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22 February 2006 Intracellular dynamics observed by mode switching of microscope with a light incidence to the interface at alternate angles through the ultra high NA objective
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Abstract
In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Cα (PKCα) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCα fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.
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Yoshihiko Wakazono, Takashi Sakurai, Mica Ohara-Imaizumi, Shinya Nagamatsu, Seiji Yamamoto M.D., and Susumu Terakawa M.D. "Intracellular dynamics observed by mode switching of microscope with a light incidence to the interface at alternate angles through the ultra high NA objective", Proc. SPIE 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV, 60881L (22 February 2006); https://doi.org/10.1117/12.647094
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