Study for fast scanning with high axial resolution of Lattice light-sheet microscopy

Abstract

Researchers in the optical field have developed light-sheet microscopy to achieve fast scan rates and low photo-toxicity. However, the conventional light-sheet microscopy uses a Gaussian beam or a Bessel beam, so that the resolution in the z-axis direction is not significantly different from the confocal microscope widely used in biology and chemistry. In general, the resolution in the z-axis direction is only about 1/3 of the resolution in the x- and y-directions, although the resolution in the x- and y- direction has been greatly improved for several decades. This is because the side lobe of the light-sheet exists. Lattice light sheet microscopy overcomes these limitations with thin light sheets using 2D optical pattern. The lattice beam produces a sheet with a much narrower core, which provides a higher axial resolution. Sheet-shaped illumination eliminates out-of-focus fluorescence emissions and enables high-speed imaging with low photo-toxicity. The Lattice light-sheet microscope can construct a low fluorescence background grating beam and all excitation light is within a narrow focus depth. Therefore, light that is out of focus is not irradiated onto the sample, so damage by light is limited to the focal plane. We also provide a high-resolution, high-contrast image of 200 to 1000 images per second. In this experiment, we will check the performance of Lattice light-sheet microscope complements the disadvantages of conventional confocal microscopy and light sheet microscopy. A sheet-like pattern could be used to create a much thinner light sheet, and it was confirmed that light above the focal plane was gathered higher than the surrounding area. This focuses the fluorescence emission only on the focal plane to prevent further damage to the sample due to phototoxicity of unfocused light. It was also confirmed that the light was uniformly distributed in the light sheet. In addition, when compared to a single-vessel beam light sheet and a lattice light sheet, it was confirmed that the side lobes actually decreased. The fact that the resolution of the Lattice light-sheet microscopy in the z direction corresponds to the range of about 317 nm to 370 nm was confirmed by photographing a fluorescent bead sample.