Two-photon light-sheet microscopy imaging method and device

Abstract

The invention discloses a two-photon light sheet microscopic imaging method and device, wherein the method comprises: using an ultrashort pulse laser source to generate an ultrashort pulse laser in a pre-built imaging device; The short-pulse laser beam is expanded, and the cylindrical lens is used to generate the focal line; the focal line reaches the first lens in the second 4f system through the beam splitter, and then reaches the second lens of the second 4f system through the scanning galvanometer, and the space light The focal line is formed on the surface of the modulator; the focal line is phase-modulated by the spatial light modulator, reflected and propagated, and then reaches the third 4f system and the excitation objective lens through the second 4f system and the beam splitter, and forms the modulated focal line on the sample The sample is made to excite a fluorescent signal; the fluorescent signal is collected to perform light sheet microscopy imaging of the sample. The method realizes fast imaging by quickly accessing the modulation phase on the spatial light modulator, and improves the imaging speed and imaging efficiency.

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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