After absorption of light energy, all compounds release some energy as heat; fluorescent compounds represent a special case in which some energy is also given off as light. Spectrofluorimeters are optical devices that measure the amount of light that is emitted by fluorescent compounds.
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Basic Principle of Spectrofluorimeter
When light is directed at a fluorescent compound, the light absorbed by the compound is converted into light (of a lower energy) as well as heat. The brighter the light that strikes a fluorescent compound the stronger the fluorescent light emitted by the compound. The specific wavelength absorbed by compound and the wavelength that is later emitted are characteristics of each fluorescent compound.
An intense beam of light is passed through a filter (Monochromator) that allows only light of a desired band of wavelengths to proceed toward the sample. The light not absorbed by the sample passes straight through the sample without change in angle while the light emitted by the fluorescent sample is released in all directions. By positioning the detector at a 90° angle with respect to the exciting beam, the amount of stray light from the exciting beam that reaches the detector is minimized. Since the light emitted by the fluorescent sample is given off in all directions, the positioning of the detector with respect to the exciting beam doesn’t affect the relative amount of the emitted fluorescent light received by the detector.
Biomedical Applications
The high sensitivity and selectivity of fluorescence techniques allows the detection of very low amounts of a number of compounds of biological and biomedical interest.
Fluorescence methods provide important means for biomedical analysis. The following are some of the ways they are utilized in biomedical field:
The identification of Compounds in Biological Samples
Once it is established that a sample contains a fluorescent compound, a number of analytical techniques for the detection and quantitation of this compound can be employed. Several biological compounds and drugs can be detected by fluorescence analysis. Fluorescence can be used to identify these and many other biological active compounds in complex pathological specimens.
Fluorescence Polarization
The principle of fluorescence polarization involves the fact the emission of a photon is delayed by a few nanoseconds following absorption of light. During the delay in emission of light, Brownian motion (postulated by Albert Einstein) will result in the movement of molecule and smaller molecules will move more than larger molecules. Hence, molecules excited by polarized light will emit progressively depolarized light as the size of the molecule increases. Thus, if a smaller fluorescent molecule binds to a large non-fluorescent molecule, the light emitted by the bound small fluorescent molecule will become more polarized. Therefore, the technique of fluorescence polarization allows one to measure the bindings of ligands to large molecules in real time.
Fluorescence Imaging
The availability of highly sensitive optical detection systems has allowed the localization of specific molecules in living cells. By tagging recombinant proteins with the green fluorescent protein (GFP) of jellyfish origin, one can track the expression and translocation of specific protein in living cells during hormonal responses.
The rate at which molecules move within living cells can be determined by fluorescence recovery after photobleaching, which involves the photobleaching of molecules in a small region of a cell and then monitoring of the recovery of fluorescence with time as unbleached molecules return to the bleached area.
Fluorescence energy transfer and fluorescence polarization techniques can be used to study the interaction of molecules within living cells.
Immunofluorescence
Antibodies can be prepared that specifically recognize a wide variety of molecules and microbial organisms. By labelling such antibodies with fluorescein or other fluorescent probes, one can visualize the presence of antigens at the subcellular level; this method is extensively used in molecular biology. Furthermore, one can visualize specific molecules and organisms in pathological specimens. The identification of disease causing microorganisms in pathological specimens by immunofluorescence can be used. Lastly, immunofluorescence microscopy can be used in the identification of bacteria in food products.
Also Read: Spectrophotometry Instrumental Method of Analysis
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Fluorescence in Molecular Biology
The high sensitivity of fluorescence has been used as the basis of several key techniques in molecular biology. For instance, real-time polymerase chain reaction (PCR) methods can be used to identify the amount of a particular ribonucleic acid (RNA) species in a small sample of cells. In this technique, fluorescence energy transfer is employed to detect the increase in PCR product with time.
The jellyfish green fluorescent protein (GFP) has become a key tool in molecular biology because it is fluorescent without the necessity of binding or reacting with a second molecule.
Forster Resonance Energy Transfer (FRET)
This technique involves the transfer of energy from a fluorophore, which absorbs light to a second molecule that emits light at a different wavelength. Because the efficiency of FRET depends on the distance between the absorbing and emitting molecules, FRET allows one to obtain information about the distance between two molecules. Hence, if the molecule that absorbs light binds to a molecule that emits light via FRET, one can measure the binding event via the release of light.
Related: Principle of Operation of a Medical Mass Spectrometer
