An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy

A rapid, robust method for the enumeration of total and viable microorganisms is described. A method using specific stains for viable and total cells and fluorescence light microscopy on membrane filters had been previously developed, but was sub-optimal in that some non-specific staining of the filters occurred and the filters were not flat enough for automatic image analysis methods to be employed, because not all cells in a field of view were in focus simultaneously. A new membrane filter has recently become available: the Anopore? membrane was described by the manufacturers as having a number of properties which would overcome these limitations. These include inorganic construction (giving resistance to solvents), high porosity (giving high flow rates), low surface adsorption (giving low background staining) and inherent hydrophilicity (simplifying wetting with aqueous solutions). Anopore membrane filters were found to produce very high contrast images of bacteria stained with ethidium bromide. Even with a relatively low power (magnification = 40) dry objective, these images could be easily thresholded for image analysis using only grey-level information. The methods developed here are considered to be a suitable basis for a fully automated procedure for the enumeration of total microbial populations.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.     

Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3-3'-diaminobenzidine and immunocytochemistry.

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB-stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1-0.25 microns), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed.     

High voltage microscopy of biological specimens: some practical considerations

Techniques are now available for the preparation of specimens giving excellent image contrast at high voltages, either by block-staining, staining an already embedded block or by post-staining. Further techniques are being developed for selectively staining certain features of the specimen thereby reducing the stain content of the specimen and permitting the examination of very thick specimens at adequate resolution. Specimens having inadequate contrast, particularly preparations of macro-molecules, may be imaged in one of the various dark-field modes, giving a considerable enhancement in image contrast on the viewing screen. Further enhancement of the recorded image contrast can be achieved by exposing the photographic plate to a much higher density than is normal. The complexity of image detail which occurs when the many features of a thick specimen are contracted into a two-dimensional micrograph may be elucidated by stereo-imaging techniques. Care must be taken to optimize the stereo-tilt angles, the orientation of the tilt axis and the register of the two micrographs in the stereo-pair, for the maximum depth in the specimen to be observed.     

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. (1) Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; (2) non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; (3) cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.     

Computer-aided integrating microdensitometry and specimen imaging with the Vickers M85A

A Vickers M85A flying-spot microdensitometer has been linked to a standard BBC model B microcomputer with a commercially available interface, and programs developed in which the computer controls step-wise scanning of the specimen. At each image point thirty-two absorbance readings are made and the average value is stored in the computer memory at 8-bit resolution; a complete 48×48 raster takes less than 5 s. An image of the specimen displayed on the monitor uses eight different pseudo-grey-level characters, and the manner in which the 256 possible absorbance values are represented on the screen can be altered by the user. A mask of arbitrary shape and size, drawn on the screen under key-board control, makes it possible to measure the integrated absorbance of even small, irregular and closely packed specimens such as individual bands in Drosophila polytene chromosomes. The coefficient of variation of repeated measurements of a Feulgen-stained frog nucleus is typically better than 1%. Displayed images can be printed on a dot-matrix printer, and data stored on disc for subsequent analysis. The techniques can readily be modified and adapted for other purposes, some of which are briefly discussed.     

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.     

极紫外太阳望远镜分辨率检测

在完成极紫外太阳望远镜（EUT）的装调工作之后,需要对其成像质量进行检测。将分辨率板置于平行光管的焦点处,由可见光照明该分辨率板,透射光经平行光管后成为平行光束并充满待测EUT入瞳,再经EUT成像在CCD相机上,根据所得的像可判断待测望远镜分辨率。实验结果表明EUT在可见光波段(λ=570nm)的分辨率为1.22″,接近此波段的衍射极限(1.20″)。根据可见光检测结果估算出EUT工作波段的分辨率可以达到0.32″,满足设计要求。     

斜视状态下航空遥感器像移的计算与补偿

航空遥感器成像过程中飞行器的向前飞行和姿态变化,使地面物体发出的光线相对于遥感器的光轴产生运动,从而引起共轭像点的运动,在成像介质上产生像移。运用光线矢量与光轴旋转变换相结合的方法,针对飞行器向前飞行使物体与光轴间发生的平移,以及飞行器姿态变化使光轴方向的改变,建立了光线矢量与光轴单位矢量间的关系,得到了航空遥感器在斜视状态下的像移模型。以实测遥感器的工作参数为条件,分析和计算了遥感器的像移,并给出了像移补偿的方法。实际应用证明光线矢量与光轴旋转变换相结合计算像移的方法,不仅综合了前向像移与姿态像移,而且计算简便,可推广于航空遥感器的研究中。     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

Quantitative fluorescence in confocal microscopy

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen-stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations. In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re-absorption of the excitation light and emission light respectively. In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens. Under confocal observation, as measured with the BIO-RAD MRC-500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.     

Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone-exposed rats

Alveolar type II cells in control and ozone-exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m³ for 7 days, a 50% increase in the mean number of type II cells was observed. The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time-consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.     

Automatic macroscopic density artefact removal in a Nissl‐stained microscopic atlas of whole mouse brain

Acquiring a whole mouse brain at the micrometer scale is a complex, continuous and time‐consuming process. Because of defects caused by sample preparation and microscopy, the acquired image data sets suffer from various macroscopic density artefacts that worsen the image quality. We have to develop the available preprocessing methods to improve image quality by removing the artefacts that effect cell segmentation, vascular tracing and visualization. In this study, a set of automatic artefact removal methods is proposed for images obtained by tissue staining and optical microscopy. These methods significantly improve the complicated images that contain various structures, including cells and blood vessels. The whole mouse brain data set with Nissl staining was tested, and the intensity of the processed images was uniformly distributed throughout different brain areas. Furthermore, the processed image data set with its uniform brightness and high quality is now a fundamental atlas for image analysis, including cell segmentation, vascular tracing and visualization.     

光散射在线分析系统

光散射在线分析系统是以斯坦教授的小角光散射法为理论基础，主要用于高分子材料内部结构的在线分析测试与研究。它是由硬件──光散射在线分析仪和软件──数据采集处理系统两大部分组成。该系统首创了高聚物内部结构的在线分析，并且成功地将数字图象处理技术应用于光散射图象的分析处理；由于计算机的优势和潜力，使得整个计算处理准确、快速，形象、直观，而且易于统一标准，从而提高了计算结果的可比性和可信度。     

Optimized reflection imaging in laser confocal microscopy and its application to neurobiology: Modificationsa to the biorad MRC-500

Reflection images of biological specimens recorded using laser-scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC-500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium-neon laser was not as readily absorbed as the blue and green lines of an argon-ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.     

面向细胞显微成像的虚拟染色技术的研究

细胞显微成像是生物学研究中进行细胞表型检测、获取细胞特征信息的重要手段。传统荧光成像技术是目前主要的细胞成像手段，但是荧光成像系统结构复杂、成本较高，而且特异性染色会对细胞造成损伤。针对此问题，研究了一种虚拟染色技术，使用多模态配准算法执行严格配准明场和荧光图像数据集，改进网络架构、损失函数、后处理、硬件适应性用于训练优化，并且通过虚拟染色评价标准对染色转换偏差进行验证。该方法可以降低荧光成像对荧光成像设备的依赖，无需各种复杂的染色操作，将会减轻生物研究、病理分析、疾病诊断流程的负担。     

Combined flow cytometry and image cytometry of the same cytological sample

Flow cytometry and image cytometry, two measuring techniques in the field of analytical cytology, can be used sequentially on the same cytological sample. Cells stained with a fluorochrome for the determination of for example, DNA or RNA content are first analysed in suspension by flow cytometry. The results of the fluorescence analysis of the individual cells are presented after data processing as frequency histograms of the DNA or RNA content of all the cells of the sample. In these histograms certain cell populations such as those with an increased DNA content are defined and these are then selected for further investigation. This is achieved by sorting cells of interest into centrifugation buckets by means of electrostatic deflection of the droplets containing such cells. Sorted cell populations are then centrifuged on to glass slides and stained according to the acriflavine Feulgen–SITS staining procedure, a quantitative method for DNA and protein. Image cytometry of these stained cells is performed with a computer controlled television based image analysis system (LEYTAS). With this system abnormal cells with elevated DNA content or increased chromatin contrast are automatically detected, thereby eliminating almost all artefacts and normal cells. Subsequently detected objects are stored in grey-value memories after the automated analysis for visual examination by the cytologist. The possibilities of combined flow cytometry and image cytometry are illustrated in typical examples in the field of cervical, bladder and mammary cytology.