Abstract
Fluorescence imaging is an important and efficient tool in cell biology and biomedical research. In order to observe the dynamics of biological macromolecules such as DNA, RNA and proteins in live cells, it is extremely necessary to surpass the Abbe diffraction limit in microscopic imaging. Single-molecule localization microscopy (SMLM) is a sort of super-resolution imaging technique that can obtain a large number of images of sparse fluorescent molecules by the use of photoswitchable fluorescent probes and single-molecule localization technology. The center positions of fluorescent molecules in the images are precisely located, and then the entire sample pattern is reconstructed with super resolution. In this paper, we present a single-molecule localization algorithm (SMLA) that is based on blind deconvolution and centroid localization (BDCL) method. Single-molecule localization and image reconstruction of 15,000/9990 frames of original images of tubulins are accomplished. In addition, this fluorophore localization algorithm is used to localize high particle-density images. The results show that our BDCL-SMLA method is a reasonable attempt and useful method for SMLM imaging when the imaging system is unknown.
Highlights
Optical microscopy is widely used for imaging in life science with the advantages of non-invasion, high penetrability and non-destruction to biosamples
Due to the Abbe diffraction limit [1], the spatial resolution of traditional optical microscope can only reach wavelength scale, usually 200 nm~500 nm, which enables the study of dynamic events and the fine structural details of cellular architecture, but limits its applications in studying structure, function and interaction of biological macromolecules such as DNA, RNA and proteins at nano level
We present a blind deconvolution and centroid localizationbased single-molecule localization algorithm (BDCL-SMLA) that combines maximum likelihood estimation method and centroid method to resolve the problem mentioned above
Summary
Optical microscopy is widely used for imaging in life science with the advantages of non-invasion, high penetrability and non-destruction to biosamples. Several imaging strategies to break the diffraction limit, including stimulated emission depletion microscopy (STED) [2], single-molecule localization microscopy (SMLM) [3,4] and super-resolution structured illumination microscopy (SIM) [5], have been developed with the progress of novel fluorescent probes and imaging theories. The reconstructed images show the effectiveness and practicability of the BDCL-SMLA method
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