Introduction to Fluorescence
Fluorescent Minerals
Fluorescent Microscopy
Image of Endothelial Cells
Molecular absorption of photons triggers the emission of a photon
with a longer wavelength
Photophysics
• Molecule absorbs light
• Electron in the ground state is excited to a higher energy state
• After loss of some energy in vibrational relaxation, the high energy electron returns
back to the ground state by emitting fluorescent photon.
• If the spin of electron is flipped (intersystem crossing), electron goes to the triplet
state, whose return to ground state is forbidden.
• Triplet state can result either in phophorescence or in delayed fluorescence
• Fluorophores can undergo this cycle 30,000-10,000,000 or so.
Photon Emission and Absorption Rates
See the tutorial in
http://www.olympusmicro.com/primer/java/jablonski/jabintro/index.html
Triplet State and Photobleaching
• The low probability of intersystem crossing arises
from the fact that molecules must first undergo spin
conversion to produce unpaired electrons, an
unfavorable process.
• Molecules exhibit high degree of chemical
reactivity in this state, which often results in
photobleaching and the production of damaging
free radicals.
• The ground state oxygen molecule, which is
normally a triplet, can be excited to a reactive singlet
state, leading to reactions that bleach the
fluorophore.
• Fluorophores in the triplet state can also react
directly with other biological molecules, often
resulting in deactivation of both species.
• Molecules containing heavy atoms, such as the
halogens and many transition metals, often facilitate
intersystem crossing and are frequently
phosphorescent.
Photobleaching and Blinking of Fluorophores
• Molecules with electrons in the triplet state are chemically reactive.
• Since the rate of conversion is slow, molecules stay in the triplet state a long time (spin
conversion)
• Dissolved oxygen (ground state is triplet) is highly reactive with fluorophores in the
triplet state, leading to free radicals (singlet oxygen) that are toxic to cells.
• In photobleaching, fluorophore permanently loses the ability to fluoresce due to
photon-induced chemical damage and covalent modification.
• Fluorophores in the triplet state can also react directly with other biological molecules,
often resulting in deactivation of both species.
• Molecules containing heavy atoms, such as the halogens and many transition metals,
often facilitate intersystem crossing and are frequently phosphorescent.
• Photobleaching can be reduced by limiting
the exposure time of fluorophores to
illumination or by lowering the excitation
energy (low signal)
• Solution can be deoxygenated by
antioxidative reagents (glucose oxidase,
ascorbic acid…)
• Usage of triplet state quenchers (BME,
Trolox…)
Fluorescence Lifetime and Quantum Yield
1. Extinction coefficient (ε) is an intrinsic ability of a fluorophore to absorb light at a
given wavelength (usually the absorption maxima). High ε values are 75,000-170,000.
Beer-Lambert law, A = εcl.
absorbance, A, the pathlength l and the concentration c
2. Quantum yield (Q) is the (dimensionless) ratio of photons emitted to the number of
photons absorbed. High Q values yield brighter fluorescence (typically 0.9-0.3)
Q = kf/(kf +knr)
kf is fluorescence decay rate and knr is combined nonradiative decay rate.
3. Lifetime (τ) is an average value of time spent in the excited state.
τ = 1/(kf +knr)
I(t) = Io • e(-t/τ)
Fluorescence Lifetime
The fluorescence lifetime τ = k-1 = (kf + knr)-1 depends on the
environment of the molecule through knr = ki + kx + kET + ….
Fluorescence quantum yield:
QY =
kf
k f + k nr
=
kf
k
=
τ
τr
is proportional to fluorescence lifetime.
Addition of another radiationless pathway increases knr and, thus,
decreases τ and QY.
However, the measurement of fluorescence lifetime is more robust than
measurement of fluorescence intensity (from which the QY is
determined), because it depends on the intensity of excitation nor on
the concentration of the fluorophores.
The fluorescence intensity I (t) = kf n*(t) is proportional to n*(t) and
vice versa
From Martin Hof, Radek Macháň
Fluorescence Quenching
A number of processes can lead to a reduction in
fluorescence intensity. These processes can occur
during the excited state or they may occur due to
formation of complexes in the ground state
Collisional Quenching is observed with the
collision of an excited state fluorophore and
another molecule in solution (ions, oxygen,
halogen, other fluorophores (self
quenching)), which can undergo electron
transfer, spin-orbit coupling, and intersystem
crossing to the excited triplet state without
chemical alteration, resulting in deactivation
of the fluorophore and return to the ground
state.
In Static Quenching fluorophores form a
reversible complex with the quencher
molecule in the ground state, and does not
rely on diffusion or molecular collisions.
In the simplest case of collisional quenching, the following relation, called the
Stern-Volmer equation, holds:
I0/I = 1 + KSV[Q]
where I0 and I are the fluorescence intensities observed in the absence and
presence, respectively, of quencher, [Q] is the quencher concentration and KSV is
the Stern-Volmer quenching constant
1.7
KSV = kq τ0 where kq is the
bimolecular quenching rate
constant and τ0 is the excited
state lifetime in the absence
of quencher.
I0/I
1.6
F0/F
1.5
1.4
1.3
1.2
1.1
1.0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Concentration of I- (M)
In the case of purely collisional quenching, also known as dynamic quenching,:
I0/I = τ0/τ.
Hence in this case: τ0/τ = 1 + kq τ0[Q]
In the fluorescein/iodide system, τ = 4ns and kq ~ 2 x 109 M-1 sec-1
Collisional Quenching
derivation of Stern-Volmer equation:
1
τ0 =
kf + knr
1
τ =
kf + knr + kq [Q]
presence of quencher – additional nonradiative deexcitation channel
τ 0 τ = 1 + kqτ 0[Q]
quantum yield:
QY = τ kf
I0 I = QY0 QY = τ 0 τ = 1 + K SV [Q]
Static Quenching
In some cases, the fluorophore can form a stable complex with
another molecule. If this ground-state is non-fluorescent then
we say that the fluorophore has been statically quenched.
In such a case, the dependence of the fluorescence as a
function of the quencher concentration follows the relation:
I0/I = 1 + Ka[Q]
where Ka is the association constant of the complex. Such
cases of quenching via complex formation were first described
by Gregorio Weber.
In the case of static quenching the lifetime of the sample will
not be reduced since those fluorophores which are not complexed
will have normal excited state properties.
The fluorescence of the sample is reduced since the quencher is
essentially reducing the number of fluorophores which can emit.
Static Quenching
In some cases, the fluorophore can form a stable complex with
another molecule. If this ground-state is non-fluorescent then
we say that the fluorophore has been statically quenched.
F+Q
FQ
[FQ]
Ka =
[F][Q]
[FQ] = Ka [F][Q]
I0 [F]tot. [F] + [FQ]
=
=
= 1 + K a[Q]
I
[F]
[F]
If both static and dynamic quenching are occurring in the sample
then the following relation holds:
I0/I = (1 + kq τ0[Q]) (1 + Ka[Q])
In such a case then a plot of I0/I versus [Q] will have an upward
curvature due to the [Q]2 term.
However, since the lifetime is unaffected by the presence of quencher
in cases of pure static quenching, a plot of τ0/τ versus [Q] would give
a straight line
I0/I
τ 0/ τ
[Q]
Non-linear Stern-Volmer plots can also occur in the case of purely
collisional quenching if some of the fluorophores are less accessible
than others. Consider the case of multiple tryptophan residues in a
protein – one can easily imagine that some of these residues would
be more accessible to quenchers in the solvent than other.
In the extreme case, a Stern-Volmer plot for a system having
accessible and inaccessible fluorophores could look like this:
I0/I
[Q]
The quenching of LADH intrinsic protein fluorescence by iodide gives, in fact, just such
a plot. LADH is a dimer with 2 tryptophan residues per identical monomer. One residue
is buried in the protein interior and is relatively inaccessible to iodide while the other
tryptophan residue is on the protein’s surface and is more accessible.
E1
350nm
323nm
In this case (from Eftink and Selvidge, Biochemistry 1982, 21:117) the different
emission wavelengths preferentially weigh the buried (323nm) or solvent exposed
(350nm) tryptophan.
Self quenching
The intensity of fluorescence is proportional to the concentration of the
fluorophores in a reasonable concentration range.
However, at high concentrations of the fluorophores the proportionality
is no more satisfied, because significant collisional quenching between
the molecules of the fluorophore themselves appears.
Fluorescence intensity of calcein as
a function of fluorophore
concentration
Andersson et al. Eur Biophys J 2007,
36: 621
E4
Self quenching
Typically used to study the formation of pores in vesicles caused by
membrane-active molecules – vesicle leakage assay
vesicles loaded
with 60 mM
calcein
vesicles
Triton X-100
detergent
peptide LAH4
creates pores in
the lipid bilayer,
through which the
dye can leak out
LAH4 peptide
detergent Triton
X-100 micellizes
the vesicles
Vogt and Bechinger, J Biol Chem 1999, 274: 29 115
final calcein
concentration
~ 5 µM
Applications of Quenching
Principle of enzyme detection via the disruption of intramolecular selfquenching. Enzyme-catalyzed hydrolysis of the heavily labeled substrates
relieves the intramolecular self-quenching, yielding brightly fluorescent
reaction products
Characteristics of Fluorescence Emission
Stokes shift (20-200 nm)
• Part of the excitation energy is lost in the vibrational states of the excited state
• High Stokes shift is desirable to better isolate fluorescence emission from the
excitation
Mirror Image Rule
• The emission spectrum is
independent of the excitation
wavelength as a consequence of
rapid internal conversion from higher
initial excited states to the lowest
vibrational energy level of the S(1)
excited state
• The vibrational energy level spacing is similar for the ground and excited states,
which results in a fluorescence spectrum that strongly resembles the mirror image
of the absorption spectrum
• Return transitions to the ground state (S(0)) usually occur to a higher vibrational
level
Exceptions of the Mirror Image Rule
Excitation by high energy photons leads to the population of higher
electronic and vibrational levels (S(2), S(3), etc.), which quickly lose excess
energy as the fluorophore relaxes to the lowest vibrational level of the first
excited state. Higher excitation states do not follow the mirror image rule.
Solvent Effects on Fluorescence
A variety of environmental factors affect fluorescence emission, including solvent
polarity, inorganic and organic compounds, temperature, pH, and the localized
concentration of the fluorescent species.
The high degree of sensitivity in fluorescence is primarily due to interactions that
occur in the local environment during the excited state lifetime.
A fluorophore can be considered an entirely different molecule in the excited state
(than in the ground state), and thus will display an alternate set of properties in
regard to interactions with the environment.
Polar solvent molecules surrounding
fluorophore interact with the dipole
moment of the fluorophore to yield an
ordered distribution of solvent molecules
around the fluorophore.
•Most fluorophores have larger dipole moments in the excited state than the ground
state.
•Rotational motion of small molecules occurs in ~40 ps. Fluorescence allows ample
time for solvent to reorient around the molecule in excited state (solvent
relaxation).
•Solvent polarity further induces a red shift in fluorescence.
http://www.olympusmicro.com/primer/java/jablonski/solventeffects/index.html
Trytophan Fluorescence
• Tryopthan is an aromatic amino acid side chain which is usually
buried inside a protein fold. In nonpolar environment, tryptophan
emits at 330 nm.
•Upon denaturation of a protein, the environment of the tryptophan
residue is changed from non-polar to highly polar.
• Fluorescence emission peak moves from 330 to 365 nm, due to
solvent effects.
Fluorescent Molecules
• Fluorescent probes are constructed around synthetic aromatic
organic chemicals with unconjugated double bonds.
• Designed to bind with a biological macromolecule (for example, a
protein or nucleic acid) or to localize within an organelle, such as
the cytoskeleton, mitochondria, and nucleus.
DAPI attachment to DNA
minor groove
•Fluorophores targeted at specific intracellular organelles, such as the mitochondria, lysosomes,
Golgi apparatus, and endoplasmic reticulum, are useful for monitoring transport, respiration,
mitosis, apoptosis, protein degradation, acidic compartments, and membrane.
•Many of the fluorescent probes are designed to permeate or sequester within the cell
membrane, while others must be installed using monoclonal antibodies.
•Mitochondrial probes: The mechanism of action varies, ranging from covalent attachment to
oxidation within respiring mitochondrial membranes. (MitoTracker, JC-1)
•Weakly basic permeable amines are the ideal candidates for investigating biosynthesis and
pathogenesis in lysosomes. (LysoTracker, LysoSensor)
•highly lipophilic dyes (sphingolipids) are useful as markers for the study of lipid transport and
metabolism in Golgi. (BODIPY)
Fluorescent Environmental Probes
Certain fluorophores change their wavelength of emission or intensity upon binding metal ions
(calcium, magnesium), heavy metals (enzyme cofactors), thiols, as well as pH, solvent polarity,
and membrane potential.
Calcium plays a vital role in cellular response to many forms of external stimuli. Calcium ion
concentration undergo a transient change during signalling, fluorophores are designed to
measure localized concentrations and to monitor changes when flux density waves progress
throughout the entire cytoplasm.
Two of the most common calcium probes are the ratiometric indicators fura-2 and indo-1
Fura 2, 1-100 mM Mg+2
Isosbestic Point
Requires UV excitation
Not good for cells
• Fluorophores that respond in the visible range to calcium ion fluxes are, unfortunately,
not ratiometric indicators and do not exhibit a wavelength shift.
• Isosbestic point is generated by mixture of two dyes.
• fluo-3undergoes a dramatic increase in fluorescence emission upon calcium-binding at
525 nm. Because isosbestic points are not present, it is impossible to determine
whether increase in fluorescence is due to calcium-binding or increase in dye
concentration.
• Fura red exhibits a decrease in fluorescence at 650 nanometers when binding calcium.
• A ratiometric response to calcium ion fluxes can be obtained when a mixture of fluo-3
and fura red. Isosbestic point exists when fluorophore concentration remains constant.
http://www.youtube.com/watch?v=SwuBk4l4y5E&feature=related
Modern Fluorophore Technology
fluorescence intensity remains stable for long periods of time
•High quantum yield
•High extinction coefficient
•Less pH sensitivity
•Enhanced photostability
•High duty ratio
•Less blinking
•Water solubility
•Matching absorption
maxima
•Membrane permeable
•maleimides, succinimidyl
esters, and hydrazides
•conjugated to phalloidin,
G-actin, and rabbit skeletal
muscle actin
•conjugates to lectin,
dextrin, streptavidin,
avidin, biocytin, and a wide
variety of secondary
antibodies
Fluorescent Proteins
green fluorescent protein (GFP), was isolated
from the North Atlantic jellyfish, Aequorea
victoria
GFP from PDB
In cell and molecular biology, the GFP gene is
frequently used as a reporter of expression
Green Mouse
fluorescent proteins can be fused to virtually
any protein in living cells using recombinant
complementary DNA cloning technology, and
the resulting fusion protein gene product
expressed in cell lines
Hippocampal neuron expressing GFP
Aequorea victoria
Enhanced GFP and Variants
a single point mutation (S65T) dramatically improved the spectral characteristics of
GFP, resulting in increased fluorescence, photostability and a shift of the major
excitation peak to 488nm with the peak emission kept at 509 nm
Additional mutation studies have uncovered
GFP variants that exhibit a variety of
absorption and emission characteristics
2008 Nobel Prize in Chemistry
The diversity of genetic mutations is
illustrated by this San Diego beach scene
drawn with living bacteria expressing 8
different colors of fluorescent proteins.
Roger Y. Tsien
The Fluorescent Protein Color Palette
Remaining Issues:
•Fluorescent proteins are dim and susceptible to photobleaching
•Low expression is not observable because of cell autofluorescence
•GFP cannot be fused into every gene/genetic complications
•GFP size can be an issue for certain applications
•GFP dimerization
Specific Labeling of Proteins
Functional Targets and Reactive Groups
Only a small number of protein functional groups comprise selectable targets for practical
bioconjugation methods.
Primary amines (–NH2): This group exists at the N-terminus of each polypeptide chain and in
the side chain of lysine residues.
Carboxyls (–COOH): This group exists at the C-terminus of each polypeptide chain and in the
side chains of aspartic acid and glutamic acid.
Sulfhydryls (–SH): the side chain of cysteine (Cys, C). Cysteines are joined together between
their side chains via disulfide bonds (–S–S–).
Antibodies for Labeling
ImmunoFluorescence
Genetic Tags
SNAP Tag is ~20 kD mutant of a DNA
repair protein
Mitochondrial cytochrome oxidase (red, SNAP)
Nuclei (blue, Hoechst )
Quantum Dots
The absorbed photon creates an
electron-hole pair that quickly
recombines with the concurrent
emission of a photon having lower
energy.
•Nanometer-sized crystals of purified semiconductors (CdSe)
•ZnS surface coating improves brightness
•Hydrophilic polymer shell coating (water solubility)
•Biological conjugation (antibody, 6His, Streptavidin, wheat germ agglutinin)
Advantages
Long-term photostability (20 min)
High fluorescence intensity levels (20-50 fold)
Multiple colors with single-wavelength excitation
Narrow emission spectra (30nm FWHM)
Disadvantages
Big Size (20-40 nm)
Multivalency (20-50 binding sites)
Toxicity to cells (heavy atoms)
Quantum Confinement in Qdots
The quantum confinement effect can be
observed once the diameter of the particle is
of the magnitude as the wavelength of
electron wave function.
A particle behaves as if it were free when the
confining dimension is large compared to the
wavelength of the particle.
Typically in nanoscale, the energy spectrum
turns to discrete. As a result, bandgap
becomes size dependent. This ultimately
results a blue shift in optical illumination as the
size of the particles decreases.
Fluorophores are catalogued and described according to their absorption and
fluorescence properties, including the spectral profiles, wavelengths of maximum
absorbance and emission, and the fluorescence intensity of the emitted light.
Chemically reactive forms of many dyes are commercially available