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Introduction to Fluorescence Spectroscopy

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Title: Introduction to Fluorescence Spectroscopy


1
Introduction to Fluorescence Spectroscopy
  • Chapter 1
  • Principles of Fluorescence Spectroscopy

2
Why Fluorescence?
  • Extremely sensitive method of detecting and
    observing molecules
  • Can detect femtamolar (10-15) quantities
  • Can detect single molecules
  • Time scale microsecond, nanosecond - observe
    motions in proteins

3
Experimental assays that utilize fluorescence
  • Environmental monitoring
  • DNA sequencing
  • Fluorescence in situ hybridization (FISH)
  • Flow cytometry
  • Cell localization
  • Localization of intracellular movement
  • ELISA
  • Numerous assays linked to fluorescence indicator

4
Myosin in Muscle Contraction Convert Chemical
Energy into Mechanical Work
Sliding Filaments
5
Crossbridge Cycle
Weak-Binding
Strong-Binding
Lymn and Taylor, 1971
6
Myosin Structure
Motor Domain
Actin-Binding Region
Active Site
Actin-Binding Cleft
Lever Arm
Rayment et al., 1993
7
Actomyosin Structure
FlAsH Site (residues 292-297)
Pro294

Nucleotide-binding pocket
1,5 IAEDANS
mantATP
Cys374
8
FRET mantNucleotide MV FlAsH Pair
Nucleotide-Binding Pocket More Closed in the
presence of ATP than ADP
9
IAEDANS-actin MV FlAsH FRET
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Cy3 Molecules on surface
Tracking
Single Step Photobleaching
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New Probes for Glucose Monitoring
  • 1- Brightness
  • 2- Highlight dying cells
  • 3- Pack dyes together without interference
  • Probe attached to metabolically active D-glucose
  • Measure glucose uptake in cancer cells treated
    with anticancer agent
  • Reduced uptake was dependent on the concentration
    of anticancer agent.

17
Tracking Cell Death
  • In-Vivo Imaging Emission in the near infrared
  • Quenching activation strategy

18
Overcoming Quenching
  • Intercalation maximum of one dye per every
    other base-pair
  • Did not self-quench or aggregate
  • Nanostructures used to stain T-cells
  • Non-covalently attached when dye dissociates,
    fluorescence enhances
  • Can be coupled to FRET

19
Quantum Dots
  • CdSe core with a micelle formed from pegylated
    lipid, and paramagnetic lipid
  • Useful for optical and magnetic resonance imaging
  • Extremely bright
  • Large range of excitation/emission propeties
  • Monitor multiple interactions simultaneously

20
DNA Nanosensor
  • FRET based application to detect small
    concentrations of DNA in cells
  • 50 copies or fewer of the target are present
    FRET signal is distinct

21
Fluorescence Resonance Energy Transfer FRET
  • Protein-Protein interactions in living cells
  • Designed FRET pairs of fluorescent proteins with
    the highest efficiency
  • Measure ligand preferences in real time and
    follow dynamics in real time

22
Experimental Systems?
Engineering Surfaces
Engineering cell surfaces
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Jablonski Diagram
  • Transitions between electronic states, 10-15 sec.
    too short for significant displacement of nuclei
    (the Frank-Codon principle)
  • Absorption of light causes S0?S1 transition.
  • Internal Conversion- Relaxation down to the
    lowest vibrational energy level of S1 10-12 sec
  • Fluorescence relaxation to lower energy level
    S1?S0 , the highest vibrational level of S0
  • Mirror image rule spacing of vibrational energy
    levels is similar in S0 and S1
  • Intersystem crossing - S1 spin conversion to
    triplet state (T1) or phosphorescence forbidden
    transition, longer wavelengths and lifetimes

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  • Individual emission maxima vibrational energy
    levels 1500 cm-1 apart
  • Absorption occurs from lowest vibrational energy
    level - At room temperature thermal energy is not
    adequate to populate excited vibrational states
  • Energy difference between S0 and S1 too large for
    thermal energy

30
Characteristics of Fluorescence Emission
  • Stokes shift fluorescence occurs at longer
    wavelength or lower energy than absorption
  • Rapid decay to lowest vibrational energy level of
    S1, and decay to highest energy levels of S0
  • Molecules in excited state can lose energy by
    many processes excited state reactions, solvent
    effects, energy transfer, quenching

31
Emission spectra are independent of the
excitation wavelength
  • Upon excitation to higher energy levels energy
    is quickly dissipated (lowest energy level of S1)
  • Franck-Codon Principle electronic transitions
    occur without change in position of the nuclei
  • Probability of electronic transitions similar in
    absorption and emission

32
Exceptions to mirror image rule
  • P-terphenyl different arrangement of nuclei in
    the excited state, long lived S1 state
  • Excited state reactions charge-transfer complex
    in excited state
  • Excited state complexes pyrene eximers
  • Acridine different pKa for proton dissociation
    in excited state

33
Excited State Reactions
Excited state dimers formation of pyrene dimers
at higher concentrations (Excimer).
Charge transfer reaction between anthracene and
diethylaniline (at longer wavelengths).
34
Fluorescence Lifetimes and Quantum Yields
  • Quantum Yield number of emitted photons relative
    to the number of absorbed photons, Q ?/ (?
    knr)
  • Lifetime average time molecules spends in the
    excited state prior to emitting a photon, ?
    1/(? knr)
  • Q and ? can be modified by factors that affect
    (? knr)

35
Fluorescence Quenching
  • Collisional quenching interaction with excited
    state fluorophore. Stern-Volmer equation
  • F0/F 1 KSVQ 1kq?0Q
  • Static quenching formation of
    fluorophore/quencher complex in ground state

36
Time Scale of Molecular Processes in Solution
  • Absorbance instantaneous 10-15s, average ground
    state that absorbs light
  • Length of time fluorescence molecules remain in
    excited state provides information about
    structural dynamics (protein conf. changes)
  • Solvent relaxation 10-10 s
  • Smaller changes in absorption spectra large
    changes in emission spectra

37
Fluorescence Anisotropy
  • Applications protein-protein interactions,
    fluidity of membranes, immunoassays
  • Photoselection absorption more probable when
    photons electronic vectors are aligned parallel
    to the transition moment of the fluorophore
  • Anisotropy r I - I- / (I 2I- )
  • Polarization P I - I- / (I I- )
  • Rotational diffusion free vs. bound to a
    macromolecule, r r0/(1?/?)
  • correlation time comparable to fluorescence
    lifetime

38
Resonance Energy Transfer (FRET)
  • Overlap of emission (donor) and excitation
    (acceptor) Coupling of dipole-dipole
    interactions
  • Distance between probes determines extend of
    FRET. kT (r) (1/?)(R0/r)6
  • R0 Forster distances are comparable to size of
    macromolecules

39
Steady-state and time-resolved
  • Steady-state constant illumination and
    observation
  • Time resolved - pulsed excitation and high speed
    detection

40
Why Time-Resolved Measurement
  • Important data is lost in the steady-state
    (time-averaged)
  • Example anisotropy decay size and shape of
    macromolecule
  • Detect the presence of more than one
    conformational state

41
Biochemical Fluorophores
  • Intrinsic/Extrinsic flourophores
  • Proteins-tryptophan (indole)
  • Membranes-DPH (fluorescence only in membrane
    bound)
  • DNA weakly fluorescent dye binding (EtBr,
    cationic species), labeled bases used to
    synthesize DNA
  • NADH, FAD
  • Extrinsic probes react with amino or sulfhydryl
    groups
  • Fluorescent ligands ethenoATP, mantATP
  • Fluorescence indicators pH, Ca, Mg, 02,
    other species

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PBFI- Potassium sensor
44
Molecular Information
  • Emission spectra polarity of the fluorophore
  • Quenching accessibility to solvent
  • Anisotropy volume of protein, motions of
    fluorescently labeled region
  • FRET distances between sites on a protein,
    association - dissociation

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Simplified Jablonski Diagram
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