Abstract
Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE–SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE–SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function.
Highlights
Since the first transmission electron microscope (TEM) was developed by Ernst Ruska in 1931 [1,2], it has been considered an essential tool for the ultrastructural analysis of healthy, diseased, and experimental tissue in renal histopathology, and has contributed significantly to our understanding of renal diseases [3,4,5]
Since it is known that the enlargement of subpodocyte space (SPS) increases the mechanical forces exerted on podocytes by water filtration and induces podocyte detachment from the glomerular basement membrane [14] and that BTBR ob/ob mice were characterised by a significant loss of podocytes [19,20,24,25], we identified and analysed the ultrastructure
Since it is known that the enlargement of SPS increases the mechanical forces exerted on podocytes by water filtration and induces podocyte detachment from the glomerular basement membrane [14] and that BTBR ob/ob mice were characterised by a significant loss of podocytes [19,20,24,25], we identified and analysed the ultrastructure of these cells in BTBR ob/ob mice
Summary
Since the first transmission electron microscope (TEM) was developed by Ernst Ruska in 1931 [1,2], it has been considered an essential tool for the ultrastructural analysis of healthy, diseased, and experimental tissue in renal histopathology, and has contributed significantly to our understanding of renal diseases [3,4,5]. SE–SEM is a valuable alternative to TEM when high-resolution imaging of a large volume of the sample is needed or when the pathologist does not need electron microscopy examination up considerably, with a far higher resolution than light microscopy and similar to that achieved by TEM (Figure 7), and can be applied to retrospective studies or biopsies that are already embedded in resin. The high resolving power achieved by SE–SEM was perfect for visualising and segmenting the SPS with high accuracy and precision, as well as clearly identifying glomerular cells and the podocytes, based on their morphological features This suggests that our SEM imaging approach could be used in all studies that explore the relationship between the size and numbers of glomerular resident cells and renal diseases [30,31,32]. It is our hope that our results will encourage pathologists to consider scanning electron microscopy, and in particular, the SE–SEM method, as a valuable tool that can provide insights that complement the diagnostic approach provided by TEM and that can be used in routine experimental and clinical pathology
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