Research On Focus Shifting Detection Method In Confocal Microscope System

With the development of modern micro-fabrication technology and optical superresolution technology,micro-scale space three-dimensional measurement and imaging technology is moving towards three major directions:large range,high precision and high efficiency.Space three-dimensional measurement mainly includes astigmatism,laser triangulation,scanning tunneling microscopy,mechanical probe method,atomic force microscopy,confocal microscopy and so on.Among them,confocal microscopy is widely used in materials science,biology,medical science,MEMS and so on,because it has some advantages such as three-dimensional chromatographic ability,no damage to the surface of the sample,observation of living cells,and wide measurement range.In order to improve the axial resolution of traditional confocal system,heterodyne confocal principle has been further proposed,and the problem of axially symmetrical focal shift of point detector has always been a difficulty in the realization of the system.In this paper,the technology of pupil filter is combined with the confocal microscope system,and the phase pupil filter is designed so that it can shift the focus,so as to obtain a heterodyne confocal system which can meet the requirement of miniaturization.At the same time,the transverse superresolution factor is taken as one of the evaluation parameters to ensure that the system has the transverse superresolution effect while achieving the axial focus shifting.The main tasks of the project"Research on Focus Shifting Detection Method in Confocal Microscope System"include:?1?This paper makes a detailed theoretical analysis of confocal microscopic system and the phase modulation characteristics of pupil filter,and analyzes the preconditions of soft pinhole equivalent hard pinhole,and concludes that the point expansion function h_f of imaging system introduced by soft pinhole structure will not affect the light intensity detection results of confocal system when the system is an ideal point detection under the premise of satisfying the imaging lensr_f3r_il₀/f₂.?2?By analyzing the influence of the selected pupil optimization evaluation parameters on the measurement results of confocal system,the constraints of pupil optimization are given,the optimization model of pupil filter is established,and the structure of phase-type pupil filter is optimized by genetic algorithm,so that it has the characteristics of axial focusing and transverse super-resolution.?3?In order to ensure the simulation effect,a phase alignment method of spatial light modulator based on Fresnel lens and a measurement method of phase modulation characteristics of spatial light modulator based on Young's interference are proposed in this paper,which uses spatial light modulator to simulate the modulation effect of phase-type pupil filter in the system.Finally,a relevant experimental platform is built to verify the proposed method and the measurement effect of the system.The experimental results show that the proposed alignment method of phase diagram of spatial light modulator is feasible with an alignment accuracy of 2.25?m.The spatial light modulator is approximately linear modulation,and the corresponding phase modulation range of 0-255 gray value is 0-1.5?.When using a plane mirror as the sample to be measured,the measuring lens NA₁ is 0.4,and the collector lens NA₂ is 0.25,the optical pupil shift is 5?m,the measurement range of heterodyne confocal system is±15?m,and the axial resolution is 8nm,transverse superresolution factor G_T is 1.23.