Download Fluorescence spectroscopy

Fluorescence
spectroscopy
The light: electromagnetic wave
Tamás Huber
Biophysics seminar
Dept. of Biophysics, University of Pécs
05-06. February 2014.
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Luminescence: light emission of an
excited system.
From molecules or ions: molecular luminescence
Basic phenomena are discribed by the Jablonski
termscheme.
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The types of luminescence
Chemiluminescence - Photoluminescence
1. Chemiluminescence
• light emission that is excited by the energy
from chemical reactions
(e.g.: phosphor (P) oxidation)
• acceptable for examination of metabolisms
• low intensity
• depends on physiological relations
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Bioluminescence: is the production and emission of
light by a living organism as the result of a chemical
reaction.
Examples: firefly (bug), deep-sea fishes, medusa, octopus, bacteria, planktons
2. Photoluminescence
• Light emission that is exited by direct light
radiation of certain energy (frequency) and wavelength.
• Very useful in molecular system assays, because it
carries large amount of information of the properties of
molecules, interactions and the relationship with its
environment.
1.
Luciferase catalizes the oxidation of luciferin.
2. Inactive oxyluciferin and light (h ) arise.
3. More luciferin replenish from food, or inner
synthesis.
Structure of luminescent molecules have aromatic
rings with conjugated double bounds.
• Two types:
fluorescence, phosphorescence
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Jablonski termscheme
The proof of the Kasha-rule:
Any kind of excitation wavelength
excites the molecule, the emission
spectra does not change.
 Fluorescence: the molecule
relaxes from the excited
singlet state to the singlet
ground state
Lifetime: 10-9 s
 Phosphorescence: molecule
relaxes from the excited
triplet state to the singlet
ground state (lower possibility)
Lifetime: 10-6-10 s
 Separate them by:
- the shape of the spectra,
- the time interval of the
excited state.
http://www.olympusmicro.com/primer/java/jablonski/jabintro/
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Characterization of the luminating
material
•
•
•
•
By its absorption spectrum and its
fluorescence, phosphorescence excitation
and emission spectrum
Principles of measuring fluorescence
The most important problem is to separate
the excitation light and the caused
luminescence light.
Quantum yield of the radiation
Lifetime of the excited state
Polarization degree of the emission
(anisotropy)
Practical choice of the exciting and detecting
directions
Three different compositions
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Sample
Sample
Sample
1. Detection is perpendicular to
the direction of excitation.
How to measure fluorescence?
(‘steady-state’ case)
2. Detection is parallel to the
excitation direction.
Detection of the outcoming fluorescence
from the front side.
3. Detection from the opposite
side to the excitation
direction.
!! Optical filters, monochromators!!
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The excitation spectrum
The emission spectrum
Detection at a fixed emission wavelength.
•
Measuring the intensity as function of the excitation
wavelength.
•
The shape of its function is the same as the absorption
spectrum.
Excitation
Stokes-shift ,
mirror image spectra
Emission
Wavelength
Fluorescent emission spectrum
Originates in the transition from the lowest vibrational level of
the first singlet excitation state to one vibrational level of the
ground state.
Gives information of the vibrational levels of the ground state.
Intensity
Intensity
•
Sir George Gabriel
Stokes, 1st Baronet
(1819–1903)
Excitation
Emission
Wavelength
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Effect of chemical denaturation to
excitation and emission spectra
Phosphorescence emission spectrum
Phosphofescence
emission spectrum
Excitation spectrum
GuHCl
Fluorescence intensity, (a.u.)
Fluorescence
emission spectrum
•
Wavelength, nm
•
•
During the transformation from the first triplet excitation
state to the singlet state.
At room temperature only on crystal materials.
According to the fluorescence spectrum its shifted
towards to the infrared wavelengths.
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Quantum yield
Fluorescence lifetime
Refers to the average time the molecule stays
in its excited state before emitting a photon,
or the number of excited photons decreases to
the fraction e.
= 1 / (kf + ksum)
is the ratio of the number of photons emitted to
the number of photons absorbed:
Q = Nem / Nabs < 1
f : fluorescence
sum : f + vibr. + rot. (so, f + non-radiative)
- also expressible with the rate constants:
f – fluorescence
sum – f + vibr. + rot.
(so, f + non-radiative)
Q = kf / (kf + ksum)
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• ‘Time domain measurement’
Time-Correlated Single Photon Counting
/TCSPC/
Fluorescence Intensity (cps)
How to measure lifetime?
PEVK11
IAEDANS
1000
• short excitation pulses
(~ fs)
• detection of photons in
time windows
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PEVK21
IAEDANS
1000
100
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Principles of Fluorescence Spectroscopy_Joseph R. Lakowicz.
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0
Principles of Fluorescence Spectroscopy_Joseph R. Lakowicz.
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60
80
100
Time Domain Time (ns)
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How to measure lifetime?
• ‘Frequency domain measurement’
How to measure lifetime?
• ‘Frequency domain measurement’
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Fluorescent dyes
• nativ or intrinsic fluorophores:
Tryptophan, tyrosine, phenylalanine
Advantage: no protein modification
Extrinsic fluorophores
Direct labeling with dyes:
Dansyl
Rhodamine
IAEDANS
IAF
FITC
Fluorescently labeled toxins:
Falloidin
B-scorpiontoxin
A-bungarotoxin
Macrophages
Actin is labeled by
phalloidin-Alexa 568-cal (Red)
Nuclei: DAPI (Blue)
Streptococcus aureus (Green)
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Labeling proteins with
fluorescent dyes
- quality and location can be planned
- labeling is specific for the binding
residues
- protein could be modified, we have to
test the activity
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Labeling with specific
antibodies
Primary antibody
(immunfluorescent, immunhistochemical
labeling)
•
Antigen

The antibody binds to the surface of
the recognized molecule with high
affinity.

Monoclonal and polyclonal antibodies.

Direct labeling: a fluorescent dye is

bound to the antibody
Indirect labeling: the primary
antibody is not labeled, the secondary
antibody is labeled.
Measuring phosphorescence
Fluorophore
•
•
•
Secondary antibody
Primary antibody
Antigen
The excitation light must be separated from the
phosphorescence light in time
The change of intensity during the time must be
measurable.
Must be measured at low temperature
Phosphoroskop:
After the excitation we hide the sample with an optical cylinder, then the
emitted light can get to the detector.
The time after the excitation and before the detection
depends on:
• the velocity of the rotation
• the number of the slits
Shortest reachable time has a magnitude of 10-5 s.
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The phosphoroscop
Sample
The excitation light can get
through the slit, but the
phosphorescence can not get
through the wall of the cylinder.
The sample
•
Usually a solution (protein, nucleic acid, pigment
extract, cell suspension)
•
The material of the cuvette must be nonfluorescent
Glass cuvettes (visible range only)
Special glass cuvettes (λ > 300 nm)
Plastic cuvettes
Special quartz cuvettes (measuring fluorescence)
•
•
•
•
Sample
After a quarter rotation the way
of the excitation light is closed
and the phosphorescence gets to
the detector.
•
•
•
Cuvette holders:
Temperature can be set
More places (usually 4), rotatable
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Excitation light sources
Optical filters
Selection of different wavelengths
Absorption filters
Continuous-, (heated to high
temperatures)
Lamps filled with halogen gases
Lamps filled with high pressure gases
•
Usually made of glass.
Contains organic and inorganic
components that is why light
beams with given wavelength can
go through and other
wavelengths can not.
Plastic (cheaper, lighter)
Line-, (atoms)
Intensive, monochromatic light
Lamps filled with low pressure mercury
•
•
Etc.
Dicroic
mirrors
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Optical filters
UV filters:
UV rays can not go through, but rays with longer wavelength can.
Neutral filters:
Transmission has a wide spectrum range and independent from
the wavelength.
Photochemical, and photobiological processes can be examined.
Interference filters
If
a thin transparent spacer is placed between two
semireflective coatings, multiple reflections and interference
can be used to select a narrow frequency band, producing an
interference filter.
Optical filters
Long pass filters
Allow to pass light with longer wavelengths.
Fluorescence microscopy: dicroic mirrors usually used as emission
filters.
Short pass filters
Optical interference or coloured glass filters.
Allow to pass light with shorter wavelengths.
Dicroic mirrors usually used as excitation filters.
Band pass filters
Combination of the uppers.
Lower transmittance.
Blocks everything beyond the chosen wavelengths.
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Optical filters
Monochromators
A: Light source
B: Slit
C: Collimator
D: Prism or grid
E: Mirror
F: Excitation slit
G: Sample
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The detector
Photomultiplier tube:
Very sensitive from the UV to the NIR.
The End!
Advantages of the application of
fluorescence
- Very good detection: measurable at low
concentrations
- Fluorescence is sensitive to the environment
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