Fluorescence spectroscopy

Fluorescence spectroscopy The process (1)

Energy exchange with surrounding molecules

Excitation E0 Molecular energy levels Excitation radiation is normally scanned into the spectrophotometer through the u.v. visible region (>200 nm preferable) and the emission spectrum (at lower wavelength) recorded Emission

Fluorescence spectroscopy The spectrometer (2)

Sample
Monochromator Fluorescent Light is scattered within the sample Excitation light source Monochromator

Detector

Spectral output

Fluorescence spectroscopy why ? (3)

This spectroscopy looks for changes in various aspects of fluorescence on the interaction between a drug and its receptor. These aspects may include the following Emission spectra Excitation spectra Quantum yield ~ quenching Polarisation Fluorescent lifetime

Natural (Intrinsic) and Imposed (Extrinsic) Fluorescence (4)

Natural (intrinsic) fluorescence in biological macromolecules is limited. In proteins certain residues have the ability to impart fluorescence when in a special environment (in order of importance) Tryptophan Tyrosine Phenylalanine (W) (Y) (F)

Imposed (Extrinsic) fluorescence may be introduced when a protein is modified with a fluorescent reagent known as a fluorophore (fluor) this is useful as long as the fluor may be introduced in a unique location adequately responds to the D-R interaction does not affect the binding affinity of the drug with the receptor

Limitations to Fluorescence (5)

Not all compounds fluoresce. Fluorophores require Aromatic ring The potential for increased extended conjugation At least one electron donating group OH, -NH2 attached to the aromatic ring (note : electron withdrawing groups can diminish or often destroy fluorescence
HO
HO O O HO O OH

CO2H

O O
N

CO2H

Fluorescent Fluoroscein

C S

Electrophilic Linking functional group

Fluoroscein isothiocyanate (FITC)

Spectral characteristics (6)

IF

EX

.
EM max

Concentration dependence of fluorescent emission (7)

For dilute solutions : IF = 2.303QIocl at higher concentrations the linearity may deviate significantly

IF
IF = 2.303QIocl

concentration

Quantum yield (8)

Fluor
(may be linked to protein as tag or a modified ligand)

Emerging excitation photons

X R

Incident excitation photons Emission photon Q = no.of photons emitted no. of photons absorbed changes in the environment / binding of the fluor changes Q

EM Interpretations of changes in max and quantum yield (9)

X R

Emerging excitation photons

Incident excitation photons Emission photon Fluor in a polar environment shows a shift to lower wavelength with an increase in Q as environment becomes less polar Temperature effect in polar environments Q decreases with increasing temperature non polar environment very little change

Fluorescence quenching (10)

is the loss of fluorescence due to loss of energy from the excited state by non-radiative pathways. These pathways are predominantly due to collisions with other molecules. Specific agents may be used to enhance the process e.g. I- and acrylamide.
This fluor is less accessible to the quenching agent

This fluor is more accessible to the quenching agent

Fluorescence quenching (11)

Presentation of quenching data is usually in the form of the Stern-Volmer plot
Fluoresence intensity in the absence of quencher Concentration of quenching agent

I = 1 + K D [Q ] IF
Fluoresence intensity in the presence of quencher Quenching constant

o F

A linear plot (intensity ratio vs. [Q] ) is generally indicative of a single class of fluor. If two fluor populations are present with differing accessibilities the plots deviate towards the abscissa.

Fluorescence polarisation (12)

Polarising filter
X R

Emerging polarised excited photons

Polarised incident excitation photons

Polarised emission photon Polarising analyser

C IF IF P= C IF + IF

Intensities found with the polarising filter parallel to the analyser, or perpendicular, vary. These are a function of the ability of the fluor to rotate (see arrow) at the molecular level within the time course of the experiment.

Fluorescence Polarisation spectroscopy (13)

Sample
Monochromator Polarised Fluorescent Light is scattered within the sample Excitation light source Monochromator

Polarising filter Polarising analyser

Detector

Spectral output

Advantages of Fluorescence spectroscopy (14)

Highly sensitive technique Up to 1,000 times more sensitive than UV / visible spectroscopy. Therefore often used in drug or drug metabolite determinations by HPLC with fluorimetric detector. Non-fluorescing compounds can be made fluorescent derivitisation. Selective versatile technique Since excitation and emission wavelengths are utilised, gives selectivity to an assay compared to UV / visible spectroscopy. Differing modes of spectroscopy yield wide versatility.