Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy

Song, L.; Varma, Cyril A. G. O.; Verhoeven, J. W.; Tanke, Hans J.

doi:10.1016/s0006-3495(96)79866-1

Cited by 227 publications

(220 citation statements)

References 26 publications

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“…To protect the emission of the fluorescein tag we added 100 mM mercaptoethylamine (MEA; Sigma). MEA is known to deplete the fluorescein triplet state without affecting the first singlet excited state (12). Thus, photobleaching caused by the long-lived triplet excited state is prevented without perturbation to the emission cycle.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Up to date no live-cell imaging study has been performed to investigate cell cycle dependent organization and dynamics of telomeres and telomere binding proteins. Long-term live cell imaging is seriously hampered by photodamage, mainly elicited by the light-catalyzed production of reactive oxygen species (ROS) (24,25). ROS react with a large variety of cellular components (lipids, proteins, DNA, .…”

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confidence: 99%

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

et al. 2008

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Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics. ' 2008 International Society for Advancement of Cytometry Key termstelomere; TRF1; TRF2; nuclear organization; chromatin dynamics; CLEM; live cell imaging TELOMERES are the natural ends of linear chromosomes. A mammalian telomere consists of a double stranded array of simple TTAGGG repeats ending in a single stranded overhang that folds back to form a T-loop structure (1). In combination with sufficient telomere repeats, a complex of indirect and direct telomere binding proteins, dubbed shelterin, assures proper telomere structure and function. The specificity of shelterin for telomeric DNA is due to the recognition of TTAGGG repeats by three of its components: the homodimers telomeric repeat binding factor 1 and 2 (TRF1 and TRF2) that bind the duplex part of telomeres, and protection of telomeres 1 that binds to the single stranded TTAGGG repeats present at the three-overhang and in the D loop of the T-loop configuration (2-5).The nucleus has a meticulous organization with functional relevance to the cell assuring proper gene expression, replication and genome stability (6-8). Telomeres are considered to play an important role in spatial genome organization. Whereas yeast telomeres are known to form few large silencing clusters at the heterochromatic nuclear periphery (9,10), there is no consensus on how telomeres are organized in the mammalian nucleus. From an architectural point of view, human telomeres have been proposed to be anchored to a nuclear scaffold, thereby possibly providing struc-

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“…Thus the low measuring illumination did not influence the photosynthesis significantly but at the same time provided enough excitation light to obtain a good measuring signal. Additionally, the low excitation light did not quench dichlorofluorescein significantly (Figure 2, Figure 4) as is often the case when this dye is used in fluorescence microscopy [24]. The decrease in symplastic fluorescence after 120 min (Figure 4) could be attributed to a flux of the dye from the symplast to the surrounding medium [11].…”

Section: Discussionmentioning

confidence: 91%

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

2007

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An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 µM), menadione (50 µM) + ascorbate (100 µM) or neither for control. Menadione (50 µM) caused a statistically significant increase in H 2 DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 µM) in the test medium significantly decreased apoplastic oxidative stress. 50 µM menadione caused an increase in symplastic H 2 DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed.

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Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy

Cited by 227 publications

References 26 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

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

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Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy

Cited by 227 publications

References 26 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

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

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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