Single-molecule detection fluorescence of surface-bound species in vacuum

Weston, Kenneth D.; Carson, Paul J.; Dearo, J. A.; Buratto, Steven K.

doi:10.1016/s0009-2614(99)00553-9

Cited by 91 publications

(106 citation statements)

References 19 publications

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“…On the other hand, the much shorter-lived triplet state lifetime can be prolonged upon oxygen removal because oxygen is known as very potent triplet quencher. For example, in vacuum, triplet state lifetimes of 4-100 ms have been measured with a triplet yield of 0.04% for single DiIC12 molecules (a carbocyanine derivative) [32]. Similar values were measured for fluorophores immobilized in thin polymer films such as PVA with low oxygen permeability [33,34].…”

Section: Resultssupporting

confidence: 54%

Photoswitching microscopy with standard fluorophores

et al. 2008

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We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires widefield illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

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Section: Resultssupporting

confidence: 54%

Photoswitching microscopy with standard fluorophores

et al. 2008

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show abstract

“…On the other hand, the much shorter-lived triplet state lifetime can be prolonged upon oxygen removal because oxygen is known as very potent triplet quencher. For example, in vacuum, triplet state lifetimes of 4-100 ms have been measured with a triplet yield of 0.04% for single DiIC12 molecules (a carbocyanine derivative) [173]. Similar values were measured for fluorophores immobilized in thin polymer films such as PVA with low oxygen permeability [174,175].…”

Section: Discussionsupporting

confidence: 54%

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Heilemann

Dedecker

Hofkens

et al. 2009

Laser & Photonics Reviews

249

191

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Optical microscopes, often referred to as 'light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluorophores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation "Photoswitching Microscopy". Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subcellular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.All subdiffraction-resolution fluorescence imaging methods are based on the separation of fluorescence emission in time. Thus, they critically depend on the availability of photoswitches, i.e. molecules that can be switched between a fluorescent and nonfluorescent state upon irradiation with light with high reliability. Here we describe the development and operation methods of diverse photoswitches and their application for superresolution fluorescence imaging and molecular quantification.

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“…In the case of two intensity levels with single rates for the transitions between both levels, g (2) (t) takes the form where C is called molecular contrast and k on and k off are the rates to and from the "on" level. The molecular contrast and the respective rates are connected with the intensity during the "on" and "off" periods I on and I off via 24 which can be further simplified if I on . I off :…”

Section: Resultsmentioning

confidence: 99%

Exponential and Power-Law Kinetics in Single-Molecule Fluorescence Intermittency

et al. 2004

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Perylenemonoimide chromophores, attached to a small dendron arm embedded in the polymer hosts Zeonex, poly(vinylbutyral), and poly(methyl methacrylate), are studied by means of laser-induced confocal fluorescence detection. Transient fluorescence intensity exhibits dark periods on two different time scales: on a 100 µs time scale and on a considerably longer time scale, ranging from 100 ms to as much as tens of seconds under high-vacuum conditions. The exponentially distributed short "off" times are attributed to triplet excursions of the molecule. The long-lived dark states follow a power-law distribution and are discussed in terms of the formation of radical anions/cations via electron tunneling processes.

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Single-molecule detection fluorescence of surface-bound species in vacuum

Cited by 91 publications

References 19 publications

Photoswitching microscopy with standard fluorophores

Photoswitching microscopy with standard fluorophores

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Exponential and Power-Law Kinetics in Single-Molecule Fluorescence Intermittency

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