| Biological fluorescence microscopy uses a specific wavelength of excitation light to illuminate a specific object in biological tissue,causing the labeled fluorescent substance to emit fluorescence for structural and functional imaging.In traditional biological fluorescence microscopy,the three-dimensional range of the entire biological tissue is illuminated so that specimens located outside the focal plane will also emit fluorescence,which affects the contrast and resolution of the image and significantly increases the photobleaching effect,greatly shortening the time for continuous imaging.In addition,the illumination light of the conventional biological fluorescence microscopy is continuous laser in visible light band,and the laser is severely absorbed and scattered during the propagation in biological tissue,resulting in small penetration depth,which is difficult to meet the imaging requirements.In order to solve the above problems,two-photon light-sheet microscopy emerges.The illumination light of the two-photon light-sheet microscopy is changed from continuous laser in visible light band to femtosecond pulse laser in near-infrared band.The effect of absorption and scattering of near-infrared illumination light by biological tissue is reduced,which increases the illumination depth.The illumination light focuses and excites two-photon fluorescence in biological tissue,and the excited two-photon fluorescence is confined to the illumination light focus and smaller than the illumination light focus area,so that the fluorescence outside the focus is not additionally excited.On this basis,the scanning devices quickly scan the illumination light in the lateral direction and the illumination depth direction to generate a two-photon light sheet at the biological tissue.Therefore,the two-photon light-sheet microscopy has advantages of high spatial resolution and low photobleaching effect,and gradually becomes an efficient instrument for living tissue imaging.With the development of the field of neuroscience,more comprehensive requirements for the performance of two-photon light-sheet microscopy have been put forward by biomedical researchers.The system needs to achieve 600?m×600?m imaging field while maintaining high resolution and low photobleaching effect.The scanning of two-photon light-sheet microscopy in lateral direction is performed by scanning galvanometer,and the scanning range of 600?m can be realized by setting its parameters;the scanning in axial direction(the depth direction of illumination)is done by adjusting the power of the illumination light by a super-high speed zoom lens(TAG zoom lens),which can achieve the axial movement of the two-photon focus,but cannot reach the penetration depth of 600?m.In addition,since the living biological tissue is a inhomogeneous optical medium,when the illumination light propagates to the depth of the biological tissue,the wavefront distortion occurs due to the influence of random aberrations,resulting in the decrease in the penetration depth of the illumination light,which seriously reduces the effective range of the two-photon light sheet.Therefore,the maximum imaging field of view of the current two-photon light-sheet microscopy is limited to the range of 170?m×170?m,which is difficult to meet the imaging requirements of the whole brain nerve tissue.In view of the above problems,adaptive optics technology is applied to the illumination path of two-photon light-sheet microscopy in this thesis,and expands the following research contents:The generation process of two-photon light sheet is simulated by MATLAB,and the effects of different aberrations on the thickness and intensity of two-photon light sheet are analyzed.The results show that the number of Zernike modes that need to be detected and corrected is no more than 58,which provides a basis for the design of adaptive optics.Since the TAG zoom lens cannot achieve the axial penetration depth of 600?m,bidirectional illumination is proposed so that the requirement of unidirectional illumination depth is reduced from 600?m to 300?m.However,the TAG zoom lens still cannot achieve the illumination depth of 300?m.For this reason,a sub-area illumination method using liquid crystal wavefront corrector(LCOS)to assist focus adjustment is proposed: using LCOS to load out-of-focus aberration to realize the movement of two-photon light sheet in depth direction of the illumination,thus twophoton light sheet sub-regions at different depths are formed.Different sub-regions are stitched together with the long-time exposure of the imaging camera to realize the expansion of two-photon light sheet in illumination depth direction,thereby realizing the expansion of the imaging field of view.The method of sub-area illumination expands the illumination depth on the one hand,and enables wavefront detection and correction under ultra-high-speed scanning conditions on the other hand.Since the operating frequency of the TAG zoom lens is as high as 200 kHz to 400 kHz,and the operating frequency of the wavefront detector and the corrector is only about 1kHz,the large difference in operating frequency makes wavefront detection and correction difficult to achieve.Therefore,based on the subregional illumination method and the concept of isoplanatic region,the method of parallel completing the field of view expansion and the isoplanatic region aberration correction using the same LCOS is proposed: The unidirectional illumination field of view is divided into 18 isoplanatic regions,each of which is 100?m×100?m.The adjustment of the isoplanatic region in the lateral direction is performed by the scanning galvanometer,and the adjustment in the axial direction is defocused by the LCOS.Based on above,the scanning within the isoplanatic region is completed by the scanning galvanometer and TAG zoom lens,and the aberration correction within the isoplanatic region is completed by LCOS,and the time required for scanning and aberration correction in the range of 600 ?m×300?m is 36 ms.Theoretical analysis of the field of view expansion and aberration correction performed by LCOS in parallel shows that the axial field expansion of 300?m and the biological tissue aberration correction of RMS=1.0? can be satisfied when the number of LCOS pixels is 512×512.At the same time,a wavefront detection method using two-photon focus as the detection beacon combined with the descanning technology in the illumination optical path is proposed The fluorescence signals in the same isoplanatic region are descanned and superimposed to realize the enhancement of the detection signal-to-noise ratio.In this way,although the scanning speed is as high as 200 kHz to 400 kHz,there is no need to perform point-by-point aberration detection and correction,and only one detection and correction in the range of the isoplanatic region is required,which greatly reduces the time for wavefront detection and correction.This method allows wavefront detector and corrector to detect and correct aberrations under ultra-high speed scanning conditions.However,experiments on wavefront detection and correction have found that wavefront detection is still unable to achieve because the axial scanning of the illumination path is achieved by adjusting the power of the light,when the light in different axial positions enter the Hartmann wavefront detector,different defocus aberrations are generated,which causes confusion in wavefront detection.Therefore,it is necessary to improve the axial scanning mode in the wavefront detection.Since the time-varying frequency of aberrations in biological tissues is very low,which is in minutes or hours.Therefore,the wavefront detection and correction can be separated,and a control method of first detecting aberration,then storing aberration,and then correcting aberration is proposed.The LCOS and TAG zoom lens are turned off during aberration detection,and the piezoelectric objective lens(PZT)is used to drive the illumination objective to perform axial scanning of the focus,and the descanning wavefront detection is performed in units of 100?m×100?m isoplanatic regions.Then the LCOS and TAG zoom lenses are used for axial scanning and corrected imaging after completing the aberration detection in the entire field of view.Although the operating frequency of PZT is relatively slow,the wavefront detection of the entire field of view takes about 200 ms,but it is still much smaller than the aberration variation period of the biological tissue so the subsequent wavefront correction can be completed without aberration variation.Based on the research above,a liquid crystal adaptive optical imaging system of two-photon light-sheet microscopy is designed,and the optical design and optimization of the system are completed.Based on the consideration of stable of system structure and portable,the mechanical structure of the system is optimized.The optimized system is integrated on a platform of 750mm×650mm size.Finally,the liquid crystal adaptive optical imaging system of two-photon light-sheet microscopy is built,and it is used for experimental verification of adaptive detection and correction imaging.In-vivo embryo samples of zebrafish stained with Rhodamine 6G fluorescent dye were prepared.The field-of-view splicing and aberration correction of a two-photon light sheet with an axial illumination depth of 300?m are realized,and the liquid crystal adaptive optical correction imaging of zebrafish tissue was realized.The image resolution and signalto-noise ratio of zebrafish tissues before and after correction are significantly improved.This thesis is the pioneering work of liquid crystal adaptive optics in two-photon light-sheet microscopy.By detecting and correcting random aberrations in biological tissues,the imaging system with high spatial-temporal resolution,large imaging field of view,large imaging depth and low light bleaching is formed,which makes it possible to obtain long-time,large field of view and high resolution imaging of in-vivo biological tissues. |
PDF Full Text Request