Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Kwon, Jiwoong; Park, Jong‐Seok; Kang, Minsu; Choi, Soobin; Park, Jumi; Kim, Gyeong Tae; Lee, Chang-Wook; Cha, Sangwon; Rhee, Hyun‐Woo; Shim, Sang‐Hee

doi:10.1038/s41467-019-14067-4

Cited by 35 publications

(41 citation statements)

References 69 publications

(111 reference statements)

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“…This set of experiments suggested that the observed reversible photobleaching was likely due to photoisomerization and/or photoejection of the chromophore, as previously proposed to explain the reversible photobleaching observed when labeling FAST and its variants with low concentrations of HBR derivatives 28,23 . This singular photochemical signature might be used advantageously for selective imaging techniques based on kinetic discrimination 29,30 or for single-molecule localization microscopies 31,32 .…”

Section: Resultsmentioning

confidence: 99%

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Benaissa

Ounoughi

Aujard

et al. 2021

Preprint

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Fluorescent reporters are essential tools in cell biology for imaging the dynamics of proteins in living cells and organisms with high spatial and temporal resolution. Chemogenetic systems made of a genetically encoded protein tag acting as an anchor for synthetic fluorophores combine the targeting selectivity of genetic tags with the advantages of synthetic fluorophores. Here, we present the directed evolution of a small 14-kDa protein tag with extended chromophore promiscuity capable of efficiently forming non-covalent fluorescent assemblies with a collection of membrane-permeant and membrane-impermeant fluorogenic chromophores displaying spectral properties spanning the entire visible spectrum. The ability to adapt the fluorescence color by choosing a different live-cell compatible fluorogenic chromophore enables to genetically encode blue, cyan, green, yellow, orange and red fluorescence with a single tag, providing an unprecedent experimental versatility. The possibility to form dark assemblies using non-fluorescent chromophores provides moreover an innovative way for switching off fluorescence on-demand in cells and organisms with high temporal resolution by chromophore replacement. Selective wash-free fluorogenic labeling of fusion proteins could be achieved with high efficiency in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms, allowing high contrast imaging with various advanced microscopy techniques. The remarkable labeling efficiency and fluorescence performance allowed the multicolor imaging of dynamic processes in multicellular systems. The ability to match the spectral properties to the imaging modalities and the high photostability enabled to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

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Section: Resultsmentioning

confidence: 99%

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Benaissa

Ounoughi

Aujard

et al. 2021

Preprint

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“…These limitations and the difficulty associated with in-vivo study of biological specimens is a handicap for this promising technique. On the bright side, it should be possible to design super bright non-toxic photoactivable probes that can be easily conjugated with the protein-of-interest for studying biological processes [37,38].…”

Section: Discussionmentioning

confidence: 99%

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging

Mondal

2020

PLoS ONE

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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.

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“…These limitations and the di culty associated with in-vivo study of biological specimens is a handicap for this promising technique. On the bright side, it should be possible to design super bright non-toxic photoactivable probes that can be easily conjugated with the protein-of-interest for studying biological processes [37] [38].…”

Section: Discussionmentioning

confidence: 99%

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

Mondal

2020

Preprint

View full text Add to dashboard Cite

To be able to resolve molecular-clusters it is crucial to access vital informations (such as, molecule density and cluster-size) that are key to understand disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precisions (LPs)) to reconstruct super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single molecules) that degrade image resolution. Ideally it should be possible to identify the brightest molecules (also termed as, the fortunate molecules) to reconstruct ultra-superresolution map, provided su cient statistics is available from the recorded data. POSSIBLE microscopy explores this possibility by introducing 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 molecule filtering is carried out by the Gaussian distribution function to filter out unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct 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 ± = 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.Imaging molecular processes with true molecular-resolution is the ultimate goal of light microscopy. This is largely eluded due to the di↵raction of light which sets a lower bound on the resolution-limit [1] [2]. However, recent microscopy techniques such as, STED [3]

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Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Cited by 35 publications

References 69 publications

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

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