Research On Z-axis Drift Self-calibration Imaging System Of Stochastic Optical Reconstruction Microscope

Super-resolution microscopy technology has become one of the powerful research tools in the field of life sciences because of its characteristics of breaking the optical diffraction limit and clear imaging of ultra-fine structures in cells.Stochastic optical reconstruction microscope(STORM),which is a single-molecule localization microscopic imaging technology,has the advantages of low complexity and high resolution of the imaging system compared to other super-resolution microscopic imaging.STORM needs high-power laser,high-NA Objective and scientific camera.In order to meet the needs of long time imaging,nano-scale positioning system is often needed for self-drift correction.At the same time,faster and more accurate localization algorithms are also the key to super-resolution results and affect widespread use of STORM.The main research work of this paper includes Z-axis drift self-calibration stochastic optical reconstruction microscope system and its data processing system.The work consist of relevant research on several key hardware and software parts of the system,a series of characterizations,and its applicability in the field of biological sciences.Firstly,this paper describes the principle of STORM and the system implementation method in detail,and analyzes the data processing scheme of single molecule localization.At the same time,it introduces the principle of focusing scheme in microscope and the characteristic drift calibration scheme in STORM.Next is the construction work and system characterization of the STORM system with Z-axis drift self-calibration.The system includes low-cost high-power laser,laser safety interlocking system,laser coupling optical path,microscope coupling optical path,focus detection optical path and integrated control of the entire system.Fluorescent bean samples were used to characterize its stability and self-calibration performance,demonstrating its stable Z-axis drift self-calibration capability and applicability,proving that it can be fully qualified for Z-axis drift calibration in the experimental data acquire.Then based on the original Wind STORM algorithm and the idea of parallel computation,Wind STORM PLUS data processing system was built to be used for high-speed processing of big data.By comparing the simulation data and the actual data of different sizes and different densities t,Wind STORM PLUS is 10 times faster than the original Wind STORM and faster than the fastest Phasor algorithm in Thunder STORM under the large data set,which greatly shortens the data processing time and improves the scope of application of the original algorithm.At the same time,facing the problem of background noise of complex data,a new background estimation algorithm was constructed for the preprocessing of complex data,which combines with the flashing characteristics of fluorescent dots in time and bases on the time domain iterative wavelet transform method.Finally,through this STORM system,this paper performs super-resolution imaging of cytoskeleton structure which includes the biological application of super-resolution imaging of tubulin and actin.It also demonstrates the application value and potential of the system in the field of biomedical research.