In this paper we describe an application of the confocal laser microscope (CLM) to interference reflection (IR) microscopy for analysis of the cell-to-substrate adhesion of cultured cells. The CLM in IR mode provided sharp and high-contrast images of focal contacts of cells grown on glass surfaces without interference from out-of-focus reflections. For simultaneous imaging of fluorescence and IR signals, the 488 nm line of an argon ion laser was used for illumination and a dichroic mirror was placed in front of two photomultipliers to split the light returning from the specimen into reflected (488 nm) and fluorescence (green) signals. Thus we could correlate the IR and fluorescence images of cells stained with fluorescent labels for vinculin or F-actin. To confirm the cell contact, we also examined cultured living cells perfused with a medium containing Lucifer Yellow. Fluorescent dye-excluded spots in the plane of cell adhesion corresponded well to dark spots in IR images. Because of these advantages over the conventional IR microscope, we can corroborate the usefulness of the CLM for IR analysis.

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It is now accepted that inositol trisphosphate receptors may play a key role in the coupling between cytoplasmic second-messenger and nuclear third­ messenger-triggered events. Therefore, we studied the receptor-mediated nuclear calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clones, TP67.21) by means of a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3-loaded B cells showed that the antigen-stimulated calcium signals were transferred not only to the cytoplasm but also to the nucleus. These calcium signals were observed in the absence as well as in the presence of the external free calcium ions. The nuclear calcium signals in the B cells were also observed by the addition of thapsigargin, an inhibitor of the intracellular Ca²⁺ pump. Thapsigargin is known to increase intracellular free Ca²⁺ concentration by depleting Ca²⁺ from endoplasmic reticulum without a change in the level of inositol phosphate. Thus, the present results indicate that inositol trisphosphate receptors in the nuclear membranes do not affect the regulation of the nuclear calcium signals in B cells (TP67.21).

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We developed a new image analyzer system and also devised new microspheres to determine the activity of myeloperoxidase (MPO) released into phagolysosome which was formed by phagocytosis of polymorphonuclear leukocytes (PMN). When free 3-(p-hydroxyphenyl) propionic acid (HPPA) was reacted with purified human MPO in the presence of H₂O₂, fluorescence was produced depending on increase of concentrations of HPPA and H₂O₂ in a time-dependent fashion with the optimal pH at 5.4. This indicates that HPPA acted as a substrate for human MPO. The microspheres were prepared from methacryl acid polymer having a diameter of 1.9 μm and conjugated with HPPA. When HPPA-­conjugated microspheres (HPPA-MS) were incubated with H₂O₂ in a cell-free system consisting of degranulated fluid of PMN or purified MPO, the microspheres were converted to fluorescence-­bearing microspheres to which free HPPA molecule appeared fixed. Fluorescein production as also induced by incubation of the microspheres with viable PMN. The kinetics involved in the generation of solid-phase fluorescein bound to the micro­spheres by MPO activity in microsphere-ingested PMN was also analyzed by a newly developed immuno-reaction image analyzer system IMRAS. These results indicate that HPPA-MS are useful as a probe for assay of active phagocytosis and for image analysis on the activity of MPO-released during phagocytosis in viable PMN.

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