PHYTOMap is a multiplexed fluorescence in situ hybridization (FISH) method that enables single-cell and 3D spatial analysis of gene expression in whole-mount plant tissue in a transgene-free manner and at low cost.
This pipeline processes images acquired in sequential rounds of FISH in PHYTOMap experiments.
Image registration: This step corrects shifts in a field-of-view during sample handling and imaging by a global affine alignment using random sample consensus (RANSAC)-based feature matching.
Spot detection and decoding: starfish package is used to detect single RNA molecule-derived spots in registered images, and the spots are decoded based on imaging rounds and channels they are detected. Deteiled tuorial on starfish is available here
Segmentation: Cells need to be segmented to allow single-cell analysis. This step is not covered here.
Cell by gene matrix: Decoded spots are assigned to segmented cells and counted to obtain a cell by gene matrix for downstream analysis.