Download Fluorescence

Fluorescence
1.
2.
3.
4.
5.
6.
What is fluorescence
Technical issues
Uses of Fluorescence:
Quantitation
Ligand binding
Conformational changes
Measuring distances
Fluorescence quenching
Fluorescence lifetimes
Fluorescence
What happens after a molecule has adsorbed light
exciting
light
photobleaching
*
excited
state
Lifetime 1 –10 10-9 secs
(1-10 nsecs)
(try to avoid)
heat
emission of
fluorescence light
Fluorescence
excited
state
heat
Energy
heat
ground
state
Absorption
Stokes
shift
Intensity
Abs
Flu
Wavelength (nm)
Lowest vibrational
level in excited state
Fluorescence
emission
Fluorescence of Trp
280 nm
340 nm
Intensity
Abs
Flu
Wavelength (nm)
Fluorescent amino acids: Trp, Phe, Tyr
Fluorescence of proteins is dominated by Trp
Fluorescence
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4.
5.
6.
What is fluorescence
Technical issues
Uses of Fluorescence:
Quantitation
Ligand binding
Conformational changes
Measuring distances
Fluorescence quenching
Fluorescence lifetimes
Fluorescence Instrumentation
Spectrofluorimeter
Looks at samples in solution
(Typically 3 mls)
High sensitivity ( conc 0.1 µM)
Fluorescence microscope
Looks at spatial distribution in 2D or
3D (confocal)
Source of Radiation
Arc lamp (450 watt Xenon) + monochromator
Laser (generally fixed wavelength
eg Argon-ion laser 488 nm
Detector
Phototube (can be electrically cooled)
CCD camera on fluorescence microscopes
Fluorimeter (right angle geometry)
excitation
monochromator
light
source
slit
cuvette
unabsorbed
exciting light
slit
fluorescence light
emission
monochromator
fluorescence
detector
Sensitivity
Down to ca 0.01 µM (ca 100 times more sensitive than
absorption spectroscopy
Limited by:
Scattered light (problem with cloudy samples)
Background fluorescence
Impurities in buffers (use Ultra-pure reagents)
Autofluorescence in cells
Ideal fluorescence molecule:
High extinction coefficient ε (Absorbs light well)
High quantum yield
Large Stoke’s Shift
High wavelength of emission
Usually contains multiple –C=C-
Sensitivity
Common problem : concentration too high
Inner filter
effect
Flu.
intensity
Optical Density less than 0.05
to avoid inner filter effect
cuvette
Concentration
Volume
observed
excitation
Inner Filter
Effect
Absorption of
light here leads
to low light
intensity in
volume observed
slit
Flu light
absorbed
fluorescence light
Fluorescence Parameters
Stokes
shift
Intensity
Abs
Flu
Wavelength (nm)
Wavelength of maximum absorption (equiv. to abs. Spectrum)
intensity of absorption – extinction coefficient ε
Wavelength of maximum fluorescence emission
environmentally sensitive
Fluorescence quantum yield Q (related to intensity of emission)
environmentally sensitive
(cont.)
Fluorescence Parameters (cont.)
Fluorescence quantum yield Q (related to intensity of emission)
Q= no. of photons emitted
no. of photons absorbed
Q between 0 and 1
(Trp Q ca 0.2)
heat
excited
state
heat
ground
state
Fluorescence
emission
Absorption
Fluorescence lifetime
Time molecules remains in the excited state
typically 1-10 nsecs
Fluorescence Intensity Measurements
Abs
OD
Flu
Abs=εcl
Fluorescence
Intensity
Arbitrary units
Wavelength (nm)
Fluorescence intensity is measured in ‘arbitrary units’
Size of signal depends on sensitivity of the fluorimeter
Thus need to standardize:
measure relative to some convenient standard solution
Fluorimeter (right angle geometry)
excitation
monochromator
light
source
slit
cuvette
unabsorbed
exciting light
slit
fluorescence light
emission
monochromator
fluorescence
detector
Fluorescence
1.
2.
3.
4.
5.
6.
What is fluorescence
Technical issues
Uses of Fluorescence:
Quantitation
Ligand binding
Conformational changes
Measuring distances
Fluorescence quenching
Fluorescence lifetimes
Uses of Fluorescence
Quantitate material:
Flu. Int. proportional to conc.
Environmental change:
Flu maximum can shift (spectral shift)
Q can change (intensity change)
(cont.)
Intensity
Abs
Flu
Wavelength (nm)
Uses of Fluorescence (cont.)
Conformational changes on a protein
Trp fluorescence
Labelled protein eg at a Cys residue with a fluorescence probe
Ligand binding
Changes in protein fluorescence
Changes in ligand fluorescence if ligand is fluorescent
Examples: Ca2+ binding to Ca2+ -ATPase
Calcium ATPase
Trp residues
Ca2+
binding sites
Ca2+-ATPase + Ca2+
Change in Trp fluorescence intensity
12
10
pH 8.5
∆F/F (%)
8
6
4
2
0
2
3
4
5
6
pCa
7
8
9
10
Cys residues
NBD-Cl
Bromomethylcoumarin
Cys-344
Lys-515
FITC
NBD-labelled Ca2+-ATPase
0.7 mM
Ca2+
0.5 mM
EGTA
5%
10 mM
Mg2+
Uses of Fluorescence (cont.)
Conformational changes on a protein
Trp fluorescence
Labelled protein eg at a Cys residue with a fluorescence probe
Ligand binding
Changes in protein fluorescence
Changes in ligand fluorescence if ligand is fluorescent
Examples: Ca2+ binding to Ca2+ -ATPase
Location of Trp residues in a membrane protein
Uses of fluorescence
To report on environment
Bacteriorhodopsin
polar
different λmax for Trp
hydrophobic
Trp fluorescence spectra
1.5
Intensity
W
Free
Trp
1.0
0.5
0.0
300
320
340
360
Wavelength (nm)
380
400
Fluorescence
1.
2.
3.
4.
5.
6.
What is fluorescence
Technical issues
Uses of Fluorescence:
Quantitation
Ligand binding
Conformational changes
Measuring distances
Fluorescence quenching
Fluorescence lifetimes
Uses of Fluorescence – Distance Measurements
Fluorescence Energy Transfer
A
r
D
Donor
Int.
Abs
Flu
Energy Transfer
Acceptor
Int.
Flu
Abs
Wavelength (nm)
Excite
Flu emission
(cont.)
Uses of Fluorescence – Distance Measurements (cont.)
Efficiency of Energy Transfer proportional to r6
A
D
Fluorescence emission spectra
D alone
Int.
F0
r
D+A
F
Efficiency of transfer E= 1- F/F0
Forster theory: E = R06/(r6 + Ro6)
R0 = distance of separation at which E = 50%
typically 50 Å
Measure decrease in F
for donor caused by the
presence of the acceptor
Energy Transfer
IAEDANS to BrF
25
BrF E439
40 Å
I n t e n s i ty
20
IAEDANS-BrF
IAEDANS
C670, C674
15
BrF
IAEDANS
10
5
0
400
450
Wavelength (nm)
500
550
IAEDANS-ATPase to FITC-PE
1.0
C670, C674
F/Fo
0.8
h=54A
EGTA
2+
Ca
vanadate
0.6
Mg
0.4
2+
Thapsivillosin
0.2
0
0
0.1
0.2
0.3
Mole Fraction FITC-PE
0.4
Fluorescence Energy Transfer
K-515
42
E-439
40
53
37
80
45
41
C-344 28
70
C-670/4
54
Uses of Fluorescence – Distance Measurements (cont.)
Measure distances on a protein
A
r
D
Peptide Hydrolysis
D
A
Molecular beacons
(DNA or RNA)
D
D
A
A
binding to
DNA
Uses of Fluorescence – Fluorescence quenching
Short range – requires contact between fluorophore and quencher
exciting
light
heat
*
emission of
fluorescence light
excited
state
exciting
light
*
100 %
heat
quencher
Uses of Fluorescence – Fluorescence quenching
Short range – requires contact between fluorophore and quencher
Water soluble – iodide (I-), acrylamide, O2
Hydrophobic – aliphatic bromides
Use to test accessibility
Quench from
Aqueous phase
Quench from
Lipid bilayer
Trp containing peptide
Dibromo-tyrosine containing peptide
W
DibromoQ Tyr
1.0
Fluorescence
0.8
no quenching
C14
0.6
Thickness
C18
0.4
C24
0.2
W Q
0.0
0
0.02
0.04
Mole fraction brominated peptide
quenched
Fluorescence
1.
2.
3.
4.
5.
6.
What is fluorescence
Technical issues
Uses of Fluorescence:
Quantitation
Ligand binding
Conformational changes
Measuring distances
Fluorescence quenching
Fluorescence lifetimes
Fluorescence lifetimes
exciting
light
heat
*
excited
state
emission of
fluorescence light
Lifetime 1 –10 10-9 secs
(1-10 nsecs)
Measure using pulse methods
Excite fluorescence with a short pulse of light
Measure time between excitation and emission of a
photon of light
(cont)
exciting
light
Fluorescence lifetimes (cont)
*
heat
emission of
fluorescence light
No
No. of photons
emitted at time t
Nt
Exponential
decay
Nt=Noexp(-t/τ )
τ = fluorescence lifetime
Time (t) (nsecs)
Uses of Fluorescence lifetimes
Environmentally sensitive
protein changes etc.
Protein change
Often:
Intensity
Lifetime
F1
F2
τ1
τ2
F1
F2
=
τ1
τ2
Advantage of τ : this is an absolute measurement
Disadvantage of F ; this is in arbitrary units, and varies with amount of protein