Requirements and Setup

Data Format

• Requirement: Folder containing single-slice TIF files of one coronal brain, sorted from rostral to caudal (additional pipeline provided that can help with cropping slide images and merging split-channel images).
• Important: Naming of the Files. The pipeline relies heavily on filenames for correct processing and sorting. The correct syntax for the filenames is EXP1-A2_filename_s001.tif where:
  • The part before the first underscore (e.g., EXP1-A2) is used to identify the animal (and should be unique). This can include both the experiment and animal name, separated by a hyphen - if necessary.
  • _s001 is the slice number (slice numbers must be padded to avoid incorrect sorting, e.g., _s001, _s002).
  • Optional: _Ch01 represents the channel (e.g., _Ch01, _DAPI), if using separate-channel images.

Ensure that filenames always start with the animal name (or a combination of experiment and animal name, unique!). Anything before the first underscore will be treated as the animal identifier. This is critical for correct organization and grouping of data.

Example filenames that work for the pipeline:

EXP1-A2_10x-Cortex_RFP-GFP-NeuN_s001.tif
EXP1-A3_retroAAV-GFP_DAPI_fNissl_10x_s002_Ch01.tif
EXP1-A3_retroAAV-GFP_DAPI_fNissl_10x_s002_Ch02.tif
EXP2-B5_thistext-really-doesnt-matter-as-long-as-the-rest_fits_s005_DAPI.tif
EXP2-B5_thistext-really-doesnt-matter-as-long-as-the-rest_fits_s005_Alexa488.tif

FIJI.app

• Download FIJI from the official website.
• Download the necessary Fiji folder from this repository and paste it into your FIJI.app folder. Make sure that it lands in the right subfolder (Fiji.app/macro/toolsets)

MATLAB (tested on 2023a)

• This pipeline is built on AP_histology, developed by Andy Peters, which provides tools to align histology images to the Allen Brain Atlas. We recommend following their detailed documentation for setup and use. Special thanks to the AP_histology team for making this invaluable resource available to the community.
• AP_histology: Follow the installation instructions on the AP_histology GitHub page
• MATLAB Toolboxes and Add-ons:
  • Install the Curve Fitting Toolbox.
  • Install the natsortfile add-on (Natural-Order Filename Sort Version 3.4.5 by Stephen23).
• Download the MATLAB folder from this toolbox and place it in your Windows user folder under Documents/MATLAB/ (in addition to the AP-histology required files).
• Make sure the MATLAB folder is added to your path (main menu > add to path > check all files and folders are listed, otherwise MATLAB won't find the scripts)

Running the Pipeline

FIJI - get coordinates of volume in 2D slices

1. Pre-processing of images

(Optional, creates folder containing single-slice TIF files of one coronal brain, sorted from rostral to caudal)

• Open FIJI and navigate to the toolbox by selecting >> 1_PrepareSlicesAsTif. (If you cannot find it, the files have not been copied into the right spot)
• Select the appropriate folder: Choose the folder that contains either whole-slide overview TIF files or single-slice separate-channel TIF files. If the correct filename convention is followed, the folder can contain multiple animals' data within the same folder.

• If you are working with whole-slide imaging:
  • Split the channels.
  • Then, if needed, make any adjustments to the split channel images (such as reducing background noise) in Photoshop.
  • Crop the whole-slide images by drawing rectangles around each slice you want to export.
  • At the end, the script will save each slice as a separate file in the subfolder "Slices/ANIMALNAME".
• If you are working with separate-channel images: This script will allow you to provide a folder of split-channel images. You will be prompted to select which channels to include and to specify the color for each channel in the final multichannel TIF file.
• If you are working with multi-channel images and want to exclude channels: This script also allows to remove channels from the image that you do not want and re-arrange the colors.

OUTPUT: The result will be a folder with one multichannel TIF file per section (i.e., per slice).
If multiple animals provided, there will be subfolders in the folder "Slices" according to the animal name.

2. CELL3D pipeline (semi-automatic detection of cells)

• Open FIJI and navigate to the toolbox by selecting >> 2_CELL3D_CellCoords.
• Set the folder to a folder containing sections (multichannel) of 1 or more animals (this can be the folder "Slices" created in the previous step or directly a folder containing TIF files of 1 animal. Make sure that no other tif files are in this or a subfolder).
• Preprocess slices: Rotate and flip slices as necessary
  • If necessary, flip the section using Ctrl + F.
  • Rotate the slices by drawing a line at the midline (from top to bottom).
  • To skip a particular slice, just press OK directly without placing a line.
• The script will ask 3 times to click a certain location: Press ok after clicking on that point in the image (a cross will be placed there). Far left corner of the slice (left most point), Midline, Far right corner of the slice (right most point). This will be used later to assess location to cingulate, medial or lateral cortex according to distance to midline
• Cell labeling:
  • Choose channel you want to process (C1 (red), C2 (green), C3 (blue), C4 (farred)).
  • Provide a particle size threshold and a binary threshold value if available (this may need some testing, but starting with the default can work, 150 threshold + 50 particle size for cortical neurons)
  • The script will isolate the channel of interest and automatically select neurons if possible.
    • If successful, you will see a selection in your image and a message will pop up to say that you can now fine tune your selection by clicking more cells (mouse click) or deleting wrongly detected cells (Ctrl + click on marker). Go through before clicking OK!
    • If unsuccessful, a message will pop up saying that you should do a manual selection. Click on each cell you want to label.
    • If you are unhappy with the semi-automatic labeling, try these steps:
      • to re-try thresholding with a different threshold, use button and click into the image. It will ask you for a new threshold, higher number means less cells to be detected. You can do this as often as you want to try to get a better number, but it can only do so much if there is a lot of background or high labeling outside the cortex.
      • to delete the current selection, press . You may e.g. want to do this if a) too many wrong ones are selected and you want to do it manually or b) if there are no cells to be selected but the script placed some markers
  • When all cells are selected, press ok, and the script will automatically save the coordinates of selected cells as ZIP and CSV file in a subfolder and loop to the next image.
  • Once a ZIP file has been created, the image will be skipped when restarting the script (meaning you can take a break any time and restart in between images). If you want to re-do a file, rename or delete this ZIP file!
• Cleaning mode:
  • This script allows you to clean up some of the selected cells. This will open each file with the corresponding cell selection and gives you the opportunity to delete or add more cells. It will loop through the whole folder.
• Output: Subfolders of CSV and ZIP files for each slice in which cells were detected

MATLAB - transform coordinates into CCFv3 space

1. Animal-specific transformation

TBD

2. Plotting Multiple Animals into 1

TBD

Future updates

• Remove med/lat/cing assessment for public