Infectious gastroenteritis is a common cause of hospitalization in the pediatric population. The most frequent cause of gastroenteritis is viral in origin. The purpose of this study was to compare a rapid modified negative-staining TEM method with the conventional pseudoreplica technique in detection of viral particles in fecal samples from children with viral gastroenteritis. The modified negative-staining method resulted in a significantly higher (2.5 ± 0.5, p = 0.02) viral rating score than that for the conventional pseudoreplica technique (1.7 ± 0.4). In addition, the preparation time for the negative-staining method was approximately one fifth that for the conventional pseudoreplica technique. Rapid diagnosis of viral gastroenteritis may be made by ultrastructural detection of viral particles in fecal samples using the negative staining technique.

In this article we present a method to identify and extract spherical particle images from noisy spot-scan and flood beam electron microscopy images. We use a template matching algorithm with additional image preprocessing operations to allow consistent particle selection in spot-scan and other highly spatially varying images. In addition, this algorithm incorporates an automated image-cutting and edge-sewing mechanism that allows efficient particle selection despite the large size of electron microscopy images. We have tested this template matching algorithm on various spherical virus particle images with a large range of defocus values and have found that the particles are consistently selected in an accurately centered manner. In addition, this method is able to extract spherical virus particles from 400-kV electron microscopy images with defocus values of less than 1.0 μm underfocus where the particles are not readily visible to the human eye.

Laser microsurgery and laser-generated optical force traps (optical tweezers) are both valuable light microscopic-based approaches for studying intra- and extracellular motility processes, including chromosome segregation during mitosis. Here we describe a system in use in our laboratory that allows living cells to be followed by high-resolution differential interference contrast (DIC) video-enhanced time-lapse light microscopy while selected mitotic organelles and spindle components are subjected to laser microsurgery and/or manipulation with an optical force trap. This system couples the output from two different Neodymium-YAG lasers to the same inverted light microscope equipped with both phase-contrast and de Senarmont compensation DIC optics, a motorized stage, and a high-resolution low-light-level CCD camera. Unlike similar systems using phase-contrast optics, our DIC-based system can image living cells in thin optical sections without contamination due to phase halos or out-of-focus object information. These advantages greatly facilitate laser-based light microscopic studies on mitotic organelles and components, including spindle poles (centrosomes) and kinetochores, which are at or below the resolution limit of the light microscope and buried within a large complex structure. When used in conjunction with image processing and high-resolution object-tracking techniques, our system provides new information on the roles that kinetochores and spindle microtubules play during chromosome segregation in plant and animal cells.

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about −117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at −155 to −170°C to produce semithin frozen sections (0.5 to 3 μm thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.