Mondal, PP (2020) Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging. In: PLoS ONE, 15 (11).
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Abstract
To be able to resolve molecular-clusters it is crucial to access vital information (such as, molecule density, cluster-size, and others) that are key in understanding disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precision (LP)) to reconstruct a super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single-molecules) that undermine image resolution. Ideally, it should be possible to identify the brightest molecules (also termed as the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy explores this possibility by introducing a narrow probability size-distribution of single-molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single-molecules are filtered by the Gaussian function to remove unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct an ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single-molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single-molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques. Cluster analysis indicates densely-packed HA molecules, HA-HA interaction, and a surge in the number of HA molecules per cluster post 24 Hrs of transfection. The study using POSSIBLE microscopy introduces new insights in influenza biology. We anticipate exciting applications in the multidisciplinary field of disease biology, oncology, and biomedical imaging. © 2020 Partha Pratim Mondal. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
| Item Type: | Journal Article |
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| Publication: | PLoS ONE |
| Publisher: | Public Library of Science |
| Additional Information: | The copyright for this article belongs to The Authors. |
| Keywords: | Article; diagnostic imaging; fibroblast; filtration; fortunate molecule; genetic transfection; image analysis; image reconstruction; influenza; intermethod comparison; microscopy; molecular imaging; molecular size; molecule; probabilistic optically selective single molecule imaging based localization encoded microscopy; single molecule imaging; single molecule localization microscopy; ultrasuperresolution imaging |
| Department/Centre: | Division of Physical & Mathematical Sciences > Instrumentation Appiled Physics |
| Date Deposited: | 10 Jan 2023 12:21 |
| Last Modified: | 10 Jan 2023 12:21 |
| URI: | https://eprints.iisc.ac.in/id/eprint/79028 |
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