Abstract
Super-resolution and single molecule imaging methods often rely on photon counts for quantification of precision and image processing to improve particle detection and localization. However, there is still no consensus on an accurate photoelectron calibration method for EMCCDs. In order to directly measure the light response of an EMCCD, we illuminate each sensor individually with highly attenuated laser light and compare pixel values to readout from a calibrated photomultiplier tube in photon counting mode. The data is then compared to manufacturer specification sheets as well as various published calibration methods and results from EMCCD theory. Meanwhile, smoothing and background subtraction are often used to improve the quality of single molecule data, but the effects of such routines on localization and spot-finding have not been rigorously studied. Furthermore, precision is usually calculated based on a simple model of nonlinear least-squares Gaussian fitting of the point spread function in which the effects of denoising are not at all considered. We apply several methods for image filtering and background subtraction to synthetic images representative of PALM/STORM data and measure their effects on localization precision, bias and spot-finding (quantified using Type I and Type II error rates).
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