Single-Molecule Anisotropy Imaging

Harms, Gregory S.; Sonnleitner, Max; Schütz, Gerhard J.; Gruber, Hermann J.; Schmidt, Th.

doi:10.1016/s0006-3495(99)77118-3

Cited by 145 publications

(127 citation statements)

References 40 publications

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“…7 of this thesis). Anisotropy contains information about rotational diffusion of the probe (122,123) and structural information (124). A shift of the emission spectrum reports changes in the local environment, e.g.…”

Section: Single Molecule Trackingmentioning

confidence: 99%

Membrane heterogeneity – from lipid domains to curvature effects

Semrau¹,

Schmidt²

2009

Soft Matter

102

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Section: Single Molecule Trackingmentioning

confidence: 99%

Membrane heterogeneity – from lipid domains to curvature effects

Semrau¹,

Schmidt²

2009

Soft Matter

102

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“…Using a laserbased fluorescence microscopy setup equipped with a highsensitivity and high-speed charge-coupled device (CCD) camera (Schmidt et al, 1996), SMM has successfully been applied to proteins fused to autofluorescent proteins, like GFP, providing insight into the mobility patterns of several proteins at a time resolution of ,5 ms and a positional accuracy of ,40 nm (de Keijzer et al, 2008;Harms et al, 1999;Iino et al, 2001;Lommerse et al, 2005). Initially, these studies mostly focused on membrane proteins, but in recent years, data on the threedimensional (3D) mobility of fluorescently labeled proteins in the nuclei of living cells have been extracted using this approach.…”

Section: Introductionmentioning

confidence: 99%

Androgen receptor complexes probe DNA for recognition sequences by short random interactions

Royen

Cappellen

Geverts

et al. 2014

Journal of Cell Science

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BSTRACTOwing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a consistent quantitative scheme of their action is still lacking. Using the androgen receptor (AR) as a model system, we combined three different fluorescence microscopy assays: single-molecule microscopy, photobleaching and correlation spectroscopy, to provide a quantitative model of the action of this transcription factor. This approach enabled us to distinguish two types of AR-DNA binding: very brief interactions, in the order of a few hundred milliseconds, and hormone-induced longer-lasting interactions, with a characteristic binding time of several seconds. In addition, freely mobile ARs were slowed down in the presence of hormone, suggesting the formation of large AR-co-regulator complexes in the nucleoplasm upon hormone activation. Our data suggest a model in which mobile hormone-induced complexes of transcription factors and co-regulators probe DNA by briefly binding at random sites, only forming relatively stable transcription initiation complexes when bound to specific recognition sequences.

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“…We note a similar approach to acquire steady-state anisotropy images of single molecules has been realized by simultaneous acquisition of both polarization components using a Wollaston prism polarizer. 23 The TR-FAIM extension of FLIM yields not only fluorescence lifetime maps, but also rotational correlation time maps and maps of the initial polarization anisotropy. To demonstrate this technique we have applied it to the simultaneous measurement of the rotational correlation times and fluorescence lifetimes of rhodamine 6G in solvents of different viscosity and different refractive index.…”

Section: Introductionmentioning

confidence: 99%

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

Siegel

Suhling

Lévêque‐Fort

et al. 2003

Review of Scientific Instruments

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We report a picosecond time-gated fluorescence lifetime imaging ͑FLIM͒ system extended to perform time-resolved fluorescence anisotropy imaging ͑TR-FAIM͒. Upon excitation with linearly polarized laser pulses, the parallel and perpendicular components of the fluorescence emission from a sample are imaged simultaneously using a polarization-resolved imager. The imaging technique presented here quantitatively reports the rotational mobility of a fluorophore as it varies according to the local environment. In a single acquisition run it yields maps of both rotational correlation time and fluorescence lifetime as they vary across a sample. TR-FAIM has been applied to imaging standard multiwell plate samples of rhodamine 6G dissolved in methanol, ethylene glycol, trimethylene glycol, and glycerol. The observed rotational correlation times and fluorescence lifetimes, which report the local viscosity and refractive index of the local rhodamine 6G environment, respectively, are in good agreement with previously published single point measurements. By considering the linear dependence of the rotational correlation time on viscosity up to 20 cP, we are able to obtain a two-dimensional viscosity map. Wide-field maps of rotational correlation time, and therefore viscosity, have been obtained. This illustrates the potential to image the local viscosity and fluorescence lifetime distributions of fluorophore tagged proteins in cells.

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Single-Molecule Anisotropy Imaging

Cited by 145 publications

References 40 publications

Membrane heterogeneity – from lipid domains to curvature effects

Membrane heterogeneity – from lipid domains to curvature effects

Androgen receptor complexes probe DNA for recognition sequences by short random interactions

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

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