To investigate the relationship between intracellular calcium dynamics and degranulation in single intact mast cells, we developed a method for the simultaneous observation of these two events. Rat peritoneal mast cells were doubly stained with two types of fluorescent dyes, quinacrine and Ca-indicator fluo-3. Basic quinacrine was specifically incorporated in secretory granules and the fluorescence intensity was decreased upon degranulation. With emission at 520 nm, the excitation spectra for the two dyes differed so that alternate excitation at 420 and 490 nm made it feasible to measure intracellular Ca²⁺ elevation and degranulation, with the time-resolution of 2 s. Degranulation was always accompanied by a preceding transient rise in intracellular Ca²⁺ yet an increase in intracellular Ca²⁺ did not necessarily induce degranulation. These findings suggest that intracellular Ca²⁺ elevation is a necessary but not sufficient condition for degranulation in intact mast cells.

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A technique is described to obtain real-time, reflected light confocal images of wing cells in the epithelium of the normal in vivo human cornea. The real-time confocal imaging of normal wing cells in the in vivo human cornea is proposed as a new benchmark for comparison of various real-time confocal microscopes that are used for corneal imaging. The real-time, confocal microscope eliminates motion blur from the living, moving eye by synchronization of image acquisition and the scanning slit. The photographs in this paper are photographed from a video monitor which was coupled to a video recorder. Single video frames were photographed without the necessity of frame averaging or post acquisition digital image processing.

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The 47 kD molecule of the asexual blood stage of Plasmodium falciparum (P. f) was reported to react specifically with sera from patients at the acute stage of infection (Kano et al., 1990). Confocal laser scanning microscopy was applied to the indirect fluorescent antibody test (IFAT) in order to observe the localization of the 47 kD antigenic polypeptides. Two monoclonal antibodies (mAbs) which were observed to bind specifically to the 47 kD molecule were prepared. Optical section images of the confocal micrographs of the IFAT demonstrated that these two mAbs against the same 47 kD molecule reacted with different sites within the parasites: one showed the reaction in the center of the merozoite; the other, on the surface of the merozoite. The spatial distribution of antigens were the same in geographically different Gambian and Colombian strains and Thailand-Myanmer border isolate. It was thus revealed that the 47 kD molecule consists of at least two differentiable antigenic polypeptides. Confocal laser scanning microscopy in the study of reactivities of mAbs with subcellular structures was useful for determining the localization of the antigenic molecules with the least possible modification in the parasite.

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The three-dimensional quantities of a biomolecule, such as electrostatic potential, line of electric force, and electric field, are visualized by the computer graphics software AVS (1992). In the present work, we applied this visualization technique to interpret a biochemical reaction such as metal ion-catalyzed hydrolysis of glycine methyl ester. The pictures of lines of electric force and electric field show clearly which atom would be attacked by a nucleophilic reagent. The visualization can be further enhanced by generating the stereoscopic image from different viewing angles around the molecule. The pictures should be drawn on a well-selected site. On this point, the animation techniques for creating a sequence of pictures are useful.

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Megamitochondria induced by ethanol and hydrazine in culture cells were detected under laser confocal microscopy. Mitochondria were labeled in the living state after incubation for 3 days in culture media containing either ethanol or hydrazine. During incubation, mitochondria began to enlarge and reached maximum size in 3 days in the presence of 1—3% ethanol or hydrazine at concentrations of up to 2 mM. Above these concentrations, cells became degenerated. Since megamitochondria were formed for such a short time and the number of mitochondria per cell decreased progressively, the present results also seem to support our previous proposal that megamitochondria are formed by the fusion of adjacent mitochondria. These results provide us with a much simpler experimental system for the study of the mechanism and the process of megamitochondria formation, compared with animal experiments.

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Image analyzing software aided simple quantitative analysis method for the formation of root hairs in lettuce (Lactuca sativa L. cv. Grand Rapids) seedlings was newly developed. Low-magnification microscopic images of main roots and root hairs were first recorded on a magneto-optical disk using a CCD color camera and an image recorder. Recorded images were next converted to monochromatic gray scale images, then binarized to black and white images using image analyzing software. Depending on the threshold level for the binarization, an image of either the main root plus root hairs or just the main root was extracted as a black image. Next, “exclusive or” between the image of the main root plus root hairs and that of the main root was calculated. The thus-obtained image was next inverted, and the total number of black pixels was calculated as equivalent to the total amount of root hair. The total number of black pixels included in the long slender window along the surface of the main root in the image of the binarized root hairs plus main root was used to represent the density of the root hairs. Both values showed good correlation to the values determined by manual measurement using magnified printed images. This method made it possible to analyze root hair formation quickly and accurately.

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