Fluorescence Spectroscopy

1
Fluorescence Spectroscopy

• Physical Biochemistry, November 2006
• Dr Ardan Patwardhan, a.patwardhan_at_imperial.ac.uk,
  Faculty of Natural Sciences, Imperial College
  London

2
Frank-Condon principle

• As the time-scale for an electronic transition is
  much shorter than for vibrational transitions, it
  can be assumed that inter nuclear distances will
  not change during the transition
• The transition most likely to occur is the one
  for which the initial and excited wavefunctions
  overlap the most

3
Fluorescence

• An excited molecule, e.g. in solution, may lose
  its excess vibrational energy in collisions
  (internal conversion)
• As a consequence, the emitted photon has a longer
  wavelength (lower energy) than the absorbed
  photon
• Usually requires flat rigid molecules with
  extensive conjugation and delocalization

4
Phosphorescence

• A collision may cause an excited electron to flip
  spin
• This is know as an intersystem crossing and
  leaves the molecule in a triplet state
• Transitions from triplet to singlet states via
  the emission of EMR are forbidden
• The lifetime of the triplet state is very long
  and can range from seconds to hours

5
Mirror symmetry of spectra

• Often, but not always present
• Thought to be due to the fact that if 0?n
  vibration transition most likely on absorption,
  then the same most likely on emission

6
Dynamics

• Solvent relaxation Solvent cage reorganizes
  itself to better stabilize excited state ? energy
  and lifetime of excited state may change
• Difficult to define general rules for solvent
  effects

7
Fluorescence parameters

• Quantum Efficiency Q.E. Emitted photons/
  Absorbed photons
• The Molar Extinction Coefficient, e (M-1cm-1) is
  the absorbance of a 1 molar solution in a 1 cm
  lightpath at a specified wavelength.
• Brightness (sensitivity) Q.E x e
• Absorption and fluorescence spectra are
  independent

8
Lifetime

• Fluorescence lifetime is the average time that an
  electron spends in the excited state before a
  photon is emitted
• Measurement of the fluorescence from a large
  number of molecules, following a short pulse
  excitation, will show an exponential decay
• The lifetime is given by the 1/e point of the
  decay

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Basic fluorescence spectrometer

• Source and detector at right angles

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Quenching and photobleaching

• Photobleaching (fading) permanent loss of
  fluorescence due to photo-induced chemical
  modification of molecule
• Quenching Competing processes that induce
  non-radiative relaxation of excited-state
  electrons to the ground state

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Quenching as a tool to probe fluorophore
environment

• Requires contact between fluorophore and quencher
• Typical quenchers iodide, acrylamide and O2

12
Fluorescence Anisotropy

• Sample excited with linearly polarized light
• Anisotropy, , of the
  resulting fluorescence measured
• Reorientation of the molecule within the lifetime
  of the fluorophore will lead to a reduction in
  anisotropy

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Fluorescence Recovery after Photo-bleaching (FRAP)

• Useful technique for studying transport
  properties within a cell, especially
  transmembrane protein diffusion
• Label the molecule with a fluorophore
• Bleach (destroy) the fluorophore is a well
  defined area with a high intensity laser
• Use a weaker beam to examine the recovery of
  fluorescence as a function of time
• Nature Cell Biology  3, E145 - E147 (2001)

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FRAP

• FRAP can be used to estimate the rate of
  diffusion, and the fraction of molecules that are
  mobile/immobile
• Can also be used to distinguish between active
  transport and diffusion

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Fluorescence Resonance Energy Transfer (FRET)

• Non-radiative transfer of excited state energy
  from donor molecule to acceptor molecule, i.e., a
  form of quenching!
• Donor and acceptor molecules must be in close
  proximity (lt10nm)
• Molecular dipoles must be somewhat oriented
• Absorption spectra of acceptor molecule must
  overlap to some extent with the fluorescence
  spectra of donor molecule

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FRET contd

• FRET is very sensitive to the distance between
  donor an acceptor and is therefore an extremely
  useful tool for studying molecular dynamics
• Efficiency ? R-6

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Fluorescence correlation spectroscopy

• Method for observing fluorescence from single
  molecules
• Relies on a low concentration, a small
  illuminated volume and a fast detector
• Using the auto-correlation function simplifies
  averaging over many measurements
• Useful for molecular binding and aggregation
  studies

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Time-resolved Fluorescence Spectroscopy

• Short pulse excitation followed by an interval
  during which the resulting fluorescence is
  measured as a function of time
• Lifetimes can be very sensitive to local
  environment
• Possibly linked to the refractive index of the
  environment
• Lifetime measurements are robust against
  variation in intensity, i.e., an absolute
  measurement

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Intrinsic Fluorescence

• Fluorescence spectra lie in the UV region
• The spectra for both Tyr and Trp are extremely
  sensitive to local environment

20
Solvent effects are useful for studying protein
folding
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Fluorescein as a pH sensor
Only the monoanion and dianion are fluorescent
and the dianion fluorescence dominates!
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Sodium and Potassium ion sensitive probes
Ligand pocket engineered to complex ion of
interest
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Ca2 probes (R Tsien)
Ligand binding twists the lone pairs on the
Nitrogens out of conjugation with the ring
hypsochromic shift
BAPTA
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Ca2 probes contd.
Quin
Fura-2
Conjugation increased to red-shift spectrum and
improve extinction coefficients and quantum yields
25
Main Points

• The fluorescence spectrum is shifted to longer
  wavelengths compared with the absorption spectrum
• Fluorescence spectroscopy allows in general more
  sensitive measurements than absorption
  spectroscopy
• Both the fluorescence spectrum and the lifetime
  are sensitive to the environment