Biology subjects

Hoyer, P. F.

Publications and source records attributed to Hoyer, P. F..

3 recordsLinked to original sources

bioRxiv2021

Three-dimensional Super-resolved Imaging of Paraffin-embedded Kidney Samples

Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. New technologies, including superresolved nanoscopy and expansion microscopy, as well as new sample preparation techniques, are starting to revolutionize imaging of biopsy specimens. However, our previous approaches for simple and fast three-dimensional imaging of optically cleared samples are to date not compatible with formalin fixed paraffin-embedded (FFPE) tissue, impeding application in clinical routine. Here we provide protocols that circumvent these limitations and allow for three dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. This would increase the feasibility to implement these protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

pathology↗

bioRxiv2021

Deep learning-based segmentation and quantification of podocyte foot process morphology

The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

pathology↗

bioRxiv2021

Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD. Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination.

cell biology↗