Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Abstract

Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future.

Identifying and fixing in-plane positioning and stability issues on a microscope using nanoGPS OxyO scales

“…Microscopes may also provide an increasing number of modalities that are used sequentially, and positioning reproducibility is key to obtain accurate highresolution multimodal images [9][10][11] . Correlative observations may also be performed on different instruments, and several methods have been developed to perform coordinate transfer between the coordinate systems of the two platforms [12][13][14][15] . Good positioning accuracy on both instruments is required to obtain good accuracy on the correlative observations.…”

Identifying and fixing in-plane positioning and stability issues on a microscope using machine-readable patterned position scales

“…Microscopes may also provide an increasing number of modalities that are used sequentially, and positioning reproducibility is key to obtain accurate high-resolution multimodal images [12][13][14] . Correlative observations may also be performed on different instruments, and several methods have been developed to perform coordinate transfer between the coordinate systems of the two platforms [15][16][17][18] . Good positioning accuracy on both instruments is required to obtain good accuracy on the correlative observations.…”