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Depth of field

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.

We thank K. Jalink for encouragement and support; S. Schwartz, F. Huang, J. Byars and S. Liu for assistance with experiments; and V. van Ravesteijn and P. Kruit for providing scanning electron microscope data. We appreciate the thoughtful comments of T. Young and L. van Vliet. R.P.J.N. and D.L.P. are supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organisation for Scientific Research (NWO) and which is partly funded by the Ministry of Economic Affairs, Agriculture and Innovation. K.A.L. was supported by US National Science Foundation CAREER Award #0954836 and US National Institutes of Health grant P50GM085273.

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Dec 16, 2019 — One way a lens can correct for spherical aberration is by adjusting the physical shape of the lens elements. By grinding a lens so that it ...