Algorithmic corrections for localization microscopy with sCMOS cameras - characterisation of a computationally efficient localization approach.

Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect localization precision and accuracy. We present a simplified sCMOS non-uniform noise model that incorporates pixel specific read-noise, offset and sensitivity variation. Using this model we develop a new weighted least squared (WLS) fitting method designed to remove the effect of sCMOS pixel non-uniformities. Simulations with the sCMOS noise model, performed to test under which conditions sCMOS specific localization corrections are required, suggested that pixel specific offsets should always be removed. In many applications with thick biological samples photon fluxes are sufficiently high that corrections of read-noise and sensitivity correction may be neglected. When correction is required, e.g. during fast imaging in thin samples, our WLS fit procedure recovered the performance of an equivalent sensor with uniform pixel properties and the fit estimates also attained the Cramer-Rao lower bound. Experiments with sub-resolution beads and a DNA origami test sample confirmed the results of the simulations. The WLS localization procedure is fast to converge, compatible with 2D, 3D and multi-emitter localization and thus provides a computationally efficient sCMOS localization approach compatible with most SMLM modalities.

[1]  Mengting Li,et al.  Assessing low-light cameras with photon transfer curve method , 2016 .

[2]  Using Super-Resolution Nanorulers to study the Capabilities of EM-CCD and sCMOS Cameras beyond the Diffraction Limit , 2022 .

[3]  Marcel P. Bruchez,et al.  Evaluation of sCMOS cameras for detection and localization of single Cy5 molecules , 2012, Optics express.

[4]  David Baddeley,et al.  Visualization of Localization Microscopy Data , 2010, Microscopy and Microanalysis.

[5]  Peng Yin,et al.  Optical imaging of individual biomolecules in densely packed clusters , 2016 .

[6]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[7]  Bo Huang,et al.  Active microscope stabilization in three dimensions using image correlation , 2013, Optical Nanoscopy.

[8]  H. Flyvbjerg,et al.  Optimized localization-analysis for single-molecule tracking and super-resolution microscopy , 2010, Nature Methods.

[9]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[10]  Michael W. Davidson,et al.  Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms , 2013, Nature Methods.

[11]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[12]  S. Ram,et al.  Localization accuracy in single-molecule microscopy. , 2004, Biophysical journal.

[13]  M. Heilemann,et al.  Direct stochastic optical reconstruction microscopy with standard fluorescent probes , 2011, Nature Protocols.

[14]  Hongqiang Ma,et al.  Localization-based super-resolution microscopy with an sCMOS camera. , 2011, Optics express.

[15]  Joerg Bewersdorf,et al.  Optical nanoscopy: from acquisition to analysis. , 2012, Annual review of biomedical engineering.

[16]  Tobias M. P. Hartwich,et al.  Video-rate nanoscopy using sCMOS camera- specific single-molecule localization algorithms , 2013 .

[17]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[18]  S. Hess,et al.  Precisely and accurately localizing single emitters in fluorescence microscopy , 2014, Nature Methods.

[19]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[20]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[21]  E. Betzig,et al.  Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics , 2008, Nature Methods.

[22]  David Baddeley,et al.  4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues , 2011, PloS one.