Measuring image resolution in optical nanoscopy
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Fluorescence Polarization; Fluorescent Dyes; Imaging, Three-Dimensional; Microscopy, Fluorescence
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Bioimaging and Biomedical Optics | Biophysics | Cell Biology | Molecular Biology | Nanotechnology | Structural Biology
Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample's spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.
Nanoscale biophysics, Super-resolution microscopy, Fluorescence imaging, Cellular imaging
DOI of Published Version
Nieuwenhuizen RP, Lidke KA, Bates M, Puig DL, Grünwald D, Stallinga S, Rieger B. Measuring image resolution in optical nanoscopy. Nat Methods. 2013 Jun;10(6):557-62. doi: 10.1038/nmeth.2448. Link to article on publisher's site
Nieuwenhuizen, Robert P. J.; Lidke, Keith A.; Bates, Mark; Puig, Daniela Leyton; Grunwald, David; Stallinga, Sjoerd; and Rieger, Bernd, "Measuring image resolution in optical nanoscopy" (2013). Biochemistry and Molecular Pharmacology Publications and Presentations. 162.