Application of low-vacuum scanning electron microscopy for renal biopsy specimens.

Low-vacuum scanning electron microscopy (LV-SEM) has been developed which enables the observation of soft, moist, and electrically insulating materials without any pretreatment unlike conventional scanning electron microscopy, in which samples must be solid, dry and usually electrically conductive. The purpose of this study was to assess the usefulness of LV-SEM for renal biopsy specimens. We analyzed 20 renal biopsy samples obtained for diagnostic purposes. The sections were stained with periodic acid methenamine silver to enhance the contrast, and subsequently examined by LV-SEM. LV-SEM showed a precise and fine structure of the glomerulus in both formalin fixed paraffin and glutaraldehyde-osmium tetroxide-fixed epoxy resin sections up to 10,000-fold magnification. The spike formation on the basement membrane was clearly observed in the membranous nephropathy samples. Similarly to transmission electron microscopy, electron dense deposits were observed in the epoxy resin sections of the IgA nephropathy and membranous nephropathy samples. LV-SEM could accurately show various glomerular lesions at high magnification after a simple and rapid processing of the samples. We consider that this is a novel and useful diagnostic tool for renal pathologies.

Rapid three-dimensional analysis of renal biopsy sections by low vacuum scanning electron microscopy.

Renal biopsy paraffin sections were examined by low vacuum scanning electron microscopy (LVSEM) in the backscattered electron (BSE) mode, a novel method for rapid pathological analysis which allowed detailed and efficient three-dimensional observations of glomeruli. Renal samples that had been already diagnosed by light microscopy (LM) as exhibiting IgA nephropathy, minor glomerular abnormalities, and membranous glomerulonephritis (GN) were rapidly processed in the present study. Unstained paraffin sections of biopsy samples on glass slides were deparaffinized, stained with platinum blue (Pt-blue) or periodic acid silver-methenamine (PAM), and directly observed with a LVSEM. Overviews of whole sections and detailed observations of individual glomeruli were immediately performed at arbitrary magnifications between ×50 to ×18,000. Cut surface views and surface views of glomeruli were demonstrated at the same time. On Pt-blue-stained sections, podocytes, endothelia, mesangium, and glomerular basement membranes (GBMs) could be distinguished due to the different yields of BSE signals, and pathological features were investigated in every sample. The abnormal surface appearances of podocytes with foot processes and the varying thicknesses of GBM were revealed three-dimensionally, features difficult to observe under LM and transmission electron microscopy. PAM-positive GBM alterations in membranous GN were distinctly visualized through overlying cells without cell removal under LVSEM at high magnification. Not only prominent spike formation but also slight protrusions were clearly revealed in the side views of GBM. Crater-like or hole-like structures were shown in the en face views of GBM. Accordingly, LVSEM is expected to provide a novel approach to the pathological diagnosis of human glomerular diseases using conventional renal biopsy sections.

Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue.

The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x40 to x10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.