Here, we presented a LVSEM technique. The primary advantage of this technique is that it can examine “pathological tissue paraffin sections for light microscopy” that are mounted on glass slides. Another important advantage is that SEMs can provide magnification ranging from several thousands to several tens of thousands, reaching a “high level of analytical quality achieved by electron microscopes.”
This technique uses heavy-metal staining and backscattered-electron imaging. This easily allows for stereoscopic renditions of surface and cross-sectional views. Furthermore, it can visualize underlying anatomical structures without exposing them (i.e., without removing surface tissue, such as cell layers, that overlies these structures).
Particularly, this technique is valuable for evaluating renal biopsy specimens, as it provides novel findings that cannot be obtained by conventional methods. We believe that this technique will produce a wide range of relevant pathological information, including diagnostic staging of membranous glomerulonephritis. (Due to limitations in light microscopy, this staging has been conducted using a transmission electron microscope.)
This technique is fast and inexpensive, as it utilizes paraffin sections prepared for routine diagnostic work. In addition to freshly cut sections, previously prepared specimens can be examined using this technique. Thus, images of an immunostained tissue section can be directly compared with those of the same section stained with a heavy metal 9). Retrospective analysis of tissue archives is also possible with this technique.
However, although the sample chamber of the tabletop SEM is under low-vacuum conditions, tissue sections can still dry up during long examinations. This sometimes results in shrinkage or cracking of the sections. Thus, in order to extend the use of our technique to the histopathological diagnosis of other pathological conditions, it is necessary to identify a method for preventing such damage to samples.
In addition to improving these technical issues, we must first establish an atlas and medical criteria for evaluating renal biopsy specimens using a LVSEM. This can be achieved by carefully analyzing and reviewing observations and findings from many different renal disorders. We consider this to be the most important task in the near future.
Concluding remarks
Here, we described how to examine paraffin-embedded tissue sections using a tabletop LVSEM. We also presented actual examples that evaluated not only renal biopsy specimens, but also several other histopathological tissue samples.
To date, electron microscopic diagnosis of pathological tissue has mostly used transmission electron microscopes (TEMs), and rarely employed scanning electron microscopes (SEMs).
This is because conventional SEM-based methods can only observe certain types of materials and limited (either surface or cracked surface) areas of specimens. In addition, the conventional methods require the dedicated devices and sample pretreatments (such as fixation, dehydration, drying, and metal coating).
Therefore, researchers who are currently working on electron microscopic diagnosis or who have previously used SEMs may have been surprised and had the following reaction: How could you use an SEM for paraffin sections?!
Although we initially developed our technique for the “examination of renal biopsy specimens,” it has been applied to the study of many different tissue samples, and its usefulness is now slowly becoming widely recognized.
Unlike light microscope- and TEM-based methods, our technique allows for the relatively easy acquisition of wide-range three-dimensional information of tissue samples. With renal tissue specimens in particular, the quality of stereoscopic images of glomerular microstructures was much higher than we anticipated. Similarly, in other tissue sections, we successfully visualized intracellular secretory granules and even measured their dimensions. Based on these results, we are confident that readers will now understand how our technique attains “high levels of analytical quality achieved by electron microscopes” using only paraffin sections – the goal that has never been accomplished by conventional light microscope-based methods.
“It’s only a paraffin section, but nevertheless, it’s important.” To our surprise, paraffin sections still hold plenty of unexposed useful information.
Of course, the quality of evaluation data obtained with LVSEMs may not be as high as those obtained with TEMs. However, an advantage of our technique is that it is easy to perform. Thus, clinicians themselves can operate LVSEMs to search for pathological aberrations. This enables them to treat patients quickly and effectively, while they are waiting for detailed evaluation results produced by a TEM.
Based on our experience, we feel that we can better appreciate light microscopy images of paraffin sections, once we recognize the three-dimensional basic structures of glomeruli on these sections.
Here, we have made our database available to the public. We hope that this will help more researchers understand our technique, and utilize it not only for evaluating renal biopsy specimens, but also for observing many different histopathological tissue samples and clinical materials. We also hope that this increase in case numbers will help to develop this technique as a novel electron microscope-based analytical tool for diagnosis and research that can offer both “speed” and “ease of use.”
