Abstract
The resolution and the width of view in the light microscope are quite limited in all three spatial dimensions. However, by trading one dimension off, a sectional view can be obtained with the highest possible detectability. We evaluated the sectioning property of the slit scanning method and applied it to functional studies of the cellular activity. Individual molecules of tetramethyl rhodamine were easily visualized under the slit light illumination. Narrowing the slit width from 30 to 1 μm greatly enhanced the contrast of the single molecule image. The Q-dot nanocrystals (<50 nm) were also easily visible at a single particle level. Fluorescent beads of 1 μm in diameter appeared as 0.2 μm spots when placed at the window for the slit light emergence. They appeared as rings of variable sizes when placed a few micron distant from the emergence window, indicating that the thickness of the slit light was smaller than 1 μm. Scanning the cell with the thin sheet of light, we recorded fluorescent images of the cell at the shallow and deep parts. MIN6 cells with their secretory vesicles stained with the green fluorescent protein tagged to insulin showed clear shape of the vesicles. Hippocampal neurons dissociated and cultured on a coverslip in the medium containing FM1-43 showed rapid uptake of the dye into fine vesicles in the neurites upon stimulation. These fluorescent vesicles increased in number with the number of stimulation. Later, they showed trafficking activity along the neurite shaft. [Jpn J Physiol 54 Suppl:S16 (2004)]
