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

Ground state

1. singlet

2. singlet

1. triplet

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.

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

sourceExcitation

monochromatorSample

Emission monochromator Detector

Can take absorption and emission spectrum

Fluorescence instrumentation

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).

1000

104

40 60 80100 300

Lig

ht i

nten

sity

Fibrin fiber diameter(nm)

20 m

20 m

650 nm

0 nm

540 nm

0 nm

20 m

A

D

C

B

Fig. 6 Guthold et al.

Fluorescence microscopy (A) and Atomic Force Microscopy images of Oregon-Green-labeled fibrin fibers. Diameters range from 40 to 400 nm.

Fluorescent molecules

  Amino acid Lifetime Absorption Fluorescence

Wavelength Absorptivity Wavelength Quantum

Tryptophan 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

• Three amino acid have intrinsic fluorescence

• 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/PhotochemCAD/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 effectsGeneral 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:

*

a f 3

2 P( )E E

a

P P( ) P( n )

2

2

n 1High frequency (electron) polarizability: P( n )

2n 11

Low frequency polarizability (molecular dipole reorientation): P( )2 1

Fluorescence decay

Absorption N(0) molecules with get excited.

Fluorescence intensity is proportional to number of

excited molecules.

Flu

ores

cenc

e In

tens

ity

time

maxI

e

maxI

Decay of excited molecules is a first-order process, with lifetime.

Decay can happen via three pathways:

i. Fluorescence with associated intrinsic lifetime o.

ii. Conversion to triplet state (phosphorescence and non-radiative decay).

iii. Non-radiative decay.

kt

t

dN tk N t

dt

N t N 0 e

N 0 e

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.

Quantum yield

0

# of quanta emitted by fluorescenceQ

# of quanta absorbed

is lifetime of all molecules in excited state, 0 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

Qrel = 1.00

Qrel = 0.46

Qrel = 0.23

Application: Staining of DNA in gels.

Fluorophores with good DNA binding

affinities (often intercalation), extremely

large fluorescence enhancements upon

binding nucleic acids (some >1000-fold),

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)

Increased quantum yield upon binding Changing quantum yield upon binding

Extinction coefficients were determined for free dye in aqueous solution.

  Nucleic Acid Stain

Sensitivity for dsDNA

Extinction Coefficient(cm-1 M-1)

Quantum Yield

Bound to dsDNA

Fluorescence Enhancement

on Binding dsDNA

PicoGreen® Reagent

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

 

Quantum yield depends very much on environment

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.

Clever example: Molecular Beacons

used to detect presence of a certain DNA sequence in solution or cells (show on white board).

transfer 6

0

1E

r1

R

Efficiency of transfer:

Close together FRET signal

Far apart (further than Förster radius) no FRET signal

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

(De-)Polarization of emitted light depends on:1. Orientation of emitting transition dipole relative to absorbing transition dipole2. Amount of molecular rotation during fluorescent lifetime! Depolarization of emitted light

Absorption is best for those molecules whose transition dipole is parallel to plane of polarization.

Linear polarization of fluorescence

Fluorescence anisotropy:

I Ir=

I 2I

1. Assume molecules don’t rotate while being excited

depolarization due only to random orientation of molecules with respect to incoming light, , and angle

Depolarization is described in terms of:

20

1r ( 3cos 1)

5

If there is no molecular rotation, anisotropy will vary between 2/5 (absorbing and emitting trans. dipoles are parallel) and-1/5 (dipoles are perpendicular).

Anisotropy for fluorescence of rhodamine as a function of of exciting light

Linear polarization of fluorescence2. Now assume molecules tumble (rotate) before emitting.

depolarization due rotation of molecules.

Two extremes: i) molecules don’t rotate before emission r = r0

ii) molecules randomly orient before emitting: r = 0

Time-resolved fluorescence provides a convenient way to measure rotational motion of biological molecules.

t /0r t r e

… correlation time

information about size &shape of molecule

large slow tumbling large molecular weight

Flu

or. a

niso

trop

y r

time

0r

e

0r

Linear polarization of fluorescence

Large slow rotation large molecule

Small faster rotation compact molecule

Perrin plotsInstead of pulse illumination, use continuous illumination to measure anisotropy will get average anisotropy ravg.

HW 11.6

B

0

k T1 11

r r V T

… lifetime

… viscosity

T … temperature

V … volume of molecule

1

r

T

T

B

0

kslope:

r V

0

1intercept:

r

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 double-

stranded 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/