The Research Of Large Depth Of Field Three-dimensional Nanometer Resolution Single-particle Tracking Based On Double-helix Point Spread Function

In recent years, far-field super-resolved fluorescence microscopy have been developed,which can break the diffraction barrier and increase resolution to about 10-20 nm in all three dimensions。So, we can observe the intracellular hyperfine structure, dynamic process and function in living cells at the molecular scale. This technology will lead to unprecedented visualization and deeper understanding of various life process, thereby greatly promote the development of life sciences and other fields. Although far-field super-resolved fluorescence microscopy has made great progress, there are still challenges to be faced for thick specimen imaging, such as, how to further improve the depth of field of the system for acquirement of the complete dynamic feature information inside living cell. The present method can be implemented with dynamic imaging within intact cells are mostly based on improved multi focal plane microscopy(MFM). E.g., the MFM based on the distorted grating and the MFM based on the multi-detectors. The low coefficient of utilization for light energy, high complexity of systems, or high cost restricts the application widely of these methods. Our project group put forward a new method to image the whole cell at nanometer resolution. In order to realize nanoscale imaging in extended depth of field, the double-helix point spread function(DH-PSF) microscopy are combined with the distorted grating. The multiple particles in the whole living cell were tracked successfully at high-precision by this three-dimensional(3D) super-resolved system. The main work is summarized as follows:1.The theories and technologies about DH-PSF super-resolved fluorescence microscopy that are pertinent closely to the research work are illustrated.2.Summarized the work done by our project group about designing and fabricating a novel bifunctional phase plate combines a distorted diffraction grating with a DH-PSF, which can accomplish a large depth-of-field 3D nanoscale single molecule imaging method. And introduced the large depth-of-field 3D nanoscale single molecule imaging system based on the bifunctional phase plate..3.The theoretical localization accuracy of the nanoscopy is compared to that for 3D localization by DH-PSF microscopy and MUM by the theory fisher information. The experiment results show that our system results in almost constant localization precision in all three dimensions for a 10 mm thick depth of fields.4.The applications of the system to 3D single particle tracking for single fluorescent bead in agarose solution and inside a living macrophage cell in three dimensions are explored preliminarily. The experiments prove the capability of our setup for the study of fast and dynamic process in thick samples.