#6669 ANALYSIS OF KIDNEY BIOPSIES AND URINE SEDIMENT USING SCANNING ELECTRON MICROSCOPY (SEM) AND ATOMIC FORCE MICROSCOPY (AFM)
Authors: Gigliotti Giuseppe , Nigro Michelangelo , Gigliotti Andrea , Borriello Gianmarco , Massa Antonio , Mancini Antonio , Mariarosa Scognamiglio , Reverchon Ernesto , Viggiano Davide 
Published: June 2023

Abstract

Background and Aims
The classification of renal diseases is dependent on the evaluation of kidney biopsies using light microscopy techniques. Ultrastructural details (e.g. podocyte foot processes, amyloid fibrils, Fabri's intracellular inclusions) need transmission electron microscopy (TEM) which has high costs, requires specialized personnel and is time consuming. Therefore, alternative techniques are highly desirable. Here we report our experience with scanning electron microscopy (SEM) and atomic force microscopy (AFM).

Method
Paraffin-embedded kidney sections (3μm thick) were examined after dewaxing and Jones-methenamine methane staining for type IV collagen fibers. Dried urine sediments were prepared using standard methods for urine cytological analysis. Analysis was then performed using atomic force microscopy (nGauge system) on dried specimens and with Scanning Electron Microscopy (Zeiss) after gold-spattering of the surface. Reference images were obtained by standard microscopy.

Results
The combination of SEM and thin paraffin sections allows the observation of ultrastructural details in kidney biopsies with a very simple and fast preparation. AFM is even faster than SEM, as it does not require any preparation of the surface of the sample. An important advantage of SEM is the possibility to view the sample at low magnification, which makes it easier to identify the location of the glomeruli. It is thus possible to switch from small magnifications (100x) to very extreme magnifications (10000x). Notably, the interpretation of the images requires knowledge of the artifacts induced by paraffin inclusion. Specifically, structures and cells containing lipids (for example foamy cells) appear empty. Chromatin is also condensed in the nuclei around the periphery of the nucleus and in spike structures whit large empty areas. The recognition of nuclei in SEM and AFM is important for the subsequent interpretation of the images. Podocyte pedicles fusion can be identified relatively easily in SEM and in AFM. Both techniques add relevant ultrastructural details of urine sediment cells, from epithelial cells to isolated tubular cells. Also, casts show many ultrastructural features which are not evident in classical optical microscopy. It is also possible to measure the thickness of the basement membrane with great precision both in SEM and with AFM. The major limitation of this approach is the limited experience of pathologists with these methods and the absence of an atlas of normal and pathological kidney.

Conclusion
Considering the affordable costs of SEM and AFM instruments and the simple preparation of samples for their analysis, these should be considered a valid alternative to TEM. SEM and AFM are alternative methods, simple and relatively cheap and very fast to obtain ultrastructural details of renal biopsies. We thank ANED and AST for the great support given to research.