13 February 2012 Optimization of precision localization microscopy using CMOS camera technology
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Light microscopy imaging is being transformed by the application of computational methods that permit the detection of spatial features below the optical diffraction limit. Successful localization microscopy (STORM, dSTORM, PALM, PhILM, etc.) relies on the precise position detection of fluorescence emitted by single molecules using highly sensitive cameras with rapid acquisition speeds. Electron multiplying CCD (EM-CCD) cameras are the current standard detector for these applications. Here, we challenge the notion that EM-CCD cameras are the best choice for precision localization microscopy and demonstrate, through simulated and experimental data, that certain CMOS detector technology achieves better localization precision of single molecule fluorophores. It is well-established that localization precision is limited by system noise. Our findings show that the two overlooked noise sources relevant for precision localization microscopy are the shot noise of the background light in the sample and the excess noise from electron multiplication in EM-CCD cameras. At low light conditions (< 200 photons/fluorophore) with no optical background, EM-CCD cameras are the preferred detector. However, in practical applications, optical background noise is significant, creating conditions where CMOS performs better than EM-CCD. Furthermore, the excess noise of EM-CCD is equivalent to reducing the information content of each photon detected which, in localization microscopy, reduces the precision of the localization. Thus, new CMOS technology with 100fps, <1.3 e- read noise and high QE is the best detector choice for super resolution precision localization microscopy.
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Stephanie Fullerton, Stephanie Fullerton, Keith Bennett, Keith Bennett, Eiji Toda, Eiji Toda, Teruo Takahashi, Teruo Takahashi, } "Optimization of precision localization microscopy using CMOS camera technology", Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280T (13 February 2012); doi: 10.1117/12.906336; https://doi.org/10.1117/12.906336

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