Two-photon ray-plate microscopic imaging method and device

Abstract

The invention discloses a two-photon ray-plate microscopic imaging method and device. The method comprises the following steps: producing ultra-short pulse laser by utilizing ultra-short pulse laser source in a pre-constructed imaging device; performing beam expanding on the ultra-short pulse laser through a first 4f system, and generating a focusing line by utilizing a cylindrical lens, wherein the focusing line arrives a first lens in a second 4f system through a beam splitter, and then arrives a second lens of a second 4f system through a scanning galvanometer, thereby forming the focusingline on a surface of a spatial light modulator; performing phase modulation on the focusing line through the spatial light modulator, and then reflecting and propagating the focusing line, thereby arriving a third 4f system and an excitation objective lens through the second 4f system and the beam splitter; and collecting a fluorescence signal so as to perform ray-plate microscopic imaging of a sample. Through the method disclosed by the invention, the quick imaging is realized by quickly accessing modulated phase on the spatial light modulator, and the imaging speed and the imaging efficiencyare improved.

Description

technical field [0001] The invention relates to the field of optical technology, in particular to a two-photon light sheet microscopic imaging method and device. Background technique [0002] Light Sheet Microscopy (LSM) is a new type of optical microscopy imaging method. Compared with confocal fluorescence microscopy, light sheet microscopy greatly reduces photobleaching and improves imaging data throughput. In recent years, It has been widely used in biomedical microscopy imaging. [0003] However, considering the influence of biological tissue scattering and other factors, conventional light sheet microscopy based on single-photon excitation has shallow penetration depth and low imaging signal-to-noise ratio, which limits the wide application of this technology. Two-photon fluorescence excitation is based on the nonlinear effect of two-photon absorption. Its excitation wavelength is twice the wavelength of the corresponding single-photon excitation light, thereby reduci...

AI Technical Summary

Problems solved by technology

However, it is worth noting that the conventional method uses the method of moving the objective lens or sample in the axial direction of the excitation objective lens for scanning, and its mechanical inertia results in a low imaging rate, which cannot meet the application requirements of biological dynamic imaging.

Although people use fast focusing systems, such as electrically tunable lenses or ultrasonic tunable lenses (Cell Research 25, 254–257 (2015)), to increase the scanning speed of the excitation objective lens in the axial direction, the above methods can only perform continuous scanning

However, for some biological dynamic observations in the axial direction of the excitation objective lens, only the region of interest needs to be observed. At this time, continuous scanning will not only reduce the imaging speed, but will also increase the photodamage of the tissue.

[0005] Two-photon light sheet imaging usually uses push-broom linear excitation combined with (virtual) confocal detection to achieve imaging, but the mechanical inertia of the scanning device severely limits the imaging speed. Therefore, it is an urgent problem for those skilled in the art to solve The technical problem is: how to design a two-photon light-sheet microscopic imaging system that scans rapidly in the axial direction of the excitation objective lens and is selective for the target excitation field of view

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