Singlemolecule fluorescence spectroscopy

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Single-molecule fluorescence spectroscopy

• Lecture 12
• October 11th

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Announcement(s)
The students will attend the seminar that will
be held by the theoretical chemist Harold
Scheraga (Cornell), in Chemistry on October 11th,
400 PM, Room 1-019, Center for Science
Technology
The term paper submission will be accompanied by
a short comprehensive talk (20 min 5 min qns)
that represents an overview in the field. This
will be a good practice for other future
occasions. Two future sessions will be devoted
to your talks!
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why study biology at the single-molecule level??
conformational change of a protein
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why study biology at the single-molecule level??
- observation of new science in unexplored regimes
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Outline

• What is fluorescence??
• Fluorescent molecules
• Equipment for single-molecule fluorescence
  experiments
• Some applications examples

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fluorescence from molecules physical fundaments
photon
molecule in excited state
molecule in ground state
photon
light can induce transitions between electronic
states in a molecule
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fluorescence the Jablonski diagram
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fluorescence properties that can be measured

• spectra (environmental effects)
• fluorescence life times
• polarization (orientation and dynamics)
• excitation transfer (distances -gt dynamics)
• location of fluorescence

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fluorescence requirements for a good fluorophore

• good spectral properties
• strong absorber of light (large extinction
  coefficient)
• high fluorescence quantum yield
• low quantum yield for loss processes (triplets)
• low quantum yield of photodestruction
• small molecule / easily attachable to
  biomolecule to be studied

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1.7 Fluorescence quantum yield
S1
knr
kr
S0
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fluorescence chromophores intrinsic or
synthetic??

• common intrinsic fluorophores like tryptophan,
  NAD(P)H are not good enough
• chlorophylls flavins work
• in most cases extrinsic fluorophores have to be
  added
• genetically encoded (green fluorescence
  protein)
• chemical attachment of synthetic dyes

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fluorescence a typical synthetic chromophore
tetramethylrhodamine
580
550

• extinction coefficient 100,000 Molar-1 cm-1
• fluorescence quantum yield 50
• triplet quantum yield lt1
• available in reactive forms (to attach to amines,
  thiols) and attached to many proteins and other
  compounds (lipids, ligands to proteins)

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the fluorescence of a single TMR can be measured
easily
extinction coefficient (e) 100 000 M-1 cm-1

• area of an opaque object with the same that
  blocks the light as good as the molecule
• dI/I (sCNAv/1000)dL
• dI/I e2.303dL

absorption cross section (s) s e 2303 /
N0 410-16 cm2
excitation power 100 W/cm2
excitation photon flux power / photon
energy 2.5 1020 photonss -1cm-2 photon
energy hc/l
excitationsmolecule-1s-1 exc
fluxs 105 photonss -1cm-2
emitted photonsmolecule-1s-1 em excQY
105 photonss -1cm-2
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single-molecule fluorescence microscopy

• excitation source laser
• Lasers cw (ion), pulsed (Nd-YAG, Ti-sapphire,
  diodes

• optical system with high collection
  efficiency high NA objective

• optics to separate fluorescence from
  excitation light filters / dichroic mirrors
• monochromators, spectrographs filters colored
  glass, notch holographic, multidielectric

• detector - CCD camera, PMT - eyes
  PMT, APD, CCDPhotoMultiplier Tube, Avalanche
  PhotoDiode,
• Charge Coupling Device (signal is usually weak)
  electronics

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rotation of F1-ATPase
Adachi, K., R. Yasuda, H. Noji, H. Itoh, Y.
Harada, M. Yoshida, and K. Kinosita, Jr. 2000.
Proc. Natl. Acad. Sci. U.S.A. 977243-7247
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folding / unfolding of RNA (Tetrahymena
ribozymes)
X. Zhuang, L. Bartley, H. Babcock, R. Russell, T.
Ha, D. Herschlag, and S. Chu Science 2000 June
16 288 2048-2051.
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FLUORESCENCE MEASUREMENTS

• Information given by each property of
  fluorescence photons- spectrum- delay after
  excitation (lifetime)- polarization

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Spectra
Fluo. intensity
Sample
Detector
Laser ?exc
?fluo
?exc
?fluo
Spectrograph
Excitation spectrum Fluorescence spectrum
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Solvent effects
Energy
Non-polar solvent
Polar solvent
S1
S1
S1
S0
S0
Static molecular dipole moment
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Fluorescence Lifetime
Sample
number
Pulsed laser
Filter
delay, t
Detector
Laser pulses
time
delay
photons
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Polarization
Fluid
Rigid
depolarized
polarized
Polarization memory during the fluorescence lifeti
me fluo. anisotropy
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Fluorescence Resonance Energy Transfer (FRET)
Dipole-dipole interaction (near-field)
Donor
Acceptor
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Transfer Efficiency

• Fraction of excitations transferred to acceptor
• R0 Förster radius, maximum 10 nm for large
  overlap

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Förster Resonance Energy Transfer



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FRET studies of interaction and
dynamics(molecular ruler)
Association of two biomolecules
Dynamics of a biomolecule
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Other specific labeling and imaging

• Possibility to specifically label certain
  biomolecules, sequences, etc. with fluorophores
• Staining and imaging with various colors
• Detection of minute amounts (DNA assays)
• Fluorescence lifetime imaging (FLIM)
• Fluorescence recovery after photobleaching

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multicolor2-photonmicroscopy
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specific labeling with various colors
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Fluorescence Correlation Spectroscopy
Keeps track of the fluctuations of the
fluorescence intensity.
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Single molecule spectroscopy

• Single molecule tracking
• dynamics of single enzyme
• sp-FRET
• orientation fluctuations
• lifetime measurement

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5 mm
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Immobilized Molecules
Signal/Background ratio must be large enough
or
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Microscopy images

• Counting, stoichiometry, colocalization
• Orientation

Comparison of polarization modulation for
epi-illumination and total internal reflection.
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Orientation and position of single molecules
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CONCLUSION

• Versatile (many labels)
• Non-invasive
• Sensitive
• Gives information on both structure and dynamics