Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.

Peck, Konan; Stryer, Lubert; Glazer, Alexander N.; Mathies, Richard A.

doi:10.1073/pnas.86.11.4087

Cited by 172 publications

(89 citation statements)

References 15 publications

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“…Many new fluorescent dyes have become available to interact with and identify specific biological targets [73], and fluorescent-activated cell sorters can be used to separate white blood cells from other cells found in blood. The high potential sensitivity and specificity of fluorescence spectroscopy techniques is clearly indicated by the ability to detect and monitor single molecules [74] even under physiological conditions [75].…”

Section: Fluorescence Theorymentioning

confidence: 99%

Cross-connection detection in Australian dual reticulation systems by monitoring inherent fluorescent organic matter

Hambly

Henderson

Baker

et al. 2012

Environmental Technology Reviews

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New housing developments are now commonly incorporating dual reticulation water systems for the redistribution of recycled water back to households for non-potable use. Within such distribution systems is the potential for cross-connections between recycled and drinking water pipelines, and a number of such events have been documented both in Australia and internationally. While many cross-connections are unlikely to present a source of health concern given the high level of recycled water treatment, they do have the potential to negatively impact public confidence in dual reticulation systems. A rapid and highly sensitive method of cross-connection detection is required to increase consumer confidence in the construction and maintenance of such recycled water distribution systems. This paper reviews a number of current and potential cross-connection detection methods, highlighting the use of fluorescence spectroscopy as a highly promising analytical tool for portable cross-connection detection.

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Section: Fluorescence Theorymentioning

confidence: 99%

Cross-connection detection in Australian dual reticulation systems by monitoring inherent fluorescent organic matter

Hambly

Henderson

Baker

et al. 2012

Environmental Technology Reviews

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“…As fluorescence-labeled molecules diffuse through the focal region of the microscope, they emit fluorescence bursts, which are detected and analyzed in real time or recorded for later processing. Burst fluorescence methods go back to some of the earliest applications of single-molecule detection (2,5,6,(37)(38)(39)(40). Detection of multiple fluorescence parameters including photon arrival times, fluorescence lifetimes, fluorescence anisotropy, and emission wavelength provides additional information that can be used to identify and sort molecules (41-43).…”

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confidence: 99%

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Johnson

2006

Biochemistry

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Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca 2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca 2+ -ATPase.Single-molecule fluorescence methods have the potential to resolve details about protein conformations, interactions, and dynamics that were previously hidden by ensemble averaging. As the practice of single-molecule methods expands, it is useful to examine what new information can be obtained about biomolecules and what the limitations of the methods are. This paper will examine these questions in the context of recent applications of single-molecule fluorescence techniques in the author's laboratory to the conformations, dynamics, and interactions of the calcium signaling protein calmodulin (CaM) (see Fig. 1).Proteins are dynamic molecules. Multiple conformational states are required for them to function. CaM is no exception, and appears to sample an unusually wide range of conformations (1). How can the distribution of such conformations be characterized? Conventional bulk measurements detect the average properties of many (typically > 10 10 ) molecules and therefore do not resolve the range of conformations that are actually present. One way to attack this problem is to detect and characterize molecules one at a time.Introduced in the early 1990s (2-6), single-molecule fluorescence methods can now address real problems in biology (see recent reviews (7-10)). Conformational states and conformational motions can be detected that may be missed by other methods. Dynamic processes can be probed under either equilibrium or non-equilibrium conditions by following the fluctuations of an appropriate single-molecule spectroscopic signal. In kinetic *To whom correspondence should be addressed. E-mail: ckjohnson@ku.edu. † We acknowledge support for this research from NIH R01 GM58715, the American Heart Association (99513262 and 0455487Z) and a Research Corporation Research Opportunity Award. B.D.S. and J.R.U. acknowledge support from training grant NIH GM08545, and E.S.P. acknowledges support from t...

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“…15). Although the most general single molecule application relies on the use of fluorescent dyes, which are attached to the biomolecule of interest, the intrinsic fluorescence emission of biomolecules has also been utilized in some cases (16)(17)(18).…”

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confidence: 99%

Two-photon excitation microscopy of tryptophan-containing proteins

Lippitz

Erker²,

Decker³

et al. 2002

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

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We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

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Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.

Cited by 172 publications

References 15 publications

Cross-connection detection in Australian dual reticulation systems by monitoring inherent fluorescent organic matter

Cross-connection detection in Australian dual reticulation systems by monitoring inherent fluorescent organic matter

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Two-photon excitation microscopy of tryptophan-containing proteins

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