Probing Individual Molecules with Confocal Fluorescence Microscopy

Nie, Shuai; Chiu, Daniel T.; Zare, Richard N.

doi:10.1126/science.7973650

Cited by 590 publications

(392 citation statements)

References 31 publications

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“…With the use of single-molecule techniques it is possible to overcome the problem of ensemble averaging of traditional functional biochemical methods (4)(5)(6)(7)(8). Furthermore, the activities of different molecules do not need to be synchronized to follow fluctuations.…”

mentioning

confidence: 99%

Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein

Peleg

Ghanouni

Kobilka

et al. 2001

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

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Single-molecule studies of the conformations of the intact ␤2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the ␤2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.G protein-coupled receptors (GPCRs) represent one of the largest families of integral membrane proteins, and GPCRs are responsible for most transmembrane signal transduction by hormones and neurotransmitters, as well as for the senses of vision, smell, and taste. GPCRs are characterized by seven transmembrane (TM) domains with an extracellular N terminus and a cytoplasmic C terminus (1). For many GPCRs, such as the ␤ 2 adrenergic receptor (␤ 2 AR), small molecular weight ligands bind to sites within the hydrophobic core formed by the TM ␣-helices. The ␤ 2 AR ( Fig. 1) belongs to the major subfamily of GPCRs that includes rhodopsin, for which a high-resolution crystal structure is now available (2). The binding of agonist ligands to a receptor induces conformational changes that facilitate interactions between the receptor and its cognate G protein.We have developed a method for site-specifically labeling Cys-265 in the ␤ 2 AR with fluorescein-5-maleimide (FM; ref. 3) to yield what we call FM␤ 2 AR. Cys-265 is adjacent to a G protein-coupling domain at the end of TM6 (see Fig. 1). In ensemble measurements, we demonstrated previously that FM bound to Cys-265 sensitively reports conformational changes, with agonists inducing a decrease in the fluorescence intensity of FM␤ 2 AR in proportion to the biological efficacy of the agonist. By using exogenous quenchers localized to different environments, we determined that during agonist-induced activation, the domain adjacent to Cys-265 rotates and͞or tilts to a more buried environment, which is closer to both the surface of the micellar compa...

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mentioning

confidence: 99%

Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein

Peleg

Ghanouni

Kobilka

et al. 2001

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

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“…Such substrates would be suited for the fluorescence detection of specially labelled ligands at membranes (Wise & Wingard, 1991;Zhou et al, 1991). Fluorescence techniques would offer a superior sensitivity, giving detection of single molecules (Eigen & Rigler, 1994;Nie et al, 1994); however, the inherent disadvantage is the necessity of labels. The detection limit of label-free integrated optics measurements is much higher, but the sensitivity of the method presented can certainly be increased by improving the chemical stability of the waveguide material and thus reducing drift effects.…”

Section: Discussionmentioning

confidence: 99%

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

et al. 1995

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A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface.In a first step, phospholipids carrying head-group thiols were covalently immobilized onto Si02-Ti02 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A , which corresponds to the thickness of a fluid bilayer membrane.Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipidanchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopeptide concentration in the outer monolayer.

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“…To calculate the DE% given the path length, the detection volume was assumed to be a cylinder with a height to diameter ratio of 3.5 (23,24). This height-to-diameter ratio was determined using theoretical calculations of the diffraction limited beam waist and the confocal detection volume with an excitation wavelength of 532 nm, a 1003 microscope objective with a numerical aperture of 1.25, and a refractive index of 1.33 (23,24). The detection volume was found to be 72 fL, which was 17% (61%) of the total possible detection volume defined by the capillary inner diameter and the path length.…”

Section: Detection Efficiencymentioning

confidence: 99%

Design and characterization of a compact dual channel virus counter

2005

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Background: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the ''gold standard.'' Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. Methods: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels.

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Probing Individual Molecules with Confocal Fluorescence Microscopy

Cited by 590 publications

References 31 publications

Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

Design and characterization of a compact dual channel virus counter

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Probing Individual Molecules with Confocal Fluorescence Microscopy

Cited by 590 publications

References 31 publications

Single-molecule spectroscopy of the β 2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule spectroscopy of the β 2 adrenergic receptor: Observation of conformational substates in a membrane protein

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

Design and characterization of a compact dual channel virus counter

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Product

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Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule spectroscopy of the β ₂ adrenergic receptor: Observation of conformational substates in a membrane protein