Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

Abstract

Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse. This goal is still important today and therefore has a strong influence on instrument design. However, modifications and extensions of the early designs allow for the recovery of much more information from the detected photons and enable entirely new applications. Here, we present a new instrument that captures single photon events on multiple synchronized channels with picosecond resolution and over virtually unlimited time spans. This is achieved by means of crystal-locked time digitizers with high resolution and very short dead time. Subsequent event processing in programmable logic permits classical histogramming as well as time tagging of individual photons and their streaming to the host computer. Through the latter, any algorithms and methods for the analysis of fluorescence dynamics can be implemented either in real time or offline. Instrument test results from single molecule applications will be presented.

Gold Nanostar Substrates for Metal-Enhanced Fluorescence through the First and Second Near-Infrared Windows

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Time-correlated single-photon counting (TCSPC), which is considered as the most sensitive digital technique for determining photoluminescence lifetimes, was applied to acquire fluorescence decay curves for AF750 and AF790. 47 Lifetime data were collected using the Fluorolog Tau 3 system, equipped with a DeltaDiode TM -C1 controller (Horiba Scientific). Samples were excited using the DeltaDiode TM 730L…”

Wide-field TCSPC: methods and applications

“…Poisson noise which is given by the square root of the number of counts [34]. It is important to note that due to Poisson statistics, there is always a chance to detect two photons after one excitation pulse-this probability can never be exactly zero, and it is independent of the time scale used, picoseconds or microseconds, and it is also independent of the technological implementation of TCSPC, using a single TAC, multiple TACs, [35], TDCs [36,37] or imaging. It is just a question of how much pile-up can be tolerated, for low peak counts more than for very high peak counts [38].…”

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

“…27 The difference in the two time-tagging modes ͑T2 and T3 modes͒ is primarily in the handling of sync events from, e.g., a pulsed laser. In T2 mode, all detector signal inputs are functionally identical.…”

“…Accommodating the high sync rates in T3 mode is achieved by means of a sync divider as introduced previously. 27 The event records in T3 mode are composed of two timing figures: ͑1͒ the start-stop timing difference between the photon event and the last sync event, and ͑2͒ the arrival time of the event pair on the overall experiment time scale ͑the time tag͒. The time tag is obtained by counting sync pulses.…”

“…Ideally, this is done on independent channels, such that detector dead time effects can be eliminated by cross correlating the signals. 27 …”