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).

Manual cMIP Searches in FIJI#

Step 1. Download the cMIP image library here. For searching against GAL4 libraries, download the Gen1_GAL4_R_VT_JRC2018U folder. For searching against LEXA libraries, download the Gen1_LexA_VT_R_JRC2018U folder.

  • Note: These files are, by nature, quite large. The lab also has a shared external SSD driver containing the cMIP image libraries.
  • Note: When searching for LEXAs, the folder includes a collection of VT-LEXA drivers that are mostly unavailable to order

Step 2. Install the “Color MIP Mask Search” plugin, found here. 

  • Once downloaded, open FIJI and simply drag and drop in the ColorMIP_Mask_Search.jar file. Restart FIJI.

Step 3. Open the collection of interest by dragging and dropping the entire folder (e.g., Gen1_GAL4_R_VT_JRC2018U) into FIJI. Ensure that you check the line to open as a “Virtual Stack”.

Step 4: Create a mask using one of three methods:

  • Option 1: (better) Find your neuron of interest in the Hemibrain (e.g., LC33), open in NeuronBridge (NB), and download the cMIP image of your neuron to be used as a Mask.
    • You will want to remove the color-bar in the top right corner by opening the mask in FIJI, using the freehand selection tool to circle just the neuron, and selecting edit>clear outside.
    • Save a copy of the image to your computer for future reference (e.g., LC33_mask.tif).

  • Option 2: Find your neuron of interest by clicking through the virtual stack to find an image that you want to use to create a mask
    • Note that the images are in alpabetical order, with VT lines at the very end of the stack. 
    • Hit “Shift+d” to open the image in a new window. Make sure the “duplicate entire stack” option is unchecked in the pop-up window before hitting “OK.”
    • Use the free form selection tool to outline the cells/neuropils you want to search for. Then select Edit > “Clear Outside” to set the rest of the pixels in the image to zero. 
    • Save a copy of the image to your computer for future reference (e.g., LC33_mask.tif).
  • Option 3: Create a mask of your neuron of interest from a confocal stack (OR em skeleton) that you have.
    • To generate a colorMIP image from a light level image, you first need to register this image to a standard brain template(JRC2018 Central Brain Unisex, available here). See the tetra page on Registration using CMTK plugin in FIJI or https://github.com/wilson-lab/nat-tech
    • To go from em skeleton to colorMIP, you must first transform the em skeleton to a stack (that then can be processed identically to a light level stack. To do this you can use the function “flywireid_to_nrrd” which can be found in the R/startup folder in the nat-tech repository. This function takes neuron IDs (flywire) and outputs the nrrd stacks. This is useful especially if your neuron is not available in the datasets described above (many ascending neurons fall in this category)
    • Once your image is registered to this template, you can use the Color MIP Generator Fiji Plugin. Follow the instructions to install and run the plugin.
    • Note: in order to get the plugin to work, the ‘Automatic Brightness adjustment’ box needs to be unchecked. Your settings in the Color MIP Generator should look like this

  • The plugin will ask you to designate the folder where your aligned confocal files are, and a directory where your generated color MIP image will be saved
  • Note: If you used a confocal stack, the registered image might have the wrong dimensions so you need to adjust the dimensions to match the virtual stack. This can be done by going to Image > Adjust > Size… in Fiji where you can set the pixel dimensions to match the dimensions of the virtual stack you want to search.

Step 5. Run the cMIP search.

  • Open both your (i) search mask and the (ii) virtual stack of your cMIP library in FIJI.
  • Select Plugins > “ColorMIP Mask Search” to begin. Make sure that the Mask (your mask) and the Data (your collection library) look good. In general, the default parameters should work relatively well, so leave them as-is for now.
  • Hit “OK” to begin the search. Depending on the parameters and collection size this can take anywhere from a few seconds to a few minutes.
  • When the search is finished, your results (“hits”) will open in new window. Save them as a .tif file for future reference.  You should get between 20-200 hits (which includes duplicates)

Troubleshooting: If you feel like you are getting too few results, try one of the following – 

  • Checking “yes” next to the “Add mirror search” option 
  • Lowering the “Threshold for data” value
  • Decreasing the “% of positive PX” value 
  • Drawing a new ROI using a different driver line (e.g. one that’s less busy, or has stronger expression in your cells/region of interest)
Backward FAFB v14/CATMAID/walled garden Electrophysiology Forward