Application of a new fluorescent microscopic system for research in radiation biology

We developed and herein introduce a new microscopic system for automatic acquisition of time-lapse images of both brightfield images and four kinds of fluorescence images. We combined an inverted incident-light fluorescence microscope with an incubation system, a Hoffman Modulation Contrast device, a color chilled 3CCD camera, and an automatic excitation-filter exchanger. The light source switching for brightfield imaging was controlled automatically by a computer. Cells were maintained in 5% CO₂ plus 95% air at 37° C on a microscopic stage with an incubation system, which consists of a gas-regulatory system and two separate temperature-control devices. The fluorescence of annexin V (AV)-enhanced green fluorescent protein (EGFP), AV-fluorescein isothiocyanate, and propidium iodide (PI) diminished 3%, 77%, and 28%, respectively, at 24 hours during time-lapse observation using this system. Time-lapse observation of individual Jurkat cells using this system confirmed that anti-Fas antibody-mediated apoptotic cells occurred at first budding and were stained with AV-EGFP alone, and thereafter were also stained with PI when the cells became flat as a result of ruptures of the plasma membrane. These results present direct evidence that apoptotic cells undergo secondary necrosis, which could be recognized based on PI staining. There was a significantly long time interval between the staining of individual cells with AV-EGFP, indicating apoptosis, and the staining of these cells with PI, which indicated the occurrence of secondary necrosis. On the other hand, most of the cells that underwent necrosis were detected simultaneously with AV-EGFP and PI. The microscopic system we developed allows time-lapse and simultaneous observation of individual cells for changes in both morphology and staining pattern of fluorescent dyes that allow the visualization of cellular function. This system might be a useful tool for research in radiation biology.