Hessian single-molecule localization microscopy using sCMOS camera

Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single-pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain, and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian-SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.