Control System Of Laser Scanning Confocal Microscopy And The Study Of Light Sheet Fluorescence Microscopy With High Signal-to-noise Ratio

The far-field optical microscopy can achieve far-field,noninvasive imaging of living biological tissues and real-time detection,which plays an important role in the development of the life science.Laser scanning confocal microscopy(LSCM)is one of the most widely studied and used scientific instruments.Moreover,it is the basic platform for further research of the super-resolution imaging technologies such as stimulated emission depletion(STED)Microscopy.The software control system is the key technology for the operation of the LSCM.This paper mainly studies the control system of the laser scanning confocal microscope.Also,considering the future development of the typical super-resolution technologies like STED method based on the confocal microscope,the control system is designed and programmed.In this software control system,the modularization software design is introduced.The software system includes scanning control module,photon number recording module,system operation module and data processing module.The scanning control module uses three-dimensional(3D)nano-positioning stage to realize the movement of the sample,and the step size at nano-scale is achieved when controlling nanopositioning stage through the API interface functions.S-scanning manner or Z-scanning manner is applied during the experiments.In the data acquisition module,avalanche photodiode(APD)detector is used to accept the weak fluorescence signal and the counting card of time-corrected single photon counting(TCSPC)is used to count photons.The principle of photon counting card achieving the photon number acquisition is integrating the output signal of APD in each pixel time.In the system operation module,the main research work is focused on the time synchronization control of the TCSPC card and 3D nano-positioning stage.In the data processing module,the photon number of each pixel can be displayed in real time,image construction and data storage can be realized.In this paper,an intelligent software system with point scanning imaging is developed and laser scanning confocal imaging can be achieved.And the 3D nano-positioning stage,TCSPC data acquisition card and single photon APD detector can work together through successful communicating among various functional modules of scanning manner control,photon number acquisition,data storage and image construction.Taking the advantages of high axial resolution and wide field of view,the Bessel beam is an important illumination method for light-sheet fluorescence microscopy(LSFM).But the fluorescence from the out-of-focus region excited by sidelobes of a Bessel beam reduces the signal-to-noise ratio of the image.Here,we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination.In the proposed method,the simple is imaged twice using the extended solid Bessel beam and the ring-like Bessel beam.By subtracting between the two images with similar out-of-focus blur,the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast and signal-to-noise ratio.