Fluorescence spectroscopy Review Lecture

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Fluorescence spectroscopyReview Lecture

• Lecture 13
• October 18th

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Schedule term papers/talks

• First session talks (11-29-05)
• Diana Gavlitchi (SUNY Upstate Medical
  University)
• Membrane proteins
• 2. David Quint (SU Physics) Multiple pathways
  in protein folding
• 3. Lin Caiping (SUNY - ESF) Biodegradable
  polymers. Applications in drug
• delivery

• Second session talks (12-01-05)
• 1. Bogdana P. Gorianova (SUNY ESF) Different
  approaches for the DNA delivery
• 2. Luca Giomi (SU Physics) Kinetics
  thermodynamics in single molecule biological
  physics
• 3. Benjamin R. Lundgren (SU SB3) Bacterial
  adhesion. Biophysical aspects
• 4. Arsen A. Simonyan (SUNY ESF) Polymeric
  materials in drug delivery

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Intrinsic fluorescence of proteins
Fluorescence by aromatic side chains is much more
sensitive than absorbance.
However, the change in fluorescence varies in an
unpredictable manner.
Chromophores are components of molecules which
absorb light.
They are generally aromatic rings
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Fluorescence by aromatic side chains is much more
sensitive than absorbance.
However, the change in fluorescence varies in an
unpredictable manner.
Because fluorescence can change in an
unpredictable manner, the magnitude of change is
not very informative. If fluorescence is used to
monitor protein folding, the amount of change
in fluorescence may not correspond to anything
meaningful. The fact that there is a change can
be used as a probe for alterations in the folded
state of the protein.
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A better indicator of perturbations in folding is
the wavelength of the emitted light.
For example, Trp residues that are exposed to
water fluoresce maximally at a wavelength of 350
nm, while totally buried Trp residues emit at
about 330 nm. (Eftink et al., Biochemistry 26
8338-8346 (1987).
Fluorescence by a protein is complex when there
is more than one aromatic side chain. The
proximity of aromatic groups in a folded protein
results in very efficient energy transfer
between these groups. Light absorbed by one
chromophore is transferred to another that
absorbs at a longer wavelength, which can then
emit the energy as fluorescence.
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The proximity of aromatic groups in a folded
protein results in very efficient energy
transfer between these groups.
Absorbance Max Phenylalanine 257.4
nm Tyrosine 274.6 nm Tryptophan 279.8 nm
The absorbance wavelengths of amino acids are in
the order of PheltTyr,Trp. Proteins containing
all three aromatic residues emit fluorescence
light typical of Trp Proteins containing only
Tyr and Phe emit light typical of Tyr. Proteins
containing only Phe emits light typical of Phe.
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The Fluorescence Process
In addition to exploiting the natural
fluorescence properties of aromatic amino acid
side chains, one can introduce fluorescent
markers to study dynamic behavior of proteins in
solution. Fluorescence is the result of a
three-stage process that occurs in certain
molecules (generally polyaromatic hydrocarbons
or heterocycles) called fluorophores or
fluorescent dyes. A fluorescent probe is
a fluorophore designed to bind to a specific
region of a biological specimen or to respond to
a specific stimulus.
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The Fluorescence Process
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.
Singlet States
Triplet States
Stage 1 Excitation Stage 2 Excited-State
Lifetime Stage 3 Fluorescence Emission
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 2 Excited-state lifetime These processes
have two important consequences. First, the
energy of S1' is partially dissipated, yielding a
relaxed singlet excited state (S1) from which
fluorescence emission originates. Second, not
all the molecules initially excited by absorption
(Stage 1) return to the ground state (S0) by
fluorescence emission. Other processes such as
collisional quenching, fluorescence energy
transfer and intersystem crossing may also
depopulate S1.
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A simplified Jablonski Diagram
Stage 2 Excited-state lifetime
Number of emitted photons Number of absorbed
photons
?
Quantum Yield
The fluorescence quantum yield, which is the
ratio of the number of fluorescence photons
emitted (Stage 3) to the number of photons
absorbed (Stage 1), is a measure of the relative
extent to which these processes occur.
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Fluorescence quantum yield
S1
knr
kr
S0
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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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Fluorescence Spectra
The entire fluorescence process is cyclical.
Unless the fluorophore is irreversibly
destroyed in the excited state (an important
phenomenon known as photobleaching), the same
fluorophore can be repeatedly excited and
detected. For polyatomic molecules in solution,
the discrete electronic transitions represented
by hvEx and hvEM are replaced by rather broad
energy spectra called the fluorescence
excitation spectrum and fluorescence emission
spectrum, respectively.
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The Fluorescence Spectra
With few exceptions, the fluorescence excitation
spectrum of a single fluorophore species in
dilute solution is identical to its absorption
spectrum. Under the same conditions, the
fluorescence emission spectrum is independent of
the excitation wavelength, due to the partial
dissipation of excitation energy during the
excited-state lifetime.
Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescence Intensity
Wavelength
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Fluorescence Instrumentation
Fluorescence microscopes resolve fluorescence as
a function of spatial coordinates in two or
three dimensions for microscopic objects (less
than 0.1 mm diameter).
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular
Dichroic Filter
Objective
Emission Filter
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Fluorescence Instrumentation
Flow cytometers measure fluorescence per cell in
a flowing stream, allowing subpopulations within
a large sample to be identified and quantitated.
Flow Cytometry involves the use of a beam of
laser light projected through a liquid stream
that contains cells, or other particles, which
when struck by the focussed light give out
signals which are picked up by detectors.
These signals are then converted for computer
storage and data analysis, and can provide
information about various cellular properties.
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Fluorescence Instrumentation
Spectrofluorometers and microplate readers
measure the average properties of bulk (µL to
mL) samples. Fluorescence scanners resolve
fluorescence as a function of spatial
coordinates in two dimensions for macroscopic
objects such as electrophoresis gels, blots and
chromatograms.
Each type of instrument produces different
measurement artifacts and makes different
demands on the fluorescent probe. For example,
although photobleaching is often a significant
problem in fluorescence microscopy, it is not a
major impediment in flow cytometry because the
dwell time of individual cells in the excitation
beam is short.
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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 molar extinction coefficient
• A absorbance or optical density (OD) a
  dimensionless quantity

n molecules
d
s absorption cross section
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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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Excitation Saturation

• The rate of emission is dependent upon the time
  the molecule remains within the excitation state
  (the excited state lifetime tf)
• Optical saturation occurs when the rate of
  excitation exceeds the reciprocal of tf
• Molecules that remain in the excitation beam for
  extended periods have higher probability of
  interstate crossings and thus phosphorescence
• Usually, increasing dye concentration can be the
  most effective means of increasing signal when
  energy is not the limiting factor (I.e. laser
  based confocal systems)

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Fluorescence Signals
Because fluorescence quantitation is dependent on
the instrument, fluorescent reference standards
are essential for calibrating measurements made
at different times or using different instrument
configurations. A spectrofluorometer is
extremely flexible, providing continuous ranges
of excitation and emission wavelengths. Laser
scanning microscopes and flow cytometers,
however, require probes that are excitable at a
single fixed wavelength. In contemporary
instruments, the excitation source is usually the
488 nm spectral line of the argon-ion laser.
Biological samples labeled with fluorescent
probes typically contain more than one
fluorescent species, making signal-isolation
issues more complex. Additional optical signals
may be due to background fluorescence or to a
second fluorescent probe.
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Background Fluorescence
Fluorescence detection sensitivity is severely
compromised by background signals, which may
originate from endogenous components in the
sample (referred to as autofluorescence) or
from unbound or nonspecifically bound probes
(referred to as reagent background). Detection
of autofluorescence can be minimized either by
selecting filters that reduce the transmission
of the unwanted signal or by selecting probes
that absorb and emit at longer wavelengths.
Using filtrs that reduce emission from sample
contaimants can also compromises the overall
fluorescence intensity detected.
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Multicolor Labeling Experiments
A multicolor labeling experiment involves the
deliberate introduction of two or more probes to
simultaneously monitor different biochemical
functions. This technique has major
applications in flow cytometry, DNA sequencing,
fluorescence in situ hybridization and
fluorescence microscopy. Maximizing the
separation of the multiple emissions facilitates
signal isolation and data analysis.
Consequently, fluorophores with narrow spectral
bandwidths are particularly useful in multicolor
applications.
.
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Photobleaching

• Defined as the irreversible destruction of an
  excited fluorophore
• Some pathways include reactions between adjacent
  dye molecules, making the process considerably
  more complex in labeled biological specimens than
  in dilute solutions of free dye. In all cases,
  photobleaching originates from the triplet
  excited state, which is created from the singlet
  state (S1) via an excited-state process called
  intersystem crossing
• Methods for countering photobleaching
• Scan for shorter times
• Use high magnification, high NA objective
• Use wide emission filters
• Reduce excitation intensity
• Use antifade reagents (not compatible with
  viable cells)

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Quenching
Not a chemical process Dynamic quenching -
Collisional process usually controlled by mutual
diffusion Typical quenchers oxygen Aliphatic
and aromatic amines (IK, NO2, CHCl3) Static
Quenching Formation of ground state complex
between the fluorophores and quencher with a
non-fluorescent complex (temperature dependent
if you have higher quencher ground state complex
is less likely and therefore less quenching
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Antifade Agents

• Many quenchers act by reducing oxygen
  concentration to prevent formation of singlet
  oxygen
• Satisfactory for fixed samples but not live
  cells!
• Antioxidants such as propyl gallate,
  hydroquinone, p-phenylenediamine are used
• Reduce O2 concentration or use singlet oxygen
  quenchers such as carotenoids (50 mM crocetin or
  etretinate in cell cultures) ascorbate,
  imidazole, histidine, cysteamine, reduced
  glutathione, uric acid, trolox (vitamin E
  analogue)

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typical fluorophores

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Probes for Proteins
Probe Excitation Emission
FITC 488 525 PE 488 575 APC 630 650
PerCP 488 680 Cascade Blue 360 450 Coum
erin-phalloidin 350 450 Texas
Red 610 630 Tetramethylrhodamine-amines 550
575 CY3 (indotrimethinecyanines) 540 575 CY5
(indopentamethinecyanines) 640 670
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Probes for Nucleic Acids

• Hoechst 33342 (AT rich) (uv) 346 460
• DAPI (uv) 359 461
• POPO-1 434 456
• YOYO-1 491 509
• Acridine Orange (RNA) 460 650
• Acridine Orange (DNA) 502 536
• Thiazole Orange (vis) 509 525
• TOTO-1 514 533
• Ethidium Bromide 526 604
• PI (uv/vis) 536 620
• 7-Aminoactinomycin D (7AAD) 555 655

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Specific Organelle Probes
Probe Site Excitation Emission
BODIPY Golgi 505 511 NBD
Golgi 488 525 DPH Lipid 350 420 TMA-DPH
Lipid 350 420 Rhodamine
123 Mitochondria 488 525 DiO Lipid 488 500 di
I-Cn-(5) Lipid 550 565 diO-Cn-(3) Lipid 488
500
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole DPH
diphenylhexatriene
TMA - trimethylammonium
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absorption emission of Cy3
wavenumber (cm-1)
S1?S0
S1?S0
vibronic
vibronic
S2?S0
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pH Sensitive Indicators
Probe Excitation Emission

• SNARF-1 488 575
• BCECF 488 525/620
• 440/488 525

C27H19NO6
C27H20O11
SNARF-1 Benzenedicarboxylic acid, 2(or
4)-10-(dimethylamino)-3-oxo-3H-
benzocxanthene-7-yl-
BCECF Spiro(isobenzofuran-1(3H),9'-(9H)
xanthene)-2',7'-dipropanoic acid,
ar-carboxy-3',6'-dihydroxy-3-oxo-
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Other Probes of Interest
GFP - Green Fluorescent Protein GFP is from
the chemiluminescent jellyfish Aequorea
victoria Excitation maxima at 395 and 470 nm
(quantum efficiency is 0.8) Peak
emission at 509 nm Contains a
p-hydroxybenzylidene-imidazolone chromophore
Generated by oxidation of the Ser-Tyr-Gly
at positions 65-67 of the primary
sequence
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Pump-dump-probe spectroscopy on GFP
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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.
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Fluorescence Resonance Energy Transfer
Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Intensity
Absorbance
Absorbance
Wavelength