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.
It is well known that fluorescence microspectroscopy is a powerful tool in the study of biological objects. Fluorescence lifetime imaging is one of the modern and promising techniques applied in these studies. The method implies the detection of not only spectral parameters of some molecules and molecular structures but also the fluorescence lifetimes. Thus, one can easily distinguish between two fluorophores with similar spectral properties but having different fluorescence lifetimes. Additional possibilities are offered by the method for stray light rejection and detection of Raman scattering because of the difference in time-correlation of the excitation, fluorescence, and Raman photons. We suggest the application of an original position-sensitive micro-channel plate photomultiplier as a key element of a newly developed fluorescence microspectrometer designated for the study of biological objects (cells, subcellular structures and organells). We present the first results of the experiments with the test objects. The results obtained clearly show that the new system is rather promising in the studies of living cells and tissues.
Novel MCP detectors for time- and space-correlated single photon counting (TSCSPC) spectroscopy, featuring delay-line (DL) or quadrant anode (QA), are employed in microscopic fluorescence lifetime imaging on the picosecond time scale. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100 micrometer FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 by 400 pixel at 40 micrometer resolution. The detectors have a temporal IRF of 75 ps (DL) and 120 ps (QA) FWHM, sufficient for 10 ps time resolution. First results on TOTO-fixed cell systems are presented, demonstrating 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 is made that serve as reference system.
Simultaneous acquisition of time- and space-information in time-domain single photon counting spectroscopy became feasible by a recent advance in microchannel-plate photomultiplier-tube technology: we present a novel MCP-PMT detector, featuring a space- sensitive delay-line anode. The detector is characterized by temporal and spatial instrument response functions of 75 ps and 100 micrometer FWHM, respectively, at 200 space channels and a dynamic range of 105. By employing a two-dimensional multichannel analyzer with transputer, 70.000 cps through-put or higher is possible. No photons are lost at the exit slit of the monochromator, as in standard, one-channel time-correlated single photon counting spectroscopy, and sensitive biological samples can be studied at reduced excitation energies. We applied the novel detector to study the basic photophysics of DAPI and its interaction with DNA.
An ultrasensitive detection method of CCK in aqueous solution was developed. This method utilizes intrinsic native fluorescence properties of CCK-molecules. A new delay-line multichannel plate photomultiplier (MCP PMT) was used to measure both time and wavelength resolved fluorescence properties simultaneously. Both CCK4 and CCK8 showed biexponential fluorescence decay. While the short lived fluorescence decay time components were rather similar, the long-lived components differed by about 1 ns. In the case of CCK8 the long-lived component was red-shifted by 30 nm. Based on these photophysical data an experimental setup for an ultrasensitive detection was developed. The lowest detection limit was achieved by investigating the fluorescence intensity within a time gate (gate duration 2 ns) in the fluorescence maximum. In combination with a confocal setup, using a Cassegrainean configuration, CCK could be detected up to 10-12 M.
The fluorescence properties of fluorophores relevant in tissue metabolism (NADH, flavines, etc.) are characteristic of the clinical states of tissues. Especially the differentiation of healthy, cancerous, and necrotic tissue states is of large interest in lung-tumor diagnostics, e.g. to ensure that biopsies are taken from non-necrotic areas. In contrast to the common fluorescence detection our approach provides both a combination of spectral and time information from autofluorescence and the simultaneous detection of two fluorophores in order to improve differentiation between various tissues. The basis of analysis of autofluorescence is knowledge of the photophysical parameters of the fluorophores. Aqueous solutions of NADH, flavines and their mixtures have been investigated using the method of time-correlated single photon counting. The fluorescence was recorded with a new 'delay-line' microchannel-plate photomultiplier tube, that enables time- and wavelength-resolved measurements simultaneously. Nicotine-adenine-dinucleotide (NADH) and flavine-adenin-dinucleotide (FAD) display their characteristic temporal behavior (NADH: (tau) 1 equals 250 ps, (tau) 2 equals 660 ps; FAD: (tau) 1 equals 160 ps, (tau) 2 equals 2.25 ns, (tau) 3 equals 4.6 ns) in aqueous solution. In a mixture of NADH and FAD both components could be isolated by using global analytical methods. Time-gated fluorescence measurements on lung-tissue samples of 12 patients immediately after surgical resection have been performed with a fiber- based fluorescence detector. It could be demonstrated that NADH measurements are suitable for differentiating tumorous and necrotic tissue while flavine measurements are suitable for differentiating healthy and non-healthy tissue types. Applications of optical fibers facilitate simple combinations of the detection method with common surgical instruments (e.g. biopsy needles).
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