To perform fluorescence measurements

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To perform fluorescence measurements The
molecules HAVE TO FLUORESCE FLUORESCENT
LABELING
Susana Sanchez Laboratory for Fluorescence
Dynamics
4th Annual Principles of Fluorescence Techniques,
Genova, Italy, Jun 19-22, 2006
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How to choose the labeling protocol?
In vivo or in vitro
Spectroscopy or Microscopy
Light source available
Lifetime and Spectral Properties of the
fluorescent probe
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Labeling proteins
Labeling DNA
Labeling membranes
Quantum dots
Ions indicators
Labeling in vivo
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Naturally Occurring Fluorophores in Proteins
(a) aromatic amino acids
Phenylalanine (Phe F) Ex/Em 260 nm/282 nm
Tyrosine (Tyr Y) ex/em 280 nm/305 nm
Tryptophan (Trp-W) ex/em 280, 295nm/ 305-350 nm
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(b) Enzymes Cofactors
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Synthetic Fluorophores in Proteins
Tryptophan derivatives
Advantage absorbance spectrum of these analogues
is red-shifted with respect to that of
tryptophan. Therefore it is possible to
selectively excite them, in proteins, in the
presence of tryptophan of other proteins or DNA
bases.

• quantum yield similar to that of tryptophan .

• small and solvent-insensitive Stokes shift

• low quantum yield and a large Stokes shift in
  water

Protein Science (1997), 6, 689-697.
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Excitation wavelength
Protein Science (1997), 6, 689-697.
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Fluorescent proteins
Phycobiliproteins
-Intensely fluorescent proteins from red algae
and cyanobacteria (blue-green algae).
-Absorb strongly between 470 and 650 nm.
- In intact phycobilisomes, they are only weakly
fluorescent, due to efficient energy transfer to
photosynthetic reaction centers. Highly
fluorescent in vitro.
Four main classes of phycobiliproteins.
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Fluorescent Protein (FP)- example GFP
- from the bioluminescent jellyfish Aequorea
victoria. - Obvious ?-barrel structure, with
chromophore housed within the barrel. -
Remarkably, the chromophore is formed
spontaneously (from Ser-65, Tyr-66, Gly-67) upon
folding of the polypeptide chain, without the
need for enzymatic synthesis. - As a result,
it is possible to insert the gene for GFP into
cells and use the resulting protein as a reporter
for a variety of applications.
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Extrinsic probes (not present in the natural
molecule/macromolecule)
Non-covalent Attachments
bis-ANS binds to hydrophobic patches on proteins
Mant-GDP
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Covalent Attachments
Available reactive group in the protein
Light source Lifetime of the fluorescent
group Spectral properties Autofluorescence
Labeling should not change the biological
activity of the protein.
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Dansyl chloride
IAEDANS (360/480) t 15 ns

Pyrenebutyric acid
Texas Red
NBD
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Targeting amino groups

Lysine Arginine
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Targeting thiol groups

Cysteine
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General labeling protocol for extrinsic labeling
Protein in buffer
Activity measurements SDS or native
gel Denaturation exp etc.
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Characterization after the labeling Absorption
spectra
Absorbance
Bradford, Lowry, etc
Absorbance
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Labeling DNA
http//info.med.yale.edu/genetics/ward/tavi/n_coup
ling.html
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Nick translation
DNase nicks the double stranded DNA. E.Coli Pol
I has 5-3 exonuclease activity has 5-3
polymerizing activity End labeling of fragments
dUTP

200-500 bp
DNase I, which in the presence of Mg ions
becomes a single stranded endonuclease creates
random nicks in the two strands of any DNA
molecule. E. coli polymerase I, it's 5'-3'
exonuclease activity removes nucleotides "in
front" of itself. the 5'-3' polymerase activity
adds nucleotides to all the available 3' ends
created by the DNase . This exonuclease/polymera
se activity, moves (or "translates") any single
stranded nick in the 5'-3' direction. When nicks
on opposite strands meet, the DNA molecule breaks
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PCR
Taq Pol incorporates nucleotides along the entire
length of the DNA. Higher labeling efficiency by
PCR. Requires decreased amount of probe.
100-5,000 bp
Two single stranded DNA primers (18-30 bp long),
one forward and one reverse are synthesized
(yellow arrows). After adding the primers, the
Taq polymerase, the reaction mix is denatured
Then, the primers are allow to anneal (Fig. 2a)
to their target sequences (annealing step).
Then Taq polymerase synthesize the new DNA
strands (extension step, Fig 2b).
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Labeled dUTP
labeled nucleotides are synthesized by chemically
coupling allylamine-dUTP to succinimidyl-ester
derivatives of 1- fluorescent dyes 2-
haptenes (Biotin, Digoxigenin, Dinitrophenyl -
these require fluorescently-labeled antibodies or
specific proteins for visualization/detection).

Commercially labeled dUTP
fluorescein-aha-dUTP from Molecular Probes
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Labeling membranes
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Fatty acids analogs and phospholipids Sphingolipi
ds, sterols,Triacylglycerols etc. Dialkylcarbocya
nine and Dialkylaminostyryl probes. Other
nonpolar and amphiphilic probes. Laurdan, Prodan,
Bis ANS
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Membrane probes
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Laurdan
Weber, G. and Farris, F. J.Biochemistry, 18,
3075-3078 (1979) .
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Laurdan Generalized Polarization (GP)
Parasassi, T., G. De Stasio, G. Ravagnan, R. M.
Rusch and E. Gratton. Biophysical J., 60, 179-189
(1991).
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GP in the cuvette MLVs,SUVs,LUVs
Lipid Phase Transition
Parassassi, Stasio, Ravaganan, Rusch, Gratton
(1991) Biophys. J. 60, 179
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GP in the microscope (2-photon)
Measurement of Laurdan in the GUVs using SimFCS
software
ch1 Blue filter
ch2 Red filter
GP image
GP histogram
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DOPC/DPPC 11mol/mol
Hella
Erythrocytes
Living T. brucei (ec)
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Quantum dots
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Quantum Dot Size
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core
composed of cadmium sulfide (CdS), cadmium
selenide (CdSe), or cadmium telluride (CdTe).
The semiconductor material is chosen based upon
the emission wavelength, however it is the size
of the particles that tunes the emission
wavelength .
shell
biomolecule
In the cores emission is typically weak and
always unstable. 1-Reorganization of crystalline
imperfections and defects results in sites known
as traps. These sites provide a non-productive,
non-emissive, pathway. 2- The re-organized
surfaces tend to be very reactive and they easily
become polluted by solvent molecules, air
molecules, impurities, etc., The shell material
(typically ZnS in Qdots) has been selected to be
almost entirely unreactive and nearly completely
insulating for the core.
coating
a layer of organic ligands covalently attached to
the surface of the shell which further passivates
the core-shell and acts as a glue to the outer
layer. the outer layer is a mixed
hydrophobic/philic polymer. The hydrophobic part
interacts with the inner coating while the
hydrophilic portion interacts with the external
solvent to provide solubility in buffers. This
coating provides a flexible carboxylate surface
to which many biological and nonbiological
moieties can be attached. The resulting surface
is derivatizable with antibodies, Streptavidin,
lectins, nucleic acids, and related molecules of
biological interest.
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Their emission spectra is narrow and
symmetrical. The emission is tunable according
to their size and material composition, allowing
closer spacing of different probes without
substantial spectral overlap. They exhibit
excellent photo-stability. They display broad
absorption spectra, making it possible to excite
all colors of QDs simultaneously with a single
excitation light source and to minimize sample
autofluorescence by choosing an appropriate
excitation wavelength.
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The emission is tunable according to their size
and material composition
Samples were placed in front of a common UV hand
lamp. All samples are induced to emit their
respective colors even though a single source was
used to excite them. The colored spheres
illustrate the relative sizes of the CdSe quantum
dots in the vials.
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Example Wu et al. Nature Biotechnology  21, 41 -
46 (2002)

• Microtubules were labeled with
• 1-monoclonal anti-tubulin antibody.
• 2- biotinylated anti-mouse IgG and QD
  630-streptavidin (red).
• (B) Control for (A) without primary antibody.

• (C) Actin filaments were stained with
• 1-biotinylated phalloidin and QD
  535-streptavidin (green).
• (D) Control for (C) without biotin-phalloidin.
• The nuclei were counterstained with Hoechst 33342
  blue dye.

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Ions indicators
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Fluorescent probes for Ions
Fluorescence probes have been developed for a
wide range of ions
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How do we choose the correct probe for ion
determination?
1-Dissociation constant (Kd) Must be compatible
with the concentration range of interest. The Kd
of the probe is dependent on pH, temperature,
viscosity, ionic strength etc. Calibration is
important. 2- Measurement mode Qualitative or
quantitative measurements. Ratiometric
measurements. Illumination source available. 3-
Indicator form (salt, Cell-permeant acetoxymethyl
estes or dextran conjugate) Cell loading and
distribution of the probe. Salt and
dextranmicroinjection, electroporation, patch
pipette. AM-esters .cleaved by intracellular
esterases
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Probes For Calcium determination
UV FURA ( Fura-2, Fura-4F, Fura-5F, Fura-6F,
Fura-FF INDO ( Indo-1, Indo 5F) VISIBLE FLUO
(Fluo-3, Fluo-4, Fluo5F, Fluo-5N, Fluo-4N) RHOD
( Rhod-2, Rhod-FF, Rhod-5N) CALCIUM GREEN (CG-1,
CG-5N,CG-2) OREGON GREEN 488-BAPTA (OgB-1,
OgB-6F, OgB-5N, OgB-2) FURA
Cameleon system
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FURA-2
Ratiometric 2 excitation /1emission

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Indo-1
Ratiometric 1excitation /2emission

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Calcium Green-1


Calcium Green-2
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Probes For pH determination
Parent Fluorophore pH Range Typical Measurement
SNARF indicators 6.08.0 Emission ratio 580/640 nm
HPTS (pyranine) 7.08.0 Excitation ratio 450/405 nm
BCECF 6.57.5 Excitation ratio 490/440 nm
Fluoresceins and carboxyfluoresceins 6.07.2 Excitation ratio 490/450 nm
LysoSensor Green DND-189 4.56.0 Single emission 520 nm
Oregon Green dyes 4.25.7 Excitation ratio 510/450 nm or excitation ratio 490/440 nm
LysoSensor Yellow/Blue DND-160 3.56.0 Emission ratio 450/510 nm
Table 20.1 Molecular Probes' pH indicator
families, in order of decreasing pKa
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BCECF
In situ calibration can be performed by using the
ionophore nigericin (N1495) at a concentration of
1050 µM in the presence of 100150 mM potassium
to equilibrate the intracellular pH with the
controlled extra cellular medium
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Example 1
K.Hanson, M.J.Behne, N.P.Barry, T.M.Mauro,
E.Gratton. Biophysical Journal. 831682-1690.
2002.
Dye in DMSO is applied to the a live animal and
incubated for some time
Labeled skin is removed imaging
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K.Hanson, M.J.Behne, N.P.Barry, T.M.Mauro,
E.Gratton. Biophysical Journal. 831682-1690.
2002.
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Example 2
Martin Behne. University Medical Center. Hamburg,
Germany.
Calcium Green-5N
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tape
Lifetime image phasor
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Labeling in vivo
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Genetic Incorporation GFP FLAsh Halotags Mechanic
al incorporation
Labeled proteins Labeled DNA Qdots Genetic
material
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GFP-fusion proteins
GFP encoding plasmid
Your gene (example. P2b)
GFP
Introduction into different organisms
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FP-fusion proteins
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GFP-fusion proteins
The human histone H2B gene fused (GFP) and
transfected into human HeLa cells
Current Biology 1998, 8377385
Homogeneous labeling Regulation of the expression
can be a problem for FCS
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Novel Fluorescent Proteins (NFPs),
Novel Fluorescent Proteins derived from new
species of reef coral and jelly fish
Broadest spectrum of fluorescent proteins,
covering the emission spectra between 489 nm and
618 nm. Novel fluorescent proteins are
incorporated into many of the our popular
vectors, designed for constitutive fusion
protein expression in mammalian cells,
subcellular localization of organelles or
targeting of fusion proteins to a specific
location, transcriptional reporting bacterial
expression and many other special purposes
NFPs give similar or better performance than the
original Enhanced Fluorescent Proteins (EFP)
family. NFP monomer proteins are extremely
stable, allowing fluorescence monitoring over
long periods of time. The NFP family also
includes pTimer, which changes color, enabling
monitoring of cellular events over time.  
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FLASH-EDT2 labeling (FLASH tag)
Griffin et al. SCIENCE VOL 281, 1998, 269-272
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Electroporation
Electroporation is the process where cells are
mixed with a labeled compound and then briefly
exposed to pulses of high electrical voltage.
The cell membrane of the host cell is
penetrable thereby allowing foreign compounds to
enter the host cell. (Prescott et al., 1999).
Some of these cells will incorporate the new
DNA and express the desired gene.
Non-homogeneous labeling Transfected cells have
to be selected
Source http//dragon.zoo.utoronto.ca/jlm-gmf/T03
01C/technology/introduction.html
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Microinjection
Microinjection is the process of directly
injecting foreign DNA into cells. By
examination with a microscope, a cell is held in
place with gentle suction while being manipulated
with the use of a blunt capillary. A fine pipet
is then used to insert the DNA into the cytoplasm
or nucleus. (Prescott et al. 1999) This
technique is effective with plant protoplasts and
tissues.
-Photo of a Microinjection apparatus(courtesy of
A. Yanagi)
Source http//dragon.zoo.utoronto.ca/jlm-gmf/T03
01C/technology/introduction.html
Non-homogeneous labeling Transfected cells have
to be selected
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Agrobacterium-mediated transformation
This method of transformation is the most
widely used to introduce foreign genes into plant
cells. A. tumefaciens contains a Ti plasmid
(tumour-inducing) which normally infects
dicotyledon plant cells, making the bacteria an
excellent vector for the transfer of foreign DNA.
(De La Riva et al., 1998) By removing the
tumour inducing genes and replacing them with the
genes of interest, efficient transformation can
occur. As a vector of gene transfer, it has
advantages over other traditional methods in that
relatively large segments of DNA can be
transferred with little rearrangement, and
integration of low numbers of gene copies occurs
in plant chromosomes.  
Source http//dragon.zoo.utoronto.ca/jlm-gmf/T03
01C/technology/introduction.html
Non-homogeneous labeling Transfected cells have
to be selected
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Biolistics  
                                                  
       Biolistics is currently the most widely
used in the field of transgenic corn production.
The DNA construct is coated onto fine
gold/tungsten particles and then the metal
particles are fired into the callus tissue.
(Rasmussen et al., 1994) As the cells repair
their injuries, they integrate their DNA into
their genome, thus allowing for the host cell to
transcribe and translate the gene. Once the
transformation process has been completed, those
cells expressing the gene must be selected for.
Traditionally, this is done on the basis of the
selectable marker that was inserted into the DNA
construct (Brettschneider et al., 1997).
Traditional selectable markers confer
resistance (antibiotic or herbicide) with
Kanamycin one of the most popular markers used.
Source http//dragon.zoo.utoronto.ca
Non-homogeneous labeling Transfected cells have
to be selected
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Nanocrystal targeting in vivo
Blood vessels express molecular markers that
distinguish the vasculature of individual organs,
tissues, and tumors. Peptides that recognize
these vascular markers have been identified,
purified and attached to a Q-dot. Each of the
peptides directed the Qdots to the appropriate
site in the mice, showing that nanocrystals can
be targeted in vivo with an exquisite specificity.
                                                
                   Fig. 1.   Schematic
representation of Qdot targeting. Intravenous
delivery of Qdots into specific tissues of the
mouse. Qdots were coated with either peptides
only or with peptides and PEG. PEG helps the
Qdots maintain solubility in aqueous solvents and
minimize nonspecific binding.
Åkerman et al.PNAS October 1, 2002 vol. 99
no. 20 12617-12621
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Can the inappropriate labeling induce errors in
interpretation? Experimental considerations
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Correct labeling for the chosen technique
Example dimer dissociation
Spectroscopy Polarization measurements
Measuring a population of molecules
Microscopy FCS measurements
Number of molecule change 2
D dimer/D monomer 1.2
Measuring single molecules level
Number of molecule change 1
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Too many labeled particles
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Illumination Volume and sample concentration
FCS One or two-photon
Spectroscopy
0.4x0.4x1cm 0.16 cm3 (160uL160x10-6L)
1x10-15 L
Number of molecules in the excitation volume
1 uM 9.7x1013
603
10 nM 9.7x1011
6
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Observing populations or particular behavior