Time-resolved fluorescence correlation spectroscopy

Böhmer, Martín; Wahl, Michael; Rahn, Hans-Jürgen; Erdmann, Rainer; Enderlein, Jörg

doi:10.1016/s0009-2614(02)00044-1

Cited by 160 publications

(153 citation statements)

References 27 publications

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“…Comparing DnaK and Ssc1, the characteristics of the ATP-bound conformation are very similar with interdomain separations of 47 and 43 respectively. In the ADP-bound conformation, the nucleotideand substrate-binding domains of DnaK are separated by 84 , which is further than the domain separation measured for Ssc1 in either the ADP-bound (62 ) or ADP/P5 state (75 ). Upon binding of P5 to DnaK, the interdomain distance slightly decreases to 77 , corresponding to the value measured for ADP/ P5 Ssc1.…”

Section: Mfd-pie Analysis Of Dnak Conformationmentioning

confidence: 70%

“…As we have a mixture of fluorescently labeled DnaK and free dye, it was necessary to utilize a species-selective correlation analysis [61] to only analyze the labeled DnaK. Another approach would be to use species-filtered FCS, [35] an approach similar to fluorescence lifetime correlation spectroscopy, [62] where the information available from MFD can be used to separate out the different species and the autocorrelation function from the individual subpopulations determined.…”

Section: Evaluation Of Acceptor Photo-induced Dark States Using Fluormentioning

confidence: 99%

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Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

et al. 2012

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Section: Mfd-pie Analysis Of Dnak Conformationmentioning

confidence: 70%

Section: Evaluation Of Acceptor Photo-induced Dark States Using Fluormentioning

confidence: 99%

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

et al. 2012

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“…A further contrast enhancement may be obtained by separating the different dye species also by their possibly different fluorescence lifetimes, using Fluorescence Lifeteime Correlation Spectroscopy (FLCS) (Böhmer et al, 2002). FLCS allow discrimination of fluorescence from different emitters with different fluorescence lifetimes by applying temporal filters based on the fluorescence decay.…”

Section: Figurementioning

confidence: 99%

Studying Ion Exchange in Solution and at Biological Membranes by FCS

Widengren

2013

Methods in Enzymology

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By FCS, a wide range of processes can be studied, covering time ranges from subnanoseconds to seconds. In principle, any process at equilibrium conditions can be measured, which reflects itself by a change in the detected fluorescence intensity. In this review, it is described how FCS and variants thereof can be used to monitor ion exchange, in solution and along biological membranes. Analysing fluorescence fluctuations of ion sensitive fluorophores by FCS offers selective advantages over other techniques for measuring local ion concentrations, and in particular for studying exchange kinetics of ions on a very local scale. This opens for several areas of application. The FCS approach was used to investigate fundamental aspects of proton exchange at and along biological membranes. The protonation relaxation rate, as measured by FCS for a pH-sensitive dye can also provide information about local accessibility/interaction of a particular labeling site and conformational states of biomolecules, in a similar fashion as in a fluorescence quenching experiment. Beyond protonation kinetics, the same FCS concept can also be applied to ion exchange studies using other ion sensitive fluorophores, and by use of dyes sensitive to other ambient conditions the concept can be extended also beyond ion exchange studies.3

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“…By filtering the photon events according to their TCSPC time before the correlation procedure, one can obtain separate FCS curves for each species. 23 Another application of timetagged TCSPC is fluorescence lifetime imaging ͑FLIM͒, for which the spatial origin of the photons must be recorded in addition to the TCSPC data. FLIM systems using on-board histogramming suffer limits in the recordable image size.…”

Section: Introductionmentioning

confidence: 99%

Scalable time-correlated photon counting system with multiple independent input channels

Wahl

Rahn

Röhlicke

et al. 2008

Review of Scientific Instruments

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Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information (time tagging), thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized to picoseconds of relative timing and yet operate independent of each other. This is achieved by means of a modular design with independent crystal-locked time digitizers and a central processing unit for sorting and processing of the timing data. The modules communicate through high speed serial links supporting the full throughput rate of the time digitizers. Event processing is implemented in programmable logic, permitting classical histogramming, as well as time tagging of individual photons and their temporally ordered streaming to the host computer. Based on the time-ordered event data, any algorithms and methods for the analysis of fluorescence dynamics can be implemented not only in postprocessing but also in real time. Results from recently emerging single molecule applications are presented to demonstrate the capabilities of the instrument.Scalable time-correlated photon counting system with multiple independent input channels Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information ͑time tagging͒, thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized ...

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Time-resolved fluorescence correlation spectroscopy

Cited by 160 publications

References 27 publications

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

Studying Ion Exchange in Solution and at Biological Membranes by FCS

Scalable time-correlated photon counting system with multiple independent input channels

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