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.

Berg-2 Hello:#

  1. Turn on laser; turn on light for sign outside.
  2. Turn on air tank and switch on air knob.
  3. Connect IR to power cord.
  4. Make sure the PMT shutter is closed, blueish light on PMT button.
  5. Prepare you set up with your fly on the rig.
  6. Close the rig.
  7. Make sure PMT shutter is open, blueish light on PMT off.
  8. Wombat and Cassowary are not password protected.

Berg-2 Setup:#

  1. Check laser calibration at the start of each day. Should see a near linear curve.
  2. If you are ball tracking using fictrac:
  3. Position your fly correctly
  4. Take a snapshot on the fly and save as an image file
  5. Run >> get_fly_angle An angle value is printed in the console
  6. Use a fictrac config file that has been created for this angle, or near enough
  7. Raise the panels around the fly
  8. Unplug any ethernet cameras, as for some reason their use can crash the system
  9. Type into the Matlab command line, to start ScanImage, >> scanimage
  10. ScanImage uses a standard machine data file, which you should not need to change
  11. And a ScanImage configuration file, which you can make copies of and modify as you like, to preset some parameters.
  12. Reset Panel Control, restart Panel Control Board if needed, see “FAT OK”
  13. Calibrate the three-wheel controller, change the sensitivity if needed.
  14. Find your sample using LED lights
  15. Turn on LED driver, “out->camera” to locate the brain;
  16. Use the LED light path
  17. Lower the objective
  18. You can use your eyepiece to locate the sample at a low zoom
  19. Turn off LED driver, “in->PMT”
  20. Lower the curtain
  21. Find your neuron expression using two-photon imaging
  22. ScanImage: Main -> FOCUS, to try and find your neurons
  23. ScanImage: Main -> ZOOM, manipulate until your structure fills the imaging window. You should leave a little buffer space surrounding you region of interest, to help combat drift.
  24. ScanImage: Main -> GRAB, does a test acquisition following the configurations you have specified. Works best with Image Control: tiled.

Berg-2 Run:#

  1. Into the Matlab command line on Cassowary: >> panels_exp_server
  2. Cassowary will wait for a signal from Wombat before proceeding with imaging.
  3. Into the Matlab command line on Wombat, you need to execute our script for the experiment.
  4. Michael uses a GUI to organise and execute his experimental trials, called panels_exp_GUI.mlapp.

Berg-2 While imaging:#

  1. You can correct for x,y drift between imaging sessions, using the controller.
  2. You need to gently and slowly correct for z drift during an imaging session, meaning that you have to babysit each and every imaging session.

Berg-2 Goodbye:#

  1. Make sure laser is off, turn on PMT shutter, and raise the curtain;
  2. Ctrl+C stop Fictrac running;
  3. Raise objective, lower the panel, and take out the fly holder;
  4. Switch off the panel;
  5. Detach the IR light from power cord;
  6. Switch off the air knob;
  7. Turn off 2P laser (if nobody is using later); turn off light for sign outside
  8. Turn off the yellow air tank (if the other Bergs are not using it);
  9. Turn off carbogen tanks if no one else is using it

Extrac for virtual reality usage:#

During initial setup:

  1. Plug in side-view camera cables (one orange ethernet and one grey GPIO)
  2. Turn panels power switch on (note that up = OFF, down = ON). Wait for numbers to appear on top and bottom rows of panels, then turn on my panels controller (95% sure it’s the lower of the two panels controllers on the rack; they also may have a sticker on them labeled with our names). The LEDs should disappear from the panels after a second or two.

 

Wombat GUI setup:

  1. Run panels_exp_GUI from Matlab (in theory should work with either version, but I always use 2016b).
  2. Choose root save directory. A subdirectory for your specific experiment will be created within it.
  3. Enter an experiment number (intended to start at one and increment for each experiment in a given day)
  4. Enter an experiment name (will be included in the subdirectory name; I usually use an abbreviation of the fly’s genotype)
  5. Experiment directory will be created automatically if necessary, but if you want to save a screenshot from the positioning camera to calculate the fly’s angle there you can click the “Create experiment directory” button to do so manually.
  6. Enter trial duration in seconds and select the appropriate FicTrac config file
  7. Check “Using 2P” and “Using panels” boxes
  8. Select pattern file. For a standard closed-loop bar, use pattern #17 (100% brightness) or #19 (13% brightness). For a clockwise open-loop bar rotation, use patterns #1-6 (brightness % specified in the pattern file name)
  9. Set display rate to “50” and initial pattern position to “[1, 1]”
  10. Set panels to desired mode
  11. If using open-loop with one of patterns #1-6, choose position function #001 for X and position function #002 for Y.

 

Misc notes:

  • After the end of a trial, the FicTrac data files in your experiment directory on Wombat will be automatically renamed and moved into a subdirectory called “FicTracData”. This process takes a few seconds, and sometimes if you try and start a new trial before the files have been moved it can cause issues, so you should keep an eye on the save directory and make sure they’ve been moved before you click Start (usually only takes between 4-6 seconds)
  • Very rarely the code to kill FicTrac execution at the end of the trial fails to do so for mysterious reasons. If this happens you’ll just have to stop it manually with Ctrl-C, and then move the FicTrac data files into their subdirectory yourself.
  • The “ScanImage and operating Bergamo” Tettra page that Melanie and I wrote has some useful general reference info, and troubleshooting suggestions if you encounter any issues with ScanImage.
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