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Content archived on 2024-04-30

Picosecond fluorescence lifetime imaging microscopy - application to single molecule dynamics

Objective



Research objectives and content: Objectives: To set up a time- and space-correlated single photon counti (TSCSPC) spectroscopy system by using (a) the pico-second pulsed laser system of the host and (b) the TSCSPC detection system of the guest. There will be two spectroscopy systems: (a) delay-line (DL) microchannel plate (MCP) photomultiplier (PMT) attached to a polychromator and (b) quadrant-anode (QA) MCP-PMT attached to a low-background microscope, where DL- and QA-MCP-PMT are linear and imaging detectors of 10 ps time resolution. respectively. Content: To perform time-resolved spectroscopy of individual dye molecules, relevant in fundamental spectroscopy, biotechnology, and medical research. The kinetics of individual molecules may differ significantly from the average values of an ensemble. Individual kinetics of probe molecules will be of considerable interest in structural molecular biology. The antifungal agent nystatin, for instance, is an intrinsically fluorescent molecule that stresses membranes. The population of the antibiotics is believed to be divided in two sub-populations having different locations and fluorescence lifetimes, probably corresponding to different aggregation states Ensemble average measurements prevent any detailed structural inforrnation of such an heterogeneous system. The use of a sensitive time-resolved spectroscopy will solve a long time open question: What is the role of different aggregation states in the biochemical mode of action of nystatin? The use of TSCSPC spectroscopy would also be very welcome to study the binding of the hemagglutinin (HA) protein from Influenza virus to sialic acid residues. The binding results in strong conformational changes that modify the distance between the sialic acid/Trp residues. Derivatisation of the sialic acid with a proper dye would allow to follow single molecule conformational dynamics by FRET (Forster resonance energy transfer) using the TSCSPC brought by the applicant to the host institute. No such techniques are available in the country of the host institute. Training content (objective. benefit and expected impact) Objectives: introduction of the novel techniques Time- and Space-Correlated Single Photon Counting spectroscopy and Single Molecule Microscopy Elucidation of: i) the biochemical mode of action of the antifungal agent nystatin by individually studying each type of aggregate fonned at the lipid bilayers level, ii) the association of the HA protein of the influenza virus to the host cell receptors in vitro by FRET using the TSCSPC technique. Benefit: enhancement of existing technology at the host institute, training of young scientists m a technology of the future, front-line research, dissemiation of knowlege. Impact: spread of the novel technique in fundamental research, development of new applications, and enhancement of industrial feedback. Links with industry / industrial relevance (22) The applicant is founder managing director, and senior scientist of EuroPhoton GmbH, a young company, dedicated to the development of ultra-sensitive detectors on the picosecond timescale. The host institute is currently collaborating with Biovector Therapeutics (Toulouse France) in the structural characterisation of Drug Delivery Systems and vaccine formulations. The interaction between the host institute and this industry would benefit from the application of the new abovementioned techniques.

Programme(s)

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Funding Scheme

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Coordinator

Aristotle University of Thessaloniki
EU contribution
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Address
Analytical Chemistry Lab
54006 Thessaloniki
Greece

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Total cost

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