Super-resolved optical imaging, reconstruction, and spatial analysis of whole mouse glomeruli via the Glomerulus Mapping and Analysis Pipeline.

INTRODUCTION

Kidney glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate ultrastructure. However, these techniques are limited by their focus on thin sections and specific regions. This rests their capacity to evaluate whole glomeruli as holistic 3D functional units. We lack a unified, spatially integrated view of the glomerulus as a whole system to better understand structural and cellular changes across different conditions.

METHODS

We developed GloMAP (Glomerulus Mapping and Analysis Pipeline), a novel workflow that integrates tissue expansion, super-resolution optical microscopy, and computational reconstruction to generate high-resolution (about 100 nm) 3D models of entire mouse glomeruli. GloMAP integrates both manual and machine learning-assisted segmentation to precisely annotate and quantify glomerular structures, including compartmental volumes, surface areas, membrane thickness distribution, and cellular morphometric properties.

RESULTS

Using GloMAP, we reconstructed and analyzed 24 glomeruli from healthy adult, aged, and model diseased mice. Our method enabled volumetric and spatial quantification of key compartments and distributions of four primary glomerular cell types. GloMAP revealed previously inaccessible features such as global membrane thickening patterns and compartmental shifts associated with aging and focal segmental glomerular sclerosis.

CONCLUSIONS

The pipeline's compatibility with commonly available optical microscopes and its potential to integrate molecular labeling and other super-resolution techniques make it a scalable and versatile tool. GloMAP provides a scalable, accessible platform for comprehensive 3D structural and cellular analysis of whole glomeruli at 100 nm resolution. This integrative pipeline fills a critical gap between traditional light and electron microscopy and holds potential for broader application in translational nephrology research, offering new opportunities to identify structural biomarkers and pathophysiological mechanisms in kidney disease.