Python 3.9 and above
Libarary: numpy, roifile, matplotlib, opencv, pandas, scipy, scikit-learn, scikit-image, joblib (parallel computing), PyImageJ (which also requires OpenJDK 11 and Maven) https://py.imagej.net/en/latest/Install.html
Use either "conda install numpy" or "pip install pyimagej" in the Anaconda terminal
Download and install fiji.app https://imagej.net/software/fiji/ Make sure the newest fiji contains: “…\plugins\Descriptor_based_registration-2.1.8.jar” in the "plugins" folder
Pre-process the raw images from the Seq run and output processed images in a folder that is ready for color transformation
Image rename, filtering, binning, background normalization, magnification correction, image registration and cropping, illumination correction
Before running it, first make sure Fiji.app is downloaded and installed PyImageJ, OpenJDK 11 and Maven are all properly installed Then open the script and make sure JAVA_HOME is set to the correct directory in your PC os.environ['JAVA_HOME']='C:\Program Files\Microsoft\jdk-11.0.21.9-hotspot' and same for the fiji parth fijipath='...your directory/Fiji.app'
Last, run the file in the terminal with "-i +your_raw_image_folder"
& C:/Users/...your directory/python.exe "...your directory/ClusterSeqIP_v2.py" -i "/your_path_to/raw_image_folder"
Python will call fiji algorithm to perform the image registration, requires users to drag the cropping rectangle to center and manually adjust the threshold to ~0.08
Raw .tif image folder path (don't put things that are not .tif files in that folder)
2_preprocess: this folder stores preprocessed images (for debugging, and manual registration) 2_Regis: this folder stores the registered images for enduse to visualize and check the registration quality 2_processed_final: this folder stores the final processed images for next step color transformation, pass this directory to the color_transform.py once you believe all the images are properly processed and would like to move on.
ClusterSeqIP_v2_IJauto.m is the matlab version of the ClusterSeqIP_v2.py that will complete the entire image processing pipeline:
MATLAB R2020 and above
Download and install fiji.app https://imagej.net/software/fiji/ MATLAB imageJ: mij.jar, ij.jar Make sure the fiji contains: “…\plugins\Descriptor_based_registration-2.1.8.jar”
First, Add mij.jar, ij.jar and Descriptor_based_registration-2.1.8.jar to the script path
ImgFolderPath='Your Raw Image Path Folder'; %Replace this with the folder path of your raw images
Click “Run” button, the processed images will be output in the same directory of your raw image path.
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