Super‐Resolution Imaging with Small Organic Fluorophores

Heilemann, Mike; Linde, Sebastian van de; Mukherjee, Anindita; Sauer, Markus

doi:10.1002/anie.200902073

Cited by 386 publications

(378 citation statements)

References 41 publications

(50 reference statements)

Supporting

Mentioning

375

Contrasting

Unclassified

Order By: Relevance

“…Wombacher et al successfully labeled the histone protein H2B-eDHFR fusion with a TMP-ATTO655 conjugate. The fluorophore ATTO655 shows inherent photoswitching catalyzed by the reducing environment of the cell as a function of excitation laser power [91,92]. Pronounced photoswitching of ATTO655 under physiological conditions and a high stability of the "off" state has been observed.…”

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

confidence: 98%

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: 98%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

View full text Add to dashboard Cite

show abstract

“…(We typically reach a mean localization accuracy of 10 to 20 nm, depending primarily on background level and signal photon count.) The photochemical principle behind the phenomenon of reversible bleaching is still poorly understood for most fluorophores (26,38,43).…”

Section: Microscope and Operation Modesmentioning

confidence: 99%

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

et al. 2016

View full text Add to dashboard Cite

Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker gH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with

show abstract

“…Each emitter can be localised to a precision between 10 and 30 nm. Common strategies for the temporal separation of molecules involve intra-molecular rearrangements to switch from dark to fluorescent states or the exploitation of non-emitting molecular radicals 11,12 . These strategies are typically pursued using photoactivatable or photoconvertible fluorescent proteins or small molecule probes coupled with a reducing buffer and immunostaining protocols 13 .…”

mentioning

confidence: 99%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

View full text Add to dashboard Cite

Correspondence should be addressed to patrick. rubin-delanchy@bristol.ac.uk and dylan.owen@kcl.ac.uk Single-molecule identification-based super-resolution microscopy techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. While many algorithms exist for the identification and localisation of molecules from the raw image data, methods for analysing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here, we present the first model-based Bayesian approach to evaluate molecular cluster assignment proposals which, in this article, are generated by analysis based on Ripley's Kfunction. The method is also the first to take full account of the individual localisation precisions calculated for each emitter. The technique is validated using simulated and experimental data from which we characterise the clustering behaviour of CD3ζ, an important subunit of the CD3-T cell receptor complex required for T cell function, in resting and activated primary human T cells.Conventional fluorescence microscopes produce images of the distribution of fluorophores in the sample convolved with the microscope Point Spread Function (PSF). Due to diffraction, this PSF typically has a width of hundreds of nanometres meaning the resulting image has a resolution, as assessed by the Rayleigh criterion, of ~200 nm. Several strategies now exist to circumvent this resolution limit 1 . Some of these, such as Stimulated Emission Depletion (STED) microscopy, rely on narrowing the excitation spot of a confocal microscope by means of a toroidal depletion beam and the process of stimulated emission 2,3 . Despite the increased resolution, these produce conventional fluorescence images, i.e., arrays of pixels with values representing the fluorescence intensity at those locations. Quantification can be performed in the same way as for conventional microscopes.Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.The position of each fluorophore can then be estimated from the centres of the PSFs. Many algorithms are available to extract the x-y coordinates of the molecules [8][9][10] . Each emitter can be localised to a precision between 10 and 30 nm. Common strategies for the temporal separation of molecules involve intra-molecular rearrangements to switch from dark to fluorescent states or the exploitation of non-emitting molecular radicals 11,12 . These strategies are typically pursued using photoactivatable or photoconvertible fluorescent proteins or small molecule probes coupled with a reducing buffer and immunostaining protocols 13 . We refer to all such strategies as SMLM.Unlike non-pointillist microscopy methods, SMLM imaging does not produce a conventional image. Instead, the raw data is a list of the...

show abstract

Super‐Resolution Imaging with Small Organic Fluorophores

Cited by 386 publications

References 41 publications

Chemical tags: Applications in live cell fluorescence imaging

Chemical tags: Applications in live cell fluorescence imaging

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

Bayesian cluster identification in single-molecule localization microscopy data

Contact Info

Product

Resources

About