Fluorescence Spectroscopy

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Fluorescence Spectroscopy

• Part I. Background

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Perrin-Jablonski diagram
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S is singlet and T is triplet. The S0 state is
the ground state and the subscript numbers
identify individual states.
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Energy level of MO
n ? p lt p ? p lt n ? s lt s ? p lt s ? s
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Singlet Triplet
DS?0
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Characteristics of Excited States

• Energy
• Lifetime
• Quantum Yield
• Polarization

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Stokes shift

• The Stokes shift is the gap between the maximum
  of the first absorption band and the maximum of
  the fluorescence spectrum

loss of vibrational energy in the excited state
as heat by collision with solvent
heat
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Example 7-amino-4-methylcoumarin (AMC)
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Example
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Example fluorophores
fluorescein
ethidium bromide bound to DNA.
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Lifetime
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Lifetime

• Excited states decay exponentially with time
• I I0e-t/t
• I0 is the initial intensity at time zero,
• I is the intensity at some later time t
• t is the lifetime of the excited state.
• kF 1/ t, where kF is the rate constant for
  fluorescence.

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Quantum Yield

• Quantum Yield FF
• FF number of fluorescence quanta emitted
  divided by number of quanta absorbed to a singlet
  excited state
• FF ratio of photons emitted to photons
  absorbed
• Quantum yield is the ratio of photons emitted to
  photons absorbed by the system

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Quantum Yield
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Quantum Yield Structure rigidity
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Polarization

• Molecule of interest is randomly oriented in a
  rigid matrix (organic solvent at low temperature
  or room temperature polymer). And plane polarized
  light is used as the excitation source.
• Degree of polarization is defined as P

I and I are the intensities of the observed
parallel and perpendicular components, a is the
angle between thee mission and absorption
transition moments. If a is 0 than P
1/2, and if a is 90 than P -1/3.
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Experimental Measurements
Steady-state measurements F, I Time-Resolved
measurements t
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Instruments
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Inner Filter Effect

• At low concentration the emission of light is
  uniform from the front to the back of sample
  cuvette.
• At high concentration more light is emitted from
  the front than theback.
• Since emitted light only from the middle of the
  cuvette is detected the concentration must be low
  to assure accurate FF measurements.

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Inner Filter Effect
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Measurement of fluorescence quantum yields
fraction of intensity emitted at that particular
wavelength
fraction of total fluorescence that is detected
If (?em) IAbs (?ex). ?f . f(?em). K
fluorescence quantum yield
absorbed intensity at ?ex
If A?0
measured intensity of fluorescence at ?em
If we measure the sample and a standard under
the same experimental conditions, keeping ?ex
constant
Standards
Quinine sulfate in H2SO4 1N ?f 0.55
Fluorescein in NaOH 0.1N ?f 0.93
Important the index of refraction of the two
solvents (sample and standard) must be the same
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Measurement of fluorescence lifetimes
pulsed source
Start PMT
?t
Time correlated single photon counting
exc. monochromator
The TCSPC measurement relies on the concept that
the probability distribution for emission of a
single photon after an excitation yields the
actual intensity against time distribution of all
the photons emitted as a result of the
excitation. By sampling the single photon
emission after a large number of excitation
flashes, the experiment constructs this
probability distribution.
Stop PMT
emission monochromator
sample
different excitation flashes
. . . .
events
t (nsec)
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Intrinsic Fluorescence of Proteins and Peptides
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Tryptophan

• Tryptophan, the dominant intrinsic fluorophore,
  is generally present at about 1mol in proteins.
  A protein may possess just one or a few Trp
  residues, which facilitates interpretation of the
  spectral data.
• Tryptophan is very sensitive to its local
  environment. It is possible to see changes in
  emission spectra in response to conformational
  changes, subunit association, substrate binding,
  denaturation, and anything that affects the local
  environment surronding the indole ring. Also, Trp
  appears to be uniquely sensitive to collisional
  quenching, either by externally added quenchers,
  or by nearby groups in the protein.
• Tryptophan fluorescence can be selectively
  excited at 295-305 nm. (to avoid excitation of
  Tyr)

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Example Tyrosine and its derivatives
I
II
III
IV
V
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II
I
III
V
I
IV
V
III
IV
II
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• The position and structure of the fluorescence
  suggests that the indole residue is located in a
  completely nonpolar region of the protein. These
  results agree with X-ray studies, which show that
  the indole group is located in the hydrophobic
  core of the protein.
• In the presence of a denaturing agent, the TrpP
  emission loses its structure and shifts to 351nm,
  characteristic of a fully exposed Trp residue.
• Changes in emission spectra can be used to follow
  protein unfolding

Emission spectra of Pseudomonas fluorescens
azurin Pfl. For 275-nm excitation, a peak is
observed due to the tyrosine residue(s)
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Example Time-resolved protein fluorescence
I(?,t)??i(?)exp(-t/?i)
i
Resolution of the contributions of individual
tryptophan residues in multi-tryptophan proteins.
Fluorescence intensity (A.U.)
wavelength (nm)
?12ns, ?2 5ns
? ?em
Fluorescence intensity (A.U.)
t (ns)
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Green fluorescent protein (abbreviated GFP
Isolated from the Pacific jellyfish Aequorea
victoria and now plays central roles in
biochemistry and cell biology due to its
widespread use as an in vivo reporter of gene
expression, cell lineage, protein protein
interactions and protein trafficking One of the
most important attributes of GFP which makes it
so useful in the life sciences is that the
luminescent chromophore is formed in vivo, and
can thus generate a labeled cellular
macromolecule without the difficulties of
labeling with exogenous agents.
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• The structure of GFP eleven-strand beta-barrel
  wrapped around a central alpha-helix core. This
  central core contains the chromophore which is
  spontaneously formed from a chemical reaction
  involving residues Ser 65, Tyr 66, and Gly 67
  (SYG)
• There is cyclization of the polypeptide backbone
  between Ser 65 and Gly 67 to form a 5-membered
  ring, followed by oxidation of Tyr 66.
• The high quantum yield of GFP fluorescence
  probably arises from the nearly complete
  protection of the fluorophore from quenching
  water or oxygen molecules by burial within the
  beta-barrel.

Ribbon diagram of the Green Fluorescent Protein
(GFP) drawn from the wild-type crystal structure.
The buried chromophore, which is responsible for
GFP's luminescence, is shown in full atomic
detail.
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Wild type GFP from jellyfish has two excitation
peaks, a major one at 395 nm and a minor one at
475 nm with extinction coefficient of 30,000 and
7,000 M-1 cm-1, respectively. Its emission peak
is at 509 nm in the lower green portion of the
visible spectrum. For wild type GFP, exciting
the protein at 395 nm leads to rapid quenching of
the fluorescence with an increase in the 475 nm
excitation band. This photoisomerization effect
is prominent with irradiation of GFP by UV light.
In a wide range of pH, increasing pH leads to a
reduction in fluorescence by 395 nm excitation
and an increased sensitivity to 475 nm
excitation.
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Melittin
GIGAVLKVLT TGLPALISWI KRKRQQX
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Example Carboxyfluorescence
Biochemical Education 28 (2000) 171173
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Example Carboxyfluorescence
Quenching Effect
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Example Carboxyfluorescence
pH Effect