Abstract
Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.
Highlights
The advent of super-resolution (SR) microscopy has revolutionized life science research by providing visual access to specific biological structures at the nanoscale.[1−4] The
Extensive efforts have been made in this direction for the methods STORM/PALM7−22 mostly based on either (i) a priori knowledge of the blinking dynamics or the number of localizations per fluorescence marker or (ii) on an initial calibration directly within the sample by using isolated localization clusters originating from an assumed number of fluorescent molecules as a reference
In the context of DNA-PAINT, DNA origami have been extensively used for creating nanometer patterns of docking strands” (DSs) as ideal benchmarking systems for the obtainable spatial resolution of the used microscope.[6,32,33]
Summary
The advent of super-resolution (SR) microscopy has revolutionized life science research by providing visual access to specific biological structures at the nanoscale.[1−4] The.
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