Methods Fluorescence

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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)

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
                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 high-
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.

                    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

                                                 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
         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

Efficiency of energy transfer:                  Distance, Å
         R 06
E = --------------------
       R 06 + r 6        r = distance between Donor and Acceptor
Example: Protein Interactions
            Nucleic Acid Detection

Detection of nucleic
acids by fluorescently
labeled oligo-

Common dyes:
Cy3 & Cy5

• Molecular Beacons
  (see figure)
• cellular imaging
• microarrays
• etc.

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