Wolfgang Becker

High-count-rate multichannel TCSPC for optical tomography

An improved Time-Correlated Single Photon Counting (TCSPC) technique features high count rate, low differential nonlinearity and multi-detector capability. The system has four completely parallel TCSPC channels and achieves an effective overall count rate of 20 MHz. By an active routing technique, up to eight detectors can be connected to each of the TCSPC channels. We used the system to record optical mammograms after pulsed laser illumination at different wavelengths and projection angles.

Multiwavelength TCSPC lifetime imaging

We present a novel time-correlated single photon counting (TCPSC) imaging 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 time, the x-y coordinates of the scanning area and the detector channel number. The histogramming process avoids any time gating or wavelength scanning and therefore yields a near-perfect counting efficiency. Applied to resonance energy transfer (RET) measurements, the setup is capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.

Picosecond fluorescence lifetime microscopy by TCSPC imaging

A new Time-Correlated Single Photon Counting (TCSPC) imaging technique delivers combined intensity-lifetime images in a two-photon laser scanning microscope. The sample is excited by laser pulses of 150 fs duration and 80 MHz repetition rate. The microscope scans the sample with a pixel dwell time in the +s range. The fluorescence is detected with a fast PMT at the non-descanned port of the laser scanning microscope. The single photon pulses from the PMT and the scan control signals from the scanning head are used to build up a three-dimensional histogram of the photon density over the time within the decay function and the image coordinates x and y. Analysis of the recorded data delivers images containing the intensity as brightness and the lifetime as colour, images within selected time windows or decay curves in selected pixels. The performance of the system is shown for typical applications such as FRET measurements, Ca imaging and discrimination of endogenous fluorophores or different dyes in living cells and tissues.

How fast can TCSPC FLIM be made

The acquisition time of TCSPC FLIM depends on the number of pixels of the image, on the required lifetime accuracy, and on the count rate available from the sample. For samples with high fluorophore concentrations, such as stained tissue or plant cells the available count rates may come close to the maximum counting capability of the currently used TCSPC FLIM techniques. We describe the behaviour of TCSPC at high count rates and estimate the size of counting loss and pile-up effects. It turns out that systematic lifetime errors are smaller than previously believed. TCSPC FLIM can therefore be used to record fast sequences of fluorescence lifetime images. Fast sequential FLIM will be demonstrated for the measurement of chlorophyll transients in living plant tissue.

Fast-acquisition multispectral FLIM by parallel TCSPC

Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 50•106 s-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.

Lifetime imaging with the Zeiss LSM-510

The Zeiss LSM-510 NLO laser scanning microscope can be combined with a new TCSPC (time-correlated single photon counting) lifetime imaging technique developed by Becker & Hickl, Berlin. This technique is based on a three-dimensional histogramming process that records the photon density over the time within the fluorescence decay function and the x-y coordinates of the scanning area. The histogramming process avoids any time gating and therefore yields a counting efficiency close to one. Upgrading The LSM-510 for TCSPC imaging does not require changes in the microscope hardware or software. A fast detector is attached to the fibre output of the scanning head, and synchronisation of the TCSPC module with scanning is achieved via the user I/O of the scan controller. With an MCP-PMT as a detector, fluorescence decay components down to 30 ps can be resolved. The capability of the instrument is shown for the separation of chromphores by their fluorescence lifetime and for CFP/YFP FRET.

Characterization of a time-resolved non-contact scanning diffuse optical imaging system exploiting fast-gated single-photon avalanche diode detection

We present a system for non-contact time-resolved diffuse reflectance imaging, based on small source-detector distance and high dynamic range measurements utilizing a fast-gated single-photon avalanche diode. The system is suitable for imaging of diffusive media without any contact with the sample and with a spatial resolution of about 1 cm at 1 cm depth. In order to objectively assess its performances, we adopted two standardized protocols developed for time-domain brain imagers. The related tests included the recording of the instrument response function of the setup and the responsivity of its detection system. Moreover, by using liquid turbid phantoms with absorbing inclusions, depth-dependent contrast and contrast-to-noise ratio as well as lateral spatial resolution were measured. To illustrate the potentialities of the novel approach, the characteristics of the non-contact system are discussed and compared to those of a fiber-based brain imager.

Multidimensional time-correlated single photon counting

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.

Time- and Wavelength-Resolved Autofluorescence Detection by Multi-Dimensional TCSPC

We present a multi-dimensional TCSPC technique that simultaneously records the photon distribution over the time in the fluorescence decay, the wavelength, and the coordinates of a two-dimensional scan. We demonstrate the application of the technique to single-point autofluorescence measurements of skin, to multi-spectral fluorescence lifetime microscopy, and ophthalmic imaging.

Dynamic Mapping of the Human Brain by Time-Resolved NIRS Techniques

Dynamic mapping of the human brain by time-resolved near-infrared-spectroscopy (trNIRS), or functional NIRS (fNIRS), is based on the injection of picosecond or sub-nanosecond laser pulses into the head and the measurement of the pulse shape and the intensity after diffusion through the tissue. By analysing the pulse shape and the intensity of the signals at different detector and source positions and different wavelengths, changes in the oxy- and deoxy-haemoglobin concentration are obtained for extracerebral and intracerebral tissue layers and for different depth in the brain. The technique can by combined with the injection of a bolus of an exogenous absorber. By recording either absorption or fluorescence, the in- and outflow of the absorber in different brain compartments can be monitored. The in- and outflow dynamics reveal differences in the blood flow caused by impaired perfusion or stroke. In this chapter, we describe the technical principle of TCSPC-based fNIRS and the associated data processing techniques. Typical results are shown for the haemodynamic response of the brain on visual-cortex stimulation, and for brain perfusion measurement by ICG bolus injection.