We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

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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.

A confocal laser-scanning microscope for ultrasensitive fluorescence lifetime imaging on surfaces is presented. The system employs a compact electronics for time-correlated single-photon counting (TCSPC), allowing for measuring fluorescence lifetime with 40 ps time resolution, and for continuously recording photon arrival times with 100 ns time resolution. Additionally developed driver electronics serve for synchronization of scanning and data acquisition, which is significant for achieving high spatial image resolution. The capabilities of the measurement system are demonstrated on imaging single molecules immobilized on glass substrates. Finally, it is shown how the TCSPC capabilities of the system can be used not only for lifetime imaging but also for multichannel measurements.

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.