Investigation On Total Internal Reflection Fluorescence Microscopy With Elliplical Mirror Based Theory

Total internal reflection fluorescence microscopy is a fluorescent microscopy imaging technique based on evanescent wave illumination.The evanescent wave generated by total internal reflection is used to illuminate in total internal reflection fluorescence microscopy.The excitation depth of the evanescent field is nanometer-scale,and the fluorescent sample within the excitation thin layer is excited.Microscopy has the advantages of high imaging quality and high signal to noise ratio.Therefore,it has been widely used in the field of bioimaging,such as single-molecule imaging,endocytosis and exocytosis,and intercellular signal transduction,making it an important branch of fluorescence microscopy.However,with the increasing demand for microscopy imaging quality and t he acquisition of three-dimensional spatial structure of cells,traditional total internal reflection fluorescence microscopy can not meet its imaging demands.The goal of the research project “The method and theory of fluorescence microscopy imaging based on elliptical mirror total internal reflection” is seeking one method which can achieve both multi-direction uniform illumination and excitation depth adjustable,providing theory and method to solve the problem of artifacts in the total internal reflection fluorescence imaging and extend the illumination range of the excitation depth.The research results can be further designed into a modular device,combined with wide field microscopy,structured light microscopy and dark field microscopy to implement multiple functions conveniently.The main research work of this topic is as follows:Firstly,the geometric focusing model of the elliptical mirror and the apodization function with the aperture angle ? are established.The vector focusing characteristics under different polarized light illumination based on the Debye vector diffraction theory are analyzed.Theoretical analysis shows that under the action of depolarization,two new electric field components appear.With the effect of the two electric field components whose intensity is approximate and the direction is vertical,the energy density of the total electric field appears a horizontal flat single spot at the focal plane near the focus of the elliptical reflector.Secondly,the combination of the basic theory based on total internal reflection and the focusing characteristics of an elliptical mirror based on the Debye vector diffraction theory is achieved,the light field distributions of evanescent waves in microscopy with different structures under different polarizations were systematically analyzed under objective-based and ellipsoidal mirror-based total internal reflection fluorescence imaging,respectively.Through simulation comparison,it has been proved that the method based on the elliptical mirror has better illumination and a wider range of excitation depth.Thirdly,the method of total internal reflection fluorescence imaging based on an elliptical mirror is studied.Through the study of the incident angle of total internal reflection,the manufacturing and processing parameters of an ellipsoidal mirror are given.According to the ellipsoidal reflector,an artifact weakening module and three kinds of controllable excitation depth modules are designed to build a total internal reflection fluorescence imaging optical path based on an ellipsoidal reflector;In addition,the imaging module was designed and manufactured from the perspective of engineering modularization.Finally,experimental verification of samples under elliptical mirror-based total internal reflection fluorescence microscopy was performed.In the microsphere sample experiment,the illumination distribution of the sample is uniform,and the artifact effect caused by the unidirectionality of illumination is significantly suppressed;in the experiment of biological samples,this method has high signal to noise ratio and more sample details;in the experiment of controlled excitation depth,different diameters of fluorescent microspheres indirectly prove that t his method can achieve a wide range of excitation depth adjustable function.