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Epi-illumination is form of Kohler Illumination:
Objective is also condenser
White light (regular Kohler)
Brightfield, phase, etc
Light is focused
At back aperture
Of the objective,
Conjugate to
condenser
aperture
Different illumination
And image paths
Lamp or
laser
lens
detector
Detect at 90 degrees
Split with dichroic mirror
Greatly increases S/N
Epi-illumination separates
light source,
Fluorescence signal
First barrier filter
Selects excitation
Arc
lamp
Second
barrier filter
Selects signal
From background
dichroic
mirror
objective lens
specimen
General Jablonski Diagram
Typical molecular timescales:
S2
Absorption: instantaneous (10-17 s)
Vibronic Relaxation ~ 10-12 s
S1
T1
Fluorescence: 10-6 -10-12 s
10-9 most typical
Singlet to a singlet (strong)
S0
Fluorescence always from
relaxed level of S1
Phosphorescence: 10-3 -10-6 s
Triplet to singlet (weak)
Magnitude of Extinction Coefficients
Extinction coefficient ε:
Beer’s Law A= εcl
Strong absorbers (dyes) have ε between 20,000-100,000
Absorption cross section is also used: 1x 10-16 cm2 = 23,000 ε
Brightness= absorption coefficient* QY
Oscillator strength is integral of the absorption band
Sum rule: oscillator strength, f,
for one electron over all transitions is:
1x 10-16 cm2 eV
Emission spectrum is independent of of path excitation
(both spectral wavelength and width): always from S1
Emission intensities will be different
Due to different absorption probability:
Franck-Condon Principle
Franck-Condon Principle
Consider electronic states anharmonic oscillators: bond length
Most probable transitions are “vertical”
Big geometry change=broad spectrum (smaller maximum absorption)
Both for absorption and emission: conserve oscillator strength
Geometry and Absorption and Emission Spectra
Bigger Stokes shifts provide better signal to noise
In fluorescence because of filter efficiencies,
(dichroics, low pass, high pass)
But usually involve large geometry change: lower intensities
Spreading out oscillator strength
Fluorescence Quantum Yield φ: important for dyes
Ratio of emitted to absorbed photons
Quantum Yield:


kf
k f  k isc
(k is rate,
Inverse of time)
kf
k f  k isc  k nonrad
 1  k f  kisc
Measured lifetime is sum of
natural lifetime and
non radiative decay paths
Lifetime is useful contrast
Probe of environment
B. Fluorescent Probes
• Molecular Probes (Invitrogen)
– www.probes.com
– Catalog contains thousands of fluorescent
probes, with valuable technical information.
Organelle Probes
Target, Name
Mitochondria
MitoTracker dyes
Golgi
BODIPY FL and TR
C5-ceramide
Lysosomes
LysoTracker
LysoSensor
Endoplasmic reticulum
DiOC6, DiIC6
Fluorescent Brefeldin A
Excitation
Emission
490/578/551
516/599/576
505/589
511, 620 /617
Various
Various
Various
Various
484/549
501/565
Comments
MT Green accumulates in
mitochondria regardless of
MB potential, Red and
Orange in active
mitochondria; aldehyde
fixable
TR is better for double
labeling, since no green
emission
DiO and DiI also stain
membranes generally
Inhibitor of protein transport
Immunofluorescence Imaging – Detect Proteins
IgG
Fab
Fc
Fluorescence in situ Hybridization (FISH)
– Detecting Nucleic Acids
Fluorescein: most common dye for microscopy
Blue green
Xanthene family
“green fluorophore”
e80,000, φ~0.9
Brightness ~ ef
Many functionalized forms
for cell imaging: pH, ion sensing
•High quantum yield
•General purpose
•But degrades quickly
•Small Stokes shift
(filter bleedthrough)
Rhodamine 6G
Green Red
Xanthene family
“red fluorophore”
Internal Donor-acceptor pair
Red-shifts the spectra relative to
fluorescein
Many functionalized forms
for cell imaging
•High quantum yield
•General purpose
•Good stability
•Also Small Stokes shift
Green Fluorescent Protein (GFP)
Fluorophore made of
Ser65, Tyr66 and Gly67
•
•
•
•
Requires no co-factor or substrate.
Works in almost any organism.
Easy to quantify.
Genetically modifiable.
Tsien, Ann.Rev. Biochem. 67, 509 (1998)
Many fluorescent proteins:
Jellyfish, Coral Reefs
Colored Proteins allow labeling of
multiple specific organelles
Variants of Fluorescent Proteins
BFP
EBFP, Sapphire, T-sapphire
CFP
ECFP, mCFP, Cerulean, CyPet, AmCyan, Midoriishi Cyan
GFP
EGFP, Azami Green, TurboGFP, ZsGreen, Emerald
YFP
EYFP, Topaz, Venus, mCitrine, YPet, ZsYellow1, PhiYFP
OFP
mBanana, Kusabira Orange, mOrange
RFP
dsRed, tangerine, dTamato, mStrawberry, AsRed2, mRFP, mCherry,
mRasberry, mPlum, JRed, HcRed
GFP Chromophore
Aequorea: FSYGVQ
Renilla:
p-hydroxybenzylidene-imidazolidone
Ser - dehydroTyr - Gly
FSYGDR
Chromophore Maturation
Takes ~ 30 min for wild type GFP.
GREEN FLUORESCENCE PROTEIN
GFP
Jelly fish
isolate DNA
encoding GFP
cellular protein
cellular protein
couple gene for GFP
with gene for protein of interest
GFP
transform cell with
altered protein
Completely general and versatile
Problems with Fluorescent Protein
• Size comparable to the target. Might
interfere with the function of the target
protein
• Maturation time
• Probably not 100% fluorescent
• PH dependence
• Many variants mis-fold when fused to
another protein
Linearly Polarized Light
s= horizontal
p= vertical
For propagation
Parallel to floor
Polarizer is device that selects polarization
Can be crystal or film (Polaroid)
Operation of Analyzer (Birefringent)
Light transmitted at angle  relative to angle of 2
Crossed polarizers
2
I  I 0 cos 
Combining linear polarized light
IN PHASE
Linear Polarization
OUT OF PHASE
Elliptical Polarization
Circularly Polarized Light
• Decompose to linear polarized
light with 1/4 phase shift.
• No direction (always pass 50%
through polarizer in dependent of
polarizer orientation)
• NOT the same as unpolarized
light.
• Can be converted back to linear
polarized light with birefringent
materials (1/4 wave plate).
Half wave plate rotate the polarization direction of light
Absorption is polarized
Fluorescence is also polarized
GFP Crystal
Anisotropic sample
- Fluorescent intensity is dependent on the polarization _and_ the
orientation of the molecules
Isotropic sample
- Fluorescent intensity is independent of excitation polarization
- Fluorescence is polarized if the excitation is polarized.
Fluorescence anisotropy
r < 0.4
Microscopic Measurements of
Anisotropy
r = r0 / ( 1 +  /  )
Use Small Numerical Aperture
Fluorescence Resonance Energy Transfer (FRET)
Förster Radius
The distance at which energy transfer is
50% efficient (i.e. 50% of excited donors
are deactivated by FRET) is defined by the
Förster Radius (R0).
Applications of FRET in Biology
Survey of FRET-Based
Assays
•
•
•
•
•
•
•
Protease activity
Calcium Ion measurements
cAMP
Protein tyrosine kinase activity
Phospholipase C activity
Protein kinase C activity
Membrane potential
FRET probes conformational changes
Different conformation gives
Different FRET signature
Inter and Intramolecular
Forms of FRET with
Proteins
CFP-YFP good combo
FRET increases
In both cases
Protein-Protein Interactions
In cytoplasm and
membranes
When FRET Occurs
No FRET for
No overlap of donor emission,
acceptor absorption
No FRET for
Orthogonal dipole
orientation
No FRET for molecules
more than 10 nm apart
Number of FRET Publications since 1989
Fluorescence Resonance Energy Transfer Detection of Probe Proximity
F  FD
FA  F
  D
FRET 
 Max

0
0
FD
FA  FA
 D0
0
D
0
A
0
D
R0 typically 40-50 Angstroms
50% transfer
R06
FRET  6
6
R  R0
Typical Values of Ro
green
Donor
Fluorescein
Acceptor
Ro (Å)
Tetramethylrhodamine 55
IAEDANS
EDANS
Fluorescein
Fluorescein
DABCYL
Fluorescein
46
33
44
BODIPY FL
Fluorescein
Cy3
CFP
BODIPY FL
QSY 7 dye
Cy5
YFP
57
61
53
50
red
GFPs and other colored “FPs have transformed FRET microscopy
FRET Considerations:
1. Spectral overlap
2. Chromophore orientations
3. Distance dependence (Eff.  1/R6)
4. How to quantify?
E
E
f acceptor
f donor
f acceptor  f baseline  f A spillover
f donor  f D  spillover
Practical Challenges to FRET Quantitation
•
•
•
•
Emission from A contaminates D channel (filters)
Emission from D contaminates A channel
Unknown labeling levels for D and A
Signal variation due to bleaching
– Complicates kinetic studies
– Bleaching rate of D can actually be slowed by FRET
Solutions:
• Separately labeled D and A controls to define
bleedthrough
0
F
• Acceptor destruction by photobleaching to establish D
• Dual wavelength ratio imaging to normalize away
variations in label levels and bleaching effects
Ca2+ Release During Shrimp Egg Activation
• From Lindsay et al. (1992). Extracellular Mg2+ Induces
an Intracellular Ca2+ Wave During Oocyte Activation in
the Marine Shrimp Sicyonia ingentis. Dev. Biol. 152:94102.
Low quantum yield with no Ca2+, big increase
When binds Ca2+: up to 50 fold increase
Not absolute concentration of ions, measure relative changes: easier
Fluo- dyes
By Tsien
Choose depending on desired
Range of sensing
Blue Ca2+ Indicators: Fluo-3 has
single Ex and Em wavelengths
• A visible light excitable
dye (488 nm), so Argon
laser can be used.
• Emission at 525 nm.
• OK for qualitative
detection but not
quantitative.
Calcium Sensing Indo-1
Ratiometric using single excitation, dual emission
Excite 338 nm, collect 405, 485 nm fluorescence
Determine absolute calcium concentration by imaging
Free Ca2+ Concentration in a Purkinje
Neuron from Embryonic Mouse Cerebellum
• Neurons were loaded with
fura-2.
• Neurons were stimulated
with glutamate receptor
agonist.
• The composite image
represents the ratio of
images obtained with
excitation at 340 nm and
380 nm.
Membrane Potential
Membrane Potential Is Due to
Charge Imbalance
Depolarized
Bis-oxonol
Very sensitive: 1%/mV
Anionic dye crosses into
Mitochondria when depolarized
(high potassium)
Resting potential (~-300 mV)
Voltage Sensitive Styryl Chromophore
Fast dye
Stains membrane
e Max~30,000
Large geometry change,
Charge shift upon absorption:
Makes spectra sensitive to electric fields
Charge shift in styryl dyes
Excited state
-
+
+
-
Ground state
Voltage sensitivity of membrane potential dyes
(electrochromism / Stark Effect)

E
S1 d

E

E 0
d
d
d
E0
d
S0 d
Depolarized
Blue shift
intra
extracellular
d
d
d
d
d
Hyper-polarized
Red shift
d
 1
h   G   E   E   G   E E 2
2


 depends upon dipole moment, polarizability, field strength and orientation
Mechanism of Voltage-dependent
Spectral Shifts in Styryl Dyes ‘Electrochromism’
Red shift
Blue shift
Ratiometric approach at
Inflection points
for Highest sensitivity
1) Single excitation, dual emission:
Laser excitation
2) Dual excitation, same emission:
Arc lamp excitation
F/F: normalizes
Also more sensitive,
Changes are small-10%/100 mV
F/F: normalizes for geometrical factors, bleaching:
Ratiometric approach used for many types of dyes