Michael Wahl

Fluorescence Lifetime Correlation Spectroscopy

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

Time-resolved fluorescence correlation spectroscopy

A new method of performing fluorescence correlation spectroscopy (FCS) measurements for mixtures of several fluorescent molecular species is introduced. It uses time-resolved fluorescence detection for separating the different FCS-contributions from the different species. This allows simultaneous and independent monitoring of the diffusion of several molecular species in one sample, or performing multi-label cross-correlation measurements. In this way, the proposed method is equivalent to dual- or multi-color FCS. However, it is simpler to implement experimentally, because it requires only single wavelength excitation and detection. This Letter outlines the theoretical basis and presents experimental results of the method.

Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting

Fluorescence correlation spectroscopy (FCS) is a powerful spectroscopic technique for studying samples at dilute fluorophore concentrations down to single molecules. The standard way of data acquisition, at such low concentrations, is an asynchronous photon counting mode that generates data only when a photon is detected. A significant problem is how to efficiently convert such asynchronously recorded photon count data into a FCS curve. This problem becomes even more challenging for more complex correlation analysis such as the recently introduced combination of FCS and time-correlated single-photon counting (TCSPC). Here, we present, analyze, and apply an algorithm that is highly efficient and can easily be adapted to arbitrarily complex correlation analysis.

An ultrafast quantum random number generator with provably bounded output bias based on photon arrival time measurements

We report the implementation of a quantum random number generator based on photon arrival times. Due to fast and high resolution timing we are able to generate the highest bitrate of any current generator based on photon arrival times. Bias in the raw data due to the exponential distribution of the arrival times is removed by postprocessing which is directly integrated in the field programmable logic of the timing electronics.

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

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.

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 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.

TCSPC upgrade of a confocal FCS microscope

We extended the measurement capabilities of the Carl Zeiss ConfoCor 1 FCS microscope by (a) using pulsed picosecond diode lasers instead of a continuous wave (CW) laser excitation, (b) introducing a fast single photon avalanche diode detector, and (c) exploiting the capabilities of the PicoQuant TimeHarp 200 board. When the time-tagged time-resolved (TTTR) mode of the TimeHarp is utilized, the complete fluorescence dynamics are recorded. That is, the time-evolution of the fluctuations and the fluorescence decay kinetics are captured simultaneously. Recording individual photon events (without on-the-fly data reduction like in hardware correlators) preserves the full information content of the measurement for virtually unlimited data analysis tasks and provides a much more detailed view of processes happening in the detection volume. For example, autocorrelation functions of dyes in a mixture can be separated and/or their cross-correlation can be investigated. These virtual two-channel measurements are perf...

Hardware solution for continuous time resolved burst detection of single molecules in flow

Time Correlated Single Photon Counting (TCSPC) is a valuable tool for Single Molecule Detection (SMD). However, existing TCSPC systems did not support continuous data collection and processing as is desirable for applications such as SMD for e.g. DNA-sequencing in a liquid flow. First attempts at using existing instrumentation in this kind of operation mode required additional routing hardware to switch between several memory banks and were not truly continuous. We have designed a hard- and software system to perform continuous real-time TCSPC based upon a modern solid state Time to Digital Converter (TDC). Short dead times of the fully digital TDC design combined with fast Field Programmable Gay Array logic permit a continuous data throughput as high as 3 Mcounts/sec. The histogramming time may be set as short as 100 microsecond(s) . Every histogram or every single fluorescence photon can be real-time tagged at 200 ns resolution in addition to recording its arrival time relative to the excitation pulse. Continuous switching between memory banks permits concurrent histogramming and data read-out. The instrument provides a time resolution of 60 ps and up to 4096 histogram channels. The overall instrument response function in combination with a low cost picosecond diode laser and an inexpensive photomultiplier tube was found to be 180 ps and well sufficient to measure sub-nanosecond fluorescence lifetimes.

Integrated multichannel photon timing instrument with very short dead time and high throughput

Precisely timed detection of single photons plays an important role in the field of quantum information processing and fluorescence sensing. The method of time-correlated single photon counting is therefore constantly evolving and the associated instrumentation is being improved with new ideas and technologies. Simultaneous, time tagged readout of multiple detector channels is invaluable in many applications, spanning from fluorescence lifetime imaging in biology to the measurement of quantum optical correlations in basic research. Here we present a new integrated design, providing up to three independent input channels, a very short dead time, very high throughput, and a timing resolution of 25 ps at reasonable cost and small size. Apart from design features and test results of the instrument, we show an application in quantum optics, namely, the measurement of the photon statistics of a heralded single photon source based on cavity-enhanced spontaneous parametric down-conversion.

Simple Near-Infrared Time-Correlated Single Photon Counting Instrument with a Pulsed Diode Laser and Avalanche Photodiode for Time-Resolved Measurements in Scanning Applications

A simple apparatus for time-correlated single photon counting (TCSPC) measurements in the near-infrared (near-IR) region for scanning-type applications has been constructed and examined. The apparatus consisted of five major components including a pulsed diode laser source (lasing wavelength = 780 nm; repetition rate 80 MHz; power = 5 mW; pulse width = 150 ps), an integrated microscope, a large-photoactive-area avalanche photodiode (APD), a TCSPC PC-board including the electronics, and a Windows-based software package for accumulating the fluorescence decay profiles. The instrument response function (IRF) of this assembly was found to be 460 ps, which is adequate for measuring lifetimes with τf ⩾ 500 ps. Due to the small size of the device, it also allowed implementation into scanning experiments where lifetimes were measured. To demonstrate this capability, we scanned a three-well microscope slide containing a near-IR dye. The decay profile of the near-IR dye, aluminum 2,3-naphthalocyanine, was collected and analyzed to obtain its lifetime, which was found to be 2.73 ns, in close agreement with the literature value for this particular dye. In addition, a three-dimensional plot showing the decay profiles (time vs. photocounts) and scan position of aluminum 2,3-naphthalocyanine fluorescence was acquired by scanning the microscope head over this three-well glass slide. In the scanning mode, the IRFs as well as the decays of the dyes were found to be very stable. The device demonstrated a concentration detection sensitivity of 0.44 nM; however, the dynamic range was limited due to the slow time constant (passive quenching) associated with the APD.