A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

An alternative method to prepare samples of the pathogenic yeast Cryptococcus neoformans for scanning electron microscopy analysis

In this work, an alternative to conventional preparation procedures for scanning electron microscopy (SEM) analysis of Cryptococcus neoformans was performed. The cells were fixed directly in the agar culture. This method is simpler than others already reported and the morphology of the cells was well preserved.     

Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy

Wharton D. A. 1982. Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy. International Journal for Parasitology12: 335–343. A method for the preparation of the infective larvae of Trichostrongylus colubriformis for scanning electron microscopy is described. This involves freeze-substitution with methanol followed by critical-point drying. The method resulted in good preservation and the retention of the coiled posture. The morphology of ensheathed and exsheathed larvae is described and the mechanisms of coil formation discussed. Coiling was also observed in the infective larvae of nine other trichostrongyle species.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

猪主动脉脱细胞血管基质制备

目的研究脱细胞血管基质的制备方法。方法采用胰蛋白酶、低渗溶液、化学除垢剂法处理猪胸主动脉来制备脱细胞血管基质,标本作苏木素-伊红染色,大体、光镜及扫描电镜、透射电镜观察。结果经该法处理的血管细胞全部脱除,胶原纤维、弹性纤维无断裂,细胞外基质保持完好。结论酶、低渗溶液、化学除垢剂联合法是制备脱细胞血管基质的较好方法。     

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.     

Observations on the Preparation of Epoxy Embedded Tissue Samples for Energy-Dispersive X-Ray Analysis

Elemental X-ray microanalysis of biological tissues by energy-dispersive detectors attached to conventional transmission or scanning electron microscopes is a technique with many potential applications. Proper specimen preparation and consideration of problems inherent with the method are necessary to achieve satisfactory results. This report concerns some of the problems encountered in analyzing tissue samples embedded for electron microscopy in epoxy resins.     

Detection of mycoplasma contaminations in cell cultures by PCR analysis

Mycoplasma contamination is still one of the main problems in using cell cultures in biological and medical research and in the production of bioactive substances, because mycoplasma can alter nearly all parameters and products of the cell. They can persist undetected in the culture if no special detection methods are applied. In recent years, the PCR technology has become a commonly used method to analyze genomic DNA and the expression of genes, with both high specificity and sensitivity. This technique can be effectively employed for the detection and even the identification of mycoplasma contaminations in cell cultures applying primers complementary to the 16S rDNA region. Although this technique, once established, is characterized by simplicity and speed, PCR is still a complex process and its sensitivity and specificity can be influenced by a number of different parameters, e.g. inhibiting compounds originating from the preparation process of the DNA, RNA or cDNA, contamination of the solutions with PCR products, and the selection of a primer pair which does not cover all the mycoplasma species occurring in cell cultures. Thus, adequate controls have to be included to obtain reliable results. The present review examines the use of different primers of the 16S rDNA region including their specificity, the sensitivity applying various DNA or RNA preparation procedures, and the methods to detect finally the amplicons. In conclusion, basic nucleic acid preparation and PCR product detection methods offer a simple, fast and reliable technique for the examination of mycoplasma contaminations in cell cultures, provided that the indispensable control assays are implemented.     

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Transmission electron microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability.     

A Modification of the Mycoplasma Serum-Drop Growth Inhibition Test

A modification of the serologic mycoplasma growth inhibition test is described. It is based on application of a drop of antiserum directly onto the centre of an inoculated agar plate provided with a slightly concave agar surface. The special apparatus devised for the preparation of these plates is described. This modified serum-drop method is more sensitive than other growth inhibition tests used today and is equally specific.     

Correlation of Scanning and Transmission Electron Microscopy on the Same Tissue Sample

Tissue processed for scanning electron microscopy (SEM) in a critical point bomb utilizing Freon-13 showed excellent subsurface preservation when prepared for transmission electron microscopy (TEM). The critical point method is the only commercially available SEM preparation technique in which the quality of preservation will not limit microscopists in efforts to correlate SEM and TEM observations.     

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.     

Serum deprivation increases the expression of low density lipoprotein receptor-related protein in primary cultured rat astrocytes

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and "ciliogenic" cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.     

Dialysis Membrane Technique for Studying Microbial Interaction

A dialysis membrane method is described which allows (i) cultivation of fungi on an agar support, (ii) observation of growth and development by direct light microscopy, (iii) transfer of cultures from agar surfaces for subsequent treatments or for biochemical analysis, and (iv) preparation for scanning electron microscopy. The method is used routinely in studies of fungus-nematode and fungus-fungus interactions.     

High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

BACKGROUND: Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. METHODS: We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. RESULTS: With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. CONCLUSIONS: SFM is a valuable method for the evaluation of fluorescently labeled cells.     

制备高分辨植物SEM样品的微波浸软法

微波辐射浸软法是一种简单、快速制备高分辨植物扫描电镜样品的方法。经冰冻断裂处理后的样品,利用断续微波辐射浸软,既可阻止浸软溶液温度陡然升高,不使植物结构受到破坏,又可使浸软时间显著缩短,由原来的72小时变为30分钟。因为断续的微波辐射能保持较低的炉内温度,所以它不仅能很好地适用于扫描电镜样品制备过程中的浸软,而且也适用于样品固定、导电染色等。此法大大缩短了O-D-O法的制样周期。     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Application of Polyethylene Glycol on Preparation of Plant Specimen of Freeze-Fracture

We report for the first time the use of polyethylene glycol 1540 as a freeze-fracture reagent on plant specimen prepared for observation by scanning electron microscopy. This method is a good fracture technique after being successfully experimented, which in some aspect is better than the “O-D-O” method.