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Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy |
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| Affiliation: | 1. Laboratory of Animal Cell Biology and Embryology, College of Veterinary Medicine, Nanjing Agricultural University, PR China;2. College of Veterinary Medicine, Yangzhou University, PR China;1. EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium;2. Graz Centre for Electron Microscopy, Steyrergasse 17, 8010 Graz, Austria;3. Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, A-8010 Graz, Austria;1. College of Computer and Information Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China;2. College of Physics and Information Engineering, FuZhou University, Fuzhou 350002, China;3. Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, China |
| Abstract: | A sub-diffraction limit fluorescence localization microscope was constructed using a standard cooled 1.4 mega-pixel fluorescence charge-coupled device (CCD) camera to simultaneously resolve closely adjacent paired quantum dots on a flat surface with emissions of 540 and 630 nm. The images of the overlapping Airy discs were analyzed to determine the center of the point spread function after noise reduction using Fourier transformation analysis. The Cartesian coordinates of the centers of the point spread functions were compared in serial images. Histograms constructed from serial images fit well to Gaussian functions for resolving two quantum dots separated by as little as 10 nm in the x–y coordinates. Statistical analysis of multiple pairs validated discrimination of inter-fluorophore distances that vary by 10 nm. The method is simple and developed for x–y resolution of dilute fluorophores on a flat surface, not serial z sectioning. |
| Keywords: | Super-resolution microscopy Diffraction unlimited Fluorescence Localization microscopy Quantum dots CCD camera |
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