Photon-Free Calibration of CMOS Cameras for Precise Single Molecule Localization Microscopy Reconstruction

Abstract

Scientific-grade CMOS (sCMOS) cameras have evolved into a preferred detector type for single molecule localization microscopy (SMLM). Compared to EM-CCDs, sCMOS cameras do not only feature potentially increased localization precisions and framerates, but also come at typically lower cost. Transitioning to industry-grade CMOS cameras can decrease the cost much further, while in principle maintaining the advantages of sCMOS detectors. Consequently, such highly affordable cameras are recently gaining popularity in the community. However, considerable pixel-to-pixel variations in terms of noise, offset and gain which are well-known for sCMOS cameras can be even pronounced for CMOS cameras and impair the localization uncertainties. Explicit consideration during the reconstruction procedure is able to largely circumvent this effect if the detector is characterized accordingly. To this end, we present a novel approach to characterize CMOS cameras. It relies on thermally generated electrons only, but allows for similar data processing as in the traditional approach of using photoelectrons. Going without the need of external stimuli, this makes in-situ camera characterization possible. We show that industry-grade CMOS cameras which have been characterized by this approach can efficiently applied to common SMLM schemes of PALM, dSTORM and DNA-PAINT.