Microscopy Image Stitching

Setup Environment

1. Install requirments

If Squid software (https://github.com/Cephla-Lab/Squid) is not installed, run the following commands before continue to the next steps:

wget https://raw.githubusercontent.com/hongquanli/octopi-research/master/software/setup_22.04.sh
chmod +x setup_22.04.sh
./setup_22.04.sh

If Squid software is already installed, start from here:

pip install dask_image
pip install ome_zarr
pip install aicsimageio
pip install basicpy

2. Run Stitcher

Clone this repo. In terminal change to its directory and run the following command:

For graphical user interface:

python3 stitcher_gui.py

For command line usage:

python3 stitcher_cli.py -i /path/to/images

or with registration and flatfield correction

python stitcher_cli.py -i /path/to/images -r -ff --registration-channel "Fluorescence 488 nm Ex"

Guideline for GUI Usage

Select Input Folder

• The input folder should be the folder named with Experiment ID which contains all the timepoints of your acquired images, acquisition parameters.json and configurations.xml
• Make sure acquisiton parameters.json file contains the correct information for objective magnification and sensor_pixel_size_um, etc.
• The stitcher works with imaging data acquired with the latest Squid software. For data acquired with older version software, use update_coordinates.py to update coordinates.csv format. See Updating coordinates.csv file part.

Flatfield Correction

• When this option is checked, the stitcher will apply flatfield to individual images using baSiCPy when stitching

Cross-Correlation Registration

• When this option is checked, Cross-Correlation Registration will be performed. Otherwise the images will be stitched based on their coordinates in coordinates.csv file

Merge Timepoints and Merge HCS Regions

• Being implemented. Not ready yet.

Output Format

• Either OME-ZARR or OME-TIFF

View Output

• Opens filepath to visualize in napari viewer

Scripts for Data Pre-processing

Updating coordinates.csv file

Run:

python3 update_coordinates.py <input folder>

• This script updates coordinates.csv in data acquired with older version Squid software to match the format in latest version
• The input folder should be the folder named with Experiment ID which contains all the timepoints of your acquired images, acquisition parameters.json and configurations.xml
• Only works for data acquired with Wellplate Multipoint

Converting Flexible Multipoint data into Wellplate Multipoint format

Use convert_to_coordinate_acquisition.py. To be tested.