Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy

Wang, Lu; Frei, Michelle S.; Salim, Aleksandar; Johnsson, Kai

doi:10.1021/jacs.8b11134

Cited by 426 publications

(348 citation statements)

References 106 publications

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“…Membright, ER-Tracker or MitoTracker) ( Fig. 4b, centre) (77,(82)(83)(84), additional 'linker' molecules are normally required to associate SFs with the structure of interest. These linkers must bind the target structure with high affinity and specificity (e.g.…”

Section: Fluorescent Probe Development For Live-cell Super-resolutionmentioning

confidence: 99%

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Tosheva¹,

Yuan²,

Pereira³

et al. 2019

Preprint

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Super-Resolution Microscopy enables non-invasive, moleculespecific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations. Phototoxicity | Photodamage | Super-Resolution Microscopy | Fluorescence Correspondence: s.culley@ucl.ac.uk, r.henriques@ucl.ac.uk Tosheva et al. | 1-12Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted:

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Section: Fluorescent Probe Development For Live-cell Super-resolutionmentioning

confidence: 99%

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Tosheva¹,

Yuan²,

Pereira³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Therefore, there is a clear need for advanced probes suitable for selective targeting and in vivo super-resolution imaging of intracellular structures. Such membrane permeable, improved probes should address challenges such as selective conjugatability, autofluorescence, and background fluorescence [3]. Autofluorescence can be overcome by carefully selecting fluorescent frameworks that are either excitable towards the red range of the spectrum or possess large Stokes-shifts.…”

Section: Introductionmentioning

confidence: 99%

A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellular Super-Resolution Imaging of Proteins

et al. 2020

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Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.

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“…Organic dyes that are covalently linked to proteins offer superior brightness, photostability and flexibility compared to fluorescent proteins 10,22 . Many organic dyes are cell permeable and therefore suitable for intracellular labeling.…”

Section: Introductionmentioning

confidence: 99%

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Poc

Gutzeit

Ast

et al. 2020

Preprint

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Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra-and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

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Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy

Cited by 426 publications

References 106 publications

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellular Super-Resolution Imaging of Proteins

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

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