Emission spectroscopy
(Mainly fluorescence spectroscopy)
Reading: van Holde, Chapter 11
Homework: due Wednesday, April 2
van Holde 11.2. 11.3, 11.4, 11.5, 11.7, 11.8
Grad students: remember to schedule your presentations
Fluorescence microscopy.
Bovine Pulmonary Artery
Endothelilal cells
Overview:
1. What is fluorescence? (conceptual QM picture, fluorescence spectra, quantum yield,
lifetimes)
2. One powerful application: Fluorescence microscopy
3. Solvent effects
4. FRET (what is it?, Förster radius, quenching, applications of FRET)
5.
(Time permitting: Linear polarization of fluorescence, rotational motion (tumbling), Perrin plots)
Outlook:
Dr. Guthold: 4 lessons on Emission Spectroscopy, 2 lessons on Single Molecule Techn.
Emission spectroscopy
Conceptual Quantum mechanical picture
Ground state and excited states of the electrons in the
outermost occupied shell of a molecule.
Electronic energy levels
There are different types of excited states (higher energy level
states), depending on spin and angular momentum; here we
will just talk about triplet and singlet states.
Quick quiz: Why is
quantum mechanics
called quantum
mechanics?
Fluorescein
Two (highest
energy) electrons
in ground state
Emission spectroscopy
Conceptual Quantum mechanical picture
2. singlet
Different paths by which excited electrons can return to ground
state – only one path results in fluorescence. Molecules will
fluoresce if the emission process has a lifetime that is shorter than
the conversion to the triplet state or non-radiative loss of energy.
Internal
conversion
~ 10-12 s
Absorbance, Fluorescence and Phosphorescence
1. singlet
Ground state
(wait time) ~ 10-8 s
Fluorescence
Non-radiative ~10-8 s
Absorption ~ 10-15 s
~102 to 10-11 s
Non-radiative
(wait time) ~ 102 to 10-4 s
Phosphorcence
1. triplet
Some Terminology
•
Luminescence: Process, in which susceptible molecules emit light from
electronically excited states created by either a physical (for example,
absorption of light), mechanical (friction), or chemical mechanism.
•
Photoluminescence: Generation of luminescence through excitation of a
molecule by ultraviolet or visible light photons. Divided into two categories:
fluorescence and phosphorescence, depending upon the electronic
configuration of the excited state and the emission pathway.
•
Fluorescence (emission from singlet state): Some atoms and molecules
absorb light at a particular wavelength, and subsequently emit light of longer
wavelength after a brief interval, termed the fluorescence lifetime, t0.
Fluorescent molecules are called fluorophores.
•
Phosphorescence (emission from triplet state): Similar to fluorescence, but
with a much longer excited state lifetime.
Fluorescence
spectra
Vibrational
relaxation
fluorescence
Absorption
Remember :
Energy of a photon (light) :
E      h   h
c

h = 6.6·10-34 J·s (Planck’s const.)
 … frequency
In fluorescence, the return to the
ground state (almost) always
occurs from lowest state of
excited state (0’-level).
absorption
emission
Fluorescence spectra
•
Excitation: Light-induced transition, molecule goes from ground state to an excited
electronic & vibrational state.
•
Vibrational Relaxation: Molecule loses some energy, falls to lowest vibrational
state, still in excited electronic state.
•
Emission: Molecule returns to ground electronic state while emitting a higher wavelength
(lower energy) photon
Fluorescence spectra
•
Excitation Spectrum: Fluorescence intensity vs. wavelength used to excite transition
 resembles absorbance spectrum.
•
Emission Spectrum: Fluorescence intensity vs. wavelength emitted for transition back to
ground state: red-shifted with respect to excitation spectrum.
•
Quantum Yield: Ratio of (emitted fluorescence energy)/(absorbed energy). More in bit.
Fluorescence spectra
Example: Fluorescein absorption and emission spectra
Stokes shift
White board example:
Looking at the fluorescein
spectrum, what is the energy
difference between the ground
state and the excited singlet state
in fluorescein?
Looking at the absorption and emission peaks:
Fluorescein absorbs in the blue/purple (~ 490 nm) and emits in the green (~516 nm).
Steady State/Frequency Domain Fluorescence Instrumentation
Dual monochromators, one for excitation and the other for emission.
Obtain an Excitation and an Emission Spectrum.
Fluorescence decay, life-time, time-resolved fluorescence
Fluorescence Intensity
Flash sample with brief (~ns) pulse of light at ex and follow intensity vs time at em
Absorption  N(0) molecules with get excited.
 Fluorescence intensity is proportional to number of excited molecules.
I max
dN (t )
  k  N (t )
dt
N(t )  N (0)  e  kt
N max  N( 0 )
I max
e
t
N(t )  N (0)  e
time
I(t )  I (0)  e
Decay of excited molecules is a first-order process, with lifetime t.
Decay can happen via three pathways:
i.
Fluorescence with associated intrinsic lifetime to
ii.
Conversion to triplet state (phosphorescence and non-radiative decay).
iii. Non-radiative decay.


t
t
t
t
Quantum yield
When light is absorbed, only a fraction of it is emitted via fluorescence; the
rest of the excited molecules decay via other processes.
# of quanta emitted by fluorescence
Q
# of total quanta absorbed
t A
 
t0 k
t is lifetime of all molecules in excited state, t0 is intrinsic lifetime (lifetime of “fluorescence state”).
k, is fluorescence decay constant, A is Einstein coefficient
 Corollary: Fluorescence intensity is proportional to product of
absorptivity (exctinction coefficient) and quantum yield.
Experimental data - fluorescence lifetime
White board example:
If Q is 0.5 for this fluorophore, what is
the fluorescence lifetime, t, and
intrinsic fluorescence lifetime, t0 ?
Get t from slope
Flash
lamp
pulse
Factors Influencing Detected Fluorescence Intensity
F() = Io() · c · e() · l · Q · f() · d()
F=k·c·e·Q
F=k·c·S
1. Incident Light Intensity, Io ()
More photons in = more photons out. Detector reads intensity, unlike absorbance.
2. Fraction of Light Absorbed, , Beer’s law: A = e() · c · l
Depends on concentration, c; path length of optical cell, l; & molar extinction coefficient, e
3. Quantum Yield, Q
Only emitted photons count.
4. Fractional Emission , f()
Emission at a particular wavelength
5. Detector Efficiency , d()
Wavelength-dependent instrumental factor.
6. Group all instrumentation parameters, k = Io() · f() · d()
7. Sensitivity, S = e · Q
Product of extinction coefficient and quantum yield (fluorophore property)
Fluorescent amino acids
• Three amino acid have intrinsic fluorescence
Absorption
Amino acid
Lifetime
Fluorescence
Intensity
Wavelength
Exctinc. coeff.
emax
Wavelength
Quantum
Yield Q
Intensity ~
emax·Q
Tryptophan
2.6 ns
280 nm
5,600
348 nm
0.20
1120
Tyrosine
3.6 ns
274 nm
1,400
303 nm
0.1
140
Phenylalanine
6.4 ns
257 nm
200
282 nm .
0.04
8
• Fluorescence of a folded protein is a mixture of fluorescence from individual aromatic
residues. Most of the emissions are due to excitation of tryptophan
• Tryptophan:
 Highest extinction coeff. and highest quantum yield  strongest fluorescence intensity.
 Intensity, quantum yield, and wavelength of maximum fluorescence emission are very
solvent dependent. Fluorescence spectrum shifts to shorter wavelength and intensity
increases as polarity of the solvent surrounding the tryptophan residue decreases.
 Tryptophan fluorescence can be quenched by neighboring protonated acidic groups
such as Asp or Glu.
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/fluor.htm
• Tyrosine
 Like tryptophan, has strong absorption bands at ~280 nm.
 Tyrosine is a weaker emitter than tryptophan, but it may still contribute
significantly to protein fluorescence because it is usually present in larger
numbers.
 The fluorescence from tyrosine can be easily quenched by nearby
tryptophan residues because of energy transfer effects (more later – FRET).
• Phenylalanine
 Only a benzene ring and a methylene group is weakly fluorescent
(product of quantum yield and extinction coefficient is low. Phenylalanine
fluorescence is observed only in the absence of both tyrosine and
tryptophan).
A few applications:
- Determine protein concentration/presence in purifications, reactions, etc.
Can measure absorbance (at 280 nm) or emission.
- Often fluorescence changes upon binding or protein rearrangements.
Fluorescence Excitation &
Emission Spectra of
Aromatic Amino Acids
Absorption and Emission spectra of the aromatic amino
acids at pH 7 in aqueous solutions (I. Gryczynski)
Powerful application of fluorescence: Fluorescence microscopy
(shown here: epi-fluorescence illumination)
Specimen
Objective lens
Collimating
lens
Dichroic mirror
Hg – or
Xe lamp
Emission filter
Excitation
filter
Schematics from: http://www.ifr87.cnrsgif.fr/pbc/imagerie/outils/micros/microimg/mictout.gif
Camera
Note: Photobleaching: Fluorophores will “die” after a while of intense illumination.
Fluorescence microscopy
Normal African Green Monkey Kidney
Fibroblast Cells (CV-1)
(From Olympus web page: http://www.olympusmicro.com).
Immunofluorescently labeled with primary anti-tubulin mouse
monoclonal antibodies followed by goat anti-mouse Fab
fragments conjugated to Rhodamine Red-X. In addition, the
specimen was stained with DAPI (targeting DNA in the
nucleus).
Bovine Pulmonary Artery Endothelilal cells
(Justin Sigley, Guthold lab)
Labeled with three fluorescent stains. DAPI, which targets
DNA in the nucleus (blue), fluorescein-type stain
(microtubules, green), rhodamine-type stain (actin, red).
(Overlay image)
Space bar; 10 mm
Fluorescence microscopy
• Advantages:
– Can label selected features of a sample, e. g.,
nucleus, DNA, microtubules, specific proteins
– Can observe how those molecule behave over time.
– Can see (though not resolve) features on nanometer
level, even single molecules.
We use it to label and visualize
fibrin fibers (100 nm protein fibers
in a blood clot).
Liu, W. et al. (2006) Science 313, 634
B
A
Ridge
Fibrin fiber
AFM
tip
Groove
Scanning electron microscopy
image of blood clot:
Yuri Veklich & John Weisel
(Vacuum environment)
e = 70%
Background: eGFP
Quick quizz:
How are Martin Chalfie, Osamu
Shimomura, Roger Tsien?
Solvent effects
Solvents can affect the fluorescence emission spectrum enormously.
Emission wavelength can be shifted and quantum yield can change.
There are specific and general solvent effects.
Specific solvent effects: A chemical reaction of the excited state with the
solvent. Example: Hydrogen-bonds, acid-base interactions, charge transfer.
Changing Fluorescence can be used to
detect solvent interactions.
Specific solvent interaction:
2-anilinonaphthalene fluorescence was
changed to high wavelength by replacing
cyclohexan with ethanol. Ethanol forms
hydrogen bond.
Solvent effects
General solvent effects: Fluorescence depends on polarizability of solvent
 Increasing dielectric constant of solvent usually shifts fluorescence to
higher wavelength.
 Polar interactions lower energy of excited state (stabilize excited state).
Lower DE:
0rg
 DE = hc/
 Emission at longer 
Polarization Red-Shift
H20
Putting a fluorophore from cyclohexan (low dielectric constant) into water
(high dielectric constant), shifts fluorescence to higher wavelengths.
Quantum yield depends very much on environment
Increased quantum yield upon binding
Changing quantum yield upon binding
Application: Staining of DNA in gels.
Fluorophores with good DNA binding
affinities (often intercalation), extremely
Qrel = 1.00
large fluorescence enhancements upon
Qrel = 0.46
binding nucleic acids (some >1000-fold),
Qrel = 0.23
and negligible fluorescence for the free
dyes.
SYBR Green
SYBR stained dsDNA gel.
Excite with UV, emits in visible.
(DNA/SYBR Green I complex: Q~0.8;
~300-fold increase over free dye)
Fluorescence resonance energy transfer (FRET)
When two fluorophores are close together it is possible that one of them
absorbs the light (donor), then transfers the energy to the neighboring
fluorophore (acceptor), which then emits the light.
The two conditions for this to happen are:
1. Transition dipole interaction between the two fluorophores (i.e., they need
to be close together and aligned).
2. Significant overlap of the emission spectrum of the donor with the
absorption spectrum of the acceptor.
Example: Fluorescein (donor) and Alexa-546 (acceptor):
absorption
emission
absorption
emission
The absorption and
emisson spectra of
some fluorophores
Good
FRET pair
Fluorescence resonance energy transfer (FRET)
Basically, FRET is a great method to determine the distance between two
fluorophores (molecules) in the range of ~1-10 nm.
r … D-A distance
Efficiency of transfer:
Etransfer 
R0 Förster radius
1
 r 
1  
 R0 
6
Close together  FRET signal
Far apart (further than Förster
radius)  low FRET signal
Clever example: Molecular Beacons (also: more later on quenching)
 used to detect presence of a certain DNA sequence in solution or cells (show
on white board).
Fluorescence energy resonance transfer (FRET)
Donor-acceptor pairs
White board example:
a. What is the FRET
efficiency for a fluoresceinrhodamine pair, that is 2.25
nm apart?
b. If excited with 488 nm light,
what is the main emitted
wavelength?
FRET Application:
Coexisting conformations of fibronectin in cell culture imaged using fluorescence
resonance energy transfer, Baneyx et al., PNAS 98:14464, 2001
D
A
Extended conformation: Donor and acceptor are far apart: no FRET (donor emits)
Folded conformation: Donor and acceptor are close  FRET (acceptor emits)
FRET Detects Compact and Extended Fibronectin
Conformations in Cultured Cells
Region 1: Compact (and some extended) conformation
Region 2: Compact and extended conformation
Region 3: Mostly extended conformation
Quenching
Some molecules are quenchers  they suppress fluorescence when they are close to a
fluorophore
Fluorophore = fluorescein
Quencher = dabcyl
Molecular Beacons & Fluorescence Quenching
Quenched
 no light
Not quenched
 light
Applications of fluorescence to proteins
• Fluorescence microscopy
• Analytical detection of presence of proteins
• Monitor changes in quantum yield as indication of changing
environment (binding, unfolding, etc.)
• Effects of energy transfer (FRET).  Determine distance of
fluorescent groups from each other in 1-10 nm range.
• Changes in fluorescence polarization to determine shape and size
of molecules (tumbling depends on shape and size)
• Monitor (change) in fluorescence parameters to determine
stoichiometry, presence of intermediates, binding constants, etc.
Application of fluorescence to DNA
• Staining of oligonucleotides in gels
• Monitoring the unwinding of doublestranded DNA helicase
• Monitoring DNA melting
Also: there are tons of reactive fluorophores that can be used to label proteins
(Cysteines, primary amines, etc) and DNA.
See: Molecular Probes, Inc.
http://probes.invitrogen.com/
Extra slide: Using quartz optics, we can use native protein
fluorescence for UV imaging
Schematic of the DUV microscope developed for imaging tryptophan fluorescence of fibrinogen fibers.
J. Kim, H. Song, I. Park, C. Carlisle, K. Bonin, and M. Guthold “Denaturing of single electrospun fibrinogen fibers studied by deep
ultra-violet fluorescence microscopy” Microscopy Research & Technique (2011) 74, 219-224
Extra slides:
Linear polarization of fluorescence
Linear polarization of fluorescence
 Light to excite fluorophore is now linearly polarized
 Emitted fluorescent light will be depolarized
Absorption is best for those molecules whose
transition dipole is parallel to plane of polarization.
(De-)Polarization of emitted light depends on:
1. Orientation of emitting transition dipole relative to absorbing transition dipole
2. Amount of molecular rotation during fluorescent lifetime!
 Depolarization of emitted light
Linear polarization of fluorescence
Fluorescence anisotropy:
Depolarization is described in terms of:
r=
I  I
I  2I 
1. Assume molecules don’t rotate while being excited
 depolarization due only to random orientation of molecules with respect to
incoming light, q, and angle g:
1
r0  ( 3 cos 2 g  1 )
5
 If there is no molecular rotation,
anisotropy will vary between 2/5 (absorbing
and emitting trans. dipoles are parallel) and1/5 (dipoles are perpendicular).
Anisotropy for fluorescence of rhodamine
as a function of  of exciting light
Linear polarization of fluorescence
2. Now assume molecules tumble (rotate) before emitting.
 depolarization due rotation of molecules.
i) molecules don’t rotate before emission  r = r0
Two extremes:
ii) molecules randomly orient before emitting: r = 0
Fluor. anisotropy r
Time-resolved fluorescence provides a convenient way to measure rotational
motion of biological molecules.
r  t )  r0  e
r0
r0
e

t / 
… correlation time
information about size &shape of
molecule
 large  slow tumbling  large
molecular weight
time
Example: rotational correlation time for BSA
Mol Wt = 66,500 grams/mole
Drot = 8.3E6 sec-1
How long for BSA to “tumble”?
Rotational Correlation Time:
 = V h / k T = (8.1E-20)*(0.01) / [1.38E-16 * 293]
 = 2.0E-8 sec = 20 nsec
BSA rotates in ~ 10 - 20 nanonseconds
Fluorescent lifetime ~ 5 nanoseconds
Linear polarization of fluorescence
Large   slow rotation  large molecule
Small   faster rotation  compact molecule
Perrin plots
Instead of pulse illumination, use continuous illumination to measure
anisotropy  will get average anisotropy ravg.
HW 11.6
t … lifetime

1 1  k BT t
 
 1
r r0  Vh T ) 
1
r
h … viscosity
T … temperature
V … volume of molecule
1
intercept:
r0
slope:
t kB
r0V
T
h T )