Abstract
Abstract Single-molecule localization microscopy (SMLM) plays an irreplaceable role in biological studies, in which nanometer-sized biomolecules are hardly to be resolved due to diffraction limit unless being stochastically activated and accurately located by SMLM. For biological samples preimmobilized for SMLM, most biomolecules are cross-linked and constrained at their immobilizing sites but still expected to undergo confined stochastic motion in regard to their nanometer sizes. However, few lines of direct evidence have been reported about the detectability and influence of confined biomolecule stochastic motion on localization precision in SMLM. Here, we access the potential stochastic motion for each immobilized single biomolecule by calculating the displacements between any two of its localizations at different frames during sequential imaging of Alexa Fluor-647-conjugated oligonucleotides. For most molecules, localization displacements are remarkably larger at random frame intervals than at shortest intervals even after sample drift correction, increase with interval times and then saturate, showing that biomolecule stochastic motion is detected and confined around the immobilizing sizes in SMLM. Moreover, localization precision is inversely proportional to confined biomolecule stochastic motion, whereas it can be deteriorated or improved by enlarging the biomolecules or adding a post-crosslinking step, respectively. Consistently, post-crosslinking of cell samples sparsely stained for tubulin proteins results in a better localization precision. Overall, this study reveals that confined stochastic motion of immobilized biomolecules worsens localization precision in SMLM, and improved localization precision can be achieved via restricting such a motion.
Highlights
Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy (STORM) [1], This work is licensed under the Creative Commons Attribution 4.0 InternationalJ
To assess the potential stochastic motion of immobilized biomolecules in SMLM, we applied in this study singlemolecule samples containing 40-nucleotide oligomers, which are 12–20 nm in sizes, to mimic regular biomolecules, including endogenous molecules and their linkers like antibodies or probes in biological samples
In light of their nanometer sizes, we hypothesized that Alexa Fluor-647 (AF647)-conjugated oligonucleotides in imaging solution stochastically move around the immobilized sites within a confined region cycled and that the stochastic movement can be recognized by SMLM and probably deteriorate localization precision
Summary
Since endogenous biomolecules as well as the antibodies or probes binding to them exhibit sizes of tens of nanometers [2, 9, 20], confined stochastic motion of immobilized biomolecules is expected to be detected in SMLM and result in increased localization uncertainty. New analysis is needed to systematically clarify the detectability and potential impact of immobilized biomolecules in SMLM, so that effective strategies could be applied to improve image resolution To address this issue, we applied here single-molecule samples containing sparsely-immobilized AF647-conjugated oligonucleotides, which act as a quantifiable model for systematically assessing confined biomolecule stochastic motion. We applied here single-molecule samples containing sparsely-immobilized AF647-conjugated oligonucleotides, which act as a quantifiable model for systematically assessing confined biomolecule stochastic motion These oligomers are of 12–30 nm in sizes, which are within the regular range of biomolecule sizes. We revealed that enlarging molecule sizes or adding an extra step of postcrosslinking at the end of sample preparation decreased or improved localization precision, respectively, which implies strategies for optimizing localization precision and image resolution via minimizing molecule stochastic motion in SMLM
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