Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes

Kastrup, Lars; Blom, Hans; Eggeling, Christian; Hell, Stefan W.

doi:10.1103/physrevlett.94.178104

Cited by 217 publications

(218 citation statements)

References 15 publications

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“…As an added benefit, as with other scanning fluorescence correlation methods 6,7 , such as line scanning FCS 8 or circle scanning FCS 9 , immobile fractions can be identified and eliminated in RICS analysis 10 . The usefulness of FCS and RICS can be greatly enhanced by further reduction of the observation volume, for example, by using laser-induced STED, which forms the basis of STED microscopy 11,12 . Based on a confocal setup, the excitation beam is overlaid with a second, red-shifted beam inducing stimulated emission of electronically excited fluorophores (Fig.…”

Section: Resultsmentioning

confidence: 99%

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

et al. 2013

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Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from rasterscanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

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Section: Resultsmentioning

confidence: 99%

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

et al. 2013

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“…The main application of STED has been structural imaging, but the reduction of the excitation volume results in a decreased number of fluorophores diffusing through that volume, which is advantageous for the investigation of molecular mobility and interactions by fluorescence correlation spectroscopy (FCS) [17][18][19][20]. FCS is a technique that allows the diffusion coefficient of a molecule to be determined from the analysis of the fluctuations of its fluorescence emission as it traverses a known volume: The lower the number of fluorophores within the focal volume, the more accurate the measurements.…”

Section: Stedmentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

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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.

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“…Fortunately, several optical imaging techniques offer the ability to interrogate samples with resolution better than the Abbe diffraction limit. Far field optical imaging techniques based on photoswitching and saturation of fluorescence have demonstrated resolution less than 30 nm, [37][38][39] but the optical probe itself is still diffraction limited. Another SPM technique capable of breaking the Abbe diffraction limit is near-field scanning optical microscopy (NSOM), 40,41 in which incident light is typically fed through an optical fiber having an aperture at the tip of the fiber (Fig.…”

Section: Scanning Photocurrent Methodsmentioning

confidence: 99%