Single molecule fluorescence in situ hybridization using RNAscope to study hematopoietic and vascular interactions in the zebrafish embryo and larva - FIJI image analysis script example

This script is a FIJI macro used to perform basic cell segmentation and collect fluorescence intensity within objects, that can be used to analyse fluorescent in situ hybridization data. The script can be open with Fiji/ImageJ (doi:10.1038/nmeth.2019, doi:10.1038/nmeth.2089). The script is written in macro language (IJ1 macro), and was tested on version 2.3.0/1.53q of ImageJ2. This script was written using several freely available code snippets from 3D ImageJ suite website as well as collective ressources (https://mcib3d.frama.io/3d-suite-imagej/plugins/3DManager/3D-Manager-macros/, https://forum.image.sc/) and using the macro recorder from FIJI.

The script requires a tiff image containing confocal z-stack images with two channels (one to segment cells, using a nuclear or cytoplasmic soluble signal, and the fluorescent in situ hybridization channel).

This script opens consecutively all images present in a folder, performs pre-processing steps (3D gaussian blur) and 3D segmentation (3D object Counter).

The script generates masks for the segmentation (the mask is automatically saved and can be opened and modified manually using the 3D manager tool), that are used to collect fluorescence in the two channels, and save the information for each channel in 2 .csv file. CSV files can be opened with excel, R or any software of your choice for further analysis.