Transcript Emmission Spectroscopy

Emission spectroscopy
(mainly fluorescence spectroscopy)
Reading: van Holde Chapter 11
Presentation: Nicole Levi: “Probing the interaction between two single
molecules: Fluorescence resonance energy transfer between a single donor
and a single acceptor” Ha et al. PNAS 93, 2664
HW: van Holde 11.2. 11.3, 11.4, 11.5, 11.6, 11.7; due Friday, April 8
Quantum mechanics
for the purpose of
fluorescence
Quantum mechanics
for the purpose of
fluorescence
Molecules will fluoresce if the emission process has a
lifetime that is shorter than the conversion to the triplet
state or nonradiative loss of energy.
1. triplet
Ground state
2. singlet
1. singlet
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 subsequently emit light of longer
wavelength after a brief interval, termed the fluorescence lifetime.
Fluorescent molecules are called fluorophores.
Phosphorescence (emission from triplet state): Similar to fluorescence, but
with a much longer excited state lifetime.
Fluorescence
spectroscopy
Example:
Fluorescein absorption and emission spectra
Stokes shift
source
Excitation
monochromator
Sample
Fluorescence
instrumentation
Emission
monochromator
Detector
Can take absorption and
emission spectrum
Fluorescence microscopy
• Advantages:
– Can label selected features of a sample, eg.
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.
Fluorescence microscopy
(here: epi-fluorescence illumination)
(See white board)
Normal African Green Monkey Kidney Fibroblast Cells (CV-1)
(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).
Fluorescence microscopy (A) and Atomic Force Microscopy images of
Oregon-Green-labeled fibrin fibers. Diameters range from 40 to 400 nm.
540 nm
A
C
0 nm
20 m
650 nm
20 m
D
B
Light intensity
4
0 nm
20 m
10
1000
40
60 80 100
300
Fibrin fiber diameter(nm)
Fluorescent molecules
• Three amino acid have intrinsic fluorescence
Amino acid
Lifetime
Tryptophan
Absorption
Fluorescence
Wavelength
Absorptivity
Wavelength
Quantum
2.6 ns
280 nm
5,600
348 nm
0.20
Tyrosine
3.6 ns
274 nm
1,400
303 nm
0.14
Phenylalanine
6.4 ns
257 nm
200
282 nm .
0.04
• Fluorescence of a folded protein is mixture of fluorescence from individual aromatic
residues. Most of the emissions are due to excitation of tryptophan.
• Tryptophan:
 Highest absorptivity and highest quantum  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 tryptophane
residue decreases.
 Tryptophan fluorescence can be quenched by neighbouring 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 usually present in larger
numbers.
 The fluorescence from tyrosine can be easily quenched by nearby
tryptophan residues because of energy transfer effects.
• Phenylalanine
 Only a benzene ring and a methylene group is weakly fluorescent
(product of quantum yield and molar absorbtivity maximum is low.
Phenylalanine fluorescence is observed only in the absence of both
tyrosine and tryptophane.
http://omlc.ogi.edu/spectra/Photoch
emCAD/html/alpha.html
Absorption and emission spectra
One Analytical Application
• Check for presence of certain proteins, for example,
elution from high pressure liquid chromatography.
Isolation of melittin, which has one tryptophan residue.
Solvent effects
Solvents affect the fluorescence emission spectrum. Two kinds: 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.
2-anilinonaphthalene fluorescence was
changed to hight wavelength by
replacing cyclohexan with ethanol.
Ethanol forms hydrogen bond.
Solvent effects
General solvent effects: Depend on polarizability of solvent 
increasing dielectric constant shifts fluorescence to higher wavelength.
Putting a fluorophore from
cyclohexan (low dielectric constant)
into water (high dielectric constant),
shifts fluorescence to higher
wavelengths.
Solvent effects
General solvent effect is
described by Lippert equation:
2 P(    )
Ea  E f 
a3
*
 P  P(  )  P( n )
n2  1
High frequency (electron) polarizability: P( n )  2
2n  1
Low frequency polarizability (molecular dipole reorientation): P(  ) 
 1
2  1
Fluorescence Intensity
Fluorescence decay
Absorption  N(0) molecules with get excited.
 Fluorescence intensity is proportional to number of
I max
excited molecules.
I max
e
t
dN  t 
 k  N  t 
dt
N  t   N  0   e  kt
time
 N 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
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.
The quantum yield is the ratio of {total number of quanta emitted} to {the
total number of quanta absorbed}.
The quanta are related to the area under the absorption and emission
spectra.
# of quanta emitted by fluorescence
Q
# of quanta absorbed
t

t0
t is lifetime of all molecules in excited state, t0 is intrinsic lifetime (lifetime of “fluorescence state”).
 Corollary: Fluorescence intensity is proportional to product of
absorptivity (exctinction coefficient) and quantum yield.
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 stained dsDNA gel.
Excite with UV, emits in visible.
(DNA/SYBR Green I complex: Q~0.8;
~300-fold increase over free dye)
Quantum yield depends very much on environment
Extinction coefficients were determined for free dye in aqueous solution.
Sensitivity
for dsDNA
Extinction
Coefficient
(cm-1 M-1)
Quantum
Yield
Bound to
dsDNA
Fluorescence
Enhancement
on Binding
dsDNA
25 pg/mL
70,000
0.53
~2000 fold
Hoechst
33258
1-10 ng/mL
40,000
0.59
~100 fold
Ethidium
bromide
1-10 ng/mL
5,000
<0.3
~25 fold
Nucleic Acid
Stain
PicoGreen®
Reagent
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):
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.
Efficiency of transfer:
Etransfer 
1
 r 
1  
 R0 
6
Close together  FRET signal
Far apart (further than Förster
radius)  no FRET signal
Clever example: Molecular Beacons
 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
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
Depolarization is described in terms of:
Fluorescence anisotropy:
I  I
r=
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 l 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
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 BTt
 
 1
r r0  V T  
1
r
 … viscosity
T … temperature
V … volume of molecule
1
intercept:
r0
slope:
t kB
r0V
T
 T 
Application of fluorescence to proteins
• 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/