Transcript Methods: Fluorescence

Methods:
Fluorescence
Biochemistry 4000
Dr. Ute Kothe
Remember: Absorbance
Absorbance of monochromatic light reduces the intensity (I)
Measured relatively to original intensity (I0)
Depends on path length (l, often 1 cm), concentration (c) and
molar extinction coefficient (e, units: M-1 cm-1)
 Used to measure concentrations
Beer-Lambert law
log (I0/I) = A = ε l c
Is very fast & provides information only on average ground state
of molecules; energy is set free by non-radiative decay (heat)
Fluorophores
Often aromatic organic molecules
Only atoms that are fluorescent:
Lanthanides (europium, terbium)
What is Fluorescence?
Upon excitation of a
fluorophore, it re-emits light
at a longer wavelength.
Emission spectra
– typically independent of
excitation wavelength
Excitation spectra
Why Fluorescence?
Highly sensitive Detection in small quantities
non-dangerous
sensitive to environment
Information on:
• Interactions of solvent molecules with fluorophores
• Rotational diffusion of biomolecules
• Distances between sites on biomolecules
• Conformational changes
• Binding interactions
• Cellular Imaging
• Single-Molecule Detection
Intrinsic & Extrinsic Fluorophores
Intrinsic Fluorophores:
Occur naturally
• Trp, Tyr, Phe
• NADH, FAD, FMN, Chlorophyll
• Etc.
Extrinsic Fluorophores:
Added artifically to a sample
• Dyes binding DNA (ethidium bromide)
• Labelling of amino groups (dansyl
chloride, fluorescein isothiocyanate)
• Labelling of sulfhydryl groups
(maleimide dyes)
• etc.
Fluorescence Spectrometer
Light Source: Xenon Lamp or Laser
Excitation Monochromator
Sample Cell
Emission Monochromator
Detector: Photomultiplier
Fluorescence Plate Reader

For fast highthroughput
measurements
in multiple
well plates
Jablonski Diagram
Allowed singlet states:
e- in excited orbital is paried by opposite
spin to second e- in ground-state orbital
Forbidden triplet states
due to spin conversion
Franck-Codon Principle: all electronic transitions occur without
change in the position of the nuclei (because they are too fast for
siginificant displacement of nuclei).
Stokes Shift
The energy of emission is
typically less than the
energy of absorption. Thus
Fluorescence occurs at
longer wavelengths.
Fluorescence
Solvent effects
General Solvent Effects:
Fluorescence is highly dependend on solvent polarity!
 Tool to detect environement of fluorophor!
• Dipole moment of excited state larger than ground state
• solvent molecules reorient around excited dipole
• thus, solvent molecule lower the energy of the excited state
• Emission is shifted to longer wavelength
N: native state
U: unfolded state
Specific Solvent Effects:
Chemical reactions of excited state with solvent,
e.g. H-bonding, acid-base reactions etc.
Quantifiying Binding Interactions
Binding of Mant-GTP (●) and Mant-GDP (▲) to EF-G
[Ligand]
F = -----------------[Ligand] + KD
 KD determination
Environment of fluorophore, mant-nucleotide, changes upon binding to
EF-G, i.e. the polarity of the surrounding changes and thus the
fluorescence.
Resonance Energy Transfer
Transfer of energy from donor fluorophore to acceptor molecule
If donor emission spectra overlaps with acceptor absorption spectra
No intermediate photon!
D and A are coupled by
dipole-dipole interactions
Distance Dependent
Spectroscopic Ruler
Distance Dependence of Fluorescence Resonance Energy
Transfer (FRET) can be used to measure distances between two
dyes, e.g. Attached to different interacting proteins.
Förster radius (R0):
Distance of 50% energy transfer
Depends on dye pair
Typically 30 – 60 Å
E
Distance, Å
Efficiency of energy transfer:
R 06
E = -------------------R06 + r6
r = distance between Donor and Acceptor
Example: Protein Interactions
Nucleic Acid Detection
Detection of nucleic
acids by fluorescently
labeled oligonucleotides
Common dyes:
Cy3 & Cy5
Applications:
• Molecular Beacons
(see figure)
• cellular imaging
• microarrays
• etc.