Time-resolved fluorescence microscopy

Suhling, Klaus; French, Paul M. W.; Phillips, David

doi:10.1039/b412924p

Cited by 518 publications

(298 citation statements)

References 130 publications

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“…The molecule is at the center of the cluster, and we assume that it is surrounded by a spherical exclusion volume of radius 0 R that is forced to remain free of particles. The molecule is supposed to be excited by a laser pulse, and a detector outside the system records the emitted photon at each fluorescence cycle, as in actual fluorescence lifetime imaging techniques [2,4]. In the weak-coupling regime, and for an emitter with a transition dipole p = p u where u is a unit vector, the spontaneous decay rate u Γ takes the form [8]:…”

Section: Description Of the Systemmentioning

confidence: 99%

“…In this case, the decay rate (or lifetime) can be studied statistically. Although fluorescence lifetime imaging techniques using averaged measurement have become important tools in biological imaging [2], little attention has been paid so far to the lifetime fluctuations. It has been shown both experimentally and theoretically that the fluorescence decay rate in random media exhibits a wide distribution [3][4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

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Lifetime fluctuations of a single emitter in a disordered nanoscopic system: The influence of the transition dipole orientation

Froufe‐Pérez

Carminati

2008

Physica Status Solidi (a)

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We study the fluctuations of the fluorescence decay rate of a single emitter in a random cluster of nanoparticles, in a regime dominated by near‐field scattering. Configurational changes of the environment induce statistical changes of the decay rate. Two regimes are considered which differ in terms of transition dipole orientation. In one regime, the orientation of the transition dipole is assumed to remain constant while the configuration of the cluster changes randomly. In another regime, the orientation of the transition dipole is assumed unknown and continuously averaged over the three directions of space. Using exact numerical simulations and a simple analytical model, we show that the statistical distributions of the spontaneous decay rate are substantially different in both regimes. In both cases, the decay rate fluctuations are strongly dependent on the level of absorption at the nanoscale. We discuss the impact of this result in terms of imaging in complex media. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Description Of the Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Lifetime fluctuations of a single emitter in a disordered nanoscopic system: The influence of the transition dipole orientation

Froufe‐Pérez

Carminati

2008

Physica Status Solidi (a)

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“…Because the measure of lifetime does not depend in general on the concentration of the fluorophores in the sample, it often provides several advantages with respect to intensity-based methods for the measurement of FRET. Methods for measuring fluorescence lifetime are generally classified as time or frequency domain methods (36). Here we used a digital frequency domain setup, the FLIMBox (27), coupled to a confocal laser scanning microscope, to perform FLIM.…”

Section: Napi-2c and Napi-2a Interactions With Pdzk1 And Nherf-1 Measmentioning

confidence: 99%

Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters

Giral

Lanzanò

Caldas

et al. 2011

Journal of Biological Chemistry

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The sodium-dependent phosphate (Na/P i ) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P i . The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P i reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P i transporters. Pdzk1 ؊/؊ mice adapted to chronic low P i diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P i transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P i transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P i concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P i transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.The type II sodium-coupled phosphate (Na/P i ) 3 transporters are the molecules responsible for tubular reabsorption of P i and are the target of hormonal and nonhormonal mechanisms that control phosphate homeostasis. NaPi-2a (NaPiIIa) is responsible for ϳ70% of the P i reabsorbed in the adult kidney of mice, whereas NaPi-2c (NaPiIIc) handles the remaining 30% (1, 2). A low dietary P i intake induces an increase of P i reabsorption in the proximal tubule mediated by augmented apical expression of NaPi-2a and NaPi-2c transporters and consequent increased P i uptake (3-6).Despite sharing a very similar molecular structure, NaPi-2a and NaPi-2c exhibit an increasing list of physiological differences. For example, whereas NaPi-2a is electrogenic, NaPi-2c is electroneutral (4, 7). In addition, both transporters participate in the renal adaptation to changes in dietary P i with different characteristics. In response to a high P i intake, NaPi-2a abundance decreases quickly (less than 1 h), internalization takes place in a microtubule-independent way, and molecules are targeted to the lysosomes via endosomes (8, 9). In contrast, under a high P i intake, NaPi-2c abundance decreases slowly (4 h), molecules are internalized through a microtubule-dependent pathway, and rather than being degraded, they are accumulated in a subapical compartment (10). Similar differences...

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“…It is also the foundation of fluorescence lifetime imaging microscopy (FLIM), which measures it on each point of a sample to map out the environment of a molecular probe, monitor the presence or absence of a species by its effect on the lifetime of a molecular probe, or distinguish between different species having well separated lifetimes (43)(44)(45)(46)(47)(48). This latter application is for instance useful to distinguish spectrally similar green fluorescent protein (GFP)-tagged proteins, which cannot be easily separated spectrally (49,50).…”

Section: Fluorescence Lifetime Imaging Microscopy (Flim)mentioning

confidence: 99%

Detectors for single-molecule fluorescence imaging and spectroscopy

Michalet

Siegmund

Vallerga

et al. 2007

Journal of Modern Optics

116

106

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Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy.

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Time-resolved fluorescence microscopy

Cited by 518 publications

References 130 publications

Lifetime fluctuations of a single emitter in a disordered nanoscopic system: The influence of the transition dipole orientation

Lifetime fluctuations of a single emitter in a disordered nanoscopic system: The influence of the transition dipole orientation

Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters

Detectors for single-molecule fluorescence imaging and spectroscopy

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