to us, we can re-generate with the help of WellGenetics.
- Jenny has previously had success receiving VT Gal4 plasmids (and unavailable Gen1 Gal4 line plasmids) from Heather Dionne (dionneh@janelia.hhmi.org) with embryo plasmid injection done by BestGene. This saves time (weeks) and money.
VT LexA lines are not available from any stock center, and so these lines would need to be re-made if we want to use them. In this case, it may be worth requesting the plasmid from Barry Dickson; WellGenetics could then re-inject this plasmid into flies.

Split-Gal4 lines#

Many enhancer fragments from the GMR and VT collections have been converted to split-Gal4 hemidrivers.
The relevant citation is Pfeiffer et al. 2010 for the GMR hemidrivers.
The relevant citation is Tirian et al. 2017 for VT split hemidrivers, aka BJD lines.
All GMT and VT split-Gal4 hemidrivers available from BDSC are listed here.
The likely expression patterns of split-Gal4 hemidrivers can be inferred from the expression patterns of the corresponding GMR line or VT line. However, split-Gal4 hemidriver expression patterns often do not match the expression patterns of the corresponding GMR line or VT line, partly because the split-Gal4 transgenes may be inserted into a different site in the genome. This is why it is necessary to actually screen split-Gal4 combinations for the desired expression pattern (see Split-Gal4 screening).
A specific combination of a AD domain and a DBD domain is called a split-Gal4 combination. Split-Gal4 combinations created atJanelia are reported here.
Note that Janelia publications reporting new split-Gal4 combinations typically use combinations of GMR and VT hemidrivers, but the VT hemidrivers are reported as “BJD” lines. The only way to identify the relevant enhancer fragment (from the VT collection) is to ask someone inside Janelia, e.g. Heather Dionne ( dionneh@janelia.hhmi.org).

InSITE lines#

The InSITE transgenic driver line collection is described in Gohl et al. 2011 Nature Methods. The lines are designed to be “swappable”, meaning that (in principle) it should be possible to convert a given line from a Gal4 line to a LexA or Q-system line having a similar expression pattern - e.g., John Tuthill swapped LexA into 0203-Gal4 to create 0203-LexA. See also InSITE info at BDRC.
To do a quick visual screen, begin with the InSITE Database. Images have been annotated based on the brain regions where they drive expression, so you can also do some coarse searches this way.
To examine interesting expression patterns in detail, the next step is to check the confocal stacks, which are stored on the server, in the directory ‘InSite Lines’. Note that in order to view a z-stack, you need to download the mdb file (not the lsm file), and then open the mdb file using the Zeiss LSM Image Browser. You can download the LSM Image Browser here.
InSITE lines are available from the BDRC. Lines in the database which are not available from the BDRC may be available from other InSITE consortium labs (Clandinin, Luo, Scott, Waddell, Miesenböck); please ask Rachel to send a request on your behalf if you are interested in this.
It is important for us to follow the rules established by the consortium in using these lines:
1. Do not distribute any unpublished lines from this collection outside our lab. However, once any given line is published, it should be shared freely.
2. The acknowledgements section of any paper that uses these lines should include Daryl M. Gohl and Marion A. Silies, as well as Tom Clandinin; all of them contributed to creating the collection.
3. Consider the interests of the other members of the consortium. Every participating lab has made a large contribution toward the collection (either in sweat or in dollars), and their investment needs to be protected. For example, it would not be a good idea to take these lines and use them to study Drosophila gustation, because this overlaps with Kristen Scott’s work. Also, it might be good idea not to talk about specific expression patterns in the collection with people outside our lab, because you might inadvertently end up sharing information which another collaborator would prefer to keep confidential.
4. If you want to take a small number of lines with you when you leave the lab, that is probably OK, but you should ask Rachel to confirm this with Tom.

Fly Holders

Fri, 12 Mar 2021 15:35:54 +0000

Shims#

The steel shims in the fly holders are ordered from Etchit (www.etchitmn.com, Randy Johnson, Sales Manager, randy@etchitmn.com, 763-450-3130). The most popular shape is the R92 shape (rounded_head_taper_back_92pct_v07 - R92).

These designs are currently on the wilson lab github page, and are inventor files.

Cone-shaped Holders#

The cone-shaped fly holders are ordered from Protolabs (www.protolabs.com). They provide both 3D printing and CNC machining depending on what you need.

Service: CNC Machining
File Format: STEP (.stp or .step)
- There is an option in Autodesk (.ipt) to export as a CAD format
Material: acetal homopolymer delrin 150 (black), now listed as Acetal Homopolymer (POM-H) (black)
- This material is rigid and light, but does not absorb liquid

When requesting a quote, the manufacturer will likely indicate that the cone is too thin and there are edges that will not be machined properly. That is fine. Just order a handful more than you need (~5/order) with the assumption that some will turn out damaged.

Fly Food

Fri, 12 Mar 2021 15:32:07 +0000

We get fly food from Archon Scientific. This is a monthly subscription service.

A few days before the scheduled shipment, Archon will e-mail the Fly Food Czar asking how many trials of vials and bottles we want for that month. Archon will also indicate the shipment date for the following month so that we know exactly how many dates there are between the two shipments.
The Fly Food Czar responds to that e-mail by mentioning how many trays of vials and bottles we want. It might be good to add an additional reminder in the same e-mail to make sure that vials need to be stacked horizontally to prevent them from being squashed during the shipment.
Archon will confirm the order and he will send us the food via FedEx?. Once the shipment is on the way, Archon will send the tracking number.
The food will arrive in a large cardboard box in a few days, and they can be stored in the incubator or in the lab.
If we are likely to run out of food before the next shipment date, the Fly Food Czar will e-mail Archon to place an additional order (“booster shipment”). If it’s a small quantity, he can usually send it out the next day. The only catch is that the price will be a little higher than what you get from the monthly subscription.
As a default, the food will be light molasses food, and vials and bottles will be plugged (additional charge). Archon likes getting feedback from us on the quality of the food, and they will let the Fly Food Czar know if they are making any changes to the recipe (e.g. water content).

Archon Scientific, Inc. contact info:

Flycircuit database

Fri, 12 Mar 2021 15:29:57 +0000

The Flycircuit database can be accessed using the login name rachel_wilson@hms.harvard.edu and the password flypokers. For details, see Chiang et al. 2011. Note that the corresponding neurotransmitter may not be released by the Gal4-expressing neurons that are obtained in the Gal4 line that was constructed using the regulatory regions of these genes.

Note that if you limit your query by “Innervation sites”, the uppercase letters (e.g., “AMMC”) designate cells on the right side of the fly’s brain, and the lowercase letters (e.g., “ammc”) designate cells on the left side.

FicTrac Part 2: general usage

Fri, 12 Mar 2021 15:27:15 +0000

Installation#

See installing fictrac

Configuration#

There are two steps for configuring fictrac prior to running the program.

First, you must provide a text file in the fictrac/sample directory that contains the configuration parameters for your setup. At a minimum, you must define the following parameters:

src_fn - defines the image source. This can be either a video file or a camera index.
To set your camera index, check the order of your cameras in SpinView.
Note: If you receive an error or your fictrac feed is blank, try a different camera port as you may be connected to an unavailable or incorrect port.
vfov - defines the vertical field of view (angle in degrees).
This can be approximated by measuring your camera angle [tan(x) = vertical field of view in the fictrac feed/distance from your camera to the ball]. 1. Example: for a setup with a distance from the camera sensor to the ball of distance_var = 17.5 (cm), and the full height of the image used by fictrac (the rectangular ROI you select for recordings in SpinView) height_var = 0.78 (cm), the vertical field of view would be:vfov = radtodeg(2*atan(height_var/(2*distance_var))).
Note: to calculate the full height of the image used by fictrac (for the example above), take a picture with a ruler at the same focal point as the ball, and use the distance between lines (1 mm in this example) to calculate the height of whole the image.

FedEx (Federal Express)

Fri, 12 Mar 2021 15:23:22 +0000

Current Number: 393133139

If you are initiating a Fedex shipment, please log in to our account at www.fedex.com (username WilsonRach, password F1ypokers; note that that the second character in this password is the numeral “1” and not the lowercase letter “L”). (Note that “4. Billing Details: your reference” should prepopulate with CC30620, which is correct, unless you use a preset address, in which case it may prepopulate to 008196 and then create an error; the solution is to change it to CC30620.) You should enter your shipping data and print the airbill to affix to the envelope, pak (bag), or box. You can find shipping materials near the catalogs / software documentation shelf.

Fax

Fri, 12 Mar 2021 15:21:43 +0000

Use the departmental fax, outside Goldenson 420. No code is needed to use it. Do not dial “9” before the number. (Just key in 1-(area code)-(7 digit number). Use international prefix 011 for international faxes. If the machine seems unresponsive, make sure the “manual” button is not lit. The fax number for this machine is: (617) 432-1639.

FAFB v14/CATMAID/walled garden

Fri, 12 Mar 2021 15:20:37 +0000

The FAFB dataset is documented in Zheng et al., 2018.

CATMAID is a platform for collaboratively reconsructing and annotating neurons in large EM datasets. Basic instructions for viewing and tracing are available on the CATMAID page. Tools and scripts for using CATMAID to work with FAFB are archived on this GitHub repository.

The v14 instance of FAFB refers to the 14th round of alignment. This alignment was posted to a public server at Johns Hopkins. It was also used as the basis for establishing a workspace within CATMAID for multiple labs to work together. This workspace can only be joined by application, and it requires the explicit consent of all groups already in the workspace and so it it called the ‘walled garden’. Often, however, the terms v14 and ‘walled garden’ are used synonymously, because essentially everybody working in v14 is working in the walled garden.

Purchasing ethanol

Fri, 12 Mar 2021 15:14:47 +0000

For cleaning surfaces and general use (e.g. fly morgues) we can use 70% denatured ethanol (contains trace methanol).

Ethanol can be purchased in gallons from VWR using the Harvard B2P system, for example:

BDH ETHANOL 70% ACS DENATURED POLY 4 L

Catalog # BDH1164-4LP

For other uses (e.g. cell culture) or if higher concentrations are needed VWR or Fisher are both convenient, HHMI funds can be used for Fisher through P2P.

Lab Gmail (flypokers)

Fri, 12 Mar 2021 15:13:25 +0000

A lab email account as been setup for the purpose of supplying vendors / PAYPAL with this information. This also minimizes the amount of catalogs / spam that you and Rachel get in your inboxes.

email: flypokers@gmail.com

password: wilsonlab

If you’re a new user, or you’re a lab member using a new device for the first time, you’ll may need the recovery info to get past Google security procedures:

Recovery email is Rachel’s HMS account: rachel_wilson@hms.harvard.edu
Recovery phone (temporarily) Stephen Holtz’s cell 571-338-0895

Dropbox (HMS accounts)

Fri, 12 Mar 2021 15:03:14 +0000

HMS provides us with Dropbox business accounts with unlimited storage space. For more information, see

http://it.hms.harvard.edu/services/storage-data-management/dropbox-business

Data back-up

Fri, 12 Mar 2021 15:01:44 +0000

All data should be stored in two places, one of which must be off-site (i.e., not in the Warren Alpert Building). The server is the preferred location for off-site backup. Data acquisition routines and other important files should also be backed up regularly. Each person is responsible for their own backup.

HMS offers us a Dropbox business account with unlimited storage that can also be used for data backup and transfer. You can request an account from HMS IT here.

Data acquisition cards (DAQ)

Fri, 12 Mar 2021 15:00:31 +0000

PCI(e) cards#

Most rigs use the NI PCI-6251 (single breakout box) data acquisition card. This has the older PCI slot (long gold strip) and will not fit into many modern computers. However, there are NI PCIe-6321 cards, which is essentially the same card but uses a PCIe slot (two short gold strips) rather than the rapidly disappearing legacy PCI slot.

If you are ordering a new card however then you are recommended to use NI PCIe-6351. Its specs can be found here.

Czardoms

Fri, 12 Mar 2021 14:59:12 +0000

Assignments#

Name	Czardom
Alex Bates	Fly Incubator Czar Connectomics Database Czar Party Train Czar
Jingxuan Fan	Coherent Laser Co-Czar
Stephen Holtz	Infrastructure and Organization Co-Czar
Bakari Moitt	TBD
Mo Osman	Website Czar
Diego Pacheco Pinedo	Safety Czar
Janki Patel	Pipette Puller Czar Millipore Water Czar
Noah Pettit	Data and Server Management Czar
Pablo Reimers	Gas Czar Lab Meeting Food Czar
Sophia Renauld	Forcep Sharpening Czar Sink and Paper Towels Czar Outgoing Fly Shipments Czar
Victoria Rockwell	Back Bench Czar
Carl Wienecke	Coherent Laser Co-Czar
Helen Yang	Fly Food Czar
Wenyi Zhang	Spectraphysics Laser Czar Office and Printer Supply Czar
Yunzhi Zhao	Flystocks Database Czar

Description of Responsibilities:#

Back Bench
- Keep the back bench area clean and orderly.
Coherent Laser
- Maintain laser and manage necessary repairs.
Fly Incubators
- Maintain and replace the mite paper on all shelves
- Clean the interior of the incubators every 6-8 months
Fly Food
- Order, unpack, and store all standard fly food (molasses and corn) vials and bottles
- Order all specialty fly food (e.g., molasses and german food packets)
Fly Traps
- Regularly empty and re-fill fly traps throughout the lab
Forcep Sharpening
- Coordinate forcep sharpening order shipping and payment via Corte Instruments
Gas
- Order all standard (CO2, Carbogen, Medical Air) gas tanks for the main lab and 2P room
G4 Arenas
- Keep tabs on the status of any unused arenas, and our stock of spare parts. Understand how the G4 arenas work and how to troubleshoot them.
Lab Meeting Food
- Order and coordinate pickup/delivery for lab meeting food
Office and Printer Supply
- Restock office supplies as necessary.
- Fix printer problems.
- Replace the printer every few years, when it dies.
Outgoing Fly Shipments
- Prepare vials of flies for shipment to other labs, as requested.
- Pack and ship vials when they are ready to ship.
Party Train
- Coordinate, reserve, and pay for all “Party Trains”.
Safety
- Serve as main liaison/point-person between the lab and EH&S.
- Implement any safety-related changes in the lab.
Sink and Paper Towels
- Re-fill paper towel holders.
- Ensure dirty glassware or mold does not accumulate in the main lab sinks.
Spectraphysics Laser
- Maintain laser and manage necessary repairs.

Conferences

Fri, 12 Mar 2021 13:57:35 +0000

Past Conference Reviews:

CSHL Neurobiology of Drosophila
- Basic info: every other year (odd years) in early October from Tuesday evening to Saturday lunch. Single track
- Matt: By far my favorite, strongly recommend folks go every time. Presented a poster, very well attended with a lot of good questions and feedback.
- Stephen: Good for people trying to get familiar with Drosophila neuroscience for the first time and for veterans/people looking to move into a different part of Droso neuro. It is a single track conference (everyone sees the same talks) with very good coverage across many fields and a very well attended poster session where you will likely get some feedback. Recommend.
- Helen: Except for 2 keynote lectures, all the talks are short format (12-15 minutes) and almost all of them are given by trainees or junior faculty members; talks are usually very good. Started with a strong neurodevelopment focus but has become more physiology and circuits focused each year. I strongly recommend attending this conference every time it is held. If you attend one conference in a year, this one would be at the top of my list. For senior grad students, consider asking Rachel to nominate you for the Elkins lecture, the keynote graduate student talk.
- Sophia: Went as a first year grad student (no pres or poster), found it approachable and a good introduction to the science and people of drosophila neuro. Would recommend
CSHL Neuronal Circuits
- Info: every other year (even years) in mid-March from Wednesday evening to Saturday lunch. Single track
- Matt: Good, more general neuroscience audience but a lot of fly folks. Presented a poster, ok attendance but a lot of good questions and feedback.
- Helen: smaller and shorter than the CSHL Drosophila meeting and focused on systems/circuits neuroscience across species. Good meeting but lower priority than Drosophila one. For senior grad students, consider asking Rachel to nominate you for the Larry Katz lecture, the keynote graduate student talk.
COSYNE
- Info: held annually at the end of February/beginning of March. Divided into single track main meeting and workshop after main meeting
- Matt: Least favorite, talks were not presented for general audience and many of the questions/comments were obviously sexist. Much of the theory presented was presented for “insiders” rather than a “general audience”, making the conference not really worth my time. Presented a talk which went well, though relatively little engagement afterwards.
- Stephen: In the past (pre 2018) was a place to hear interesting ideas and there was a fair amount of strong modeling of quantitative neuroscience mixed in among far more computational topics. The workshops are a really nice way to get familiar with a field, and it is a nice environment for meeting people/networking more easily relative to the conference. I would probably do both or just the conference though. Recommend if computationally curious, but I am unsure if the vibe has shifted as others have noted.
- Helen: I last attended in 2023 and did not like it for the same reasons as Matt. I did get to catch up with some of my grad school classmates and some others in the field, so it wasn’t a complete waste of time. Maybe someone with a stronger background in theory and computation would get more out of it, but for the vast majority of talks/posters, I did not come away with an understanding of why I should care.
ASCONA https://www.asconacircuits.org/
- A ‘fancy’ and small conference that has a lot of systems neuroscience. A lot of solid, interesting work presented usually by PIs. It is held in a very nice, beautiful swiss location just north of Italy, and like it or not, you will rub shoulders with glam journal editors. Probably good for a more mature project, only one person per lab is allowed and it can be competitive based on the year and how recently someone from the lab has attended. A good experience for meeting people and maybe for networking/setting up future connections. Recommend (stephen)
NCM (Annual meeting of the Society for the Neural Control of Movement)
- Info: held annually in ~April in a different interesting location each time. Single track. Mostly human and primate work, with some rodent
- Helen: I attended this to learn about what the vertebrate people thinking about when it comes to the neural control of movement and was one of 2 fly people at a conference of ~400 people. If you work on movement, not a bad conference to attend once in a while to expand your horizons
- Sophia: I attended this meeting May 2025. This meeting was 1/3, 1/3, 1/3 human, primate, rodent work, and again, I was one of two fly people. Despite this, my poster was more well attended than I had expected, and folks were generally still interested in work in flies. In particular, I was impressed by some of the more translational/clinical research presented. If you work on motor control, are thinking about how potential projects in this lab can contribute to the broader field, and/or are thinking of a more translational aspect to your career at any point, I would recommend attending.
Modulation of Neural Circuits and Behavior Gordon Conference
- Info: held every other year (odd years) towards the end of May. Small, single track conference. Like all Gordon conferences, has an associated seminar meeting in the 2 days before for trainees.
- Helen: Systems neuroscience, generally, but check the program for the specific conference since there is a theme each time. 50-75% of talks given by PIs. Lots of networking opportunities.
Crete meeting (officially, Workshop on Neural Circuits and Behavior of Drosophila)
- Info: held approximately every other year in June at the Orthodox Academy of Crete, fundraising permitting. Small (~60-80 people), single track meeting, that lasts 6-7 days. Everyone who attends gives a short talk (15-20 minutes). Must apply to attend, and given the size of the meeting, usually 1 person per lab. Senior grad students and postdocs at the late stage of their project. Usually info about this meeting spreads by word of mouth, but apparently, they’ve made a website now.
- Helen: I really like this meeting. The location is gorgeous, and there are plenty of opportunities to just hang out and talk science with fly circuits people. Vibe is very casual, and includes stray cats that will wander into the conference hall during the presentations. On the other hand, because these people are the ones closest to us in the field, expect detailed and vigorous discussion. From a networking perspective, probably best for senior grad students and postdocs who will be staying in the fly circuits field.
Neuroethology (International Congress of Neuroethology)
- Info: every two years held somewhere new, often in an interesting place. A wide variety of species studied with an emphasis on non-model systems and behaviors observed in nature. Large poster session(s) with big and small talks (may have changed since I went - Stephen).
- Stephen: Talks are usually great because presenters are a) aware they are talking to outsiders for their field, and b) used to explaining their idiosyncratic system. It can be a nice place to gain an appreciation for animal behavior beyond the mouse and fly. I think it is a nice thing to attend any time in the lab for a shot in the arm or to get some different perspectives on behavior, there is disproportionate neuro droso (and insect vision/navigation) attendance, so you may get more relevant feedback if you do present a poster than you might think. There are awards for graduate students which lab alum Tots got, so maybe look into that if you’re attending and senior.

Misc Smaller Themed Conference Series

Cleanliness

Fri, 12 Mar 2021 13:56:03 +0000

Transfer your fly cultures every 14-21 days. (Occasionally stocks with deleterious mutations may require longer to mature; in these cases, you can of course make exceptions.) Do not keep old cruddy cultures around. This encourages mites and other parasites to destroy our stocks.

Wipe down your dissecting area and the fly-pushing area frequently with ethanol. This also discourages parasitic infestation. (Wiping down with a few drops of concentrated acid gets rid of saline corrosion spots on the benchtop.)

Custodial Services

Fri, 12 Mar 2021 13:52:23 +0000

For questions or problems with custodial services, contact the custodial supervisor, Teovane Martins (617-432-2923). The building manager for the Warren Alpert Building and the Goldenson Building is Jerico Johnston (617-432-5378).

You can also contact Neurobio Research Operations at neuro_ops@hms.harvard.edu

If there’s an equipment emergency, contact Neuro Ops before contacting custodial services.

 [617-432-2251](tel:617-432-2251)
 

 

 
 
 
 
 

 [617-432-2251](tel:617-432-2251)

Address format for shipping

Fri, 12 Mar 2021 13:49:18 +0000

Our shipping address (for packages) is:

[your name - important!]

Department of Neurobiology

Harvard Medical School

200 Longwood Ave.

WAB320

Boston, MA 02115

Note: 200 Longwood is the Warren Alpert loading dock.

Our mailing address (for letters and invoices) is:

[your name - important!]

Department of Neurobiology

Harvard Medical School

220 Longwood Ave.

Boston, MA 02115

Our HHMI Purchase Shipping/Billing Address (The same just with HHMI):

[your name]/Rachel Wilson

HHMI/Harvard Med School

Color-depth MIP searches

Fri, 12 Mar 2021 13:47:10 +0000

About cMIP Searches#

MIP stands for Maximum Intensity Projection. Color-MIP (“cMIP”) is a way of displaying 3D neural morphologies so that different neurons and drivers can be easily compared. Otsuna et al. 2018 created a database of cMIP images and a Fiji plugin for searching them.

When publish papers that use color-depth MIP serches, we should cite: Otsuna et al. (2018) for the color depth MIPs and search tools, Jenett et al. (2012) for the Janelia Gen1 GAL4 collection, Tirian and Dickson (2017) for the VT Gen1 GAL4 collection and/or for the VT hemidrivers, and Dionne et al. (2018) for Janelia hemidrivers (if we are using these hemidrivers in the publication in question).

Purchasing chemicals/drugs

Fri, 12 Mar 2021 13:45:20 +0000

Our default chemical supplier is Sigma-Aldrich, which has punch-out catalogs in both the HHMI P2P and HMS B2P purchasing systems. Questions re: pricing and products can be referred to 508-238-9865.

For drugs (pharmacological agents), as opposed to chemicals, you can sometimes get better prices or a larger selection from other vendors. Check out Tocris (http://www.tocris.com, customer # HAR002A), or Calbiochem (http://www.emdbiosciences.com/html/CBC/home.html). These vendors also provide more extensive information about a compound’s biological actions, solubility, storage.

Balances

Fri, 12 Mar 2021 13:39:51 +0000

If you move either of the balances (even a few inches), check the leveling index above the rear right-hand leg. You may need to adjust the height of the rear legs (using the wheel under each leg) to re-level the balance. The air bubble should be inside the black circle. Always use the coarse balance for coarse measurements.

Appliances

Thu, 11 Mar 2021 16:25:42 +0000

If ordering from Best Buy (1-888-BEST BUY, http://www.bestbuy.com), use customer number 71161 for tax exemption.

Antibodies

Tue, 09 Mar 2021 14:52:51 +0000

To keep our antibodies fresh and well-documented, is important that everybody follows the following rules:#

ALWAYS UPDATE THE ANTIBODY INVENTORY LOG when you order/receive antibodies or move an aliquot between different storage locations. The log is kept as a Google Document called ImmunoLog.
When you receive an antibody, please aliquot it immediately. Do NOT freeze unaliquotted stock tubes unless the storage instructions explicitly say it should not be aliquotted. Label aliquot tubes INDIVIDUALLY, preferably with the small round freezer dot stickers on the tops of the vials. Under no circumstances should an unlabeled tube be placed in any of the freezers. When you’re done, place the aliquots in one of our storage boxes with individual tube slots and update that information in the ImmunoLog. Do NOT store aliquots inside of a Falcon tube (which wouldn’t fit in the appropriate boxes anyway). Don’t add a new box to the freezer unless all existing boxes are completely full—there is probably plenty of space for your aliquots in an existing box. Note that you can use the ImmunoLog to count the total number of aliquots in a particular box without opening the freezer.

Safety

Tue, 09 Mar 2021 14:00:21 +0000

Call 911 if there is a potentially life-threatening accident.

Laser eye damage#

Use caution to shut down the laser system or put it in a safe state.
Immediately call Harvard University Operations at (617) 495-5560 for transportation to Mass Eye and Ear Infirmary. Do not drive yourself to the hospital.
Remain in a sitting position to minimize further damage to the retina.
Notify a colleague and ask for assistance.
Ask a colleague to contact Rachel immediately (home phone: 617-487-5301; in case you cannot reach Rachel, leave a voicemail at her home number and also email her).
Report the accident to Harvard Radiation Safety Services (617-496-204, xiaowei_yan@harvard.edu).

Urgent care#

In a non-life-threatening situation, Harvard employees and students should seek urgent care at HUHS in Vanderbilt Hall (M/Th 9–6:30; T/W/F 9–5) During weekends and evenings, urgent care is available from HUHS in Cambridge (huhs.harvard.edu/urgentcare). Lab members who are not Harvard employees or students (e.g., HHMI employees) should go to Brigham and Women’s Emergency Room.

Fly food

Fri, 12 Mar 2021 15:31:42 +0000

Helen is our current Fly Food Czar.

Food from Archon Scientific#

We purchase most of our fly food from Archon Scientific via a monthly subscription service. The food is paid for by a blanket PO issued by HHMI. The blanket PO for FY23 is HHPO1513060.

A few days before the scheduled shipment, Archon will e-mail the Fly Food Czar asking how many trials of vials and bottles we want for that month. Archon will also indicate the shipment date for the following month so that we know exactly how many dates there are between the two shipments.
The Fly Food Czar responds to that e-mail by mentioning how many trays of vials and bottles we want. It might be good to add an additional reminder in the same e-mail to make sure that vials need to be stacked horizontally to prevent them from being squashed during the shipment.
Archon will confirm the order and he will send us the food via FedEx?. Once the shipment is on the way, Archon will send the tracking number.
The food will arrive in a large cardboard box in a few days, and they can be stored in the incubator or in the lab.
If we are likely to run out of food before the next shipment date, the Fly Food Czar will e-mail Archon to place an additional order (“booster shipment”). If it’s a small quantity, he can usually send it out the next day. The only catch is that the price will be a little higher than what you get from the monthly subscription.
As a default, the food will be light molasses food, and vials and bottles will be plugged (additional charge). Archon likes getting feedback from us on the quality of the food, and they will let the Fly Food Czar know if they are making any changes to the recipe (e.g. water content).
Our contact at Archon is “Joe” Joseph Daniels (josephdaniels@archonscientific.com), 919-450-6744

ATR Molasses food#

The link to this protocol can be found here. Currently we make this once a week, approximately.

Billing Codes

Fri, 12 Mar 2021 13:43:20 +0000

For internal HMS charges (library document delivery service, garage parking for visitors, etc.), we need to supply a 33-digit billing code.

Most small miscellaneous charges of this type should be charged to “startup”, and the code for this is:

520-45318-XXXX-025881-730001-0000-66271

For personnel whose work is related to R01-dopamine, use this code:

R01 Dopamine - 520.45318.6600.151296.377729.0001.66271

If you’re not sure if this applies to you, check out this page: /purchasing-billing-shipping/personnel-funding-assignments/

Tax Certificates

Mon, 22 Nov 2021 15:54:33 +0000

~~Massachusetts ST2~~

~~Massachusetts ST5~~

~~Federal 501(c)(3)~~

New location of ST-2 form: https://hu.sharepoint.com/sites/FAD/Web/Forms/AllItems.aspx?id=%2Fsites%2FFAD%2FWeb%2FOC%2DTax%2FTax%20Forms%2Fcertificate%5Fst%5F2%2Epdf&parent=%2Fsites%2FFAD%2FWeb%2FOC%2DTax%2FTax%20Forms&p=true&ga=1

When setting up a new account with a vendor, be sure that we are not charged sales tax. (HMS is a tax-exempt institution.) Some vendors need evidence of our Massachusetts ST-2 form, which documents this tax-exempt status. Our ST-2 certificate number is 042-103-580. If the vendor requests it, can can provide this certificate by fax and/or as an e-mail attachment.

Lab meeting schedule

Mon, 15 Mar 2021 18:16:40 +0000

Rachel’s assistant keeps this page updated, but anybody is free to edit it.

If you anticipate being unavailable for a given week, please add this to “notes” so that the conflict is clear.

Open embedded document

Fly stock maintenance

Fri, 12 Mar 2021 15:50:46 +0000

Please follow these guidelines for maintaining your stocks:

Maintain all your stocks in duplicates. A stock can be easily lost due to mold or just random infertility events. You must keep 2 copies of all your stocks (i.e., 2 separate vials or bottles). The flipping schedule for the 2 copies should be staggered, ideally by one-half the time between flips. This provides robustness to random events (e.g. a poor batch of food) and allows you to always have a vial to scrape a couple virgins from if you get desperate.
You should keep your stock collection at room temperature or at 18 deg C. You’ll have to flip less often if you keep them at 18 deg C, but unhealthy stocks may not reliably make it at that temperature.
We generally maintain our stocks in white food vials, though very sick stocks may do better on molasses.
With our current food, stocks on white food at room temperature should be flipped every 4-5 weeks. On molasses at room temperature, the ideal time between flips is probably 3-4 weeks. The time between flips is ideally the max amount of time that still allows you to reliably propagate all your stocks. Reliably as in losing both copies of a stock is something that happens a handful of times during your tenure in the lab, and losing a transgene (i.e. all the stocks with that transgene die) is vanishingly rare.
Stocks that you are using for current experiments and/or crosses should generally be kept in bottles or vials separate from your stock collection. Bottles if you need a lot of flies and vials if you don’t. Your stock collection just maintains that genotype of flies from generation to generation, and the less you handle them, the lower the chance that they become contaminated or that you accidentally kill them by taking too many virgins.
It’s a good idea to look at your stocks under the microscope every few months to make sure that the appropriate genetic markers still exist.

New Lab Member Guide

Tue, 04 Jul 2023 19:13:53 +0000

Compiled by Emily Kellogg and Rachel Wilson

Hello! Welcome to the Wilson lab!

Useful links#

Lab Meeting Schedule
Lab Away Dates
Immunohistochemistry Reagents Log
Ask Rachel to share her calendar.
Ask Rachel for a key.
Ask NeuroOps for a locker
Read the HMS Neurobio FAQs
Familiarize yourself with the contents of this WilsonLabWiki! (And keep it updated!)
Make sure you get an HMS email and HUID as soon as possible, if you don’t already have one
Check out the new postdoc orientation guide. This is pitched to new postdocs, but it has some items of interest for all new lab members.

Getting Started#

Here are some basic topics you’ll need to master in the first couple of weeks of experimental work:

Split-Gal4 screening (crosses)

Fri, 12 Mar 2021 12:29:57 +0000

USEFUL STOCKS#

yw UAS-mCD8::GFP; S/CyO; +
10XUAS-mCD8::GFP; + ; TM2/TM6B

Step 1A. Screening hemidriver combinations #

Combine each AD and DBD combination together with UAS-GFP. The goal here is to visualize the expression pattern of this hemidriver combination. The result is not a stable stock, however, so this is for screening purposes only. Here it is convenient to begin with virgins having UAS-GFP on the X chromosome and double balanced on the second chromosome.

New Research Assistant Guide

Mon, 13 May 2024 14:56:52 +0000

Main job responsibilities#

The main responsibilities of this job are the dissection of Drosophila central nervous system, screening hemidriver combinations for neurons of interest for different lab members with confocal microscopy, other immunohistochemistry experiments, stock maintenance, making of various fly foods, running behavioral experiments for lab members, and computational neuroanatomy.

Fly food#

We have various protocols for different types of fly foods. The main foods you will make are ATR molasses and German food. Depending on what’s going on with Genesee, you might have to make this food from scratch or from a packet. This page goes over the different types of food and their protocols.

WellGenetics services

Mon, 15 Mar 2021 18:48:59 +0000

WellGenetics is our preferred vendor for transgenic fly stocks.

Our primary contact is Dr. Yawen Chen (yawen.chen@wellgenetics.com). Say hi if you see her at the CSHL Neurobiology of Drosophila Conference!

Before ordering flies, make sure that BDSC and Janelia don’t have the flies made already. If the flies are potentially available at Janelia, you could ask Rachel to contact Barry Dickson lab for the flies and/or the plasmids that contain the enhancer fragment of interest.

Ordering/importing flies

Sun, 14 Mar 2021 11:40:57 +0000

Bloomington (BDSC)#

Sign up for your own account (click here) and select “Add User to Existing Account”.
User Number (BUN): 866
Account name: Rachel Wilson
Use your own account when placing orders (not Rachel’s account).
Do NOT select DOA (dead on arrival) unless you are replacing stocks that were actually DOA.
Bloomington now accepts payments by credit card, use the HHMI Pro-card for this. You will be alerted a few days after the order that you must pay, and can log back in, navigate to “orders” and click “Pay Now”
All stocks at Bloomington (including stocks not yet on Flybase) are here.
Bloomington stock subtypes are all listed here, including RNAi lines distinct from the VDRC stocks.
Bloomington does not ship during the second half of December.

WellGenetics#

When sending flies to WellGenetics, you’ll need get a Taiwan import permit from them and then attach it to your shipping package.

KDRC (Korea)#

URL: kdrc.kr
Email: flypokers@gmail.com
Password : flypokers_wilsonlab123
On checkout upload the LONPR (attached below)
As of 4/2025 stocks are free, and we just need to provide the FedEx billing code (in the account already)
Update the shipping address on checkout! It does not populate properly by default.

VDRC (Vienna)#

URL: http://stockcenter.vdrc.at
User ID: WilsonLab
Email: flypokers@gmail.com
Password : flypokers_wilsonlab123
DO NOT use the HHMI card! International charges are not allowed. If you want to use HHMI funds reach out to our current science ops contact (as of 10/2023 this is Cindy Truong).
You can pay using the HMS P-card; VDRC will need to know the last 4 digits of the SSN for the P-Card (4990)
importation requires a USDA shipping label and a USDA “letter of no jurisdiction - see below
VDRC does not ship during the second half of December

Janelia FlyBank (split-Gal4)#

URL: https://flybank.janelia.org/
User ID: flypokers@gmail.com
Password: wilsonlab

Exelixis (Harvard)#

User name: Wilson Lab
Password: flypokers
pay using the P-card

NIG#

URL: https://shigen.nig.ac.jp/fly/nigfly
Shares login with DGRC (below)

DGRC (Kyoto)#

URL: http://kyotofly.kit.jp/cgi-bin/stocks/index.cgi
User ID: 2711
Password : dvek7m
invoices are sent via email just after flies ship
try to pay using the PCard; if you obtain an error message regarding payment just ask Rachel to enter her personal credit card number, or else use your own personal credit card and file for reimbursement
importation requires a USDA shipping label and a USDA “letter of no jurisdiction - see below
To search by NP number, select Quick Search and enter the full NP designation (e.g., NP3060), or select Advanced Search and enter the number (minus the preceding “NP”, e.g. 3060) in the Original Number area. Be sure select the correct hit (e.g., P{GawB}NP3060).

Importation#

You’ll need to deal with importation paperwork if you’re requesting flies from any source outside the US (whether VDRC, DGRC, or an individual lab outside the US). To import Drosophila melanogaster that harbor only “routine” transgenic constructs, we no longer need a permit. However, we need to supply a “Letter of No Permit Required” (LONPR) from the USDA. Please email thie letter to the sender/vendor so they can print it out and include it with the shipment.

When your flies arrive

Fri, 12 Mar 2021 15:44:55 +0000

When you receive a vial a flies, they need to be logged and then quarantined.

Open the Fly Stocks Database stock entry form and record all known info on this stock (see below for instructions).
Write the date of arrival on the vial and inspect it carefully for mites: look especially at the pupal cases.
Tap adults onto a CO2 pad and inspect their bodies for mites. (Also: make sure that any genetic markers in the stock are consistent with the stock description.) Then, transfer some clean male and female adult flies to a fresh culture. Wipe the CO2 pad with ethanol and thoroughly wash benchtop, microscope, transfer funnel, and all instruments with ethanol.
If there are no visible mites anywhere, then place the original vial and also the fresh culture with “clean” adults in a small box marked with your name on the shelf near the chemicals, in the back of the lab. If you see mites at any point, then all vials containing any flies from this stock should be stored in a box lined with mite paper on the same shelf, and should be checked frequently.
Transfer the “clean” adults 2 more times to clean vials, once every 2 days. Discard the intermediate vials when you remove flies from them. At the end of 4 days you should be done with this procedure.
About 5 - 10 days later, when you see plenty of larvae in the last of these “clean” vials, inspect the original shipping vials one more time for mites, then discard. If you have seen no mites at any stage of this process, then the stock is “clean”.
There should be at least two bottles or vials of each stock in the lab at all times once quarantine is finished.
If you need to use the stock to start a cross right away, then pick out a small number of adults with no visible mites to use for the cross, and keep the cross in the fume hood until you are confident the received stock was clean.

Note: Mites can crawl through sponge plugs or loose cotton, so if the original vial was capped with one of these closures, then it should not be stored inside the lab anywhere if the stock had mites. (Consult Rachel about how to handle this.) Mites supposedly cannot crawl through dense cotton plugs.

Fly stock crediting, distribution, and shipping

Fri, 12 Mar 2021 15:39:15 +0000

Any fly stocks you hold should be in the Fly stocks database, and the database entry should refer to the immediate origin of the stock as well as their original provenance, as well as any restrictions or requirements attached to publications that use the stock. For example, the database might note when there are authorship restrictions or unusual acknowledgement crecits that must accompany a particular stock.
If you get an unpublished stock from another lab, make sure Rachel is aware of it so she checks to see if there are any strings attached.
Do not distribute any flies to other labs without checking with Rachel. In general, the lab that originated the stock must also be consulted (by Rachel, generally) to make sure they are aware of the distribution. This is typically just a courtesy, but occasionally becomes critical.
It is your responsibility to acknowledge the originator lab in any talk or manuscript on your work.

Shipping flies to other labs#

When shipping to other labs, the following is recommended: Add adults to a fresh molasses vial and leave them for 2-3 days until there are a large number of eggs. Flip adults to a fresh molasses vial and ship both vials. If it is a particularly unhealthy stock, then wait another few days to ship an additional flip (total of 3 vials). In the winter, insulation is ideal and if it is particularly cold then you can add a long acting single-use handwarmer. Use overnight shipping.

Mites

Fri, 12 Mar 2021 17:03:41 +0000

If you find a mite in any culture,

Determine if it’s a food mite (“media mite”) or a fly predator. The food mites are white with long hairs; the fly mites are red or brown with short hairs.
Remove a handful of “clean” adults (~20 females and a few males) from the infested culture and place them on the CO2 pad. Inspect them carefully and discard any that carries a mite on its body. Divide them among two clean vials, and place vials in a separate box on mite paper. Discard the original infested culture in the biohazard bag, knot the bag, and remove it to a hallway box. Saturate the CO2 pad in ethanol, set the pad aside to dry (with a note), and clean the entire area around the flypushing station with detergent and ethanol.
Transfer the supposedly clean adults again after 2 days to fresh vials, discarding the previous vials. Continue to keep the new vials on mite paper.
Once again, transfer the supposedly clean adults after 2 more days, again discarding the previous vials. This new pair of duplicate cultures should now be clean.
Inspect these “clean” vials again carefully just before you discard them.

Tips:

Adult fly body anatomy

Wed, 01 Jun 2022 11:30:40 +0000

The Atlas of Drosophila Morphology is an excellent resource, find a pdf on the lab server in the “Atlas of Drosophila Morphology” folder.

Relationship of the CNS, circulatory system, and digestive system (credit: Joe Brock, Francis Crick Institute)

Nutrient control of Drosophila longevity

Circulatory system (A. Miller. The internal anatomy and histology of the imago of Drosophila melanogaster. In M. Demerec, editor, Biology of Drosophila, CSHL Press, p. 444, see hard copy for labels)

Landing sites for transgenes

Mon, 13 Sep 2021 18:26:25 +0000

When making transgenic flies using the PhiC31 system, transgene can be inserted into different locations in the chromosome. Here are some common landing sites.

from Knapp…Simpson (Genetics, 2015)

Here’s a list of landing site flies that BestGene already owns. WellGenetics also has the common ones, such as attP40 and attP2, but you might need to ship them a stock of landing site flies if you’re trying out a less common site.

Learning to do fly crosses

Thu, 18 Mar 2021 02:09:26 +0000

If genetics is very new to you, here are two brief reference sheets, one on classical genetics and one on molecular biology, that you may find helpful.

📎 A Brief Introduction to Classical Genetics_v2.docx

📎 A Brief Introduction to Molecular Biology_v2.docx

Read Drosophila Workers Unite! A laboratory manual for working with Drosophila by Michele Markstein

Read chapters 1 and 4 of Fly Pushing: The Theory and Practice of Drosophila Genetics by Ralph Greenspan- we have a lab copy and here is a digital copy

Useful markers for fly pushing

Mon, 05 Jul 2021 17:26:38 +0000

Genetic tools for neuroanatomy

Fri, 12 Aug 2022 20:10:32 +0000

by Isabel D’Alessandro and Rachel Wilson

MultiColor FlpOut #

MultiColor FlpOut (MCFO) is a method for the combinatorial stochastic labeling of cells. You can read more about MCFO in the original paper (Nern et al. 2015), and in this document. The protocol for MCFO immunostaining is described in this document.

Split-Gal4 #

The Drosophila split-GAL4 system, first described in Luan et al., 2006, allows for restricted expression of regulatory targets only in cells where the two components of the split-GAL4 activator are co-expressed. The GAL4 DNA-binding domain fused to the Zip- protein-pairing domain can be expressed in one pattern, and a transcriptional activation domain fused to the Zip+ protein-pairing domain can be expressed in another pattern. The UAS reporter construct will be expressed in the intersection of the two patterns. We now have thousands of stocks that carry either 1) a construct that expresses a DNA-binding domain protein derived from either GAL4 or lexA or 2) a construct that expresses a transcriptional activation domain derived from either GAL4, VP16 or p65. Read more about the split-Gal4 system in Dionne et al. 2018, and Tirian and Dickson, 2017.

Dissections & immunostaining

Fri, 12 Aug 2022 20:12:05 +0000

by Isabel D’Alessandro

Dissections #

Here are some videos which show how to dissect the whole fly brain, with slight variations in technique:

I usually perform dissections in 3-well glass plates in saline. Following each dissection, I use a pipette to transfer the brain to a 1.5mL eppendorf tube containing 200uL PBS. After all dissections are complete, I add 24uL PFA to each tube to fix the brains. For batch processing of brains of the same genotype, I add 3-5 brains to a single tube.