Abstract
Aiming at an improved form of the evanescence microscope, we constructed a system scanning with a slit beam thinner than 1 μm in width for imaging a single exocytosis in neuronal cells stained with fluorescent dyes and proteins. The slit-scanning fluorescence (SSF) microscope consisted of an objective lens (x100 apo, NA=1.65), adjustable slits (0.1 mm width), and a 473 nm DPSS laser. The illuminated area in the focal region was approximately 1 x 10 μm/square, and about 1 μm thick when the angle of the incident light was set at the critical angle for total internal reflection. By shifting the angle below 50 degree, the beam passed through the specimen on the high refractive index glass (n=1.78). The image of glass-attached fluorescent beads of 3 μm in diameter appeared as a spot or a ring of 1 μm in diameter. This result indicates that the slit beam was indeed 1 μm or less in width and was able to form the optically-sliced images thinner than 1 μm thick, a better performance than confocal microscopy. In cultured neurons stained with FM1-43, many vesicle like structures (smaller than 0.3 μm) and their axonal movements were visible. A high resolution analysis of the synaptic function may be facilitated not only by the evanescence microscopy but also by the SSF microscopy proposed above. [Jpn J Physiol 54 Suppl:S147 (2004)]
