AUTHOR=Seidemann Suse , Salomon Florian , Hoffmann Karl B. , Kurth Thomas , Sbalzarini Ivo F. , Haase Robert , Ader Marius 

TITLE=Automated quantification of photoreceptor outer segments in developing and degenerating retinas on microscopy images across scales

JOURNAL=Frontiers in Molecular Neuroscience

VOLUME=Volume 17 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2024.1398447

DOI=10.3389/fnmol.2024.1398447

ISSN=1662-5099

ABSTRACT=The functionality of photoreceptors, rods and cones, is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during development and in adulthood of wild type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify number, size, shape and fluorescence intensity of POS, retinal cryo-sections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in 'cone photoreceptor function loss 1' mice. TEM images were used to establish the ultrastructural quantification method QuaPOS- TEM for alignment of POS membranes. Images were analyzed using a custom written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild type and two inherited retinal degeneration ('retinal degeneration 19' and 'rhodopsin knock-out') mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume and membrane coherency during wild type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All code used for the presented analysis is available open source including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in depth characterization of POS on retinal sections in developmental studies, for disease modeling or after therapeutic interventions affecting photoreceptors.