Imaging of Liver Tissues Using a Combination of Silver Impregnation and Low-Vacuum Scanning Electron Microscopy; A Simple Method for the High-Resolution Visualization of Reticulin Structures with Applicability to a Quantitative Analysis.

Background: Low-vacuum scanning electron microscopy (LVSEM) enables the detailed three-dimensional imaging of archival tissues without special pretreatments. The clinical utility of LVSEM in the assessment of liver diseases has not yet been defined. So, we investigated the utility of LVSEM imaging in morphological assessments of normal and diseased liver tissues, with a focus on reticulin structures. Methods: Formalin-fixed tissue samples of two normal livers and two hepatocellular carcinomas with background regenerative nodules/areas were stained with platinum blue stain or silver-impregnated using Watanabe's method and then comparatively observed under LVSEM. We also evaluated the applicability of LVSEM imaging of liver tissues to a quantitative analysis using a digital image analysis technique. Results: Optimal high-resolution images of reticulin structures were obtained using 10-µm-thick silver-impregnated sections. Reticulin fibers were clearly observed to run dendritically around sinusoids in normal livers, and markedly increased in regenerative nodules/areas. Normal reticulin frameworks were lost in hepatocellular carcinoma, leaving a few fragments of reticulin fibers within tumors. Moreover, when a quantitative analysis was applied to these images, we successfully demonstrated a significantly higher reticulin fiber density in regenerative nodules/areas than in the normal liver (P < 0.05). Conclusion: We not only obtained detailed three-dimensional images of reticulin structures in various liver tissues by LVSEM combined with silver impregnation but also showed their applicability to a quantitative analysis. The method presented herein may be applied to future studies for the more accurate diagnosis and better classification/risk stratification of various liver diseases.

Low-vacuum scanning electron microscopy may allow early diagnosis of human renal transplant antibody-mediated rejection.

Antibody-mediated rejection (ABMR) is an important cause of both short- and long-term injury to renal allografts. Transplant glomerulopathy (TG) is strongly associated with ABMR and reduced graft survival. Ultrastructural changes in early-stage ABMR include TG as a duplication of the glomerular basement membrane (GBM), which can be observed only by transmission electron microscopy (TEM). Low-vacuum scanning electron microscopy (LVSEM) is a new technique that allows comparatively inexpensive, rapid, and convenient observations with high magnification. We analyzed human renal transplants using LVSEM and evaluated the ultrastructural changes representing TG in ABMR. GBM duplication was more clearly visible in the LVSEM images than in the light microscopy (LM) images. In the ABMR group, the cg score of the Banff classification was higher in 54% (7/13) of specimens for LVSEM images than for LM images. And 4 specimens exhibited duplication of the GBM analyzed by LVSEM, but not by LM. In addition, three-dimensional ultrastructural changes, such as coarse meshwork structures of GBM, were observed in ABMR specimens. The ABMR group also exhibited ultrastructural changes in the peritubular capillary basement membranes. In conclusion, analyses of renal transplant tissues using LVSEM allows the identification of GBM duplication and ultrastructural changes of basement membranes at the electron microscopic level, and is useful for early-stage diagnosis of ABMR.

Aerosol droplet delivery of mesoporous silica nanoparticles: A strategy for respiratory-based therapeutics.

A highly versatile nanoplatform that couples mesoporous silica nanoparticles (MSNs) with an aerosol technology to achieve direct nanoscale delivery to the respiratory tract is described. This novel method can deposit MSN nanoparticles throughout the entire respiratory tract, including nasal, tracheobronchial and pulmonary regions using a water-based aerosol. This delivery method was successfully tested in mice by inhalation. The MSN nanoparticles used have the potential for carrying and delivering therapeutic agents to highly specific target sites of the respiratory tract. The approach provides a critical foundation for developing therapeutic treatment protocols for a wide range of diseases where aerosol delivery to the respiratory system would be desirable. FROM THE CLINICAL EDITOR: Delivery of drugs via the respiratory tract is an attractive route of administration. In this article, the authors described the design of mesoporous silica nanoparticles which could act as carriers for drugs. The underlying efficacy was successfully tested in a mouse model. This drug-carrier inhalation nanotechnology should potentially be useful in human clinical setting in the future.

Morphological diagnosis of Alport syndrome and thin basement membrane nephropathy by low vacuum scanning electron microscopy.

Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetic disorders caused by mutations of the type IV collagen genes COL4A3, COL4A4, and/or COL4A5. We here aimed to investigate the three-dimensional ultrastructure of the glomerular basement membrane (GBM) in order to introduce a novel method of diagnosing AS and TBMN. The subjects were 4 patients with AS and 6 patients with TBMN. Conventional renal biopsy paraffin sections from AS and TBMN patients were stained with periodic acid methenamine silver (PAM) and observed directly under low vacuum scanning electron microscopy (LVSEM). The PAM-positive GBMs were clearly visible under LVSEM through the overlying cellular components. The GBMs showed characteristic coarse meshwork appearances in AS, and thin and sheet-like appearances in TBMN. At the cut side view of the capillary wall, the GBMs in AS appeared as fibrous inclusions between a podocyte and an endothelial cell, while the GBMs in TBMN showed thin linear appearances. These different findings of GBMs between AS and TBMN were easily observed under LVSEM. Thus, we conclude that three-dimensional morphological evaluation by LVSEM using conventional renal biopsy paraffin sections will likely be useful for the diagnosis of AS and TBMN, including for retrospective investigations.

A novel approach to the histological diagnosis of pediatric nephrotic syndrome by low vacuum scanning electron microscopy.

Despite intensive treatment, steroid-resistant nephrotic syndrome (NS) often progresses to endstage renal disease. Therefore, a more accurate and quick histological diagnosis is required to properly treat such patients. The aim of this study was to introduce a novel approach to the histological diagnosis of pediatric NS by low vacuum scanning electron microscopy (LVSEM) and to describe the morphological differences in glomeruli between steroid-sensitive and steroid-resistant NS specimens. The subjects were three patients with steroid-sensitive NS and four patients with steroid-resistant NS. Conventional renal biopsy paraffin sections were stained with platinum-blue (Pt-blue) or periodic acid methenamine silver (PAM) and directly observed under LVSEM at magnifications between ×50 and ×10,000. The Pt-blue-stained sections showed three-dimensional structural alterations in glomerular podocytes and foot processes. PAM-stained sections showed changes in the structure and thickness of the glomerular basement membrane (GBM). Consequently, many round-shaped podocytes and elongated primary foot processes were exclusively recognized in steroid-resistant NS, although irregularities in foot process interdigitation, fusions, effacements, and microvillus transformations were observed in both steroid-sensitive and steroidresistant NS. Irregularities in thickness and the wrinkling of GBMs were clearly detected in steroid-resistant NS. The evaluation by LVSEM is probably useful for the renal histological diagnosis of pediatric NS.

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.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Three-dimensional helical coiling structures and band patterns of hydrous metaphase chromosomes observed by low vacuum scanning electron microscopy.

Helical coiling structures and band patterns of hydrous metaphase chromosomes were documented three-dimensionally by low vacuum scanning electron microscopy (SEM). Fixed or unfixed isolated Chinese hamster metaphase chromosomes were stained with platinum blue (Pt blue) and observed in the backscattered electron mode for low vacuum SEM without any hypotonic treatment or drying processes. Fibrous structures were shown both in the fixed and unfixed hydrous chromosomes; helical chromatid coils and their subcoils were clarified especially in the fixed chromosomes having contrasting alternative bands of light and darkness, while the translucent perichromosomal matrix and compact fibrous structures were recognized in the unfixed chromosomes. The helical coils were more clearly represented in a loosened chromatid of metaphase chromosomes. Treatment with a tris-HCl buffer solution and Pt blue staining in a hydrous condition successfully produced banding patterns similar to G-bands on metaphase chromosomes. These banded chromosomes observed by low vacuum SEM were also analyzed stereoscopically by field emission SEM after critical point drying. These findings indicate that: 1) native or unfixed chromosomes maintain the compact arrangement of high-order helical structures covered with the peri-chromosomal matrix; 2) helical coiling appearances of chromatids frequently observed in previous papers might be caused by loosening of the final level of the high-order structure of the metaphase chromosome; and 3) banding patterns might be produced by the rearrangement or reorganization of chromatin fibers at the 30 nm fiber level after the extraction of some chromosomal components including the peri- or intra-chromosomal materials during the banding procedure.