Research On 920nm Multi-modal Nonlinear Microscopy System

Nonlinear microscopy uses the nonlinear interaction between the light source and the sample to generate a signal for imaging.This imaging mode limits the excitation of the signal light to the focus of objective,eliminating the effects of stray light at other locations,and significantly increasing the resolution of the image.Two-photon microscopy is one of the most widely used nonlinear imaging methods in medical diagnostics,biological research and other fields.With the emergence of various variants of green fluorescent protein,and 920 nm excitation is suitable for these proteins.Therefore,two-photon microscopy operating at 920 nm is especially important.However,in the traditional two-photon microscopy system,the excitation light path and the detection light path are coaxial,it is susceptible to adverse effects such as photobleaching and phototoxicity.And the way of point scanning for three-dimensional imaging resulting in a slower imaging speed,then it is not easy to capture rapidly changing images such as nerve impulse transmission.One effective solution is to combine light-sheet illumination with two-photon imaging,only the imaging plane is illuminated by the excitation laser.And it is a flat imaging,which greatly increases the imaging speed.In order to ensure high imaging speed during light-sheet imaging,the axial resolution is often too low.The system of this thesis adds point detection to obtain axial high resolution imaging.In addition,the Gaussian light-sheet has the problem of uneven field of view,then system introduces Bessel illumination to balance the imaging field of view.This thesis builds a 920 nm multi-modal two-photon nonlinear imaging system.The illumination light path uses a femtosecond pulse laser in the 920 nm as a light source,and the spatial light modulator can realize two illumination modes of Gaussian light and Bessel light,and two direction scanning is performed using two galvanometers.The detection path has two modes of light-sheet detection and point detection.For the lightsheet detection mode,the x galvanometer realizes the sheet illumination,and the z galvanometer combines the high speed tunable lens for axial scanning to finally realize the three-dimensional scanning.For the point detection mode,the two galvanometers realize the two-dimensional scanning of the x-z field of view.The timing relationship between the two galvanometers and the point detector is synchronized to obtain an accurate two-dimensional image.In the experiments,the system was first used to imaging fluorescence microspheres to test the field of view and resolution.According to the imaging results,the actual imaging field of view was calculated to be 770?m×770?m×324?m.The light-sheet mode has a horizontal resolution of 0.767?m and an axial resolution of 5.83?m,while the point detection mode has a resolution of 2.12?m,which improves the axial resolution of the system.The zebrafish of EGFP-labeled vessels was then imaged to verify the highresolution imaging capabilities of the system,imaging results of zebrafish heart verified the high-speed imaging capability.Finally,it was verified that the Bessel light illumination extends the fluorescence excitation originally concentrated in the center of the field of view to the entire imaging field of view.