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2D Fluorescence lifetime imaging of subcellular structures

Obiettivo



The present proposal is designed as preparatory phase in view of the participation of EuroPhoton Ltd. and Lambda Ltd. in an RTD project, i.e. an envisaged demonstration project with title "Two- and Tree-Dimensional Subnanosecond Fluorescence Lifetime Imaging of Subcellular Structures", to be submitted in September 95, in Area 6.1.2 of ù Biotechnology. Potential par to be convinced are: Prof. J. Bereiter-Hahn (Frankfurt, DE), Prof M. Robert-Nicoud (Grenoble, FR), Prof. J. Coppey (Paris, FR), Prof. D. Phillips (London, GB), Prof. S. Schneider (Erlangen, DE), a SME electronics company (DE), and one of the established microscope manufacturers, such as Zeiss or Leica. The aim of the present study is the construction of a 2D prototype, in order to demonstrate the usefulness of (sub)nanosecond time information in cell-biological fluorescence microscope studies.
Fluorescence dynamics will be acquired by applying time-correlated single photon counting (TCSPC) spectroscopy, a well established method for the acquisition - of fluorescence dynamics on the (sub)nanosecond time-scale of very weakly emitting sources, distinguished by its unsurpassed dynamic range. A recent advance in micro-channel plate (MCP)-PMT technology, i.e. the introduction of a delay-line (DL) anode, resulted in the exciting possibility of simultaneous acquisition of time and space information, i.e. in timeand space-correlated single photon counting (TSCSPC) spectroscopy, at temporal and spatial IRFs characterised by FWHM of 80 ps and 100 ,um, respectively, at 200 space channels along the x-axis. By scanning the exciting laser beam along the y-axis, a 2D lifetime imaging device will result. Due to the inherent advantages of the DL principle, the capacity of acquiring photons is about 200 times larger than with standard, single-channel MCP-PMTs, at full preservation of confocality, by simply replacing the observing confocal pinhole by a confocal slit. Superposition of lifetime image and standard CCD image will result in enhanced contrast and depth of information.
Due to the necessary use of pulsed, high-repetition rate lasers with their specific wavelengths, novel dyes have to be developed that are adjusted to laser lines and repetition rates, i.e. absorption maxima and fluorescence lifetimes have to be tailor-made.
Energy transfer, collisional quenching, rotation of the fluorescent probe, intercalation or association of probe with DNA has profound impact on fluorescence lifetime. Observation of prompt or delayed luminescence kinetics in context with fluorescence in-situ hybridisation, will give new impetus to genome cartography. Due to the utilisation of the time domain as additional carrier of information, a new horizon will open to look at elasticity of DNA, association of DNA with dyes and drugs, or macromolecular association within the cytoplasm of living cells. Thus, fluorescence lifetime constitutes a further source of information in cellular biology, and affords a new tool for the investigation of macromolecular complexes in their natural environment.

Campo scientifico (EuroSciVoc)

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Coordinatore

EuroPhoton GmbH - Gesellschaft für Optische Sensorik
Contributo UE
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Indirizzo
27,Mozartstrasse
12247 Berlin
Germania

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