Multi-parameter fluorescence lifetime imaging microscopy is a powerful tool to investigate the spatial dependence of the
fluorescence decay signal of chromophores in living cells. A multi-channel detection system based on a position
sensitive Quadrant Anode photomultiplier and time-correlated single photon counting (TCSPC) was applied to monitor
the fluorescence decay in individual living cells of the cyanobacterium Thermosynechococcus elongatus. The
fluorescence lifetime imaging system was used to detect at a frame-rate of 1 Hertz the recovery after photobleaching of
Phycobilisomes (PBS), a photosynthetic pigment-protein complex with light harvesting functions. Simultaneous
monitoring of the fluorescence decays of the PBS- and the Chlorophyll a (Chl a)-containing antenna systems unveils a
heterogeneity of the cyanobacterial population with respect to the fluorescence lifetime. Furthermore the FLIM images
clearly show that the decay of the fluorescence signal in the targeted area becomes longer after the bleaching at 633 nm
for the whole measuring time. The results suggest that the diffusing PBSs, which replace the bleached ones in the
targeted area, can only partially reestablish excitation energy transfer to Chl a.
Novel proximity-type Time- and Space-Correlated Single Photon Counting (TSCSPC) crossed-delay-line (DL)- and
multi-anode (MA)-systems of outstanding performance and homogeneity were developed, using large-area detector
heads of 25 and 40 mm diameter. Instrument response functions IRF(space) = (60 ± 5) μm FWHM and IRF(time) = (28
± 3) ps FWHM were achieved over the full 12 cm2 area of the detector. Deadtime at throughput of 105 cps is 10% for
"high-resolution" system and 5% in the "video"-system at 106 cps, at slightly reduced time- and space resolution. A
fluorescence lifetime of (3.5 ± 1) ps can be recovered from multi-exponential dynamics of a single living
cyanobacterium (Acaryochloris marina). The present large-area detectors are particularly useful in simultaneous
multichannel applications, such as 2-colour anisotropy or 4-colour lifetime imaging, utilizing dual- or quad-view image
splitters. The long-term stability, low- excitation-intensity (< 100 mW/cm2) widefield systems enable minimal-invasive
observation, without significant bleaching or photodynamic reactions, thus allowing long-period observation of up to
several hours in living cells.
Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary
processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes
of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be
sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the
reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field
FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate
photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under
minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by
presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss
leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.
Franz-Josef Schmitt, Christoph Theiss, Karin Wache, Justus Fuesers, Stefan Andree, Andrianto Handojo, Anne Karradt, Daniela Kiekebusch, Hans Joachim Eichler, Hann-Jörg Eckert
KEYWORDS: Picosecond phenomena, Luminescence, Antennas, Energy transfer, Molecules, Proteins, Single photon, Visible radiation, Molecular energy transfer, Time metrology
The phototrophic cyanobacterium Acaryochloris marina discovered in 1996 has a unique composition of the light
harvesting system. The chlorophyll (Chl) antenna contains mainly Chl d instead of the usually dominant Chl a and the
Phycobiliprotein (PBP) antenna has a simpler rod shaped structure than in typical cynobacteria [1].
The interaction of the photosynthetic subunits and especially the mechanisms regulating the energy transfer under
different stress conditions are presently interesting and open fields in photosynthesis research.
In this study we use time- and wavelength-resolved single photon counting to investigate the excited states dynamics in
living cells of A.marina. The fluorescence dynamics is synchronistically monitored in the visible and near infrared
spectrum with high signal to noise ratio and short data acquisition times while using low excitation light intensities.
These attributes are necessary to investigate photosynthetic processes in sensitive biological samples, when the light
emission varies due to metabolic changes.
The results suggest a fast excitation energy transfer kinetics of 20-30 ps along the PBP antenna of A.marina followed by
a transfer of about 60 ps to the Chl d antenna.
Cells of A. marina which are stored at 0°C for some time show a decoupling of the PBP antenna, which is partially
reversible when the sample is kept at 25 °C for a short time. Decoupling effects appearing after strong illumination with
white light (1600 W/m2) suggest a mechanism which removes the PBP antenna at different stress conditions to avoid
photo damage of the reaction center of Photosystem II (PS II).
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