1

singlesingle--moleculemoleculefluorescencefluorescencespectroscopyspectroscopy

1 introduction

michael börsch09/05/2003

2

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

3

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

4

5

history IIsingle-molecule biophysics

• review by S. Weiss 09/2000

6

• 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

7

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

8

fluorescence(1) absorbance of one photon (fs)

9

(2) vibrational relaxation (ps)

fluorescence

10

fluorescence

(3) fluorescence parameters:spectrumquantum yieldlifetimeanisotropy

11

fluorophores

12

fluorophores II

rhodamine 6 G

tetramethylrhodamine

cyanine 5

13

fluorophores III

green fluorescent protein GFP(Uni Leiden; spectra: Clontech)

14

fluorophores IV

QuantumDotCorp 565-585-605-streptavidin in kidney cells

NanorodsT. NannUni Freiburg

nanocrystalssemiconductor quantum dots,

CdSe with capping

15

fluorescence spectrometer

16

sample chamber SLM 8100

17

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

50

100

150

200

250

300

350

400

450

500

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

0

100

dark counts H2O, 11 µW 532 nm H2O, 45 µW 532 nm

phot

on c

ount

s / 5

00 m

s

wavelength / nm

18

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

19

principles of single-molecule detection

S

S

T

0

1

1

photon burst

trajectory of a molecule

detection area

detection area

20

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

21

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

22

ObjectiveScanning

Photo DiodeSPAD

ConfocalAperture

EmissionFilter

DichroicMirror

SemireflectingMirror

Excitation Filter

Drop of solvent

Laser

PiezoActuator

Set-up for SMD

23

R6G in H2O

24

H2O

25

methods for SMD IIFCS flow cell microdroplets

confocal microscopewidefield or TIRFplus CCD camera near field microscope

26

imaging by confocal scanning

x, y

z

27

line scanning 2D with 'fixed' dyeCy5, NSOMN. van HulstUni Twente

LH2photobleachingR. HochstrasserPenn. Uni

rotating R6GA. Meixner

28

methods for SMD IIIFCS flow cell microdroplets

confocal microscopewidefield or TIRFplus CCD camera near field microscope

29

imaging with CCD, widefield, TIRF

ribozyme folding,single-molecule FRET,X. Zhuang,Harvard Uni

membrane-embeddedproteinG. SchützUni Linz

ATP bindingto myosinT. Yanagida

DsRED

30

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

31

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: michael.boersch@physchem.uni-freiburg.de