Study On Three-dimensional Super-resolution Fluorescence Microscopy And Its Application In Chromatin Conformation

A living organism is composed of cells with different kinds of functions.Although the genome in nucleus is the same,various three-dimensional(3D)chromatin organizations expressed different in transcription regulations and cell identity determinations.The study on the distribution and interactions of chromatin organizations in single cells is important to understanding molecular functions,gene expressions,as well as to uncover the underlying mechanisms of life.Characteristic size of chromatin conformations is at nanometer level,far beyond the diffraction limit of optical systems.Several super-resolution fluorescence microscopies have broken the diffraction limit,providing the spatial resolution of 10-20 nm in the lateral and 20-50 nm in the axial,which satisfies the resolution acquirements of studying on chromatin conformation in single cell.Recently super-resolution fluorescence microscopy has been utilized in imaging genomic domains with a resolution limit of 4.9 kb.However,such methods are mainly constrained to short non-repetitive elements of interested or short unique DNA sequences.In this thesis,a simple FISH method that uses molecular beacon(MB)probes has been developed to facilitate the probe-target binding,while minimizing non-specific fluorescence.Three-dimensional stochastic optical reconstruction microscopy(3D-STORM)was used under optimized imaging conditions to efficiently distinguish sparsely distributed Alexa647 molecules from background of cellular auto-fluorescence.Utilizing only 29–34 individual MB probes,3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome have been observed,respectively.What have done in this thesis is listed as follows.1.Completed the overall theoretical analysis of 3D super-resolution imaging by STORM such as single molecule localization,3D localization based on astigmatism,the mechanism of photo-switchable fluorescent probe and the depth of excitation layer under inclined illumination.Combined an anti-drift system and three drift-corrected algorithms to keep a high stability of the 3D nano-resolution imaging microscopy.Based on all of these,we accomplished imaging of the microtubule in N2 a cell and compared the variation of lipid raft between different kinds of cell in nanometer level.2.In order to visualize shorter sequence,we developed a simple FISH method with super-resolution that used molecular beacon(MB)probes(MB-FISH)to specifically bind with target sequences at a relatively low hybridization temperature.The design principle of MB probes was also optimized.MB-FISH has enable labelling the unique short non-repetitive DNA in situ by designing different MB probes for tiling along the target sequence and optimizing the hybridization conditions.3.Based on a statistical technique to model the distribution of effective photons,a reasonable method was built to classify fluorescent signal.According to this method,3D-STORM imaging conditions was optimized to efficiently distinguish sparsely labeled samples from background cellular auto-fluorescence.This method in single molecular localization contributed in distinguishing specific nanostructures and high resolution imaging.4.Observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome,respectively.Utilizing 3D-STORM and only 29–34 individual MB probes,we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome,respectively.This MB-based FISH method was capable of visualizing the shortest non-repetitive genomic sequence so far in 3D at super-resolution.The main innovation in thesis is as follows:1.Developed a simple FISH method that used molecular beacon(MB)probes to facilitate the probe-target binding,while minimizing non-specific fluorescence.Completed target non-repetitive genomic sequence in situ at relatively low hybridization temperature.2.Developed an effective method based on statistic model and applied it in single molecular localization for identifying single fluorescent probe more efficiently.3.Utilizing 3D-STORM and only 29–34 individual MB probes,we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome respectively,as the first time visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.