Research On Parabolic Mirror-based TIRF Microscopy With Shadowless Illumination And Adjustable Penetration Depth

Total internal reflection fluorescence(TIRF)microscopy utilizes the evanescent wave to illumination the fluorescence labeled samples.Since the evanescent wave intensity decays exponentially along the axial direction,the excitation of the out of focus fluorescent molecules is greatly suppressed,resulting in a good contrast image with high signal to noise ratio.Thus,TIRF microscopy has a wide range of applications in single molecular fluorescence imaging,surface biochemistry,cell dynamics and other fields.However,in the current schemes,the prism-based TIRF microscopy with a large range of incident angle adjustment but suffers the illumination shadows and interference fringes caused by unidirectional illumination,while the objective-based solution can easily solve the unidirectional illumination problem but it owns a small adjustable incident angle range inherently limited by the objective structure which is disadvantageous for research.This subject aims at solving the incompatible problems of unidirectional illumination and the small adjustable incident angle range in traditional solutions,and carries out the study of the introduction of parabolic mirrors(PM)with large numerical aperture(NA)into the TIRF imaging system.Taking advantage of PM's rotational symmetry to produce a cone shape illumination with all-azimuthal-direction illumination components in spatial domain to solve the unidirectional illumination problem,and the large NA property to obtain large-range adjustment of incident angle,this project simultaneously achieve the shadowless illumination and a large range of adjustable incident angle.The main research work is as follows,(1)In the framework of classical electromagnetic theory,the propagation modes and propagation characteristics of total reflection evanescent waves have been deduced,and the polarization state of evanescent waves has been studied.The relationship between the evanescent wave intensity at the interface and the incident angle has been given under the conditions of p-wave illumination and s-wave illumination respectively,providing theoretical support for the later study of the project.(2)A novel ring-shape beam generation device with finely adjusting beam radius ability based on a combination set of a concave and a convex cone mirrors has been proposed.Based on the device,a new type of PM based TIRF imaging device has been conceived to simultaneously achieve shadowless illumination and a wide range of adjustable incident angle(65 degree to nearly 90 degree).(3)According to the modeling of the of the illumination method of the PM-based TIRF imaging system,the influence analysis of the multi-angle incidence on the penetration depth of the evanescent wave has been given.A method of measuring the evanescent wave penetration depth has also been studied to obtain the axial intensity distribution of the evanescent wave and to achieve the calibration of the penetration depth.(4)The method of applying the PM to the TIRF imaging system has been studied and a concrete implementation scheme has been designed.Then an experiment system for the PM based TIRF microscopy has been built,and the refractive index(RI)matching liquid was prepared to eliminate the harmful scattering effects caused by the RI mismatch between the microbeads and the environment liquid.Finally,the shadowless illumination effect and adjustable penetration depth property of the system has been experimentally verified,and the adjustment fineness of the evanescent wave penetration depth was demonstrated better than 0.2 degree(evaluation in the perspective of incident angle change).