Transcript Slide 1 - The Fluorescence Foundation

To perform fluorescence measurements
the molecules
HAVE TO FLUORESCE
FLUORESCENT LABELING
Susana Sanchez
Laboratory for Fluorescence Dynamics
3rd Annual Principles of Fluorescence Techniques, Genova, Italy, Sept. 13-15, 2005
How to choose the labeling protocol?
Light source available
In vivo or in vitro
Lifetime of the fluorescent probe
Spectroscopy or Microscopy
Can the wrong labeling
induce errors in
interpretation?
Experimental considerations
Illumination Volume and sample concentration
FCS
One or two-photon
Spectroscopy
0.4x0.4x1cm
0.16 cm3
(160uL=160x10-6L)
1x10-15 L
Number of molecules in the excitation volume
1 uM
10 nM
9.7x1013
9.7x1011
603
6
Correct labeling for the chosen technique
Example: dimer dissociation
Spectroscopy: Polarization measurements
Measuring a
population of
molecules
Microscopy: FCS measurements
D dimer/D monomer
1.2
Number of molecule
change
2
Number of molecule
change
1
Measuring
single
molecules
level
Too many labeled particles
7
6
Particle Number
5
4
3
2
1
0
0
100
200
300
time (s)
g  
Dcoef
g 0
log 
1
N
g  

400
500
Observing populations or particular behavior
Large Unilamellar Vesicles
50nm a 400nm
(0.05-0.4 mm)
Giant Unilamellar Vesicles
10-100mm
80 um
Measuring a
population of
liposomes
Measuring
single
liposomes
Labeling proteins
Labeling DNA
Labeling membranes
Coffee break
Quantum dots
Ions indicators
Labeling “in vivo”
Labeling proteins
Naturally Occurring Fluorophores in Proteins
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
Enzymes Cofactors
NADH
(oxido-reductases)
Ex/Em 340/460 nm
FAD
(metabolic enzymes
(ex/em 450nm/540 nm)
Porphyrins
(ex/em 550 nm/620 nm),
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 solventinsensitive Stokes shift
5-Hydroxytryptophan
ex/em 310nm/339 nm
•low quantum yield
and a large Stokes
shift in water
7-azatryptophan
ex/em 320nm/403nm
Protein Science (1997), 6, 689-697.
Fluorescent proteins
Phycobiliproteins
-Intensely fluorescent proteins from red algae
and cyanobacteria (blue-green algae).
red 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.
cyanobacteria
Four main classes of phycobiliproteins.
Green Fluorescent Protein (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.
Extrinsic probes
(not present in the natural molecule/macromolecule)
Non-covalent Attachments
bis-ANS
binds to hydrophobic patches on proteins
Mant-GDP
Ethidium bromide
Interact with
dsDNA & IgG/Antigen
Covalent Attachments
Fluorescent
group
Reactive
group
Light source
Reactive
group
aa
Available reactive
group in the protein
Lifetime of the
fluorescent group
Spectral properties
Autofluorescence
Labeling should not change the biological activity of the protein.
Fluorescent
group
Reactive
group
Reactive
group
aa
FITC
(488/512)
=4.05
Dansyl
chloride
BODIPI
(493/503),
=
IAEDANS
(360/480)
 =15 ns
Pyrenebutyric acid
Texas Red
N(CH3)2
NBD
O
S
Cl
O
ANS
(374/454)
 (EtOH)= 8 ns
Targeting amino groups
Fluorescent
group
Reactive
group
+
Reactive
group
+
Lysine
Arginine
aa
Targeting thiol groups:
Fluorescent
group
Reactive
group
+
Reactive
group
+
Cysteine
aa
General labeling protocol for extrinsic labeling
Removal of the free dye
Protein
in buffer
Incubation time
Addition of the
fluorescent dye
Labeling ratio
[protein]
[fluorescent dye]
Sample characterization
Absorption spectra
Protein determination
Biological testing
Activity measurements
SDS or native gel
Denaturation exp etc.
Labeling DNA
http://info.med.yale.edu/genetics/ward/tavi/n_coupling.html
Nick translation
5’
3’
A
3’
5’
E.Coli Pol I has 5’-3’ exonuclease activity
has 5’-3’ polymerizing activity
dUTP
5’
3’
B
3’
DNase nicks the double stranded DNA.
200-500 bp
End labeling of fragments
5’
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/polymerase activity, moves (or "translates") any single stranded nick
in the 5'-3' direction. When nicks on opposite strands meet, the DNA molecule breaks
PCR
5’
5’
3’
A
3’
5’
5’
dUTP
Taq Pol incorporates nucleotides along
the entire length of the DNA.
5’
B
3’
5’
5’
Higher labeling efficiency by PCR.
Requires decreased amount of probe.
5’
C
5’
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).
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
Labeling membranes
Fatty acids analogs and phospholipids
Sphingolipids, sterols,Triacylglycerols etc.
Dialkylcarbocyanine and Dialkylaminostyryl probes.
Other nonpolar and amphiphilic probes.
Laurdan, Prodan, Bis ANS
Membrane probes
DPH (D202),
NBD-C6-HPC (N3786),
cis-parinaric acid (P36005),
BODIPY 500/510 C4
N-Rh-PE (L1392),
bis-pyrene-PC (B3782),
DiI (D282),
DiA (D3883)
C12-fluorescein (D109).
Laurdan
Weber, G. and Farris, F. J.Biochemistry, 18, 3075-3078 (1979) .
Emission Intensity
1.2
Liquid
crystalline
phase
Gel
phase
1.0
0.8
0.6
0.4
0.2
0.0
350
400
450
500
wavelength
550
600
Laurdan Generalized Polarization (GP)
Ex=340 nm
1.2
IB
Emission Intensity
1.0
IR
0.8
0.6
0.4
0.2
0.0
400
450
500
550
600
wavelength
IB  IR
GPex =
IB  IR
-0.2
loose
lipid
packing
0.6
tight
lipid
packing
Parasassi, T., G. De Stasio, G. Ravagnan, R. M. Rusch and E. Gratton. Biophysical J., 60, 179-189 (1991).
GP in the cuvette
MLVs,SUVs,LUVs
Lipid Phase Transition
DPPC
DPPS:DPPC (2:1)
DPPG:DPPC (2:1) +
DMPA:DMPC (2:1)
DPPG:DLPC (1:1)
DPPC:DLPC (1:1)
GP
Temperature (°C)
Parassassi, Stasio, Ravaganan, Rusch, & Gratton (1991) Biophys. J. 60, 179
GP in the microscope
(2-photon)
70
140x10
3
60
120
50
% Transmittance
40
80
30
60
20
Measurement of
Laurdan in the GUVs
using SimFCS software
40
10
0
350
Fluorescence (au)
100
20
400
450
500
550
600
Wavelength (nm)
ch1
Blue filter
ch2
Red filter
GP image
GP histogram
0%chol
0.6
0.4
0.4
0.2
0.2
0.0
0.0
37
25
GP
GP
25
-0.2
C
-0.4
-0.6
31% chol
0.6
-0.2
C
-0.4
T
-0.6
-0.8
37
T
-0.8
-1.0
-1.0
0
10
20
30
40
50
60
70
0
10
20
temperature (celcius)
Temperature
(Celsius)
-1
30
40
50
Temperature
(Celsius)
Temperature(celcius)
GP
1
60
70
DOPC/DPPC 1:1mol/mol (31% mol chol)
24.8°C, rHDL 96Å
0.70
7000
6000
0.65
5000
0.60
GP
0.55
3000
0.50
2000
750
0.30
500
0.25
250
0.20
0
-20
0
20
40
60
80
100
time (minutes)
120
140
160
Number of pixels
4000
Coffee Break
Quantum dots
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
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 nonproductive,
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.
fuorescein
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.
Typical water-soluble
nanocrystal (NC) sample in
PBS
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.
Quantum Dot Size
Example
Wu et al. Nature Biotechnology 21, 41 - 46 (2002)
(A) 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.
(A) (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.
Ions indicators
Fluorescent probes for Ions
Fluorescence probes have been developed for a wide range
of ions:
Cations:
H+, Ca2+, Li+, Na+, K+, Mg2+, Zn2+, Pb2+ and others
Anions:
Cl-, PO42-, Citrates, ATP, and others
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 dextran…microinjection, electroporation, patch pipette.
AM-esters ….cleaved by intracellular esterases
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
FURA-2
Ratiometric: 2 excitation /1emission
Indo-1
Ratiometric: 1excitation /2emission
Calcium Green-1
Calcium Green-2
Probes For pH determination
Parent Fluorophore
pH
Range
Typical Measurement
SNARF indicators
6.0–8.0
Emission ratio 580/640 nm
HPTS (pyranine)
7.0–8.0
Excitation ratio 450/405 nm
BCECF
6.5–7.5
Excitation ratio 490/440 nm
Fluoresceins and
carboxyfluoresceins
6.0–7.2
Excitation ratio 490/450 nm
LysoSensor Green DND-189
4.5–6.0
Single emission 520 nm
Oregon Green dyes
4.2–5.7
Excitation ratio 510/450 nm or
excitation ratio 490/440 nm
LysoSensor Yellow/Blue DND160
3.5–6.0
Emission ratio 450/510 nm
Table 20.1 — Molecular Probes' pH indicator families, in order of decreasing pKa
BCECF
In situ calibration can be performed by using the ionophore nigericin (N1495) at a concentration of
10~50 μM in the presence of 100~150 mM potassium to equilibrate the intracellular pH with the
controlled extra cellular medium
Example 1
K.Hanson, M.J.Behne, N.P.Barry, T.M.Mauro, E.Gratton. Biophysical
Journal. 83:1682-1690. 2002.
Dye in DMSO is applied to the a live animal and incubated for some time
Labeled skin is removed
imaging
80
300
100
1000
100
1000
100
1000
100
1000
100
2000
c
20 mm
4.0
Depth (mm):
8.0
0
1.7
3.4
5.1
6.8
10.2
SC-SG Junction
Average pH
7.0
6.8
6.6
6.4
0
5
10
Depth (mm)
15
K.Hanson, M.J.Behne, N.P.Barry, T.M.Mauro, E.Gratton. Biophysical Journal. 83:1682-1690. 2002.
Labeling “in vivo”
Genetic Incorporation
GFP
FLAsh
Mechanical incorporation
Labeled proteins
Labeled DNA
Qdots
Genetic material
GFP-fusion proteins
ApaLI (178)
CE N6 /ARSH4
SwaI (5688)
GFP encoding plasmid
PstI (400)
ApaLI (5398)
NcoI (623)
URA3
AmpR
pUG35
6231 bp
KpnI (2009)
CY C1
ApaLI (4152)
NcoI (2294)
yG FP
NcoI (2818)
ori
SacI (3444)
ClaI (3005)
M ET2 5
HindIII (3010)
EcoRI (3022)
GFP
Your gene
P2b
PstI (3032)
(example. P2b)
AvaI (3034)
XmaI (3034)
SmaI (3036)
BamHI (3040)
ApaLI (178)
CEN6/ARSH4
SwaI (6006)
PstI (400)
ApaLI (5716)
NcoI (623)
URA3
AmpR
pUG35-P2b
Introduction into
6549 bp
KpnI (2009)
ApaLI (4470)
different
organisms
CYC1
NcoI (2294)
ori
yGFP
SacI (3762)
NcoI (2818)
ClaI (3005)
MET25
BamHI (3358)
HindIII (3010)
EcoRI (3022)
P2b
P2b
GFP
GFP-fusion proteins
GFP-fusion proteins
The human histone H2B gene fused (GFP) and
transfected into human HeLa cells
Current Biology 1998, 8:377–385
Homogeneous labeling
Regulation of the expression can be a problem for FCS
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.
FLASH-EDT2 labeling (FLASH tag)
Tetra-cys motif
receptor domain composed of as few as six
natural amino acids that could be genetically
incorporated into proteins of interest,.
a small (,700-dalton), synthetic, membranepermeant ligand that could be linked to various
spectroscopic probes or crosslinks.
bis-arsenical fluorophore FLASH-EDT2
The ligand has relatively few binding sites in
nontransfected mammalian cells but binds to the
designed peptide domain with a nanomolar or
lower dissociation constant.
An unexpected bonus is that the ligand is
nonfluorescent until it binds its target, whereupon
it becomes strongly fluorescent.
Hela cells transiently transfected with a gene for Tetracysteine-calmodulin.
Labeled 36 hours late vwith 1µM FLSH-EDTA2 for 1 hour
Griffin et al. SCIENCE VOL 281, 1998, 269-272
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/T0301C/technology/introduction.html
Microinjection
Microinjection is the process
injecting foreign DNA into cells.
of
directly
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/T0301C/technology/introduction.html
Non-homogeneous labeling
Transfected cells have to be selected
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/T0301C/technology/introduction.html
Non-homogeneous labeling
Transfected cells have to be selected
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
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
Final Coffee
Example 2
Martin Behne. University Medical Center. Hamburg, Germany.
Calcium Green-5N
Phasor
A
Phase delay
emission
m
Φ
g
excitation
C
t2
t2 min
Phasor
A
B
Phase delay
excitation
t1 max
t1
f2
m
Φ
g
C
s
1
Experi
f1
1
tape
Lifetime image
phasor
Calibration curve
1.0
B
Buffer + Cell Extract
Buffer + 10% BSA
0.8
0.6
100 uM Ca
2+
0.4
100 uM Ca
2+
0.2
0.0
0.0
0 uM Ca
0 uM Ca
0.2
0.4
0.6
2+
2+
0.8
1.0
lifetime shorter than the lifetime in zero calcium in buffer
Quenching?
Location: in the granules in the puppy skin