The Squassh protocol (Segmentation and Quantification of Sub-cellular Shapes) provides a robust and versatile workflow for detecting, segmenting, and quantifying sub-cellular markers in fluorescence microscopy images and analyzing their co-localiztion. The protocol is particularly tuned to the needs of high-content high-throughput assays and directly performs all the necessary statistical significance tests.
The Squassh protocol is based on a novel image-segmentaiton theory from the MOSAIC Group, which provably provides globally optimal segmentation results with sub-pixel resolution. As an additional feature, the method accounts for the point-spread function of the microscope, hence producing optimally deconcolved and denoised results.
The Squassh workflow critically depends on Fiji as its software processing pipeline. The method, the Fiji software, and its application are described in:
A. Rizk, G. Paul, P. Incardona, M. Bugarski, M. Mansouri, A. Niemann, U. Ziegler, P. Berger, and I. F. Sbalzarini. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh. Nature Protocols, 9(3):586–596, 2014.