Introduction to Fluorescence Spectroscopy

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Introduction to Fluorescence Spectroscopy

• Chapter 1
• Principles of Fluorescence Spectroscopy

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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

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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

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

Nucleotide-binding pocket
1,5 IAEDANS
mantATP
Cys374
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FRET mantNucleotide MV FlAsH Pair
Nucleotide-Binding Pocket More Closed in the
presence of ATP than ADP
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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.

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Tracking Cell Death

• In-Vivo Imaging Emission in the near infrared
• Quenching activation strategy

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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

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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

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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

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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

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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

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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

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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

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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

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Excited State Reactions
Excited state dimers formation of pyrene dimers
at higher concentrations (Excimer).
Charge transfer reaction between anthracene and
diethylaniline (at longer wavelengths).
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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)

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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

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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

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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

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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

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Steady-state and time-resolved

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

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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

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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
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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