10
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Fluorescence methods in life sciences
Ctirad Hofr
Extrinsic fluorescence
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Is intrinsic protein fluorescence usefull
for concentration determination?
• Only to a very limited extent and for certain proteins
(dependence of tryptophan emission on the position in a
protein structure, mutual fluorescence quenching of
amino acids by energy transfer)
• Concentration determination is more accurate with
external labeling
0
2
4
6
8
220 240 260 280 300 320
λ (nm)
ε(M
-1
cm
-1
)
0
0,5
1
250 300 350 400
λ (nm)
FluorescenceIntensity
Absorbance Fluorescence
Trp
Tyr
Phe
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Extrinsic fluorophores
External, alias extrinsic fluorophores are more used than
intrinsic fluorophores.
Fluorescent labels – are added and covalently bound to
a sample. They bind to proteins and nucleic acids via
amine, sulphydryl or histidine side chains and thiol groups.
Fluorescent probes – are bound noncovalently to a
sample and then change their fluorescent properties (e.g.
intensity, position of em. maximum)
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Absorbance of biological material
• Biological material absorbs relatively less in the range 500 - 600 nm
• The lowest natural fluorescence background is in the range 400 - 500 nm
• That is also the reason why probes and labels with exc. and em. maximum in this range
are used
• To enable study of labeled biomolecules even in the presence of unlabeled proteins,
excitation and emission wavelength of labels and probes must be higher than that of
intrinsic fluorophores (aromatic AA): 400-600 nm
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Fluorescent labels
A proper label should have following
parameters to be covalently bound to a
biomolecule:
- high fluorescence intensity
- stability during continuous light exposure
- minimal effect on biological properties of
a molecule
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Brightness
• Is given by a product of quantum yield and
molar extinction coefficient ε
Bs = Q ε
• Describes efficiency of a label to transform
excitation light to fluorescence
• A covalent bond of a label to a
biomolecule leads often to a significant
change of brightness
• Proper brightness of a label is:
Bc>5000
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Examples of extrinsic fluorophore
brightness
Fluorophore ε (cm-1 M-1)
Quantum
yield (Q)
Brightness
(Bs)
Oregon Green®
488
87 000 0.9 78 300
BODIPY FL 91 000 0.9 81 900
Fluorescein
(FAM)
79 000 0.9 71 100
JOE 71 000 0.6 42 600
TAMRA 103 000 0.2 20 600
Rhodamine RedX
(ROX)
82 000 0.7 57 400
Texas Red 139 000 0.9 125 100
http://www.promega.com/geneticidproc/ussymp8proc/21.html
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Examples of fluorescent labels
• dansyl chloride (DNS-Cl; 5-dimethylaminonapthalene-1-sulfonyl chloride)
• fluorescein-5-isothiocyanate (FITC)
• 5-iodoacetamidofluorescein (5-IAF)
• tetramethylrhodamine-5(a 6)-isothiocyanate (TRITC)
• 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl; 4-chloro-7-nitrobenzofurazan)
• 6-acryloyl-2-dimethylaminonapthalene (Acrylodan)
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Dansyl chloride
• One of the first and for this reason also the most representative fluorescent labels in literature
• Is used often for protein labeling, it is useful especially for measuring of anisotropy
• Very suitable fluorescence decay time τ ~ 10 ns
• Is excited at 350 nm, where proteins almost do not absorb
• The emission spectrum is sensitive to solution polarity and has mostly a maximum around 520
nm
• Reacts with free amino groups of proteins
0
2
4
6
8
220 240 260 280 300 320
λ (nm)
ε(M
-1
cm
-1
)
Absorbance of proteins
Trp
Tyr
Phe
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Fluorescein and rhodamines
• Belong to the most widely used fluorescent
labels Abs.max. Em. max.
fluorescein (490nm) (520)
rhodamines (500-600 nm) (530-620)
• Sensitive to solvent polarity and pH
• High value ε ~ 80 000 M-1cm-1
• High quantum yield Q ~ 0.3-0.9
• Fluorescence decay time ~ 4 ns
• A large number of derivatives is synthesized,
derivates that are used for labeling of proteins
and DNA through NH2 nebo SH groups
• The fluorescence intensity depends on pH
• They tend to photobleaching
FITC
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BODIPY
• Successor to fluorescein and rhodamine labels
• Derived from the fluorophore which contains
Boron
• Emission maxima 510-675 nm
• Extremely high quantum yield Q~ 1!
• They are not sensitive to solvent polarity and pH
• Emission spectrum is narrow and emission is
thus concentrated on a narrow range of
wavelengths and more different labels in the
mixture can be differentiated
• Disadvantage: small Stokes shift resulting in the
relatively small value of Förster distance at
resonance energy transfer (R0=57 A)
• Thanks to significant overlap of emission and
absorption spectrum, selfquenching occurs at
high concentrations of labeling (when the
molecules of fluorophores are closer than R0)
• Not suitable for FRET applications
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• Very popular
• The number means the length
of the chain of conjugated
bonds between two aromatic
rings
• Suitable for areas from 550 nm
further
• Relatively small Stokes shift
• They are used for FRET studies
Cy labels
http://www.cytographica.com/animations/Cy3Cy5FRET.html
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Alexa Fluor
• High quantum
yield-> high
brightness
• Improved water
solubility
• Small dependence
of fluorescence on
pH
• Photostable !
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Photostability of fluorophores
• Photobleaching comes to
each fluorophore after a
certain time
• Photostability is the most
important in microscopy,
where high intensities of
excitation light are used
• Alexa probes show the
highest photostability
• There has not been
observed any connection
between the structure of
fluorophores and their
photostability yet
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The effect of degree of labeling on
the intensity of fluorescence
• Photobleaching occurs often in a
classic fluorescein and
rhodamines at the high degree of
labeling (fluorophore molecules
are located at distance about R0)
• In the case of Alexa fluorophores,
photobleaching does not occur in
such extent and hence the
emission intensity is higher in the
case of higher degree of labeling
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Real-time PCR
detection of DNA amplification
Quencher
Emitter
F primer
R
primer
1. The labeled probe hybridizes to a complementary
sequence. Radiation of emitter is quenched and is not
observed.
2. The probe is replaced by a new chain during polymerization.
3. At each amplification cycle, the polymerase cleaves
the emitter. The emission intensity increase is detected.
4. Polymerization is completed. The intensity of
the emitted radiation is directly proportional to the
quantity of amplified DNA.
Bi9310
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Fluorescent labels for RT-PCR
• Which of these
labels are the best to
use?
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Fluorescent probes
• Fluorescent probes are extrinsic
fluorophores, that are bound noncovalently
to a monitored structure and
alter often their fluorescent properties.
Fluorescent probes are themselves very
little fluorescent in the solution usually.
However, their fluorescence significantly
increases after binding to proteins or
DNA.
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Probes sensitive to environment
polarity
1-anilinonapthalene-8-sulfonate (ANS) a 2-p-toluidinonapthalene-6-sulphonate (TNS) are typical
probes for dynamic polarity. The table shows the ANS fluorescence parameters in various solvents, it
follows that with increasing polarity of the solvent the ANS fluorescence emission maximum shifts to red
region and simultaneously quantum yield and time decay decreases. When ANS binds to apomyoglobin,
ANS is bound into a non-polar binding site for heme, emission maximum shifts to 454 nm and
fluorescence quantum yield increases to 0.98. In this way, it is possible to study the structure and degree
of polarity of different binding sites on proteins including possible displacing of fluorescent probes from
this bond or changes induced by e.g. enzyme activation, etc. ANS was used e.g. for studying of polarity of
the binding site for heme in apomyoglobin and apohemoglobin or conformational changes in muscle and
in nerve endings during action potential. TNS was used eg. to study conformational changes after
activation of chymotrypsinogen and changes of conformation of nerve membranes.
solvent λem
max
(nm)
quantum
yield
ime decay (ns)
octanol 464 0,646 12,3
propanol 466 0,476 10,2
methanol 476 0,216 6,05
water 515 0,004 0,55
Parameters of fluorescent probe 1-anilinonapthalene-8-sulphonate (ANS) at different
solvent polarity
polarity
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Change of fluorescence of serum
albumin in the presence of ANS
• By increasing the ratio
of molecules ANS: SA
there is a shift of the
emission maximum
from 350 nm to 480 nm
• This will increase the
intensity of light which
we see by the eye at
excitation of 280 nm
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DNA probes
• Intercalators EB, AO, TOTO intercalate
between base pairs
• Hoechst, DAPI bind to a DNA minor
groove
• EB increases the intensity of
fluorescence after binding 30x
and τ extends from 2 to 20 ns
• DAPI increases the intensity of
fluorescence most near AT pairs
• TOTO (Thiazole Homodimer)
increases the intensity of fluorescence
after binding 1100x
• Probes with high affinity as EB
homodimer (binds 10 000x tighter than
monomer EB) and positively charged
TOT0 remain bound to the DNA during
electrophoresis and are used for
visualisation of DNA on a gel to help
increase sensitivity upto 500x
compared to conventional EB staining
• How is it possible to reduce the
consumption of the probe?
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Intercalation
Benzpyrene TOTO
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Syber Green
• Selectively binds to ds DNA
into minor groove
• Detection from 1 ng/mL
• Use in RT-PCR to quantify
amplified DNA
Taq
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Comparison of probes for quantification
of dsDNA
The extinction coefficients were determined for the free probe in aqueous solution
Probe
Sensitivity
for dsDNA
Extinction
Coefficient
(cm-1 M-1)
Quantum
yield after
binding to
dsDNA
Increase in
fluorescence
intensity
upon
binding to
dsDNA
PicoGreen
25
pg/mL
70,000 0.53 ~2000x
Hoechst
33258
1-10
ng/mL
40,000 0.59 ~100x
Ethidium
bromid
1-10
ng/mL
5,000 <0.3 ~30x
http://www.promega.com/geneticidproc/ussymp8proc/21.html
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Protein probe
• Increase fluorescence intensity upon binding
to the protein
• The most sensitive fluorescent probes for
staining proteins in the gel are from the
group of organometallic compounds SYPRO
• SYPRO Red, Orange, Tangerine, Rose
• High sensitivity ~ ng/mL
• Acidic fixation is important before use
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Sypro probes
• Primarily used for
staining of proteins in
the gel
• Use in criminology
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Simultaneous staining of DNA and
proteins in the gel
• DNA is
stained by
Syber
Green
• Protein
SYPRO
Ruby
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Ion indicators
• Fluorescence measurement of changes of intracellular ions is possible
through probes that change their spectral properties upon binding of the ion.
Ca2+ is measured most commonly and is related to a number of books. The
indicators are usually derivatives of chelators Ca2+, Mg2+, Na+ or K+ as EGTA
and BAPTA that have appropriate affinity for the studied ion. When selecting a
suitable indicator we take into account :
• form of the indicator (salt, acetoxymethyl ester, dextran conjugate) that
affects the way how to get into cells (microinjection, electroporation, infusion
from the patch pipette, passive diffusion) and intracellular distribution
• measurement method - some indicators exhibit spectral absorption or
emission shift upon binding of the ion (measure the ratio of intensities at
different wavelengths of excitation or emission), other exhibit change in
fluorescence intensity
• dissociation constant - must be comparable with the measured
concentration of cations (concentration less than one tenth or bigger than ten
times of the dissociation constant cause too small changes in the observed
signal)
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Indication of Ca2+ in nerve cells
using Fluoro-3
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Quantum dots
• Semiconductor material is the
base
• Particles in the order of nm
• They have a narrow range of
symmetric
• No photobleaching!
• The emission wavelength is
determined by the diameter
and by the particles material
• Wide range of emission from
UV to IR
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The binding of biomolecules to
Qdots
• The core is made of the semiconductor CdS for UV, CdSe for Vis
and CdTe for IR
• Its size is comparable with the dimension of GFP
• The core is covered with a casing (shell), which allows connection
of the core with outer hydrophilic layer (Polymer coat)
• The outer hydrophilic layer provides solubility and enable binding of
biological molecules
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Properties QDots
• The width of the emission spectrum is 20 to 30 nm,
which is about 1/3 of the value of "classic"
fluorophores
• Quantum yield 0.35-0.5
• Absorb at each wavelength (semiconductor)
• They may emit at different wavelength at the same
excitation radiation!
• Ultraphotostable 100 times more resistant to
photobleaching than the "classic" organic
fluorophores
• Decay time ~ 100 ns
• High ε ~ 10 000 000 M-1cm-1
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Size comparison of QDot
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Applicability of fluorescent labeling
• The difference
between the
human and
monkey
karyotype
• Fluorescent
labeling helps to
answer the basic
scientific
questions
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Literature
• Lakowicz J.R.: Principles of Fluorescence
Spectroscopy. Third Edition, Springer +
Business Media, New York, 2006
• Haugland R.P.: Handbook of Fluorescent
Probes and Research Products. Ninth Edition,
Molecular Probes, 2002
• Fišar Z.: FLUORESCENČNÍ SPEKTROSKOPIE
V NEUROVĚDÁCH
http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
Graphics from the book Principles of Fluorescence was for the purpose of
this lecture kindly provided by Professor JR Lakowitzem.
Acknowledgement