| The cell is the basic structural and functional unit of all life activities,but most of their activities occur in nano-scale organelles.Although technologies such as electron microscopy and atomic force microscopy have improved the resolution to the order of several nanometers,they suffer from large damage to samples,difficulty in distinguishing different structures,and inability to observe dynamic samples,and therefore are not suitable for studying biological samples,especially for fast three-dimensional(3D)observation of living cells.On the contrary,the optical microscopy has the potential advantages of less damage,high specificity,fast imaging speed,and 3D imaging capacity.It is the only imaging technology that can be used for rapid 3D observation of living cell samples.However,due to the optical diffraction limit,the lateral resolution of traditional optical microscopy is generally limited to the half of light wavelength.The axial resolution is even lower,which can only reach about one-third of the lateral resolution.The introduction of the concept of optical super-resolution imaging has greatly improved the resolution of contventional optical microscopy,and was awarded the Nobel Prize in Chemistry 2014.Neverthless,the existing super-resolution imaging methods still have some shortcomings,which are mainly reflected in the difficulty in balancing parameters such as lateral resolution,axial resolution,imaging speed,and imaging depth,so as to achieve large depth and fast 3D super-resolution imaging of subcellular structures.Considering the requirements of the three-dimensional spatitemporal resolution,the methods proposed in this thesis are based on wide-field illumination modulation.Several novel axial and 3D super-resolution techniques based on evanescent wave illumination and variable-angle modulation were proposed.And structured illumination modulation theory was developed to achieve faster and more flexial lateral and 3D super-resolution imaging.In addition,I designed and built a novel multi-modal and multi-dimensional galvanometer-based optical super-resolution microscopy system,and proposed corresponding optimization,calibration,and quantification procedures to demonstrate these theories.With these efforts,multi-color,large-depth(several micrometers of whole-cell scale),fast(tens of frames per second),long-term(more than one hour),and three-dimensional super-resolution(tens of nanometers)imaging were achieved.The brief content of each chapter is as follows:Chapter 1 explains some concepts related to this article,summarizes and compares the existing lateral and axial soper-resolution techniques,and points out the limitations and the corresponding developments performed in this thesis.Chapter 2 proposes a ring-illuminated total internal reflection fluorescence microscopy system based on a set of fast scanning galvanometer,and the quantitative system calibration methods,including scanning ring correction,incident angle measurement and polarization control.Chapter 3 proposes two methods based on variable-angle evanescent field illumination to further improve the axial resolution of total internal reflection fluorescence microscopy,and shows simulation and experimental results.Chapter 4 combines variable-angle total internal reflection fluorescence microscopy and structured light microscopy to simultaneously improve the three-dimensional spatial and temporal resolution,and also proposes the corresponding three-dimensional fast super-resolution imaging system based on compound two sets of scanning galvanometers.Chapter 5 further develops the above system to achieve large-depth and live-cell three-dimensional super-resolution imaging through three-dimensional structured illumination microsocopu.Chapter 6 proposes a workflow combining three-dimensional structured illumination microsocopu and single-molecule localization micrsocpy for data correlation.Besides,the parameters that affect the quality of the reconstruction results are analyzed detailedly. |
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