Research On Two-layer Two-photon Fluorescence Microscope Based On Confocal Principle

Two-photon fluorescence microscopy imaging technology has been widely used in biological research because of its natural tomographic capability and its suitability for thick tissue imaging.A typical two-photon fluorescence imaging technique employs a point-by-point scanning strategy.Although a faster scanning device can be used to image a single plane to achieve a video frame rate,when imaging multiple planes distributed in the axial direction,the scanning speed is slow and limits the speed of acquisition of different plane image data.One way to solve this problem is to use electrical tunable lenses and other devices to increase the axial scan speed.Another strategy is parallel scanning which acquire images of multiple planes simultaneously.In this paper,from the perspective of increasing the scanning speed by means of parallel scanning,a two-photon fluorescence imaging method based on the confocal imaging principle is proposed.The incident light beam is divided into two beams,and by adjusting the divergence and convergence of one of the lights,two focal points which are in different axial positions are formed at the sample position.By using customized apertured mirror to separate two-photon fluorescence signals from two focal points,simultaneous two-layer two-photon imaging is achieved.Based on this imaging method,a two-photon fluorescence imaging system with simultaneous bifocal imaging was established.The distance between the two focal points can be set by the beam expanding system in the optical path.The influence of the parameters such as the thickness,tilt angle and pinhole of apertured mirror on the fluorescence separation of bifocal surface was analyzed.The control software of imaging system was written based on Lab VIEW.This paper further describes the debugging method of apertured mirror,tests the resolution of bifocal-plane two-photon imaging,signal crosstalk problem of two focal plane imaging is analyzed and reduced by misaligning two excitation lights horizontally.The imaging experiments of the test samples and brain slices show that the system can achieve simultaneous excitation and detection of the bifocal plane and obtain two different levels of two-photon fluorescence images simultaneously.