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By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information—such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond—for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.
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Spectroscopic single-molecule localization microscopy: applications and prospective
Abstract Single-molecule localization microscopy (SMLM) breaks the optical diffraction limit by numerically localizing sparse fluorescence emitters to achieve super-resolution imaging. Spectroscopic SMLM or sSMLM further allows simultaneous spectroscopy and super-resolution imaging of fluorescence molecules. Hence, sSMLM can extract spectral features with single-molecule sensitivity, higher precision, and higher multiplexity than traditional multicolor microscopy modalities. These new capabilities enabled advanced multiplexed and functional cellular imaging applications. While sSMLM suffers from reduced spatial precision compared to conventional SMLM due to splitting photons to form spatial and spectral images, several methods have been reported to mitigate these weaknesses through innovative optical design and image processing techniques. This review summarizes the recent progress in sSMLM, its applications, and our perspective on future work. Graphical Abstract
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- Award ID(s):
- 1954430
- PAR ID:
- 10420877
- Date Published:
- Journal Name:
- Nano Convergence
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2196-5404
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Osiński, Marek; Kanaras, Antonios G. (Ed.)Single-molecule localization microscopy (SMLM) strategies based on fluorescence photoactivation permit the imaging of live cells with subdiffraction resolution and the high-throughput tracking of individual biomolecules in their interior. They rely predominantly on genetically-encoded fluorescent proteins to label live cells selectively and allow the sequential single-molecule localization of sparse populations of photoactivated fluorophores. Synthetic counterparts to these photoresponsive proteins are limited to a few remarkable examples at the present stage, mostly because of the daunting challenges in engineering biocompatible molecular constructs with appropriate photochemical and photophysical properties for live-cell SMLM. Our laboratory developed a new family of synthetic photoactivatable fluorophores specifically designed for these imaging applications. They combine a borondipyrromethene (BODIPY) fluorophore and an ortho-nitrobenzyl (ONB) photocage in a single molecular skeleton. The photoinduced ONB cleavage extends electronic delocalization to shift bathochromically the BODIPY absorption and emission bands. As a result, these photochemical transformations can be exploited to switch fluorescence on in a spectral region compatible with bioimaging applications and allow the localization of the photochemical product at the single-molecule level. Furthermore, our compounds can be delivered and operated in the interior of live cells to enable the visualization of organelles with nanometer resolution and the intracellular tracking of single photoactivated molecules.more » « less
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Abstract The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1186/s40580-023-00363-9
