Cyanide removal of gold from SEM specimens.

The cyanide method is highly recommended for the purposes of the removal of gold coatings from SEM specimens prior to (a) further treatment before further SEM, and (b) staining and mounting for LM.     

Electron-count imaging in SEM

We propose a method and a system for counting secondary electrons and for displaying micrographs from the SEM in real time. Evaluating the images obtained by the new system, we confirm that electron counting images have a higher signal to noise ratio than analogue images. This tendency is remarkable when the secondary electron signal is weak. We present the method and show experimental results obtained with our novel system for the detection of the secondary electron signal.     

Some methods for more efficient processing of SEM specimens

Three techniques are described for increasing the quantity of specimens which may be processed for SEM while maintaining the quality of the final product. They are: a simple stainless steel holder for safe manipulation of coverslips during incubation and fixation; a coverslip carrier which permits four or more coverslips to be dehydrated and critically point dried at the same time; and a simple pattern for making baskets to hold relatively large but delicate specimens during processing for SEM. Processing soft tissues for scanning electron microscopy (SEM) presents many problems depending on the size, shape and type of specimen. Cohen (1974) presented an overall view of this problem. Methods for handling cells in suspension have been suggested by Baker & Princen (1975), Newell & Roath (1975) and Rostgaard & Christensen (1975). Tissue culture specimens are also difficult to process for SEM. These may be long-term cultures such as fibroblasts or kidney cells, or short-term preparations such as the collecting of white blood cells or bacteria on coverslips. Nemanic (1972) described a method using Tygon tubing with slits to hold the coverslips after incubation. Perecko et al. (1973) gave details of a holder for processing six small (6 mm) coverslips after incubation. Rice et al. (1976) designed several complex multipurpose carriers for 9 × 22 mm coverslips and for cells on silver or cellulose membrane filters. Broers et al. (1975) used a mesh receptacle for bacteria attached to silicon dioxide slivers. Many investigators simply process pieces of plastic cut from the tissue culture dish itself. Using the plastic directly avoids the problems of incubating cells on glass coverslips. Coverslips are fragile and difficult to remove from the culture dish or flask. Each coverslip must be processed individually, increasing the risk of breakage and limiting the number which can be done at one time. For processing larger tissues there are commercial baskets available. Cohen (1974) illustrated mesh baskets for odd sizes and shapes of soft tissues. We have devised a simple holder which facilitates the use of 12 mm 0 grade coverslips in several types of culture dishes. We have developed a multipurpose carrier which increases the number of coverslips which can be processed for SEM and reduces the number of manipulations needed for each individual coverslip. This carrier can be used for histological and histochemical studies as well as SEM processing. We have modified Cohen's technique (1974) for mesh baskets for larger soft tissue specimens.     

Lock-in technique applied to cathodoluminescence of biomedical specimens in the SEM

Very low signals or disturbances by unwanted, foreign signals often lead to a restriction in the application of the cathodoluminescence (CL) method in the scanning electron microscope (SEM). This is even true if one uses an optimal CL detection system. We, therefore, introduced the lock-in-amplification technique, which has proved very successful in investigations of semiconductor materials into the biomedical field. After attaching the lock-in system to our SEM which has a special CL equipment, we found that this technique could remove the disturbance caused by the light emitted from the heated filament, which can be reflected into the CL detector. Specimens on polished Al-stubs or on Au-coated glass slides could be imaged with improved contrast. The same was true if we measured the wavelengths of the CL. A general improvement of the signal-to-noise ratio in all specimens could not be detected. However, the beam current could often be reduced when using the lock-in technique without a decrease in the quality of the CL image. A disadvantage of the commercially available lock-in amplifier is that pictures need a longer exposure time than without lock-in amplification.     

Vacuum-sealed specimen stage for scanning microscopy of air-sensitive materials

A vacuum-sealed specimen stage for investigation of air-sensitive materials has been designed. This stage is useful for instruments equipped with a vacuum antechamber and allows facile sample mounting in a dry box or glove bag. The sample cell is subsequently introduced into the antechamber of the instrument and the appropriate measurement or photograph is taken. Sample integrity can be maintained in the cell for subsequent investigations.     

Iron-polyethylene glycol impregnation: An attempt to reduce shrinkage of specimens in SEM preparation

Glutaraldehyde-fixed HeLa cells were soaked in a mixture of fine cationic iron colloid and polyethylene glycol, immersed in tannic acid solution containing guanidine hydrochloride, and stained with osmic acid. The treated cells showed little shrinkage in the scanning electron microscope even after ethanol dehydration and CO₂ critical point drying. On the assumption that every HeLa cell maintained contact with each other, preservation rate was computed as 0.975 × 0.0033 in linear dimension. Microvilli on the cell surface were well preserved, and few undersirable deposits were noted on the specimen surface. This treatment was also applicable to bulk staining of tissue blocks, such as rat kidneys. The podocyte foot processes and endothelial micropores of the glomerulus were well preserved; the epithelial cells of the Bowman's urinary capsule were not collapsed; the microvilli of the brush border of the proximal convoluted urinary tubule kept their ordinary length (2 μm).     

STEM imaging of thick specimens with off-axis detectors

For scanning transmission electron microscopy (STEM) images obtained with relatively small objective aperture sizes, the contrast of small objects contained within thick specimens may be considerably enhanced by using an off-axis detector aperture situated on the edge of the central beam spot. The effect is demonstrated for both crystalline and amorphous specimens. The effect arises because the detector collects part of the small angle inelastic scattering and is modified by refraction effects for specimens of rapidly changing thickness.     

生物体内部结构三维显微成像技术研究

概述了生物体内部结构的几种传统的成像技术 ,介绍了一种新的生物体内部结构三维显微成像方法。该方法对生物体作连续切片 ,由 CCD显微摄像系统获取切片的序列图像 ,然后由计算机进行三维图像重构 ,最终得到生物体内部结构的三维显微图像     

Automated high-resolution three-dimensional fluorescence imaging of large biological specimens

We describe a novel automated technique for visualizing the three‐dimensional distribution of fluorochrome‐labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome‐labelled microdamage within an 8‐mm diameter cylinder of trabecular bone at a voxel size of 3 × 3 × 8 μm³. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three‐dimensional visualization. The three‐dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

分散类样品的扫描电镜图像缺陷原因及解决方法

在介绍扫描电子显微镜(SEM)的工作原理及特点基础之上,重点阐述了荷电效应、边缘效应、电子束损伤对分散类样品所造成的图像缺陷及相应的解决方法。     

Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging

Cryoelectron microsopy is a widely used technique to observe biological material in an almost physiological, fully hydrated state. The sample is prepared for electron microsopy observation by quickly reducing its temperature to ?180 °C. The high‐speed cooling induces the formation of vitreous water, which preserves the sample conformation. However, the way vitrification occurs is still poorly understood. In order to better understand the phenomenon, we have used a stroboscopic device to visualize the interaction between the electron microscopy grid and the cryogen. By blocking the free fall of the plunger once the grid has penetrated the coolant by half its diameter, we have elucidated the way in which vitrification propagates. The findings were confirmed by numerical simulation. In addition, according to our observations, we now present an alternative way to prepare vitreous specimens. This new method, with the grid parallel to the liquid cryogen surface, decreases evaporation from the sample during its free fall towards the coolant and at the same time achieves a more uniform vitrification over the entire surface of the specimen.     

Automatic focus correction for spot-scan imaging of tilted specimens.

The variation in defocus within an image of a highly tilted specimen can be a serious source of artifact. Spot-scan imaging can be combined with dynamic focusing to greatly reduce this range of defocus. A protocol is described for determining the parameters required for the automatic focus compensation during the recording of a spot-scan image. Images of a gold test specimen demonstrate the efficacy of this procedure in extending the area of the image that contains high-quality data. In case the tilt angle or resolution is high enough that the height difference of the specimen within each small illuminated area is larger than the depth of field, the image must be treated to compensate for the focus variation. The same principle is used as was developed for compensation of conventional images of tilted specimens.     

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron–specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 μm) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.