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Emission spectroscopy (Mainly fluorescence spectroscopy). Reading : van Holde Chapter 11 Homework : due Wednesday, April 18 van Holde 11.2. 11.3, 11.4, 11.5, 11.6, 11.7; Midterm 2 : Friday, April 20. Overview:

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slide1

Emission spectroscopy

(Mainly fluorescence spectroscopy)

Reading: van Holde Chapter 11

Homework: due Wednesday, April 18

van Holde 11.2. 11.3, 11.4, 11.5, 11.6, 11.7;

Midterm 2: Friday, April 20

  • Overview:
  • What is fluorescence? (conceptual QM picture, fluorescence spectra, quantum yield, lifetimes)
  • One powerful application: Fluorescence microscopy
  • Solvent effects
  • FRET (what is it?, Förster radius, quenching, applications of FRET)
  • Linear polarization of fluorescence, rotational motion (tumbling), Perrin plots
slide2

Emission spectroscopy

Conceptual Quantum mechanical picture

Two (highest energy) electrons in ground state

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.

Quick quiz: Why is quantum mechanics called quantum mechanics?

slide3

Emission spectroscopy

Conceptual Quantum mechanical picture

Internal conversion ~ 10-12 s

Intersystem crossing

~ 10-8 s

Fluorescence

(wait time) ~ 10-8 s

Non-radiative ~10-8 s

Non-radiative

~102 to 10-11 s

Phosphorcence

(wait time) ~ 102 to 10-4 s

Absorption ~ 10-15 s

2. singlet

Absorbance, Fluorescence and Phosphorescence

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.

1. singlet

1. triplet

Ground state

some terminology
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
Fluorescence spectra

fluorescence

Absorption

h = 6.6·1034 J·s (Planck’s const.)

n … frequency

emission

absorption

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

slide6

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
slide7

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 energy absorbed to that emitted (more in a bit).
slide8

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).

slide9

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

t

time

Fluorescence decay, life-time, time-resolved fluorescence

Flash sample with brief (~ns) pulse of light at lex and follow intensity vs time at lem

Absorption  N(0) molecules with get excited.

 Fluorescence intensity is proportional to number of excited molecules.

  • Decay of excited molecules is a first-order process, with lifetime t.
  • Decay can happen via three pathways:
  • Fluorescence with associated intrinsic lifetime to
  • Conversion to triplet state (phosphorescence and non-radiative decay).
  • Non-radiative decay.
quantum yield
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.

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.

slide12

Experimental data - fluorescence lifetime

White board example:

If Q is 0.5 for this fluorophore, what is the fluorescence lifetime?

Gett from slope

Flash lamp pulse

slide13

Factors Influencing Detected Fluorescence Intensity

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

F = k · c · e · fF

F = k · c · S

  • Incident Light Intensity, Io (l)
    • More photons in = more photons out. Detector reads intensity, unlike absorbance.
  • Fraction of Light Absorbed, e(l) c l
    • Depends on concentration, c; path length of optical cell, l; & molar extinction coefficient, e
  • Quantum Yield, fF
    • Only emitted photons count.
  • Fractional Emission , f(l)
    • Emission at a particular wavelength
  • Detector Efficiency , d(l)
    • Wavelength-dependent instrumental factor.
  • Sensitivity, S
  • Product of extinction coefficient and quantum yield
fluorescent amino acids
Fluorescent amino acids
  • 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 exctinctioncoeff 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 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

slide15

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.
slide16

Fluorescence Excitation & Emission Spectra of Aromatic Amino Acids

Absorption and Emission spectra of the aromatic amino acids in pH 7 in aqueous solutions (I. Gryczynski)

slide17

Specimen

Specimen

Objective lens

Objective lens

Collimating lens

Collimating lens

Dichroic mirror

Dichroic mirror

Hg – or Xe lamp

Hg – or Xe lamp

Emission filter

Emission filter

Excitation filter

Excitation filter

Camera

Camera

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

Schematics 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.

slide18

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).

fluorescence microscopy
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.
solvent effects
Solvent effects

Solvents can affect the fluorescence emission spectrum hugely.

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 effects1
Solvent effects
  • General solvent effects: Fluorescence depends on polarizability of solvent
  • Increasing dielectric constant of solvent shifts usually fluorescence to higher wavelength.
  • Polar interactions lower energy of excited state (stabilize excited state).

Lower DE: DE = hc/l

 Emission at longer l

Polarization Red-Shift

0rg

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
Quantum yield depends very much on environment

Changing quantum yield upon binding

Increased quantum yield upon binding

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.

Qrel = 1.00

Qrel = 0.46

Qrel = 0.23

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
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:
  • Transition dipole interaction between the two fluorophores (i.e., they need to be close together and aligned).
  • Significant overlap of the emission spectrum of the donorwith the absorption spectrum of the acceptor.

Example: Fluorescein (donor) and Alexa-546 (acceptor):

slide25

Fluorescence resonance energy transfer (FRET)

Efficiency of transfer:

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

R0 Förster radius

Close together  FRET signal

Far apart (further than Förster radius)  no 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).
slide26

Fluorescence energy resonance transfer (FRET)Donor-acceptor pairs

  • White board example:
  • What is the FRET efficiency for a fluorescein-rhodamine pair, that is 2.25 nm apart?
  • If excited with 488 nm light, what is the main emitted wavelength?
slide27

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)

slide29

Quenching

Some molecules are quenchers  they suppress fluorescence when they are close to a fluorophore

Fluorophore = fluorescein Quencher = dabcyl

slide30

Quenched

 no light

Not quenched  light

Molecular Beacons & Fluorescence Quenching

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:
  • Orientation of emitting transition dipole relative to absorbing transition dipole
  • Amount of molecular rotation during fluorescent lifetime!
  •  Depolarization of emitted light
slide32

Linear polarization of fluorescence

Depolarization is described in terms of:

  • 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:

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

 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).

slide33

Linear polarization of fluorescence

Fluor. anisotropy r

r

time

  • 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.

  • … correlation time

information about size &shape of molecule

  • large  slow tumbling  large molecular weight
example rotational correlation time for bsa mol wt 66 500 grams mole
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:

r= V h / k T = (8.1E-20)*(0.01) / [1.38E-16 * 293]

r= 2.0E-8 sec = 20 nsec

BSA rotates in ~ 10 - 20 nanonseconds

Fluorescent lifetime ~ 5 nanoseconds

linear polarization of fluorescence1
Linear polarization of fluorescence

Large r  slow rotation  large molecule

Small r  faster rotation  compact molecule

perrin plots
Perrin plots

Instead of pulse illumination, use continuous illumination to measure anisotropy  will get average anisotropy ravg.

HW 11.6

  • … lifetime

h … viscosity

T … temperature

V … volume of molecule

applications of fluorescence to proteins
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
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/