ii. fluorescent dyes - huang labhuanglab.ucsf.edu/lectures/2012 ucsf fluorescence dyes.pdfii....
TRANSCRIPT
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Bo Huang2012.03.27
II. Fluorescent dyes
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The fluorescence process
hνhν
Absorption (Spontaneous)Emission
Vibration relaxation
S1
S0
WavelengthTransition prob
ability
Stokes shift
Absmax Emmax
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Extinction coefficient
hν
AbsorptionS0 +hν→ S1
S1
S0
l =1cmI0
I1
Abs=‐ log10(I1/I0)
ε =Abs/(l × c)
Typically50,000~200,000M‐1cm‐1
1µM,1cm A≈0.1(T=80%)
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Fluorescence Lifetime
hνhν
Absorption
Emission
S1 → S0 +hν
Rateconstant=kfl
Lifetimeτfl=1/kfl
τfl time
[S1]
Fluorescence=kfl[S1]
Vibration relaxation
τfl ~ns
<ps
S1
S0
1/e
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Quenching
hνhν
Non‐radioactive decay:
Absorption
Emission
S1
S0
Nonradioactivedecay
Energy dissipation through structural flexibility
DiI
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Quantum Yield
hνhν
Absorption
Emission
QE=kfl /(kfl +knr)=1/(1+τfl knr)
τfl ⇑,QE⇓knf ⇑,QE⇓
emittedphotonabsorbedphoton
QE=
Fluorescence
Nonradioactive decay
kfl
knrS1
S0
Nonradioactivedecay
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Quantum Yield
hνhν
NonradioactivedecayAbsorption
Emission
Fluorophore QE
Fluorescein in ethanol 0.97
Tryptophan, pH 7.2 0.14
EGFP 0.60
EGFP chromophore by itself 0.0005
S1
S0
emittedphotonabsorbedphoton
QE=
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The brightness of a fluoropore
hνhν
NonradioactivedecayAbsorption
Emission
S1
S0
S0 +hνk1 S1
S0 +hν1
S0 +heatknr
kfl
Brightness≈
EC(wavelength)
×QE
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Brightness can be affected by the environment
↔ ↔ ↔
FH3+ FH2 FH– F2–
Sjoeback et al., Spectrochemica Acta Part A (1995)
Fluorescein
Low pH High pH
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Brightness can be affected by the environment
Invitrogen
↔ ↔ ↔
FH3+ FH2 FH– F2–Fluorescein
Low pH High pH
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The Full Jabłonski Diagram
hνhν
Emission
Intersystem crossing
Triplet State
Vibration relaxation
hν
PhosphorescenceTriplet
Quenching
Lifetime ≈ μs ‐ms
Absorption
S1
S0
T1
Non‐radioactivedecay
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Photobleaching
hν
Absorption
Triplet State
Vibration relaxation Intersystem crossing
Most common cause: O2
S1
S0
T1
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Photobleaching is our enemy
For all fluorescence imaging:• Kills signal• Alters the relative contrast
Live imaging:• Limits observation duration• Phototoxicity from reactive
oxygen speciesMichael Davidson
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Fight against photobleaching
• Choose “great” dyes• “Anti‐fade” mounting media
– Glycerol– Oxygen scavengers– Free‐radical scavengers and triplet quenchers
• Trolox, mercaptoethanol, etc.
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Fight against photobleaching, cont.
• Labeling as densely as possible• Budget the photons
– Only expose when observing– Minimize exposure time & excitation power– Use efficient filter combinations– Use high QE, low noise camera
Fluorescencesignal =
Excitationenergy
Fluorophorebrightness
Detectionefficiency
Detectorsensitivity× × ×
Intensity × Exposure time Objective, Filters, Light path
Fluorophoreconcentration×
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Choice of fluorophores
Tsien lab
Fluorescent proteins
Natural fluorescenceOrganic dyes
Inorganic fluorophores
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Inorganic fluorophores
• Quantum dots– Extremely bright and photostable– Broad excitation, narrow emission spectra– Large, difficult for specific labeling (10 – 20 nm)
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Inorganic fluorophores
• Quantum dots• Lanthanides
– Very large Stokes or anti‐Stokes shift (UV Red or RedGreen)
– Sharp emission peaks– Extremely long life time (µs ‐ms)
Gahlaut et al., Cytometry A, 2010
Bruce Cohen
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Lavis & Raines, ACS Chem Biol, 2008
Small Molecule Fluorophores
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Lavis & Raines, ACS Chem Biol, 2008
Amax = 280 nm, Ext = 5,600Emmax = 348
Amax = 274 nm, Ext = 1,400Emmax = 303
Amax = 257 nm, Ext = 200Emmax = 282
Intrinsic Fluorescence from Amino Acids
Tryptophan fluorescence image
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Lavis & Raines, ACS Chem Biol, 2008
Fluorescent Enzyme Co‐factors
NADH fluorescence image
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Lavis & Raines, ACS Chem Biol, 2008
DNA Intercalating Dyes
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Lavis & Raines, ACS Chem Biol, 2008
The classics
Carbocyanine
RhodamineFluorescein
BODIPY
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The new generations
• Alexa Fluor series (Molecular Probes)• Atto series (ATTO TECH)• DyLight (Dyomics)• Many more…
• Check the experimental conditions of the claims.• Try them out.
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Small molecule dyes
• Small– e.g. DNA labeling
• Bright and photostable• Wide range of wavelengths
– UV to NIR
• Most of them lack labeling specificity by itself.– Specific probes for nucleic acid, plasma membrane, mitochondria
• Almost all “great” ones are unable to cross the membrane.
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(Specific) delivery of small molecule dyes
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Functional dyes
• Nucleic acid intercalating dyes– QE increment upon binding to DNA/RNA
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Functional dyes
• Nucleic acid intercalating dyes– QE increment upon binding to DNA/RNA
DAPI TOTO‐1
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Functional dyes
• Nucleic acid intercalating dyes• Membrane stains
– Amphiphilic dyes that partition in lipid bilayers
DiI
FM 1‐43
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Functional dyes
• Nucleic acid intercalating dyes• Membrane stains
– Amphiphilic dyes that partition in lipid bilayers
Alexis M. Stranahan
Dentate gyrus granule cells labeled with DiI
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Functional dyes
• Nucleic acid intercalating dyes• Membrane stains• Organelle (mitochondria, ER, Golgi, etc.) stains
– Based on charge and redox properties
MitoTracker Red CMXRos
MicroscopyU
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Small molecules probes
• Small molecules that bind proteins
ER Tracker Green
Phalloidin Taxol
α‐bungarotoxin
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Small molecules probes
• Small molecules that bind proteins
ER Tracker Green
Phalloidin Taxol
α‐bungarotoxin
Actin Microtubule
ER
Acetylcholine receptor
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Large molecule labeling
• In vitro reconstituted systems• Injecting labeled proteins
Waterman & Salmon, FASEB J, 1999
10% labeled tubulin 0.25% labeled tubulin
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Large molecule labeling
• In vitro reconstituted systems• Injecting labeled proteins• Fluorescence in situ hybridization (FISH)
8 color 3D FISH to show chromosome territories in human G0 fibroblast nucleus
Bolzer et al., PloS Biology (2005)
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Large molecule labeling
• In vitro reconstituted systems• Injecting labeled proteins• Fluorescence in situ hybridization (FISH)• Biotin‐avidin interaction
– Labeled avidin/streptavidin– Biotin‐dyes
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Large molecule labeling
• In vitro reconstituted systems• Injecting labeled proteins• Fluorescence in situ hybridization (FISH)• Biotin‐avidin interaction• Antibody (immunofluorescence)
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Labeling reactions
• Amine – succinimidyl ester chemistry (Lys and N‐term)• Thiol – maleimide chemistry (Cys)
NH2
+
NHS
NaHCO3
+ freeGel filtration
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Labeling stoichiometry
Protein concentration = (A280 – dye contribution) / E.C. protein@280
Dye concentration = Amax / E.C. dye@max
Dye per protein = Dye concentration / Protein concentration
Too high labeling stoichiometry can make the protein dead…and lead to self‐quenching
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Immunofluorescence
Antigen binding sites
Fab fragment Nanobody
Direct immunofluorescence
Primary antibody binding efficiency can be < 10%
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Immunofluorescence
Antigen binding sites
Fab fragment Nanobody
Indirect immunofluorescence
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Fixation methods
• Methanol– Precipitates proteins in situ, dissolve membrane lipids– Good for protein structures, can destroy organelles and extract
cytoplasmic proteins
• Formaldehyde (Paraformaldehyde)– Mild crosslinking of proteins– Most widely used, may not be strong enough crosslinking– Common for tissue fixation
• Glutaraldehyde– Strong crosslinking, preserving the ultrastructure– May mask some epitopes– May create fluorescence background (NaBH4 reduction)– Required for electron microscopy
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Fixation methods
• Methanol• Formaldehyde (Paraformaldehyde)• Glutaraldehyde
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Membrane permeabilization
• Acetone• Non‐ionic detergents
– Triton X‐100, Tween‐20• Extracting lipids, making holes on the membrane
• “Mild” detergents– Saponin
• Extracting cholesterol only, permeabilizing the plasma membrane while saving the organelle membranes
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Fixation artifactsMicrotubules
GAMethanolPFA
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Some fixation artifacts become visible in super‐resolution microscopy
Formaldehyde fixationGlutaraldehyde fixation
Projection
Section throughthe middle
Tom20 –mitochondria
outer membrane
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Target protein
The hybrid approaches
• SNAP‐tag, CLIP‐tag, HALO‐tag, TMP‐tag…– SNAP‐tag: based on human O6‐alkylguanine‐DNA‐alkyltransferase (hAGT)
benzylguanine
Target protein
SNAP‐tag SNAP‐tag
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