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>Detection of fortunate molecules induce particle resolution shift (PAR-shift) toward single-molecule limit in SMLM: A technique for resolving molecular clusters in cellular system
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Detection of fortunate molecules induce particle resolution shift (PAR-shift) toward single-molecule limit in SMLM: A technique for resolving molecular clusters in cellular system
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机译：Detection of fortunate molecules induce particle resolution shift (PAR-shift) toward single-molecule limit in SMLM: A technique for resolving molecular clusters in cellular system
Molecules capable of emitting a large number of photons (also known as fortunate molecules) are crucial for achieving a resolution close to single molecule limit (the actual size of a single molecule). We propose a long-exposure single molecule localization microscopy (leSMLM) technique that enables detection of fortunate molecules, which is based on the fact that detecting a relatively small subset of molecules with large photon emission increases its localization precision (∼r0/N). Fortunate molecules have the ability to emit a large burst of photons over a prolonged time (> average blinking lifetime). So, a long exposure time allows the time window necessary to detect these elite molecules. The technique involves the detection of fortunate molecules to generate enough statistics for a quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 and 11 nm toward single-molecule-limit (far from diffraction-limit) for an exposure time window of 60 and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33 and 3.43 molecules are found to emit in 30–60 ms and >60 ms, respectively, when compared to single molecule localization microscopy (SMLM). The long exposure has enabled better visualization of the Dendra2HA molecular cluster, resolving sub-clusters within a large cluster. Thus, the proposed technique leSMLM facilitates a better study of cluster formation in fixed samples. Overall, leSMLM technique offers a spatial resolution improvement of ~ 10 nm compared to traditional SMLM at the cost of marginally poor temporal resolution.
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