&lt;title&gt;Hardware solution for continuous time-resolved burst detection of single molecules in flow&lt;/title&gt;

Wahl, Michael; Erdmann, Rainer; Lauritsen, Kristian; Rahn, Hans-Juergen

doi:10.1117/12.307325

Cited by 32 publications

(19 citation statements)

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“…The TimeHarp 200 TCSPC board uses a modern integrated concept based on a custom solid state Time-to-Digital Converter (TDC) 6,7 . These circuits use the delay of the electric signals in the silicon or metal tracks inside integrated circuits for direct time measurement and conversion to a digital number.…”

Section: Data Acquisitionmentioning

confidence: 99%

Compact TCSPC upgrade package for laser scanning microscopes based on 375- to 470-nm picosecond diode lasers

Ortmann

Dertinger

Wahl

et al. 2004

Optical Diagnostics and Sensing IV

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We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 nm, 405 nm, 440 nm and 470 nm for fluorescence excitation which are coupled in through a sole single mode fiber. The detected signal is guided to a photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC 5,7 system, via the unique TimeTagged Time-Resolved (TTTR) 6 data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently fit the full fluorescence lifetime image. The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows not only to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for the purpose of Fluorescence Lifetime Imaging (FLIM) or advanced Fluorescence Resonance Energy Transfer (FRET) analysis. We present here some selected results acquired with standard laser scanning microscopes upgraded for the time-correlated single photon counting technique.

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Section: Data Acquisitionmentioning

confidence: 99%

Compact TCSPC upgrade package for laser scanning microscopes based on 375- to 470-nm picosecond diode lasers

Ortmann

Dertinger

Wahl

et al. 2004

Optical Diagnostics and Sensing IV

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“…This concept was originally conceived to recover the original event data on the host computer. 15 However, the present instrument uses the overflow records to solve the problem of sorting by interpreting them as boundaries of "frames" in which the availability of timing data is always valid for comparison. This does not require recovering the original event data.…”

Section: B Design Featuresmentioning

confidence: 99%

“…14 More generally, it is referred to as time-tagged time-resolved ͑TTTR͒ data collection. 15 The time-tagging concept avoids both redundancy in the data stream and loss of information. As a result, virtually all algorithms and methods for the analysis of fluorescence dynamics can be implemented.…”

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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“…in very large detection volumes of picoliters [29][30][31]. It is now performed entirely in software by using a time-correlated single-photon counting (TCSPC) card for data collection as shown in [32,33] and by others. In order to time-gating away Rayleigh scattering, the time-gate is chosen to start at a time where the amplitude of the excitation pulse is substantially decreased, e.g.…”

Section: Introductionmentioning

confidence: 99%

Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

Földes-Papp¹,

Liao²,

You³

et al. 2009

CPB

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Abstract:We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

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<title>Hardware solution for continuous time-resolved burst detection of single molecules in flow</title>

Cited by 32 publications

References 0 publications

Compact TCSPC upgrade package for laser scanning microscopes based on 375- to 470-nm picosecond diode lasers

Compact TCSPC upgrade package for laser scanning microscopes based on 375- to 470-nm picosecond diode lasers

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

Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

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