Lightsheet Brain Workflows

Code to format and preprocess whole-brain cleared brain images acquired with light-sheet fluoresence microscopy. Work in progress.

Right now this repo allows you to process lighsheet data from CZI files though BigStitcher and Brainreg

About

The goal of this repository is to build tools to allow for the scalable stitching fusing and processing of tiled lightsheet data using existing tools.

Rationale

Local usage

Installation

This repo

Download and unzip this repository somewhere

Fiji

You must have a working Fiji installation with the BigStitcher update site enabled

1. Start Fiji and go to Help > Update
2. Click on "Manage update sites"
3. Check BigStitcher
4. Click on Save and close and restart Fiji

BrainReg

You can install brainreg and the 25um Allen mouse brain with the following commands

conda create -n brainreg python==3.11 -y
conda activate brainreg
conda install -y -c conda-forge brainreg
brainglobe install -a allen_mouse_25um
pip install brainglobe-atlasapi

Use

Prepare for processing

1. From within Fiji, run the script called YamlGuiCreator.groovy
2. You will be prompted for an input YML file, select the parameters_template.yml file
3. On the GUI, fill in the necessary fields
   • Under General: Specify your user name and the directory where you would like your processing data to be saved
   • BigStitcher tab: These are the parameters for stitching and fusing the lighthseet data.
   • Brainreg tab: For local usage, specify your conda environment name that you installed using the instructions above
4. Run tab: Select one or more folders containing CZI files for processing
5. Click on Save

This will produce on YML file per CZI file in the save directory

Run processing

Open the script Run_stitching_and_fusion.groovy

Run the script and select a single YML file in your output directory. This will process the entire brain

Cluster usage

Work in progress

Brain orientation

• The brain orientation must follow the three-letters BrainGlobe image space definition (see also here), later used for atlas registration. This is in reference to the origin of the data (first, top left voxel).
• First letter: imaging planes are acquired from far to close to the objective with the brain surface facing the detection objective, so the first image will be the bottom of the brain → inferior (else superior)
• Second letter: brains are imaged vertically:
  • With olfactory bulb on top → anterior
  • Else, with olfactory bulb at the bottom → posterior
• Third letter: images are not mirrored, therefore left part of the image is the left hemisphere of the brain → left (else right)