Advanced Time-Correlated Single Photon Counting Techniques (original) (raw)
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Commercial and Biomedical Applications of Ultrafast Lasers IV, 2004
Time-correlated single photon counting (TCSPC) is based on the detection of single photons of a periodic light signal, measurement of the detection time of the photons, and the build-up of the photon distribution versus the time in the signal period. TCSPC achieves a near ideal counting efficiency and transit-time-spread-limited time resolution for a given detector. The drawback of traditional TCSPC is the low count rate, long acquisition time, and the fact that the technique is one-dimensional, i.e. limited to the recording of the pulse shape of light signals. We present an advanced TCSPC technique featuring multi-dimensional photon acquisition and a count rate close to the capability of currently available detectors. The technique is able to acquire photon distributions versus wavelength, spatial coordinates, and the time on the ps scale, and to record fast changes in the fluorescence lifetime and fluorescence intensity of a sample. Biomedical applications of advanced TCSPC techniques are time-domain optical tomography, recording of transient phenomena in biological systems, spectrally resolved fluorescence lifetime imaging, FRET experiments in living cells, and the investigation of dye-protein complexes by fluorescence correlation spectroscopy. We demonstrate the potential of the technique for selected applications.
Recent advances in time-correlated single-photon counting
Proceedings of SPIE, 2008
We re about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS). FLCS is a fundamental improvement of standard FCS overcoming many of its inherent limitations. The basic idea of FLCS is a weighting of the detected photons based on the additional picosecond timing information (TCSPC start-stop time) when using pulsed laser excitation. 2fFCS goes even further, combining Pulsed Interleaved Excitation (PIE) with a time-gated FCS analysis. The basic implementation of 2fFCS uses two synchronized but interleaved pulsed lasers of the same wavelength but of different polarisation to generate two close by excitation foci in a predetermined distance acting as a submicron ruler. In this case it it no longer necessary to have prior knowledge about the size and shape of the confocal volume. Maintaining the information about the photon's origin, the dual focus data allows a precise calculation of absolute diffusion coefficients.
Fluorescence lifetime imaging by time-correlated single-photon counting
Microscopy Research and Technique, 2003
We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.
Recent advances in time-correlated single-photon counting
Single Molecule Spectroscopy and Imaging, 2008
We report about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS).
REVIEW OF SCIENTIFIC INSTRUMENTS, 2005
We present an advanced time-correlated single photon counting ͑TCSPC͒ technique that delivers traditional fluorescence correlation ͑FCS͒ or cross correlation ͑FCCS͒ and fluorescence lifetime data simultaneously. Newly developed electronics allow for detection and registration of single photon events over time periods of hours with picoseconds accuracy. Subsequent software-correlation yields correlation curves covering more than 12 orders of magnitude in time. At the same time, the original data, containing all information accessible by single photon counting techniques, can be analyzed conventionally according to common single molecule fluorescence techniques. Potential applications of the new technique using pulsed or cw laser excitation are discussed.
Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting
Medical Photonics, 2015
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
Multiparametric Time-Correlated Single Photon Counting Luminescence Microscopy
Biokhimiya, 2019
Classic time correlated single photon counting (TCSPC) technique involves detection of single photons of a peri odic optical signal, registration of the photon arrival time in respect to the reference pulse, and construction of photon dis tribution with regard to the detection times. This technique achieves extremely high time resolution and near ideal detec tion efficiency. Modern TCSPC is multi dimensional, i.e., in addition to the photon arrival time relative to the excitation pulse, spatial coordinates within the image area, wavelength, time from the start of the experiment, and many other param eters are determined for each photon. Hence, the multi dimensional TCSPC allows generation of photon distributions over these parameters. This review describes both classic and multi dimensional types of TCSPC microscopy and their applica tion for fluorescence lifetime imaging in different areas of biological studies.
IEEE Transactions on Biomedical Engineering, 2013
In time-correlated single photon counting (TCSPC) systems, the maximum signal throughput is limited by the occurrence of pileup and other effects. In many biological applications that exhibit high levels of fluorescence intensity (FI), pileup related distortions yield serious distortions in the fluorescence lifetime (FLT) calculation as well as significant decrease in the signal-to-noise ratio (SNR). Recent developments that allow the use of high-repetition-rate light sources (in the range of 50-100 MHz) in fluorescence lifetime imaging (FLIM) experiments enable minimization of pileup related distortions. However, modern TCSPC configurations that use high-repetition-rate excitation sources for FLIM suffer from dead-time-related distortions that cause unpredictable distortions of the FI signal. In this study, the loss of SNR is described by F-value as it is typically done in FLIM systems. This F-value describes the relation of the relative standard deviation in the estimated FLT to the relative standard deviation in FI measurements. Optimization of the F-value allows minimization of signal distortion, as well as shortening of the acquisition time for certain samples. We applied this method for Fluorescein, Rhodamine B, and Erythrosine fluorescent solutions that have different FLT values (4 ns, 1.67 ns, and 140 ps, respectively). Index Terms-Fluorescence intensity (FI), fluorescence lifetime (FLT), fluorescence lifetime imaging (FLIM), time-correlated single photon counting (TCSPC).
Microscopy Research and Technique, 2006
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a highrepetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or twophoton laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, 2015
Fast frame rate CMOS cameras in combination with photon counting intensifiers can be used for fluorescence imaging with single photon sensitivity at kHz frame rates. We show here how the phosphor decay of the image intensifier can be exploited for accurate timing of photon arrival well below the camera exposure time. This is achieved by taking ratios of the intensity of the photon events in two subsequent frames, and effectively allows wide-field TCSPC. This technique was used for measuring decays of ruthenium compound Ru(dpp) with lifetimes as low as 1 μs with 18.5 μs frame exposure time, including in living HeLa cells, using around 0.1 μW excitation power. We speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could well be possible.