| Biological fluorescence microscope uses fluorescence labeling technology to image the specific structure and function of organisms,which is a widely used biological imaging research tool at present.Among them,the light-sheet fluorescence microscope has a unique advantage in the long-term and high-speed imaging of living organisms.The light-sheet microscope adopts the optical path structure of orthogonal illumination and imaging,which can quickly illuminate the biological samples with lamellar light.Compared with the traditional fluorescence microscope,the imaging speed is fast and there is no interference of non-focal plane fluorescence imaging and photobleaching.At present,the new two-photon optical sheet microscope further develops the advantages of optical sheet microscope,which is illuminated by near-infrared femtosecond pulse light which is less affected by biological scattering,and the twophoton virtual light sheet formed by rapid scanning of scanning devices.it can realize the illumination and imaging of biological deep light sheet with a large field of view.However,at present,the illumination depth of the advanced two-photon light-sheet microscope with high resolution(1?m)is limited to 170?m × 170?m.This is because living biological tissue is an optical medium with a non-uniform refractive index,which will be affected by biological aberrations in the process of two-photon illumination propagating to the depths of biological tissue,resulting in serious dispersion of the focus of two-photon illumination.the energy concentration decreases,the thickness of the optical sheet increases significantly,and even fluorescence imaging can not be excited in serious cases.To eliminate the influence of biological aberration,the adaptive optics technology of direct wavefront detection is applied to the illuminating optical path of a two-photon optical film microscope.the following research work is carried out:Aiming at the problem of the organic combination of adaptive wavefront correction and two-photon light-sheet microscope,according to the scanning characteristics of two-photon light-sheet microscope and isoplanatic region distribution characteristics of biological aberration space.An adaptive combination idea of wavefront correction imaging for the fractional isoplanatic region is proposed in the illuminating optical path of the two-photon optical microscope: localized isoplanatic scanning is realized by using Tunable Acoustic Gradient lens(TAG)and tip-tilt mirror scanning two-photon focus,and biological aberration correction in these isoplanatic regions is realized by liquid crystal spatial light modulator(Liquid Crystal On Silicon,LCOS).The global scanning correction splicing of different isoplanatic areas is completed by axial zoom,and the splicing scanning isoplanatic area is high resolution and large field of view while keeping a thin high-resolution light sheet.At the same time,a continuously adjustable beam expander is proposed to change the beam aperture of the incident beam at the pupil behind the microscope objective lens.a high-resolution splicing large-field light-sheet microscope with an adjustable thickness of 0.6?m ? 1.2 ?m combined with adaptive wavefront correction is realized.In order to solve the problem that it is difficult to realize wavefront detection of weak fluorescence signal in biological depth,a continuously adjustable beam expander is used to adjust the numerical aperture(Number Aperture,NA)of small microscope objective lens in illuminating optical path,and the two-photon excitation focus which is less affected by biological aberration is used as a fluorescence guide star.Combined with the isoplanatic de-scanning technique,the wavefront detection method of fluorescence detection signal enhancement in the same isoplanatic region is realized.In order to make full use of the limited fluorescence signal,the open-loop optical path structure and high-gain Hartmann EMCCD camera are selected,and the centroid detection algorithm is improved to improve the Hartmann detection ability under weak fluorescence and low signal-to-noise ratio.When realizing the wavefront detection of different isoplanatic regions in the whole domain,it is found that using the LCOS zoom in the existing optical path structure to complete the aberration detection of different isoplanatic regions will make the defocus aberration detected in the Hartmann detector and affect the detection ability of the Hartmann detector.Therefore,it is proposed that an achromatic zoom lens is introduced into the common optical path of the excitation optical path and the aberration detection optical path to eliminate the influence on Hartmann detection ability when the zoom moves to detect biological aberrations in different isoplanatic areas.Wavefront detection and wavefront correction can be carried out at the same time under the expansion of the field of view.To realize the organic cooperation of adaptive wavefront correction and wavefront detection,according to the characteristics that biological aberrations change slowly with time minutes or even hours levels,the detection of isoplanatic aberrations is proposed.After all the aberrations are preserved,the wavefront correction scanning imaging corresponding to each isoplanatic region in the whole domain is carried out.Because the biological shallow aberration has little effect on the two-photon optical sheet,the aberration has a significant effect on the two-photon optical sheet with the increase of illumination depth,and the size of the isoplanatic area in the deep layer is generally 30 ? 60?m.Therefore,for the above-mentioned 300?m × 300?m two-photon light sheet,the shallow layer 150?m × 300?m does not correct the biological aberration,and the latter 150?m × 300?m depth is divided into 60?m × 60?m,60?m × 60?m and30?m × 30?m.The scanning strategies of wavefront detection and wavefront correction for the 21 scanning regions are given.Finally,the scanning correction time of the singlelayer 300 ? m × 300 ? m two-photon optical film is 100 ms,which can adaptively correct the biological aberration while maintaining the advantage of fast surface imaging of the system.Combined with the above research,the design parameters of the adaptive two-photon optical sheet microscope system are theoretically analyzed and calculated;the optical design,mechanical design,and control design of the system are completed;and the adaptive two-photon optical film microscope system is built.Fluorescent beads and rhodamine fluorescent dye solution were used to test the resolution of the system,the scanning field of view,and the effectiveness of adaptive wavefront detection and correction.Finally,the biological aberration adaptive correction imaging of zebrafish embryos 48 hours after DAPI staining fertilization was carried out,and the imaging details and imaging contrast were significantly improved before and after correction,which realized the effective application of direct wavefront detection adaptive optics in the illuminating light path of the two-photon light-sheet microscope.The above research work makes it possible for adaptive two-photon optical sheet microscope to realize fast imaging observation with high resolution and large field of view in living biological tissue and provides a reference for the application of adaptive optics in three-photon light-sheet and even four-photon light-sheet fluorescence microscope... |
PDF Full Text Request