Cell-cell fusion induced by the hemagglutinin (HA) spike-glycoprotein of influenza virus has been studied extensively using transfected cells expressing the protein. The initial fusion event is the formation of a small pore connecting the two cells. Its appearance has been measured by sudden changes in capacitance by whole cell patch clamp and more recently by the sudden change in the fluorescence of a possibly potential-sensitive fluorophore. Using a new design of fluorescence video microscopy capable of capturing multiple images at differing wavelengths of light simultaneously at video rates, this fluorescence event is shown to follow exponential kinetics, with a life time, τ, of approximately 350 ms.

This supplement is a continuing step in the development of Microscopy and Microanalysis. The journal was launched in 1995 under the title JMSA, as the official journal of the Microscopy Society of America. In 1997 the journal was renamed and placed in the hands of Springer-Verlag, one of the world's leading publishers of scientific journals. In order to ensure the completeness and integrity of library collections, the scientific articles from Volumes 1 and 2 of JMSA are reprinted here as a supplement to Microscopy and Microanalysis.

Improvements in instrumentation for energy-dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have underlined the need for suitable standards for measuring performance. We report the results from several laboratories that were supplied with a test specimen consisting of a thin film of nickel oxide supported on a molybdenum grid. The Ni-Kα/Mo-Kα count ratio was used as an indication of number of stray electrons and/or X-rays in the TEM column; the Ni-Kα peak/background ratio provided a measure of the total background in the EDX spectrum, including bremsstrahlung contributions and the effect of detector electronics. By providing values typical of current instrumentation, the results illustrate how the test specimen can be used to evaluate TEM/EDX systems prior to purchase, during installation, and (periodically) during operation. The NiO films were also used to test EELS acquisition and quantification procedures: measured Ni/O elemental ratios were all within 10% of stoichiometry.

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.

Compositional imaging with electron energy loss spectroscopy (EELS) can be performed in both the energy-filtering transmission electron microscope (EFTEM) and in the scanning transmission electron microscope (STEM). Quantitative elemental distributions are obtained from core-edges produced by inner-shell excitations, although more detailed information about chemical bonding and electronic structure is also available from the fine structure associated with valence electron excitations. The fixed-beam EFTEM can provide data from large numbers of pixels very rapidly and offers an advantage for analysis of extended specimen regions containing relatively high atomic concentrations. Acquisition of entire spectra at each pixel in the field-emission STEM (spectrum-imaging technique) provides improved flexibility and accuracy despite the longer recording times. Spectrum-imaging allows post facto data processing with parameters that can be varied after acquisition is completed. In suitably thin specimens, EELS compositional mapping can provide a sensitivity of a few atoms for certain elements.

Z-contrast STEM using an annular detector can provide an intuitively interpretable, column-by-column, compositional map of crystals. Incoherent imaging reduces dynamical effects to second order so that the map directly reflects the positions of the atomic columns and their relative high-angle scattering power. This article outlines how these characteristics arise, presents some examples of the insights available from a direct image, and discusses recent developments of atomic-resolution microanalysis, direct structure retrieval by maximum entropy analysis, and Z-contrast imaging at 1.4 Å resolution using a 300-kV STEM.

Photoelectron imaging is a novel way of imaging the distribution of specific cell surface components. The basic principle is the photoelectric effect, in which electrons are ejected from the specimen by UV light. There are two primary forms of image contrast in photoelectron microscopy: material contrast and topographical contrast. Material contrast, provided by differences in photoelectron quantum yields, makes it possible to select labels that appear bright against the darker background of the cell surface. Topographical contrast ensures that label distribution can be interpreted in the context of cellular structures. By varying the wavelength of the exciting light, the contrast between label and cell surface can be controlled (tuned) to produce images ranging from a primarily topographical view of the surface to those showing only the label distribution. Being an emission-based technique, photoelectron imaging shares with fluorescence microscopy the ability to image label distributions at low magnification. However, unlike fluorescence microscopy the photoelectron image is formed by electrons, which generates a greater level of attainable resolution. Label contrast and wavelength-dependent tunability of contrast is illustrated with images of silver-enhanced immunogold-labeled cell surfaces, including selective labeling of cells in mixed-cell co-cultures, the cell surface CD44 adhesion protein on human glioma cells, fibronectin patterns on human fibroblasts, and the human transferrin receptor on MCF-7 human breast carcinoma cells.

A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 μm) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 μm) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

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.

Dynamic charging is the condition in which the magnitude of the charge on the surface of a non-conductor, such as a polymer, under electron irradiation appears to change with the incident current density. Measurements confirm that this effect does occur and show that the magnitude of the effect depends on the relationship between the incident beam energy and the energy (E2) at which the sample has a total electron yield of unity. The effect arises from changes in the leakage resistance of the sample, and two possible explanations for this behavior are suggested.

A dual excitation microfluorimeter is described for measuring rapidly changing, intracellular calcium signals. A spinning sector wheel is used in conjunction with a beam masking device to provide rapid, efficient switching between the 2 excitation wavelengths. Exposure intervals as short as 120 μs can be achieved, yielding ratio samples at a rate of 6 kHz. Emission photons are collected using a photomultiplier tube operating in counting mode. When tested using FURA-2 as the calcium reporting dye, throughput noise in the system is demonstrated to be due to the statistical fluctuation inherent in photon counting. An example of the operation of the system, using a guinea pig cardiac myocyte, demonstrates that sufficient ratio data may be acquires to fully characterize the fastest components of the intracellular calcium signal.

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.

The atomic force microscopy is increasingly being used in analytical laboratories to study material surface phenomena. Whereas its use is not free of artifacts itself, the AFM, because of the ways it produces topography images, can shed some light on problems associated with other analytical techniques. This article describes the use of Atomic Force Microscopy (AFM) in visualizing and evaluating the extent of some well-known artifacts produced by three techniques widely used in analytical laboratories. The three different types of artifacts demonstrated here are caused, respectively, (1) by a stylus profilometer used for topography characterization of the pole tip area of magnetic heads, (2) by an accumulation of an organic contamination caused by a stationary electron beam positioned on an analyzed surface during SEM/EDX analysis, (3) by an enhancement of aluminum grain structure produced by a rastered monodirectional sputtering ion beam during Auger depth profile analysis. The analytical consequences of each of the presented artifacts are discussed. The images were collected on the TOPOMETRIX TX 2000 “Discoverer” AFM, using standard 4-μm pyramidal tips and forces within a few nanometers.

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 (0.5-3 μm thick) for light microscopy, or ultrathin frozen sections (50-100 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. Since 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.

A 1/2[0001](0001) stacking fault and a planar 60° rotational domain boundary on the (0001) plane in as-grown CVD α-Si3N4 crystals have been characterized by high-resolution electron microscopy and image simulation. As reported previously, two types of coherent boundaries have been observed in this material, namely, stacking faults and rotational domain boundaries. The former involves only a translational displacement, while the latter separates two grains by a 60° rotation in addition to a translation. Inasmuch as the difference resulting from the rotational component can hardly be detected by high-resolution electron microscopy, care must be taken to analyze them first by analytical electron microscopy. In this paper, these two types of boundaries are studied and structural models are constructed that give simulated images in satisfactory agreement with observed images.

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 Å). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

Optical tweezers, or the single-beam optical gradient force trap, is becoming a major tool in biology for noninvasive micromanipulation on an optical microscope. The principles and practical aspects that influence construction are presented in an introductory primer. Quantitative theories are also reviewed but have yet to supplant user calibration. Various biological applications are summarized, including recent quantitative force and displacement measurements. Finally, tantalizing developments for new, nonimaging microscopy techniques based on optical tweezers are included.

We have used confocal laser scanning microscopy (CLSM), high-voltage electron microscopy (HVEM), and intracellular recording techniques to study volume changes in cultured Aplysia pacemaker neurons. Hyper- and hypo-tonic artificial sea water (ASW) decreased the pacemaker frequency and led to depolarization and hyperpolarization, respectively. However, when negative or positive current was injected into neurons in normal ASW, the frequency decreased with hyperpolarization but increased with depolarization. This suggests that the membrane potential is not the only factor underlying the reduction of the pacemaker activity.

Changes in cell volume were monitored with a CLSM and paralleled progressive changes in osmolarity. The neurons swelled and shrank nonuniformly, and, although an optical section through the middle of the cell was monitored every 4 s for as long as 14 min, a regulatory volume decrease or increase was never observed, indicating an osmometer-like behavior. The time course of shrinkage was faster than swelling after returning to control ASW after a hypotonic shock, reflecting a possible mechanical stress on the cytoskeleton.

Thick sections observed in the HVEM confirmed that membrane infoldings were present in our cultured Aplysia neurons. We hypothesize that a change in length of the latter in shrunken and swollen neurons could provide an explanation on the ultrastructural level for the increase and decrease in membrane surface area observed by CLSM. We conclude that by combining a confocal microscope with an electrophysiological set-up, three-dimensional morphology and physiological properties can be studied in living cells in real-time. This approach provides the means to correlate cell volume-related alterations and physiology.

An efficient method of merging images from electron crystallographic data has been developed that is several orders of magnitude faster than the method of phase shift origin search. The results of this new cross-correlation method are compared with the phase residual in terms of speed, consistency with the phase residual method, and the ease of incorporating symmetry constraints along with the image-to-image merging. The program is further accelerated by a more efficient sorting procedure.