Super resolution measurement of fluorescent dipoles via linear dichroism
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Because of its specific labeling and high imaging contrast, fluorescent microscopy has played a more and more important role in imaging sub-cellular organelles. Fluorescence contains four attributes: intensity (labeling density), wavelength (absorption and emission spectrum), time (fluorescence lifetime), and polarization (related to fluorescent dipoles). Fluorescence polarization could measure the dipole orientation of fluorophores from the absorption (linear dichroism) or the emission (fluorescence anisotropy) of fluorophores. Since the orientation of fluorescent dipoles is related to the structure of labeled samples, fluorescent polarization microscopy has been applied extensively. Whereas similar to other optical imaging methods, fluorescence polarization microscopy is barricaded by the diffraction limit. Super resolution fluorescence microscopy is able to achieve sub-diffraction resolution, whose key lies in the modulation of the fluorescence intensity. Such modulation could be either in a structured illumination manner, for example, STimulated Emission Depletion (STED) or Structured Illumination Microscopy (SIM); or in a stochastic blinking/fluctuation manner, for example, PhotoActivated Localization Microscopy (PALM)/STochastic Optical Reconstruction Microscopy (STORM). In this dissertation, the intensity modulation is based on the linear dichroism of fluorophores. Applying super resolution technique to fluorescence polarization microscopy, both the intensity and the orientation of fluorescent dipoles could be measured in super resolution. Optical lock-in is implemented to further improve the detection of polarization modulation, 3D super resolution imaging has also been realized by z-scanning of the sample. The main contents of the dissertation are as follows: Chapter 1 includes the introduction of the research background. Fluorescence polarization of fluorophores is firstly introduced, including the absorption and emission characteristics. Then, three different fluorescence polarization microscopy is introduced: Defocused Patter Recognition, Fluorescence Anisotropy, and Linear Dichroism. After that, various super resolution fluorescence imaging techniques are discussed as well as the application in fluorescence polarization microscopy. Chapter 2 introduces the principle of super resolution dipole orientation mapping (SDOM), together with its system setup and reconstruction algorithm. SDOM is compared to a similar technique (SPoD) and is verified by simulation, imaging samples of fluorescent beads, fixed cells and live cells. Chapter 3 contains further improvements of SDOM. The first section includes the application of optical lock in on SDOM. Thanks to the natural periodical intensity modulation of SDOM, optical lock in could be used for signal detection, which greatly improves the signal-to-noise ratio and increases the imaging resolution. The second section extended SDOM to 3D super resolution imaging, by z-scanning of the sample. Chapter 4 includes my other research during graduate. The first section is about light field microscopy. Two different setups are applied to fast volumetric imaging of neuronal activities and to 3D single molecule localization respectively. The second section is to use complementary optical imaging methods to study binding kinetics of proteins on the cell membrane, including biological specimen preparation, Fluorescence Recovery After Photobleaching (FRAP), and Fluorescence Correlation Spectroscopy (FCS). Chapter 5 is the discussion and conclusion of the dissertation.