Molecular Spectroscopy: Principles and Biophysical Applications
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Molecular Spectroscopy: Principles and Biophysical Applications

BiCh132 Fall Quarter 2012

Jack Beauchamp

Many of the illustrations and tables used in these presentations were taken from the scientific literature and various WWW sites; the authors are collectively acknowledged.

This presentation is adapted in part from BiCh132 lectures of Professor Barton.

Molecular Probes Handbook -11th Edition (Invitrogen)

Recommended text: “Principles of Fluorescence Spectroscopy” by J. R. Lakowicz (3rd Edition; 2006)

Introduction to Fluorescence Spectroscopy Applications

Useful probe of: environment



chemical reactions

Timescales: visible absorption~ 10-15 sec

vibrations ~ 10-14 sec

emission~ 10-9 sec for allowed transitions

10-6-10-3 sec for forbidden transitions

On these timescales, emission is sensitive to competing processes

Solvent Applications

Collisional vibrational dissipation

~ 10-12s


Intersystem crossing



10-15 s


10-9 s


10-6 – 10-3 s


Simplified Energy Level Diagram

(Jablonski Diagram)









Franck-Condon Principle for Electronic Transitions Applications

Franck–Condon principle energy diagram. Since electronic transitions are very fast compared with nuclear motions, vibrational levels are favored when they correspond to a minimal change in the nuclear coordinates. The potential wells are shown favoring transitions between v = 0 and v = 2.

Franck-Condon Principle for Electronic Transitions Applications

Schematic representation of the absorption and fluorescence spectra corresponding to the energy diagram in previous slide. The symmetry is due to the equal shape of the ground and excited state potential wells. The narrow lines can usually only be observed in the spectra of dilute gases. The darker curves represent the inhomogeneous broadening of the same transitions as occurs in liquids and solids. Electronic transitions between the lowest vibrational levels of the electronic states (the 0-0 transition) have the same energy in both absorption and fluorescence.

Franck-Condon Principle for Electronic Transitions (1926) Applications

Classically, the Franck–Condon principle is the approximation that an electronic transition is most likely to occur without changes in the positions of the nuclei in the molecular entity and its environment. The resulting state is called a Franck–Condon state, and the transition involved, a vertical transition. The quantum mechanical formulation of this principle is that the intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrational wavefunctions of the two states that are involved in the transition.

Edward Condon

Edward Condon Applications

ApplicationsF =fluorescence quantum yield

= fraction of singlets relaxing from excited state via fluorescence

# photons emitted by fluorescence

unless some catalytic chemiluminescent process

Fluorescence Intensity x # excited state molecules

x c I0

kF = rate of spontaneous emission P00 = transition probability

same path for excitation and emission


# photons absorbed

Rate constant for emission


kF + (rate constants for non-radiative pathways)

Fluorescence Quantum Yields

S Applications1







What processes compete with fluorescence?

1. Internal conversion, kic

collision with solvent

dissipation of energy through internal vibrational modes

basically transfer into excited vibrational states of S0

Note - kic increases with T

therefore FF decreases with T

2. Intersystem crossing, Applicationskis

spin exchange converts S to T

get slow spin-forbidden phosphorescence

for metal complexes often a mixture of states

so “luminescence”

3. Collision with quencher, kq

e.q. S1+Q S0+Q*

molecules can quench excited state by:

energy transfer

spin exchange (paramagnetic + spin orbit coupling)

electron transfer or proton transfer (+ energy)








So, what matters are the rates of these competing processes Applications

Note - kF is not temperature dependent but all else is

Decay Kinetics Applicationsof S1

Suppose initially have concentration in S1 of S1(0) then turn off light



fluorescence lifetime (measurable)

If no other processes except fluorescence,


Radiative lifetime


Practical things: Applications




Emitted light

Light source




Can measure steady state or time resolved emission

For lifetimes: - then flash and turn off light and measure decay as a function of time

- flash photolysis

- single photon counting

- streak cameras

- time resolution depends on flash

(also frequency domain measurements - phase modulation)

For quantum yields, need geometry constant and correct for emission detectors

-use standards (actinometry)

Practical (sometimes annoying) Applicationsthings:

Fluorescence Polarization / Depolarization

Principle: When a fluorescent molecule is excited with plane polarized light, light is emitted in the same polarized plane, provided that the molecule remains stationary throughout the excited state (which has a duration of 4 nanoseconds for fluorescein). If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitation


Fluorescence Polarization / Depolarization Applications

Schematic representation of FP detection. Monochromatic light passes through a vertical polarizing filter and excites fluorescent molecules in the sample tube. Only those molecules that are oriented properly in the vertically polarized plane absorb light, become excited, and subsequently emit light. The emitted light is measured in both the horizontal and vertical planes.

Fluorescence Polarization / Depolarization Applications

Here Ill is the intensity of emitted light polarized parallel to the excitation light, and I⊥ is the intensity of emitted light polarized perpendicular to the excitation light. An important property of the polarization that emerges from this equation is that it is independent of the fluorophore concentration. Although this

equation assumes that the instrument has equal sensitivity for light in both the perpendicular and parallel orientations, in practice this is not the case.

Sarah A. ApplicationsWeinreis, Jamie P. Ellis, and Silvia Cavagnero, Dynamic Fluorescence Depolarization: A Powerful Tool to Explore Protein Folding on the Ribosome, Methods. 2010 , 52(1): 57–73. doi:  10.1016/j.ymeth.2010.06.001

Anne ApplicationsGershensonand Lila M. Gierasch, Protein Folding in the Cell: Challenges and Progress, CurrOpinStruct Biol. 2011, 21(1):32–41.

Schematic depiction of a protein folding reaction in the cytoplasm of an E. coli cell, showing vividly how different the environment is from dilute in vitro refolding experiments. The cytoplasmic components are present at their known concentrations. Features of particular importance to the folding of a protein of interest (in orange) are: the striking extent of volume exclusion due to macromolecular crowding, the presence

of molecular chaperones that interact with nascent and incompletely folded proteins (GroEL in green, DnaK in red, and trigger factor in yellow), and the possibility of co-translational folding upon emergence of the polypeptide chain from the ribosome (ribosomal proteins are purple; all RNA is salmon). The cytoplasm image is courtesy of A. Elcock.

Practical Applicationsthings (for a few $ more):

Stokes shift: fluorescent Applicationsemission is red-shifted relative to absorption

Excitation Spectrum – the excitation wavelength is scanned while the emission wavelength is held constant

Emission Spectrum - the emission wavelength is scanned while the excitation wavelength is held constant

- often gives the mirror image of the absorption spectrum

Mirror generally holds because of similarity of the molecular structure and vibrational levels of S0 and S1

Given the Franck-Condon Principle, electronic transitions are vertical, that is they occur without change in nuclear positions. If a particular transition probability between 0 and 2 vibrational levels is highest in absorption, it will also be most probable in emission.

Some Exceptions Applicationsto Mirror Image Rule

1. Contaminants !!

2. Excitation to higher state(s) S2

3. Different geometry in excited state

4. Exciplexes (CT state)

5. Excimers

6. pK effects (excited state acid base properties)

Dimer excited state

Acid-base properties are modified in electronically excited states

Example- pKa for acridine in ground state= 5.5

pKa for acridine in excited state= 10.7

protonation can occur during excited state lifetime

Effects are quantified with use of the Förster Cycle

Think of some applications of this phenomenon

Förster states Cycle: Quantifies changes in acid-base properties in electronically excited states

ArOH (aryl alcohol such as napthol) – The shift in absorption spectra of the acid and its conjugate base can be used to quantify the difference in pKa in the ground and excited electronic state

Fluorescent statesProbes

Absorption and emission spectra of biomolecules. Top: Tryptophan emission from proteins. Middle: Spectra of extrinsic membrane probes. Bottom: Spectra of the naturally occurring fluorescence base, Yt base. DNA itself(---) displays very weak emission


Absorption states




max (x10-3)



F (ns)

Dansyl chloride












Normally use extrinsic probes or modified bases/ unnatural amino acids (check out the Molecular Probes Catalogue)

+ DNA ~1 20

when intercalated, yield and lifetime increase

F1 states



Fluorescence Quenching

If you have 2 fluorescent components (probes), even two bound components, they will have different rates of quenching, kq

kqfor F1 > kqfor F2

kq gives measure of accessibility of chromophore

Stern-Volmer statesAnalysis of Quenching

In the absence of quencher,

in the presence of quencher,

where quenching is the result of bimolecular collisions.

Stern-Volmer Plot states




Stern-Volmer quenching with concentration of Q, [Q]

where KSV=kq

Values of stateskq reflect

collisional frequency and bimolecular diffusion controlled rate constant, k0

Smoluchowski eqn.

R= collisional radii

D= diffusion coefficients

kq= fQk0

fQ = quenching efficiency

if fQ= 0.5, 50% of collisions lead to quenching

Can estimate D from Stokes-Einstein eqn.

expect kq’s of 1010 M-1s-1 or less

Consider equilibrium formation of a ground state complex which is not fluorescent:

Q + F FQ

The total conc. of fluorophore =


If FQ is not fluorescent, then

fraction of fluorescence

so that which is not fluorescent:

gives same KS.V. as





But could have

or even



[ which is not fluorescent:Q]




For dynamicquenching, quenching process is diffusion controlled

For staticquenching

but no change in  – not a diffusion controlled process

Singlet-Singlet Energy Transfer which is not fluorescent:

(Förster Transfer)

Singlet-Singlet Energy Transfer which is not fluorescent:

(Förster Transfer)

Singlet-Singlet Energy Transfer which is not fluorescent:

(Förster Transfer)

Very useful for “long range” distance (20-80 Å)



Donor Acceptor

Pick donor and acceptor to which is not fluorescent:have appropriately matched energy levels:



kT= rate constant for transfer




D* +A0 D0+A*


k-T is not likely given rapid vibrational relaxation









Energy transfer gives sensitized emission and donor deexcitation

Resonant interaction with acceptor excitation- weak coupling limit

Real world example: Cyan fluorescent protein/Red fluorescent protein

Absorption and emission spectra of cyan fluorescent protein (CFP, the donor) and red fluorescent protein (RFP or DsRed, the acceptor). Whenever the spectral overlap of the molecules is too great, the donor emission will be detected in the acceptor emission channel. The result is a high background signal that must be extracted from the weak acceptor fluorescence emission.

What’s the basis for the interaction? protein

-As in exciton coupling, dipole-dipole: just weak coupling limit

Can describe the potential operator

Where R is distance between A + D and are dipole moment operators

lump all geometric and orientational parameters in here- really hard to know , lots of variability

= 0-4

According to Fermi’s Golden Rule: protein

-rate of transition is proportional to the square of the expectation value for the interaction causing the excitation.

for isoenergetic D(b a) emission

A(a b) excitation

emission absorption

quantum yield

lifetime of donor w/o acceptor

frequency of transition

extinction coefficient for A

For general case, where transition involves a range of frequencies


refractive index of medium between donor and acceptor


normalized fluorescence of donor overlapping with acceptor


Naively, looks like D is emitting and A is reabsorbing but that transfer is trivial.

Also what would be effect on ?

Usual to define efficiency

get 1/R that transfer is trivial.6 dependence for E

can measure 10-100 Å distance separations depending on FRET pair

Want to measure donor-acceptor partners near R0 depending on experiment

This yields largest change in E for small changes in R that occur in the given experiment.

Very unique distance regime that transfer is trivial.

- FRET provides a spectroscopic ruler

Selected Applications of FRET that transfer is trivial.

• Structure and conformation of proteins

• Spatial distribution and assembly of protein complexes

• Receptor/ligand interactions

• Immunoassays

• Probing interactions of single molecules

• Structure and conformation of nucleic acids

• Real-time PCR assays and SNP detection

• Detection of nucleic acid hybridization

• Primer-extension assays for detecting mutations

• Automated DNA sequencing

• Distribution and transport of lipids

• Membrane fusion assays

• Membrane potential sensing

• Fluorogenic protease substrates

• Indicators for cyclic AMP and zinc

- Molecular Probes website

Different ways to carry out experiment: that transfer is trivial.

monitor quenching of donor and/or enhanced emission by acceptor

D alone


1.) Quenching of donor



E= fraction of donors deexcited

therefore 1-E= fraction of donors remaining excited

2.) Enhanced emission by acceptor

-should be sensitized emission: excite D, watch A emit

D absorb


Acceptor emission


watch here

In practice, want 3 replicas for study: that transfer is trivial.

Dalone D+A Aalone

A sensitized


donor quench

An example: Distance measurement in melittin

Depending upon solvent, can exist as monomer or tetramer, -helix or random coil

Determine overlap integral for trp/dansyl pair: that transfer is trivial.

R0= 23.6 Å

Overlap integral (shaded area) for energy transfer from a tryptophan donor to a dansyl acceptor on melittin. R0=23.6 Å

E=0.45 R=24.4 that transfer is trivial.Å

But there are issues- that transfer is trivial.

1.) 2 is not known, nor directly measurable


so even rough estimate suffices

Dale Eisinger Method- exploit the jitter



κ is related to the relative orientation of the donor/acceptor pair


Likely there is fast geometric averaging before transfer, blurring 2

often set 2=2/3 for dynamic avg. of all geometries

means uncertainty in R is < 15%

2.) Imperfect Stoichiometry that transfer is trivial.

3.) Does the probe perturb the structure?

if possible it is good to rely on intrinsic probes: in a protein tyr/trp energy transfer is possible






(monomer/ tetramer equilibrium for example)

Classic papers: that transfer is trivial.A Spectroscopic Ruler

LubertStryer and Richard Haugland

Proc. Natl. Acad. Sci USA, 58, 719-726 (1967)

A that transfer is trivial.


Without the donor, excitation is that of acceptor; that transfer is trivial.

in the presence of donor, see sensitized emission

and therefore excitation includes that of donor.

A = magnitude of excitation = a + E x d

Mapping the Cytochrome c Folding Landscape that transfer is trivial.

Julia G. Lyubovitsky, Harry B. Gray,* and Jay R. Winkler*

JACS, 2002, 124, 5481

Measurements of FRET between heme and C-terminal dansyl

There is a rapid equilibration among extended that transfer is trivial.

conformations, enabling escape from frustrated

compact structures

Some population of extended conformations,

with long distances remain even at long times.

Single Molecule Fluorescence Experiments that transfer is trivial.

Example: Nucleic that transfer is trivial.Aid Conformation and dynamics


Area detector/


Single molecule FRET study of Holliday junction by

total internal reflectance microscopy. The nucleic acid is tethered to the surface via biotin-neutravidin conjugation. The conformational dynamics is shown in the fluorescence time trace.

McKinney, Declais, Lilley, Ha, Nature Structural Biol. 10 93-97 (2003)