Axial Plane Optical Microscopy And Applications

Optical microscopy,since it was invented in the 17 th century,has become an important tool for exploring and understanding the microscopic world.By virtue of non-invasion,low damage to samples,rich imaging mechanism and other advantages,optical microscopy has been widely used in many fields such as biomedicine,bioscience,and material science.However,the conventional optical microscopy can only capture the sample information located in the lateral plane,which is perpendicular to the optical axis of the detecting objective lens.Information located in the axial plane generally needs to be obtained by scanning,which is a time-consuming process.In addition,the reconstruction is prone to misalignment.Therefore,studying the direct axial plane optical microscopy is of important significance and practical applications.This dissertation introduces the basic concept,implementation and development of axial plane optical microscopy systematically.A series of studies are carried out on the direct axial plane optical microscopy and its applications in the fields of optical manipulation,super-resolution microscopy,and live cell imaging.The main contents and innovation points of this dissertation are as follows:1.An axial plane optical microscopy(APOM)system based on the imaging plane transformation of a 45°-titled mirror is built,and the axial field of view of this system is up to 70 ?m×70 ?m under one shot.The three-dimensional theoretical model of the point spread function(PSF)of this system is constructed by using the vector diffraction integral for the first time.The special PSF of the APOM system is simulated theoretically and further verified experimentally.This dissertation summarizes the conditions that the APOM system can realize axial plane imaging,and gives the evaluation criteria and corresponding implementation methods to meet these conditions in experiment.The above work has important instruction significance for follow-up experiments.2.An experimental scheme of special beam optical micromanipulation based on axial plane imaging is proposed,and the corresponding optical system is designed and built.This system generates non-diffracting beams with a spatial light modulator in order to manipulate particles along expected trajectories.Finally,the microparticle motion is monitored in real-time both in lateral and axial plane by using the APOM system.This scheme solves the problem that the axial capture dynamics cannot be directly observed in the traditional optical tweezers.In this dissertation,the dynamics of microparticles manipulation by Bessel beam along a straight path and by Weber beam along a parabolic path are successfully observed in real-time,the transportation distances are 23 ?m and 35 ?m,respectively.The proposed technique is convenient and visualized to obtain the axial optical trapping process without scanning,and it can be applied to the study of other special beams.3.An axial plane single-molecule localization super-resolution microscopy(AP-SMLM)is proposed,and the corresponding optical system is designed and built.This system uses a single high numerical aperture objective for both generating axial light sheet and collecting fluorescence signal,and finally obtains the axial plane super-resolution images of thick samples without scanning.It avoids problems of time-consuming and misalignment caused by scanning in the traditional thick sample imaging.By using this system,the axial plane super-resolution images of different organelles in COS-7 cells and glioblastoma multiforme GBM10 cells are obtained.Incorporating with Exchange-PAINT technique,multi-target axial plane super-resolution imaging is achieved without chromatic aberration correction,successfully revealing the axial sophisticated structures and distributions of these organelles.This system can achieve the average resolution of 83 nm in lateral and 103 nm in axial dimension for such immunolabeled cell samples over a thickness of about 17 ?m.The developed technique is convenient and fast to obtain the axial information with high resolution and large field of view,and has strong compatibility with other techniques.It provides a new way for further studying the axial hyperfine structures of three-dimensional thick samples such as tissues or small organisms.4.The APOM technique is applied for imaging the fission yeast for the first time to demonstrate its practical application value.After improvement,the AP-SMLM system is used for live cell imaging of fission yeast,and the contraction dynamics of the cytokinesis ring organized during the cell division process is observed successfully.In addition,the axial plane super-resolution imaging of the fixed cells of fission yeast is performed to reveal the distribution of the regulated light chain rlc1 associated with myosin in the cytokinesis ring.This presented technique is particularly suitable for imaging samples with special axial structures,such as the yeast cytokinesis ring.This technique is conductive to further study the distributions and functions of various kinds of proteins in the cytokinesis ring.It has potential application value in fields of biochemistry and molecular genetics.