Optical Sectioning Method Based On Line-scanning Virtual Structured Modulation

In recent years,fluorescence microscopy has emerged as an important tool for biological research due to its specificity properties in labeling and detection.However,background fluorescence induced by defocus and scattering in traditional wide-field fluorescence microscopy severely interferes with 3D fine imaging of thick tissue samples.Optical-sectioning microscopy,represented by confocal microscopy,was gradually being proposed to solve this problem in principle by various means.Among them,wide-field structured illumination microscopy,as a mathematically based optical-sectioning imaging method,outstands in 3D imaging of large size samples due to its simple implementation and fast imaging speed.Though,this technique has shortcomings such as the vulnerability of signal-to-noise ratio to background fluorescence,and artifacts problem in the reconstructed image.In response to the issues above,this paper develops a new efficient robust method for structured illumination microscopy to improve image quality.Systematic research is carried out from modulation strategies,reconstruction algorithms and their application in 3D cryo imaging.This paper presents a novel optical-sectioning method based on line-scanning virtual structured modulation to significantly improve image quality in structured illumination microscopy.Different equivalence forms of modulation strategies are analyzed from classical wide-field structured illumination microscopy and principles of line-scanning virtual structure modulation are deduced.A line-scanning virtual structured modulation imaging system was designed and tested for experimental performance.The advantages of this methods over conventional wide-field structured illumination microscopy are analyzed in terms of signal-to-noise ratio,artifacts problem and imaging throughput.To solve the problems that traditional nonlinear optical-sectioning algorithms of structured illumination microscopy disrupt the linear properties of optical systems,this paper proposes a linear algorithm suitable for virtual structured modulation strategies.The problem of nonlinear reconstruction algorithms is analyzed and principles of linear proportional subtraction are deduced.A linear algorithm based on strip intensity estimation is proposed to compute the optical-sectioning image and real-time reconstruction is achieved through algorithmic improvement.The advantages of this method over nonlinear reconstruction algorithms are analyzed in terms of several key properties such as resolution,signal-to-noise ratio and reconstruction time.In this paper,the above-mentioned methods are applied to cryo imaging and a linescanning cryo micro optical-sectioning tomography system is proposed,which aims to improve the background interference problem of existing imaging method.The system uses liquid nitrogen as the refrigerant and adopts a line illumination imaging path reformed by commercial wide field microscopy,integrating several module components such as milling to achieve cryo imaging.The specific structure of the system,data acquisition process and software controls are described.Three-dimensional cryo imaging of Thy1-YFP and CD4-GFP mouse lung demonstrated the single-cell level resolution and good optical-sectioning ability of the system.