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Simultaneous acquisition of time- and space-information on the picosecond timescale became feasible by a recent advance in microchannel-plate photomultiplier-tube (MCP-PMT) technology: we present results obtained by two novel MCP-PMT detectors for time- and space-correlated single photon counting (TSCSPC), featuring a space-sensitive delay-line (DL) anode and quadrant-anode (QA), respectively, yielding continuous images.
DL-System: the DL-MCP-PMT is a linear imager with 200 pixel at 100 µm resolution. The detector has a temporal IRF of 75 ps FWHM at a dynamic range of up to 5x105. We applied time-resolved spectroscopy to study the dynamic behaviour of DNA-probes (DAPI, TOTO, C350) in solution, in micelles, complexed to DNA, protein, and fixed cells. Ageing of DAPI stock solutions was reported. A polarity model for the photophysics of DAPI was proposed. Microscope lifetime images on the picosecond timescale showed clear potential for dynamic stray-light rejection and kinetic discrimination of probe-protein and probe-DNA complexes. First results on TOTO-fixed cell systems demonstrated high-quality kinetics at subcellular resolution, with up to 6 lifetime species at higher dye concentrations, characteristically distributed among individual cell compartments. A comparison with TOTO/DNA-suspensions was made that served as reference system. The analysis of multi-exponential fluorescence decays at subcellular resolution yields new information on characteristic probe-DNA interaction within individual cell compartments [1-3].
QA-System: the QA-MCP-PMT is a 2D imager with 400x400 pixel at 15 µm resolution. The detector has a temporal IRF of 80 ps FWHM, sufficient for 10 ps time resolution, at a dynamic range of 105 of the uncooled detector. A through-put of 105 cps is possible, in burst mode, the QA-detector can achieve 106 cps. We presented first data, obtained by a 50x50 channel QA microscope system of 1 µm space- and 10 ps time-resolution, demonstrating the full potential of TSCSPC. A modified Leitz Orthoplan/Ploemopak fluorescence microscope with the QA detector at the camera image plane (camera ocular = 3.2x) was used. Laser System: as excitation source served a Ti-Saph laser, run at 76 MHz repetition rate, 1 ps pulse width, and 760 nm. Frequency doubling produced 380 nm excitation. We also presented first images of 1 µm beads, loaded with fluorescein, analysed by global analysis: the fluorescence decays were two-exponential with 1 = 1.75 ns and 2 = 4.8 ns, at A1 = (91.7 ± 1.2)%. The major fast component is attributed to self-quenching of the high-concentration sample, since the fluorescence lifetime of xanthene dyes in rigid matrix is expected to be close the radiative lifetime of about 5 ns. The data were treated by a simple 4x4 ch global analysis, work on the full 50x50 ch version is in progress. The spatial resolution was FWHM = 1.5 µm but straightforward improvements in optics should result in an improved resolution of 0.5 µm [4-5].
Simultaneous TSCSPC spectroscopy and microscopy on the picosecond times scale, as described above, are two analytic methods that allow the acquisition of time-space data of superior quality, even at ultra-low excitation levels that are suitable for the study of living cells.
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