Final Report Summary - SingleMotor-FLIN (Long-Period Observation of Single (Bio)-Molecular Motors by Minimal-Invasive Fluorescence Lifetime Imaging Nanoscopy (FLIN))
The 'Long-period observation of single (bio)-molecular motors by minimal-invasive fluorescence lifetime imaging nanoscopy(FLIN)' (SingleMotor-FLIN) project provides groundbreaking tools for the study of single molecules (SM) and single molecular motors (SMM), as well as a broad array of phenomena in nanoworld.
Objectives of technology producers:
(a) set-up of a FLIN prototype at the FLIN-Centre Berlin-Magdeburg, consisting of a commercial Nikon TE2000 body with TSCSPC detection (by EuroPhoton) and a custom-made 10 MHz Ti-Saph laser;
(b) improvements of several parameters of the current generation of TSCSPC detectors, such as (i) time-resolution, (ii) space-resolution, (iii) through-put, and v) change from LN2 to Peltier cooling;
(c) improved TIRF microscope and SM/SMM observation: (i) adaptation for multi-detector TSCSPC operation and (pseudo)-simultaneous dual-EPI/TIRF excitation/observation, (ii) development of super background-free TIRF microscope for enhanced S/N and improved observation of SM/SMM, (iii) SPR-TIRF, for obtaining an additional 100x gain in sensitivity;
(d) application of PSF-modelling for tracking of SM/SMM with high nanometre accuracy;
(e) apply dual-colour FLIN PSF-modelling method to approach 5 nm accuracy and 10 nm resolution.
Objectives of technology users: employing the novel Picosecond FLIN/FLIM method and several of its unique applications to study the behaviour of minimal model systems and artificial and biological motors.
Expected end results: construction of a FLIN prototype and its introduction to cell-biological research and the nanoworld, by studying the behaviour of single molecules and single molecular motors in living cells and artificial systems.
Intensions of use: FLIN will see a wide application in biological and medical research as well as nanotechnology.
Impact: enhancement of European cell biology and nanotechnology. The FLIN method will provide a ground-breaking tool for minimal-invasive study of delicate living systems. Improved understanding of biological SMM will help in development of artificial motors.
Major objectives:
in 1st Period:
(i) setup of functional FLIN system in Berlin at EuroPhoton GmbH by technology producers;
(ii) preparation of SMM for measurement in 2nd and 3rd periods by technology users.
in 2nd Period:
(i) set-up of multi-laser, multi-detector, multi-microscope FLIM/FLIN ensemble; (ii) preparation of SMM for measurement in 3rd periods by technology users.
in 3rd Period:
(i) partner FLIM/FLIN measurements in Berlin, using the novel prototype at EuroPhoton GmbH;
(ii) joint development of a internal and external novel multi-anode (MA) TSCSPC detector system by RRC KI and EuroPhoton GmbH.
Major results:
in 1st Period:
(i) Setup of a multilaser, multi-Detector, multi-microscope FLIN-ensemble in Berlin was performed, using project-acquired laser and microscope and existing components of the coordinator;
(ii) using novel equipment of (i), FLIN was achieved with 20 nm space- and 15 ps time-resolution, by applying the TSCSPC method, thus providing the proof-of-principle for FLIN operation;
(iii) preparation of SMM by technology users in Amsterdam (bacteriorhodopsin system), Edinburgh (artificial motor), and Magdeburg (neuronal motors with cargo) made progress. Theoretical work on light-driven artificial SMM by Praque partner resulted in a publication being in press.
in 2nd Period:
(i) set-up of multi-laser, multi-detector, multi-microscope FLIM/FLIN ensemble;
(ii) theoretical work on artificial molecular motors by Prague partner resulted in two publications;
(iii) improvement of FLIN space resolution: 10 nm;
(iv) set-up of 10x10 scanner for 1- and 2-photon excitation;
(v) operational 2-photon TIRF microscope.
in 3rd Period:
(i) partners 6 and 7 brought their samples to Berlin and performed joint FLIM/FLIN measurements, using the novel prototype at EuroPhoton GmbH;
(ii) joint development of a novel internal and external multi-anode (MA) TSCSPC detector system by RRC KI and EuroPhoton GmbH resulted in a first MA-prototype that was setup and tested at the FLIM/FLIN Centre of EuroPhoton GmbH in Berlin.
Objectives of technology producers:
(a) set-up of a FLIN prototype at the FLIN-Centre Berlin-Magdeburg, consisting of a commercial Nikon TE2000 body with TSCSPC detection (by EuroPhoton) and a custom-made 10 MHz Ti-Saph laser;
(b) improvements of several parameters of the current generation of TSCSPC detectors, such as (i) time-resolution, (ii) space-resolution, (iii) through-put, and v) change from LN2 to Peltier cooling;
(c) improved TIRF microscope and SM/SMM observation: (i) adaptation for multi-detector TSCSPC operation and (pseudo)-simultaneous dual-EPI/TIRF excitation/observation, (ii) development of super background-free TIRF microscope for enhanced S/N and improved observation of SM/SMM, (iii) SPR-TIRF, for obtaining an additional 100x gain in sensitivity;
(d) application of PSF-modelling for tracking of SM/SMM with high nanometre accuracy;
(e) apply dual-colour FLIN PSF-modelling method to approach 5 nm accuracy and 10 nm resolution.
Objectives of technology users: employing the novel Picosecond FLIN/FLIM method and several of its unique applications to study the behaviour of minimal model systems and artificial and biological motors.
Expected end results: construction of a FLIN prototype and its introduction to cell-biological research and the nanoworld, by studying the behaviour of single molecules and single molecular motors in living cells and artificial systems.
Intensions of use: FLIN will see a wide application in biological and medical research as well as nanotechnology.
Impact: enhancement of European cell biology and nanotechnology. The FLIN method will provide a ground-breaking tool for minimal-invasive study of delicate living systems. Improved understanding of biological SMM will help in development of artificial motors.
Major objectives:
in 1st Period:
(i) setup of functional FLIN system in Berlin at EuroPhoton GmbH by technology producers;
(ii) preparation of SMM for measurement in 2nd and 3rd periods by technology users.
in 2nd Period:
(i) set-up of multi-laser, multi-detector, multi-microscope FLIM/FLIN ensemble; (ii) preparation of SMM for measurement in 3rd periods by technology users.
in 3rd Period:
(i) partner FLIM/FLIN measurements in Berlin, using the novel prototype at EuroPhoton GmbH;
(ii) joint development of a internal and external novel multi-anode (MA) TSCSPC detector system by RRC KI and EuroPhoton GmbH.
Major results:
in 1st Period:
(i) Setup of a multilaser, multi-Detector, multi-microscope FLIN-ensemble in Berlin was performed, using project-acquired laser and microscope and existing components of the coordinator;
(ii) using novel equipment of (i), FLIN was achieved with 20 nm space- and 15 ps time-resolution, by applying the TSCSPC method, thus providing the proof-of-principle for FLIN operation;
(iii) preparation of SMM by technology users in Amsterdam (bacteriorhodopsin system), Edinburgh (artificial motor), and Magdeburg (neuronal motors with cargo) made progress. Theoretical work on light-driven artificial SMM by Praque partner resulted in a publication being in press.
in 2nd Period:
(i) set-up of multi-laser, multi-detector, multi-microscope FLIM/FLIN ensemble;
(ii) theoretical work on artificial molecular motors by Prague partner resulted in two publications;
(iii) improvement of FLIN space resolution: 10 nm;
(iv) set-up of 10x10 scanner for 1- and 2-photon excitation;
(v) operational 2-photon TIRF microscope.
in 3rd Period:
(i) partners 6 and 7 brought their samples to Berlin and performed joint FLIM/FLIN measurements, using the novel prototype at EuroPhoton GmbH;
(ii) joint development of a novel internal and external multi-anode (MA) TSCSPC detector system by RRC KI and EuroPhoton GmbH resulted in a first MA-prototype that was setup and tested at the FLIM/FLIN Centre of EuroPhoton GmbH in Berlin.