1 introduction - uni- · PDF file1 introduction michael börsch 09/05/2003. 2...
Transcript of 1 introduction - uni- · PDF file1 introduction michael börsch 09/05/2003. 2...
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singlesingle--moleculemoleculefluorescencefluorescencespectroscopyspectroscopy
1 introduction
michael börsch09/05/2003
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single-molecule topics• 13 years : from the beginning • imaging a single fluorophore in a solid host• diffusion in solution • fluorescence correlation spectroscopy FCS• blinking, spectral shifts, lifetime• anisotropy / polarization• fluorescence resonance energy tranfer FRET• fluorophores, quantum dots, GFP`s
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history• first single-molecule detection 05/1989• first single-molecule fluorescence detection 1990 • first book 1996• feature articles: Science 03/1999,
Chem. Phys. 09/1999• reviews by R.A. Keller and M. Orrit 12/1999
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• Probing the free-energy surface for protein folding with single-molecule fluorescencespectroscopy
• Single photons on demand from a single molecule at room temperature • Optical microscopy using a single-molecule light source• Single-molecule analysis of DNA uncoiling by a type II topoisomerase• Single-molecule optical switching of terrylene in p-terphenyl• On/off blinking and switching behaviour of single molecules of green fluorescent protein• Three-dimensional orientation measurements of symmetric single chromophores using
polarization microscopy• Quantum optics: A box for a single photon• Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase• Imaging of single fluorescent molecules and individual ATP turnovers by single myosin
molecules in aqueous solution.----------------------• Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated
Virus• Single-Molecule Enzymatic Dynamics• Discrete Intensity Jumps and Intramolecular Electronic Energy Transfer in the
Spectroscopy of Single Conjugated Polymer Molecules• Nonlinear Spectroscopy on a Single Quantum System: Two-Photon Absorption of a Single
Molecule• A Single-Molecule Study of RNA Catalysis and Folding• Single-Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells
1998 -2003 nature, science paper
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motivationdistribution of
molecular parametersprobes for the local
environmentScreening-methods
ultrasensitive analysis
• optical spectra• chemical reactivity• enzymatic activity• binding affinity
• intra-cellular biochemistry• molecular transport• protein folding• enzyme dynamics
• drug research• detection of pathogenes• medical diagnostics
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fluorophores IV
QuantumDotCorp 565-585-605-streptavidin in kidney cells
NanorodsT. NannUni Freiburg
nanocrystalssemiconductor quantum dots,
CdSe with capping
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fluorescence of R6G in H2O
• excitation volume: 5mm x 2mm x 2mm = 20 µl• R6G 200 pM ⇒ 2.4•109 Molecules• H2O 55 M ⇒ 6.6•1020 Molecules• ratio 1 : 2.8 • 1011
550 600 650 700 7500
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dark counts R6G 200 pM, 11 µW 532 nm R6G 200 pM, 45 µW 532 nm
phot
on c
ount
s / 5
00 m
s
wavelength / nm550 600 650 700 750
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dark counts H2O, 11 µW 532 nm H2O, 45 µW 532 nm
phot
on c
ount
s / 5
00 m
s
wavelength / nm
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two major problems:Rayleigh and Raman scattering
(1) high efficienyoptical filters
Background ~ V
(2) minimizing the detection volume
Background ~ λ4
(3) 'red dyes', NIR, longer wavelengths
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principles of single-molecule detection
S
S
T
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photon burst
trajectory of a molecule
detection area
detection area
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methods for SMD
in solution
on surfaces
FCS flow cell microdroplets
confocal microscopewidefield or TIRFplus CCD camera near field microscope
Courtesy: Jörg Enderlein, 1999. Forschungszentrum Jülich
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Set-up 06/11/2000Institute for Physical ChemistryUniversity of Freiburg
• laser : 532 nm, 50 mW cw• objective : 60x, N.A. 1,2 W• two SPAD
1: 545 - 610 nm2: LP 665 nm
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ObjectiveScanning
Photo DiodeSPAD
ConfocalAperture
EmissionFilter
DichroicMirror
SemireflectingMirror
Excitation Filter
Drop of solvent
Laser
PiezoActuator
Set-up for SMD
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methods for SMD IIFCS flow cell microdroplets
confocal microscopewidefield or TIRFplus CCD camera near field microscope
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line scanning 2D with 'fixed' dyeCy5, NSOMN. van HulstUni Twente
LH2photobleachingR. HochstrasserPenn. Uni
rotating R6GA. Meixner
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methods for SMD IIIFCS flow cell microdroplets
confocal microscopewidefield or TIRFplus CCD camera near field microscope
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imaging with CCD, widefield, TIRF
ribozyme folding,single-molecule FRET,X. Zhuang,Harvard Uni
membrane-embeddedproteinG. SchützUni Linz
ATP bindingto myosinT. Yanagida
DsRED
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summary• SMD starts in 1989• distribution of molecular parameters• probes the local environment• based on fluorescence / classes of fluorophores• burst of photons due fast excitation cycle• intensity, spectral range, lifetime, anisotropy• imaging, tracking • several methods: in solution, on surfaces, in host
systems, interaction between two fluorophores
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next 23/05/2003• a single molecule as reporter in a solid host • distribution of molecular parameters (physics)• probes the local environment (chemistry)• intensity, spectral range, lifetime, anisotropy• 'blinking' , 'spectral diffusion' , 'anti-bunching' ,
'photon statistcs' , 'autocorrelation function' , 'ODMR'
• www.m-boersch.org slides + literature • email: [email protected]