Live-cell dSTORM with SNAP-tag fusion proteins

Klein, Teresa; Löschberger, Anna; Proppert, Sven; Wolter, Steve; Linde, Sebastian van de; Sauer, Markus

doi:10.1038/nmeth0111-7b

Cited by 227 publications

(189 citation statements)

References 6 publications

Supporting

Mentioning

186

Contrasting

Unclassified

Order By: Relevance

“…In a recent publication, Klein et al demonstrated the use of SNAP-tag based H2B labeling with a rhodamine type fluorophore showing switching behavior in live cells similar to ATTO655. They successfully validated the capability of rhodamines for SR-imaging in live cell dSTORM experiments [93]. …”

Section: Chemical Tags For Super-resolution Imaging In Live Cellsmentioning

confidence: 88%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

View full text Add to dashboard Cite

Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

show abstract

Section: Chemical Tags For Super-resolution Imaging In Live Cellsmentioning

confidence: 88%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

View full text Add to dashboard Cite

show abstract

“…However, these methods rely on antibodies coupled to organic dyes (4,8) or on fusions with fluorescent or fluorophore-binding proteins (3,7,(9)(10)(11) and have restrictions inherent to these labeling strategies. Antibodies suffer from variable specificity depending on fixation conditions and from limited commercial availability.…”

mentioning

confidence: 99%

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Lelek

Nunzio

Henriques

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

137

135

View full text Add to dashboard Cite

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffractionlimited resolution of conventional light microscopy (∼200-300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.

show abstract

“…There are prominent differences that result from the use of photoconvertible fluorescent proteins or synthetic organic fluorophores: PA-FPs have maximal labelling efficiency and specificity, and they are smaller (the barrel in which the FP chromophore sits is 2.4×4.2 nm [123], while the size of an IgG antibody is ≈7-8 nm); on the contrary, synthetic fluorophores show higher photostability and brightness than PA-FPs, which results in higher localisation precision, and their photoswitching rates can be controlled by external means, which allows faster acquisitions. Both types of fluorophores may be used for imaging living cells; in the case of synthetic fluorophores, appropriate labelling systems and reducing conditions are required [124,125].…”

Section: Fluorescent Probes For Localisation Microscopymentioning

confidence: 99%

“…Similar to live-cell STED, live-cell STORM also requires the use of genetically encoded polypeptide tags: trimethoprim conjugates [124], HaloTag [151], SNAP-Tag [125] or tagging with small tetracysteine motifs and the fluorescein arsenical helix binder FlAsH [152,153]. Photoswitchable proteins and genetically encoded tags can be expressed simultaneously in the same cell: A recent combination of HaloTag labelling (Atto655) and expression of PATagRFP and PA-GFP has allowed triple colour STORM/PALM imaging in living cells [154].…”

Section: Fluorescent Probes For Localisation Microscopymentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

View full text Add to dashboard Cite

Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

show abstract

Live-cell dSTORM with SNAP-tag fusion proteins

Cited by 227 publications

References 6 publications

Chemical tags: Applications in live cell fluorescence imaging

Chemical tags: Applications in live cell fluorescence imaging

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Fluorescence nanoscopy. Methods and applications

Contact Info

Product

Resources

About