Dr. Larsen: 1022006 Chem 115: Instrumental Analysis

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Lecture 1Molecular Fluorescence and
Fluorescence Microscopy
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Brief History of Fluorescence

• Nicolás Monardes (1506) was the first to describe
  the bluish opalescence of the water infusion from
  the wood of a small Mexican tree.
• Galileo (1612) described the emission of light
  (phosphorescence) from the famous Bolognian
  stone, discovered by Vincenzo Casciarolo, a
  Bolognian shoemaker. Galileo wrote "It must be
  explained how it happens that the light is
  conceived into the stone, and is given back after
  some time, as in childbirth.
• Sir John Herschel (1845) made the first
  observation of fluorescence from quinine sulfate
• Sir George Stokes (1852) coined the term
  Fluorescence Stokes used sunlight to illuminate
  a quinine solution and observed the emission
  through a stained glass filter He showed that the
  fluorescence emission occurred at a higher
  wavelength (lower energy) than the excitation
  light. This displacement is now called the Stokes
  Shift
• Edmund Bequerel (1880) Showed that certain metal
  ion complexes emit radiation with a very long
  decay

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Electromagnetic Radiation
Described by means of sine wave with wavelength,
frequency, velocity and amplitude. Particle model
of radiation is necessary. Represented as
electric and magnetic fields that undergo
sinusoidal oscillations at right angles to each
other and the direction of propagation.
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Spectroscopy Interactions between EM and matter

• Absorption A process in which electromagnetic
  energy is transferred to the atoms, ions, or
  molecules composing a sample
• Promotes particles from their normal room
  temperature state (ground state) to one or more
  higher-energy states.
• Atoms, molecules or ions have a limited number of
  discrete energy levels
• For absorption to occur, the energy of the
  exciting photon must exactly match the energy
  difference between the ground state and an
  excited state of the absorbing species

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The Fate of Excited Molecules
The process responsible for the fluorescence
properties of fluorescent probes and other
fluorophores is illustrated by the simple
electronic-state diagram called a Jablonski
diagram.
Stage 1 Excitation Stage 2 Excited-State
Lifetime Stage 3 Fluorescence Emission
Singlet States
Triplet States
Vibrational energy levels
S2
Rotational energy levels
Electronic energy levels
T2
S1
ISC
ENERGY
T1
ABS
FL
I.C.
Triplet state
PH
fast
slow (phosphorescence) Much longer wavelength
(blue ex red em)
ISC
S0
Vibrational sublevels
ABS - Absorbance S 0.1.2 - Singlet Electronic
Energy Levels FL - Fluorescence T 1,2 -
Corresponding Triplet States I.C.- Nonradiative
Internal Conversion ISC - Intersystem
Crossing PH - Phosphorescence
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A Simplified Jablonski Diagram
Stage 1 Excitation The flurophore exists in
some ground state (S0). A photon of energy hvEX
is supplied by an external source such as an
incandescent lamp or a laser and absorbed by the
fluorophore. This creates an excited electronic
singlet state (S1'). This process distinguishes
fluorescence from chemiluminescence, in which
the excited state is populated by a chemical
reaction.
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A Simplified Jablonski Diagram
Stage 2 Excited-state lifetime The excited
state exists for a finite time (typically 110 -
109 seconds). During this time, the
fluorophore undergoes conformational changes and
is also subject to a interactions with its
molecular environment.
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A Simplified Jablonski Diagram
Stage 3 Fluorescence emmission
A photon of energy hvEM is emitted, returning the
fluorophore to its ground state S0. Due to
energy dissipation during the excited-state
lifetime, the energy of this photon is lower,
and therefore of longer wavelength, than the
excitation photon hvEX. The difference in
energy or wavelength represented by (hvEx hvEM)
is called the Stokes shift.
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• Stokes Shift
• is the energy difference between the lowest
  energy peak of absorbance and the highest energy
  of emission

The Stokes shift is fundamental to the
sensitivity of fluorescence techniques because
it allows emission photons to be detected
against a low background, isolated from
excitation photons.
Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescence Intensity
Wavelength
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How Does Fluorescence Occur?

• Excitation
• Energy in the form of a photon of light is
  absorbed by a fluorophore
• De-excitation
• From lowest vib. level molecule can relax to
  ground state in various ways
• 1) Spontaneous emission of a photon
  fluorescence
• 2) Internal conversion
• 3) Quenching
• 4) Intersystem crossing
• The process repeats in a cyclical manner unless
  the fluorophore is destroyed by photobleaching
  (photodegredation).

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Fate 1 Fluorescence

• Molecule emits a photon spontaneously
• Assuming no stimulated emission, i.e. no laser
  effect.
• Occurs from lowest vib. state to various vib.
  excited states of lowest electronic state.
• Intrinsic rate kF 1/?R

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Fate 2 Internal Conversion

• Non-radiative dissipation of energy.
• Occurs from collision w/solvent, internal
  vibration.
• Call rate kic. This rate increases with
  temperature. (Why? faster motions).
• Competes with fluorescence
• Fluorescence intensity descreases w/temp.
• Major intrinsic temp. dependence of Fluorescence
• Hard to monitor Fl. as a function of temp

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Fate 3 Quenching

• De-excitation from collisions with solutes (Q)
• Bi-molecular (e.g O2, Iodine, etc.),
• Other parts of molecule (Trp quenches Tyr fluor)
• Rate kQQ
• S1 Q ? S0 Q kq
• Rates often ?R 10-9 10-7 sec.
• Good quenchers are very efficient and are just
  diffusion limited, so at mM Q, collisions up to
  108 s-1. So quenching can be a big factor.
• O2 quenching of protein fluorescence (Weber)

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Fate 4 Intersystem Crossing

• Convert to excited triplet state (usually spin
  forbidden transition). kis.
• This form can a) relax to S0 either by emission
  of a photon (now at longer wavelengths
  lower energy)
• Phosphorescence
• b) relax by inter system crossing (ISC)
• In practice, phosphorescence very low intensity,
  very long lifetime (seconds or longer) vs. ns for
  other processes.
• Quenching and ISC usually decrease
  phosphorescence to negligible levels except in
  solids.

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Fluorescence Quantum Yield ?f

• Fluorescence rate is modulated by all these other
  processes.
• Define fraction of molecules that de-excite by
  fluorescence as Quantum Yield?f kf /(kf kic
  kis kqQ)
• Also is the ratio of photons emitted to photons
  absorbed, or ratio of decay times ?F ?F/?R.

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Calculations.
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Why is Fluorescence Useful to us?

• Intensity of Fl signal Presence of Fl. Groups
• Spectra Environment h-phobic/non-polar vs.
  h-philic/polar
• Polarization Size, shape
• Lifetime Motions/dynamic features of molecule
• Changes in Intensity and Spectra Ligand Binding
  / Molecular Assembly Conf. changes
• Changes in Lifetime Breathing
• Energy transfer Orientation, distances

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Photochemistry
Riboflavin
Limiflavin
lumichrome
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Fluorescence Spectrometer
M1 excitation monochrometer, M2
entission mionochrometer L light
source. s sample cell, PM photo multiplier
detector.
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Fluorescence Signals
Fluorescence intensity is quantitatively
dependent on the same parameters as absorbance.

• ln (Io/I) snd (Beer Lambert law)
• Io light intensity entering cuvet
• Ilight intensity leaving cuvet
• absorption cross section
• n molecules
• d cross section (cm)
• or
• ln (Io/I) a C d (beer Lambert law)
• aabsorption coefficient
• C concentration
• Converting to decimal logs and standardizing
  quantities we get
• Log (I0/I) ecd A
• Now e is the decadic molar extinction coefficient
• A absorbance or optical density (OD) a
  dimensionless quantity

n molecules
s absorption cross section
d
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Fluorescence Signals
Fluorescence intensity is quantitatively
dependent on the same parameters as absorbance
BUT Also have to consider the fluorescence
quantum yield of the dye the excitation source
intensity fluorescence collection efficiency of
the instrument. In dilute solutions or
suspensions, fluorescence intensity is linearly
proportional to these parameters. When sample
absorbance exceeds about 0.05 in a 1 cm
pathlength, the relationship becomes nonlinear
and artifacts such as self-absorption and
inner-filter effect may distort measurements.
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Fluorescent groups in proteins
Trp dominate fluor. spectra of proteins Tyr are
more numerous, but are quenched by Trp
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Fluorescent groups in proteins

• Green Fluorescent Protein (GFP)
• Isolated from Jellyfish
• Spontaneous chemical reaction of side-chains
  produces fluorophore in protein core

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Fluorescent groups in proteins

• Express in selected cells, or fuse to other
  proteins
• YFP, CFP, eGFP, etc.

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Fluorescence Microscopy

• The intensity of the fluorescence is very weak in
  comparison with the excitation light (10-3 to
  10-5).
• The emitted light re-radiates spherically in all
  directions.
• Dark background is required to enhance resolution

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The Stokes Shift in Microscopy

• The energy of the emitted photon is lower than
  the energy of the excitation photon due to energy
  dissipation in the excited state.
• Lower energy Longer wavelength
• The Stokes shift is important to the fluorescence
  microscopy technique because it allows the
  emission photons to be detected and isolated away
  from the excitation photons
• A single fluorophore can be repeatedly excited
  and detected.

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Introduction of Spectral Filters
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Excitation filters, cut-off filters and dichroic
beam splitters
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Transmission Fluorescence Microscopy
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Transmission Fluorescence Microscopy
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Epifluorescence Microscopy (90degree)
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Epifluorescence Microscopy (90degree)

• Specimen thickness does not interfere with
  fluorescence intensity

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Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET) is
a distance-dependent interaction between the
electronic excited states of two dye molecules.
Excitation is transferred from a donor molecule
to an acceptor molecule without emission of a
photon. FRET is dependent on the inverse sixth
power of the intermolecular separation, making
it useful over distances comparable with the
dimensions of biological macromolecules. FRET
is an important technique for investigating a
variety of biological phenomena that produce
changes in molecular proximity.
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Fluorescence Resonance Energy Transfer (FRET)
Primary Conditions for FRET Donor and acceptor
molecules must be in close proximity (typically
10100 Å). The absorption spectrum of the
acceptor must overlap fluorescence emission
spectrum of the donor (see figure). Donor and
acceptor transition dipole orientations must be
approximately parallel.
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Fluorescence Resonance Energy Transfer (FRET)
Förster Radius The distance at which energy
transfer is 50 efficient (i.e., 50 of excited
donors are deactivated by FRET) is defined by
the Förster radius (Ro). The magnitude of Ro is
dependent on the spectral properties of the donor
and acceptor dyes.