Title: Introduction to Fluorescence Spectroscopy
1Introduction to Fluorescence Spectroscopy
- Chapter 1
- Principles of Fluorescence Spectroscopy
2Why 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
3Experimental 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
4Myosin in Muscle Contraction Convert Chemical
Energy into Mechanical Work
Sliding Filaments
5Crossbridge Cycle
Weak-Binding
Strong-Binding
Lymn and Taylor, 1971
6Myosin Structure
Motor Domain
Actin-Binding Region
Active Site
Actin-Binding Cleft
Lever Arm
Rayment et al., 1993
7Actomyosin Structure
FlAsH Site (residues 292-297)
Pro294
Nucleotide-binding pocket
1,5 IAEDANS
mantATP
Cys374
8FRET mantNucleotide MV FlAsH Pair
Nucleotide-Binding Pocket More Closed in the
presence of ATP than ADP
9IAEDANS-actin MV FlAsH FRET
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13Cy3 Molecules on surface
Tracking
Single Step Photobleaching
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16New 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.
17Tracking Cell Death
- In-Vivo Imaging Emission in the near infrared
- Quenching activation strategy
18Overcoming 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
19Quantum 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
20DNA 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
21Fluorescence 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
22Experimental Systems?
Engineering Surfaces
Engineering cell surfaces
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27Jablonski 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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29- 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
30Characteristics 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
31Emission 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
32Exceptions 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
33Excited State Reactions
Excited state dimers formation of pyrene dimers
at higher concentrations (Excimer).
Charge transfer reaction between anthracene and
diethylaniline (at longer wavelengths).
34Fluorescence 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)
35Fluorescence 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
36Time 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
37Fluorescence 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
38Resonance 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
39Steady-state and time-resolved
- Steady-state constant illumination and
observation - Time resolved - pulsed excitation and high speed
detection
40Why 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
41Biochemical 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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43PBFI- Potassium sensor
44Molecular 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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48Simplified Jablonski Diagram