NMP4-2005-013880
SingleMotorFLIN
Long-Period Observation (LPO) of Single (Bio)-Molecular Motors by Novel MinimalInvasive Fluorescence Lifetime Imaging Nanoscopy (FLIN)
STREP
NMP 3.4.1.1-2
Publishable Final Activity Report
Period covered: from 1.6.2005 to 30.11.2008
Date of preparation: 7.6.2009
Duration: 42 months
Project coordinator name: Dr. Klaus Kemnitz
Project coordinator organisation name: EuroPhoton GmbH
www.euro-FLIN.de
1. Project Execution
Summary: FLIN, as recently introduced in SingleMotorFLIN (NMP4-CT-2005-013880),
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. Classical
limitations in SM/SMM studies, such as resolution, short observation times, and photodynamic reactions are overcome by minimal-invasive picosecond FLIN. FLIN is the
extension of the extremely successful fluorescence lifetime imaging microscopy (FLIM)
into the nano-domain, with down to 10 nm space-resolution. FLIN results from the
combination of nanoscopy (such as multi-colour, wide-field, point-spread- function (PSF)
modelling microscopy) with novel ultrasensitive, non-scanning imaging detectors, based
on time- and space-correlated single photon counting (TSCSPC) that allows ultra-low
excitation levels. This results, for example, in long-period (> 1 hour), minimal-invasive
observation of living cells and SM/SMM, without any cell damage or irreversible
bleaching. Minimal-invasive FLIN with global PSF-modelling allows observation of
point-source movement at 1-nm accuracy and distance determination at the 10-nm level,
while simultaneously acquiring multi-exponential pico/nanosecond fluorescence
dynamics. FLIN opens a wide avenue of novel applications, such as SMM-tracking,
FRET-verification, dual-polarisation tracking, and super-background-free 2-photon
TIRF-FLIN. SingleMotorFLIN will examine the behaviour of four types of SMM and
their dependence on energy-input. Enhanced basic understanding of biological and
artificial machines/motors will lead to advanced models and proceed one day to artificial
systems, revolutionising the interface of biological and non-biological worlds. Since
biological SMM are involved in many human disorders, the novel FLIN method will help
to show how these motors operate and how they break down in disease.
Objectives and Results:
Objectives of Technology Producers: (a) set-up of a FLIN prototype at the FLIN-Centre BerlinMagdeburg, 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) throughput, 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 dualEPI/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
nanometer 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. 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. 3rd Period: (i) partner
FLIM/FLIN measurements in Berlin, using thje 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 FLINEnsemble 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-ofprinciple 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. 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. 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.
Individual Partner Contributions: described by one Figure/Partner:
Figure 1: typical FLIN result with simultaneous nanometer-space and ps/ns-time information.
Figure 2: space-resolution in detail with ± 5 nm precision.
Figure 3: transition from non-proximity to proximity detector heads.
Figure 4: example of an internal multi-anode, in this case heptanode.
Figure 5: improved TIRF resolution
Figure 6: FLIN application, showing nm-movement of a single molecular motor (rotator).
Figure 7: chemical structure of artificial fluorescent molecular motor.
Figure 8: application of long-period observation
Figure 9: molecular modelling of molecular motor.
Figure 10: delay-line and electronics
Figure 11: joint development of MA-system by EuroPhoton GmbH and RRC Kurchatov Institute.
Fig.1: Partner 1: FLIN: The upper panel shows the green (520 nm) and red (605 nm)
fluorescence of individual Q-dots, together with the yellow emission of a hetero-dimer with
a green-red separation of 40 ± 20 nm. The lower panel displays the simultaneously acquired
3-exponential dynamics of the hetero-dimer, observed through green and red filters, thus
identifying the green and red species.
Fig.2: Partner 1: FLIN space resolution: Red-Green shifts of red-green 180 nm reference
beads: Gauss Distribution with  5 nm space resolution.
Fig.3: Partner 2: comparison of non-proximity (left) with proximity Photek-QA (right).
Fig.4: Partner 3: manufactured Heptanode, Gen.III, bottom view and side view.
Fig.5: Partner 5: Supercritical emission detection (SCE): Illustration of the image
TIRF
TIRFSCE
CTR
improvement achieved with SCE (right). Objective was x60 NA1.45oil, specimen
is a
cultured cortical astrocyte labelled with the styryl pyridinium dye FM4-64 imaged with
120 nm penetration depth in standard TIRF control (CTR, left) and SCE. Images are
scaled to identical intensites. Note the enhanced visibility of small fluorescent puncta
invisible on the CTR image, due to the reduction of background with SCE.
Fig.6: Partner 6: FLIN-application with left: clover leave-type rotational motion of Qdots bound to F1-ATPase, right: reference measurement at FLIN-Centre/EuroPhoton
GmbH.
Fig.7: Partner 7: development of artificial fluorescent molecular motor: Bodipy-labeled
molecular shuttle
Fig.8: Partner 8: example of LPO (long-period observation): Double-transfection of a
hippocampal cell culture with GFP-ProSap1 (green) and tagRFP-Mito (red)) revealed
bidirectional transport of mitochondria and the postsynaptic protein along dendrites. Depicted
Time points A-I are a selection of the entire sequence with intervals of approx. 9 min in
between.
Fig.9: Partner 9: Molecular modeling of F1 ATPase with a Qdot. Collaboration with partner 6.
Fig.10: left: DL-anode Gen II, right: ATR-19 electronics for DL-line, Gen III.
Fig. 11a: Partner 11 (joint development of internal and external MA-systems with Partner 1).
Experimental setup at EuroPhoton GmbH for testing MCP detectors. Positions: 1 – optical
bench, 2 – mask carrying enclosure, 3 – dark cylinder with lens inside, 4 – detector 40 mm with
DL applied, 5 – amplifiers/discriminators type ATP19, 6 – delay cables, 7 – crate (Minibin &
power supply), 8 – discriminator of timing signal from MCP type TC454 by TENNELEC, 9 and
10 –new electronics (9 – board with 3 TACs, 10 – motherboard), 11 – power supply for timing
electronics.
Fig. 11b: 10-ch dual purpose MA/DL electronics developed by Partner 11 and constructed and
setup in Berlin with partner 1. Top left: external QA-anode with peripheral ring, top right: 25DL
detector with external 3x3 anode, middle: electronic box, bottom: detector housing with 25DL
and 3x3 anode, mounted on an OptoSplit unit (partner 1).
2. Dissemination and Use
Section 1 - Exploitable knowledge and its Use
Exploitable
Knowledge
(description)
New Microscope
Method: FLIN
Exploitable
product(s) or
measure(s)
FLIN system
MA-System:
joint
production and
marketing
Sector(s) of
application
1. Medical
2. Biological
Timeta
ble for
comme
rcial
use
2008
2010
Patents or other
IPR protection
FLIN patent
planned for
2007
FLIN world
trademark
planned for
2007,
accepted
2008,Japanese
version below.
Owner & Other
Partner(s)
involved
Participant 1,8
Participant 1,8
Participant 1,11
Plan for using and dissemination of knowledge:
Planned/actual
Dates
2010
2009/2010
2008
2007-2008
2007-2008
2007-2008
2005
2006-2008
Type
MA-system:
joint production &
marketing
Review Article: FLIM
and FLIN
Workshop
Conferences
Exhibitions
Publications
Project web-site:
www.euro-FLIN.de
Posters
Type of audience
Count
ries
addres
sed
Size of
audience
Partner
responsible
/involved
1,11
Research and
General public
Students
Research
Industry
Research
General Public
1
Research
9, 1, 8
1, 8
9, 1, 8, 5
1, 8
Dissemination of Knowledge during full Period:
Accepted Posters and Conference Abstracts and Proceedings:
[1] Kemnitz , M. Vitali, W. Zuschratter, Optical Analisys on Biomolecular Machines, July 13-16,
2006, Berlin, Germany, Fluorescence Lifetime Imaging Nanoscopy (FLIN).
[2] K. Kemnitz , M. Vitali, W. Zuschratter, Meeting of the RTN, November 9-11, 2006, Paris, France
From FLIM to FLIN.
[3] F Nadrigny, K Kemnitz, M Vitali, S Rudolph, N Ropert, A Koulakoff, C Giaume, F Kirchhoff, and M
Oheim. (2007). 51st Annual meeting of the Biophysical Society, March 3-7, 2007, Baltimore, MD,
U.S.A. [2219-Pos/B435], Systematic Co-localization Errors between Acridine Orange and EGFP in
Astrocyte Vesicular Organelles.
[4] F Nadrigny, K Kemnitz, M Vitali, S Rudolph, N Ropert, A Koulakoff, C Giaume, F Kirchhoff, and M
Oheim. (2007. Göttingen Meeting of the German Neuroscience Society 2007, March 29 - April 1,
2007, Göttingen, Germany, [T38-2C].
Systematic Co-localization Errors between Acridine
Orange and EGFP in Astrocyte Vesicular Organelles.
[5] J. Vacek, DoD High Performance Computing Modernization Program Users Group Conference: Jun
18-22, 2007, Pittsburgh, PA, USA. Simulations of Molecular Rotors: From Isolated Systems to
Metallo-Organic Frameworks and Crystals.
[6] J. Chocholousova, MC-RTN Workshop and Meeting (tutorial), Debrecen, HU Jun 29-Jul 2, 2007,
Molecular Modelling: from Small to Large Molecular Systems.
[7] J. Chocholousova, J. Vacek, MC-RTN meeting in Paris, Nov 2006. Computer Simulations of
Molecular Machines.
[8] J. Chocholousova, J. Vacek, Vrije Universiteit Amsterdam, Nov 2006, Unidirectionality of Molecular
Rotation: Simulations of Molecular Rotors.
[9] M. van ‘t Hoff, V. de Sars, M. Oheim, MAF10 Conference: “Methods & Applications in
Fluorescence”, 2007 September 9-13, 2007. Salzburg, Austria. A programmable light engine.
[10] M. Vitali, W. Zuschratter, K. Kemnitz, 10th Conference on Methods and Application of
Fluorescence Spectroscopy, 9-12 September 2007, Salzburg. Fluorescence Lifetime
Imaging Nanoscopy (FLIN).
[11] Marco Vitali, Ian Cox, Anatoli Cherni, Martin Oheim, David Dryden, Zorica Ristic,
Alessandro Duci, Christian Goetze, Werner Zuschratter, Dirk Bald, Evgeniy Turbin,
Jaroslav Vacek, Yuri Prokazov, Sergei Stepanov, Sergei Bakhlanov, Evgeniy Drobchenko,
Klaus Kemnitz, Praha: EuroNanoForum 2009, Fluorescence Lifetime Imaging Nanoscopy.
[12] Joint STREP/MCRTN Symposium: 2nd Period Meeting Potsdam: Prague, February 13-16,
2008.
[13] 3rd Period Meeting of the Specific Targeted Research Project / STRP 013880, Potsdam,
1.10.2008 - 4.10.2008, Seminaris Seehotel, Single Motor-FLIN: Long Period Observation of
Single (Bio)-Molecular Motors by Minimal-invasive Fluorescence Lifetime Imaging
Nanoscopy (FLIN).
Publications:
[1] F Nadrigny, D Li, K Kemnitz, N Ropert, A Koulakoff, S Rudolph, M Vitali, C Giaume, F Kirchhoff,
and M Oheim. (2007), Biophys. J. BIOPHYSJ/2006/102673 AOP (advanced online publication).
Systematic Co-localization Errors between Acridine Orange and EGFP in Astrocyte Vesicular
Organelles.
[2] Vacek J., Michl J.: Advanced Functional Materials 17(5), 730, 2007. Artificial surface-mounted
molecular rotors: Molecular dynamics simulations.
[3] Vacek, J., Chocholousova J., Michl J, Proceedings of the HPCMP users group conference, IEEE
Computer Society, Los Alamitos, CA, 2007, Calculations for a Gas-Flow Driven Molecular Rotor,
accepted.
[4] J. Vacek, J. Chocholoušová, L. Kobr, J. Miller, J. Michl, Proceedings of the HPCMP users group
conference, IEEE Computer Society, Los Alamitos, CA, 2006, 193.
[5] F. Nadrigny, K. Kemnitz, A. Koulakoff, M. Vitali, S. Rudolph, N. Ropert, F. Kirchhoff, C.
Giaume, and M. Oheim, Biophys. J BioPhys. Let., ,,,,,,,,, ? Systematic Co-localization Errors
in Acridine Orange (AO)/EGFP Dual-Color Images due to AO Metachromasy.
[6] F. Nadrigny, D. Li, K. Kemnitz, N. Ropert, A. Koulakoff, S. Rudolph, M. Vitali, C. Giaume,
F. Kirchhoff, and M. Oheim, Biophys. J., 93(2007)969-980. Systematic Co-localization
Errors between Acridine Orange and EGFP in Astrocyte Vesicular Organelles.
[7] M. Vitali, Z. Ristic, A. Duci, D. Bald, W. Zuschratter, and K. Kemnitz, in preparation.
Fluorescence Lifetime Imaging Nanoscopy: Application in Study of Molecular Motors.
[8] Sergei Stepanov, Sergei Bakhlanov, Evgeniy Drobchenko, Klaus Kemnitz, in preparation.
High-Performance Widefield TSCSPC Systems with Large-Area Detectors: Applications in
Simultaneous Multichannel-FLIM.
[9] Yakovlev AV, Zhang F, Zulqurnain A, Azhar-Zahoor A, Luccardini C, Gaillard S, Mallet
JM, Tauc P, Brochon JC, Parak WJ, Feltz A, Oheim M. 2009, Wrapping nanocrystals with
an amphiphilic polymer preloaded with fixed amounts of fluorophore generates FRET-based
nanoprobes with a controlled donor/acceptor ratio. Langmuir 25(5):3232-9.
[10] van t Hoff M, Reuter M, Dryden DTF, Oheim M. 2009. Screening by imaging: scaling up
single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noisereduction techniques. Phys. Chem. Chem. Phys. DOI: 10.1039/b823155a.
[11] Erick B. Winston, Peter J. Lowell, Jaroslav Vacek, Jana Chocholou ová, Josef Michl and
John C. Price, Phys. Chem. Chem. Phys., 2008, 10, 5188, Dipolar molecular rotors in the
metal–organic framework crystal IRMOF-2.
[12] Vacek, J.; Caskey, D.C.; Horinek, D.; Shoemaker, R.K.; Stang, P.J.; Michl, J. J. Am. Chem. Soc.,
2008, 130, 7629.
[13] Caskey D.C.; Yamamoto, T.; Addicott, C.; Shoemaker, R.K.; Vacek, j.; Hawkridge, A.M.;
Muddiman, D.C.; Kottas, G.S.; Michl, J.; Stang, P. J. Am. Chem. Soc., 2008, 130, 7620.
[14] Vacek J., Chocholousova J., Michl J., Calculations of Lithium+ Carborane Complexes,
hpcmp-ugc,pp.175-179, 2008 DoD HPCMP Users Group Conference, IEEE Computer
Society, Los Alamitos, CA, 2008.
Coordinator: Dr. Klaus Kemnitz, EuroPhoton GmbH (Germany), Mozartstr. 27, D-12247
Berlin,
Germany.
Tel.:
+49-30-771-90145,
Fax.:
+49-30-771-4450,
E-mail:
KlausKemnitz@aol.com
Partners:
Partner 1 (Co-ordinator): EuroPhoton GmbH (Germany), Mozartstr. 27, D-12247 Berlin, Germany, Dr.
Klaus Kemnitz, Tel.: +49-30-771-90145, Fax.: +49-30-771-4450, E-mail: KlausKemnitz@aol.com
Partner 2: Photek Ltd., Castleham Road 26, TN38 9NS St Leonards-on-Sea, United Kingdom, Dr. Jon
Howorth, Tel.: +44-1424-850555, Fax.: +44-1424-850051, E-mail: sales@photek.co.uk
Partner 3: Petersburg Nuclear Physics Institute Russian Academy of Sciences, Orlowa Roscha 1,
Gatchina, 188300 St.-Petersburg, Russian Federation, Prof. Vladimir Nazarenko, Tel.: +7-813-7130036,
Fax.: +7-813-7136025, E-mail: vnazar@pnpi.spb.ru
Partner 5: Institut National de la Sante et de la Recherche Medicale, Rue Tolbiac 101, F-75013 Paris,
France, Dr. Martin Oheim, Tel.: +33-1-42864221, Fax.: +33-1-42864151, E-mail: martin.oheim@univparis5.fr
Partner 6: Vrije Universiteit Amsterdam, De Boelaan 1085, 1081 HV, Amsterdam, Netherlands, Dr. Rob
NeutelingsTel.: +31-20-5987301, Fax.: +31-20-6462457, E-mail: rob.neutelings@falw.vu.nl
Partner 7: The University of Edinburgh, Old College South Bridge, EH8 9YL Edinburgh, United
Kingdom, Mr. Derek Waddell, Tel.: +44-131-6509024, Fax.: +44-131-6509023, E-mail:
angela.noble@ed.ac.uk
Partner 8: Leibniz Institute for Neurobiology Magdeburg, Brenneckestr. 6, D-39118, Magdeburg,
Germany, Mr. Gerd Brandt, Tel.: +49-391-6263117, Fax.: +49-391-6263118, E-mail: zuschratter@ifnmagdeburg.de
Partner 9: Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic,
Flemingovo nam. 2, 16610 Praque, Czech Republic, Dr. Zdenek Havlas, Tel.: +420-220-183-333, Fax.:
+420-220-183-578, E-mail: uochb@uochb.cas.cz
Partner 10: Roentdek GmbH, Im Vogelshaag 8, D-65779 Kelkheim, Germany, Dr. Ottmar Jagutzki,
Tel.: +49-69-798-21600, Fax.: +49-69-798-21601, E-mail: jagutzki@atom.uni-frankfurt.de
Partner 11: Russian Research Centre Kurchatov Institute (since 8.3.2007), Kurchatov sq. 1, Moscow,
123182 Russian Federation, Dr. E.V. Turbin, E-mail: turbin.evgeniy@gmail.com, TTC-extension.