SUN-017 LOW-VACUUM SCANNING ELECTRON MICROSCOPY ANALYSIS FOR HUMAN BIOPSY SAMPLES

Abstract

One of the major goal of biological microscopy is to elucidate the structural evidence relating functional activity or pathophysiological condition. High resolution imaging by electron microscopy can provide detailed tissue architectures, but the specific and skillful technics are required and it takes long time to the sample preparation. Low-vacuum scanning electron microscopy (LV-SEM) developed recently is a simple and fast technic to observe higher resolution of histological paraffin sections prepared for conventional light microscopy. Periodic acid methenamine silver (PAM) stain is a routine staining for basement membrane in renal biopsy, and contains silver, a heavy metal, which enhances the backscattered electron signal. To evaluate the benefit of LV-SEM, we observed PAM stained glomerular basement membrane (GBM) in human renal biopsy samples. The study was performed under the declaration of Helsinki principles. The study protocol was approved by the Institutional Review Board of the Tohoku Medical and Pharmaceutical University. All participants provided written informed consent after a full explanation of the purpose of the study and the potential risk involved. The human kidneys were obtained from patients with membranous nephropathy, IgA nephropathy, Alport syndrome, or thin basement membrane disease by renal biopsy and embedded in paraffin. Four-μm sections were deparaffinized with xylene, hydrated with ethanol, and stained with periodic acid methenamine silver (PAM). Miniscope TM4000 (Hitachi High-Technologies) was used to observe the GBM by detecting backscattered electron signal. After evacuation of the specimen chamber a few minutes, the sections were observed under the electron beam accelerating voltage of 10 kV for higher magnification (x2,500 to x20,000) or 15 kV for lower magnification (x40 to x2,000) with 30 Pa. The observations at higher magnification were easily performed under LV-SEM by zooming up from x40 to x20,000. The PAM-positive GBMs were bright under LV-SEM. The GBMs showed dark electron dense deposits in membranous nephropathy and IgA nephropathy, and characteristic coarse meshwork appearances in Alport syndrome. The GBMs in thin basement membrane disease seemed relatively thinner than the others. Direct observation of conventional renal biopsy paraffin sections by LV-SEM is a simple and beneficial method for the rapid and detailed analysis of the diseases. It will likely be useful for the observation of GBM morphological change.