Research On Light Sheet Microscopy Based On Bessel Light Dual-illumination And Instrument

In the traditional Light-sheet microscope illuminated by Gaussian beam,the ability of Gaussian beam to penetrate the sample is limited,it is difficult to achieve uniform illumination in the face of samples with strong absorption and scattering ability;In addition,due to the mutual restriction between the axial resolution of Gaussian beam and the illumination field of view,it is difficult to achieve large field of view illumination and a higher axial resolution.In this paper,Bessel beam is used instead of Gaussian beam as illumination beam,and the non-diffraction characteristics and self-healing of Bessel beam are used to improve the effective illumination field of view and illumination uniformity;A line confocal detecting method combining the scanning of Bessel beam with line by line exposure of camera is proposed to suppress the background noise caused by Bessel beam’s side lobes and improve the axial resolution of the system.In addition,for some samples can absorb or scatter the illumination beam,the system can provide dualillumination to further improve the uniformity of illumination.The main works are as follows:(1)A complete light sheet microscope system is designed and built.The light sheet system uses Bessel beam for illumination.Compared with Gaussian beam illumination,it can obtain a larger illumination field of view,and the penetration ability of Bessel beam is stronger.It can also realize uniform illumination for samples with strong absorption and scattering;At the same time,the specific timing between the hardware is designed.The scanning of Bessel beam is matched with the line by line exposure of the camera to realize the line confocal scanning,effectively suppress the defocus signal excited by the side lobe of Bessel beam,and improve the image contrast and the axial resolution of the system;In addition,the system can also provide dual-illumination and uniform illumination for some samples with low transparency.Finally,the maximum field of view of a single frame can reach 832μm×832μm,the imaging frame rate under the maximum field of view can reach 40 frames / s.(2)The system control software is designed based on Lab VIEW and we use producer consumer mode to design the system software.The software can mainly realize line confocal detecting mode and wide field detecting mode;There are two slice image acquisition can be realized in both scanning modes;The software supports the independent control of sample translation table,laser,scanning galvanometer and other hardware;The software can measure intensity curve,histogram,conversion between gray image and pseudo color image display,brightness and contrast of image display,and image processing function of gamma curve correction.(3)With the line confocal detecting mode,the lateral resolution of the system can be less than1μm and the axial resolution can reach 3μm;The zebrafish samples were subjected to threedimensional tomography and three-dimensional data reconstruction;At the same time,imaging the heart and blood vessels of zebrafish can observe the movement of them,which proves that the system has the ability to image living samples.