Fluorescence - UIC Department of Chemistry

Fluorescence

1. What is fluorescence
2. Technical issues
3. Uses of Fluorescence:

Quantitation
Ligand binding
Conformational changes
4. Measuring distances
5. Fluorescence quenching
6. Fluorescence lifetimes

Fluorescence
What happens after a molecule has adsorbed light

exciting * photobleaching (try to avoid)
light
heat
excited
state emission of
fluorescence light

Lifetime 1 –10 10-9 secs
(1-10 nsecs)

Fluorescence

excited heat
state

Energy heat Lowest vibrational
Intensity level in excited state
ground
state Fluorescence
emission
Absorption

Stokes Flu
shift

Abs

Wavelength (nm)

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

1. What is fluorescence
2. Technical issues
3. Uses of Fluorescence:

Quantitation
Ligand binding
Conformational changes
4. Measuring distances
5. Fluorescence quenching
6. 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)

light excitation cuvette
source monochromator
unabsorbed
slit 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 Optical Density less than 0.05
effect to avoid inner filter effect

Flu.
intensity

Concentration cuvette Volume
observed
excitation

Inner Filter Absorption of Flu light
Effect light here leads absorbed
to low light
intensity in slit
volume observed
fluorescence light

Fluorescence Parameters

Intensity Stokes
shift

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 Q between 0 and 1
no. of photons absorbed (Trp Q ca 0.2)

excited heat
state

heat Fluorescence
emission
ground
state

Absorption

Fluorescence lifetime
Time molecules remains in the excited state
typically 1-10 nsecs

Fluorescence Intensity Measurements

OD Abs Flu Fluorescence
Abs=εcl 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)

light excitation cuvette
source monochromator
unabsorbed
slit exciting light

slit
fluorescence light

emission
monochromator

fluorescence
detector

Fluorescence

1. What is fluorescence
2. Technical issues
3. Uses of Fluorescence:

Quantitation
Ligand binding
Conformational changes
4. Measuring distances
5. Fluorescence quenching
6. 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)

Intensity (cont.)

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

∆F/F (%) Ca2+-ATPase + Ca2+
Change in Trp fluorescence intensity

12
10

pH 8.5

8
6
4
2
0

2 3 4 5 6 7 8 9 10
pCa

Cys residues Lys-515
NBD-Cl FITC

Bromomethyl-
coumarin
Cys-344

NBD-labelled Ca2+-ATPase

0.7 mM
Ca2+

0.5 mM 10 mM
EGTA Mg2+

5%

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

different λmax for Trp polar
hydrophobic

Trp fluorescence spectra

W 1.5

Free
1.0 Trp
Intensity
0.5

0.0 320 340 360 380 400
300
Wavelength (nm)

Fluorescence

1. What is fluorescence
2. Technical issues
3. Uses of Fluorescence:

Quantitation
Ligand binding
Conformational changes
4. Measuring distances
5. Fluorescence quenching
6. Fluorescence lifetimes

Uses of Fluorescence – Distance Measurements

Fluorescence Energy Transfer

Donor Int. Ar
D
Abs Flu
Energy Transfer

Acceptor Int.

Abs Flu
Wavelength (nm)

Excite Flu emission

(cont.)

Uses of Fluorescence – Distance Measurements (cont.)

Efficiency of Energy Transfer proportional to r6

Fluorescence emission spectra Ar
D alone D

F0 D+A Measure decrease in F
Int. for donor caused by the
presence of the acceptor
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

Energy Transfer IAEDANS to BrF

25

BrF E439 IAEDANS-BrF

20 40 Å IAEDANS

C670, C674

In tensity 15
BrF

IAEDANS
10

5

0 450 500 550
400 Wavelength (nm)

F/Fo IAEDANS-ATPase to FITC-PE

1.0

C670, C674

0.8 h=54A

EGTA

0.6 Ca2+

vanadate
Mg 2+

0.4 Thapsivillosin

0.2

0
0 0.1 0.2 0.3 0.4
Mole Fraction FITC-PE

Fluorescence Energy Transfer

K-515 42 E-439

53 40

37 41 C-670/4
80
C-344 28

70 54
45

Uses of Fluorescence – Distance Measurements (cont.)

Measure distances on a protein A r
D

Peptide Hydrolysis DA

Molecular beacons D A
(DNA or RNA)

DA binding to
DNA

Uses of Fluorescence – Fluorescence quenching

Short range – requires contact between fluorophore and quencher

exciting * heat
light
excited emission of
exciting state fluorescence light
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 Q Dibromo-
Tyr

1.0

Fluorescence 0.8 no quenching

0.6 C14 Thickness

0.4 C18

0.2 C24 WQ

0.0 0.02 0.04 quenched
0
Mole fraction brominated peptide

Fluorescence

1. What is fluorescence
2. Technical issues
3. Uses of Fluorescence:

Quantitation
Ligand binding
Conformational changes
4. Measuring distances
5. Fluorescence quenching
6. Fluorescence lifetimes

Fluorescence lifetimes

exciting * heat
light
emission of
excited fluorescence light
state

Lifetime 1 –10 10-9 secs
(1-10 nsecs)

Measure using pulse methods (cont)
Excite fluorescence with a short pulse of light
Measure time between excitation and emission of a
photon of light

exciting Fluorescence lifetimes (cont)

*light heat

emission of

fluorescence light

No

No. of photons Exponential
emitted at time t decay

Nt

Nt=Noexp(-t/τ )

τ = fluorescence lifetime Time (t) (nsecs)