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

Aravinth, S and Joshi, P and Mondal, PP (2022) 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. In: Review of Scientific Instruments, 93 (9).

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Abstract

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.

Item Type:	Journal Article
Publication:	Review of Scientific Instruments
Publisher:	American Institute of Physics Inc.
Additional Information:	The copyright for this article belongs to American Institute of Physics Inc.
Keywords:	Diffraction; Mobile telecommunication systems; Photons, Cellular system; Exposure-time; Localisation; Long exposures; Microscopy technique; Molecular clusters; Particle resolutions; Single molecule; Single-molecule localizations; Time windows, Molecules, article; blinking; controlled study; diffraction; human cell; human tissue; photon; protein localization; single-molecule localization microscopy
Department/Centre:	Division of Physical & Mathematical Sciences > Instrumentation Appiled Physics
Date Deposited:	08 Oct 2022 04:13
Last Modified:	08 Oct 2022 04:13
URI:	https://eprints.iisc.ac.in/id/eprint/77293

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