Emission spectroscopy(Mainly fluorescence spectroscopy)

Reading: van Holde, Chapter 11

Homework: due Wednesday, April 2van Holde 11.2. 11.3, 11.4, 11.5, 11.7, 11.8

Grad students: remember to schedule your presentations

Overview:1. What is fluorescence? (conceptual QM picture, fluorescence spectra, quantum yield,

lifetimes)2. One powerful application: Fluorescence microscopy3. Solvent effects4. FRET (what is it?, Förster radius, quenching, applications of FRET)5. (Time permitting: Linear polarization of fluorescence, rotational motion (tumbling), Perrin plots)

Fluorescence microscopy. Bovine Pulmonary Artery Endothelilal cells

Outlook: Dr. Guthold: 4 lessons on Emission Spectroscopy, 2 lessons on Single Molecule Techn.

Ground state and excited states of the electrons in the outermost occupied shell of a molecule.

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.

Two (highest energy) electrons in ground state

Emission spectroscopyConceptual Quantum mechanical picture

Quick quiz: Why is quantum mechanics called quantum mechanics? Fluorescein

Ele

ctro

nic

ener

gy le

vels

Ground state

1. singlet

2. singlet

1. triplet

Abs

orpt

ion

~ 10

-15 s

Inte

rnal

co

nver

sion

~

10-1

2 s

Intersystem

crossing

~ 10-8 s

Non

-rad

iativ

e ~1

0-8 s

Fluo

resc

ence

(wai

t tim

e) ~

10-8

s

Non

-rad

iativ

e

~102 t

o 10

-11 s

Pho

spho

rcen

ce

(wai

t tim

e) ~

102 t

o 10

-4 s

Absorbance, Fluorescence and PhosphorescenceDifferent 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.

Emission spectroscopyConceptual Quantum mechanical picture

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

Absorption fluorescence

chhE

:(light)photon a ofEnergy :Remember

absorption emission

In fluorescence, the return to the ground state (almost) always occurs from lowest state of excited state (0’-level).

h = 6.6·10-34 J·s (Planck’s const.) … frequency

Vibrational relaxation

• 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

Looking at the absorption and emission peaks:

Fluorescein absorbs in the blue/purple (~ 490 nm) and emits in the green (~516 nm).

Fluorescence spectra

White board example:

Looking at the fluorescein spectrum, what is the energy difference between the ground state and the excited singlet state in fluorescein?

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

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.

Fluo

resc

ence

Inte

nsity

timet

maxIe

max maxI N N(0 )

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.

( ) ( )

N( ) (0)

N( ) (0)

I( ) (0)

kt

t

t

dN t k N tdt

t N e

t N e

t I e

t

t

When light is absorbed, only a fraction of it is emitted via fluorescence; the rest of the excited molecules decay via other processes.

Quantum yield

0

# of quanta emitted by fluorescenceQ# of total quanta absorbedAk

tt

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.

Flash lamp pulse

Experimental data - fluorescence lifetime

Get t from slope

White board example: If Q is 0.5 for this fluorophore, what is the fluorescence lifetime, t, and intrinsic fluorescence lifetime, t0 ?

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, QOnly 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 · QProduct of extinction coefficient and quantum yield (fluorophore property)

Factors Influencing Detected Fluorescence Intensity

F() = Io() · c · e() · l · Q · f() · d()

F = k · c · e · Q

F = k · c · S

Fluorescent amino acids

  Amino acid Lifetime Absorption Fluorescence Intensity

Wavelength Exctinc. coeff.

emax Wavelength Quantum

Yield QIntensity ~ 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

• Three amino acid have intrinsic fluorescence

• 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)

Hg – or Xe lamp

Collimating lens

Excitation filter

Specimen

Objective lens

Dichroic mirror

Emission filter

CameraSchematics from: http://www.ifr87.cnrs-gif.fr/pbc/imagerie/outils/micros/microimg/mictout.gif

Powerful application of fluorescence: Fluorescence microscopy(shown here: epi-fluorescence illumination)

Note: Photobleaching: Fluorophores will “die” after a while of intense illumination.

Hg – or Xe lamp

Collimating lens

Excitation filter

Specimen

Objective lens

Dichroic mirror

Emission filter

CameraSchematics from: http://www.ifr87.cnrs-gif.fr/pbc/imagerie/outils/micros/microimg/mictout.gif

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).

Fluorescence microscopy

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 AFM tip

B

e = 70%

RidgeGroove

Fibrin fiber

A

Scanning electron microscopy image of blood clot:

Yuri Veklich & John Weisel

(Vacuum environment)

Background: eGFPQuick 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.

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.

Specific solvent effects: A chemical reaction of the excited state with the solvent. Example: Hydrogen-bonds, acid-base interactions, charge transfer.

Solvent effects

H20

0rg

Putting a fluorophore from cyclohexan (low dielectric constant) into water (high dielectric constant), shifts fluorescence to higher wavelengths.

Lower DE: DE = hc/ Emission at longer

Polarization Red-Shift

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).

Quantum yield depends very much on environment

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

SYBR Green

Changing quantum yield upon binding

Qrel = 1.00

Qrel = 0.46

Qrel = 0.23

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

absorption

emission

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.

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).

transfer 6

0

1Er1R

Efficiency of transfer:

Close together FRET signal

Far apart (further than Förster radius) low FRET signal

r … D-A distanceR0 Förster radius

Fluorescence energy resonance transfer (FRET)Donor-acceptor pairs

White board example: a. What is the FRET

efficiency for a fluorescein-rhodamine 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

Fluorophore = fluorescein Quencher = dabcyl

QuenchingSome molecules are quenchers they suppress fluorescence when they are close to a fluorophore

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 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/

Extra slide: Using quartz optics, we can use native protein fluorescence for UV imaging

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

Schematic of the DUV microscope developed for imaging tryptophan fluorescence of fibrinogen fibers.

Extra slides:

Linear polarization of fluorescence

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.

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:

Depolarization is described in terms of:

20

1r ( 3cos 1)5

g

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 fluorescence

2. 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

r … correlation time information about size &shape of moleculer large slow tumbling large

molecular weight

Fluo

r. an

isot

ropy

r

time

0re

0r

Linear polarization of fluorescence

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 nanonsecondsFluorescent lifetime ~ 5 nanoseconds

Example: rotational correlation time for BSA Mol Wt = 66,500 grams/mole

Drot = 8.3E6 sec-1

Large slow rotation large molecule Small faster rotation compact molecule

Linear polarization of fluorescence

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

HW 11.6

)B

0

k T1 1 1r r V T

th

t … lifetime

h … viscosity

T … temperature

V … volume of molecule

1r

)TTh

B

0

kslope: r Vt

0

1intercept:r