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Perhaps out of all the spectroscopy techniques used in the biological sciences fluorescence has had the greatest penetration finding many uses from molecular biology to cancer diagnosis.

2.4.1.1 Fluorescence Imaging

In molecular and cell biology fluorescence spectroscopy is often used in combination with fluorophores such as green fluorescent protein (GFP), the most widely used, that fluoresces green when exposed to an excitation in the blue region of the electromagnetic spectrum, from a laser source or a mercury discharge lamp. This fluorescent protein can be tagged onto certain molecules by incorporating the gene for GFP with the gene of the molecule of interest such that when the protein of interest is manufactured by the cell it will have the small GFP attached. This allows the influence of the tagged molecule to be charted in biomolecular processes or reactions through fluorescence imaging [28]. The unique way GFP is produced means it can also be used to investigate DNA replication or the success of transfection procedures [29].

Fluorescence spectroscopy has also been used in the diagnosis of certain cancers used in combination with a fluorescent drug. Patients ingest the drug, which fluoresces green when exposed to blue excitation, that clings preferentially to cancerous tumours. A fibre optic probe can then be used, in combination with an

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excitation source, to image tissue and look for areas of high fluorescence indicating a tumour [30]. Furthermore the use of natural laser induced auto-fluorescence has also demonstrated the ability to discriminate between healthy and neoplastic tissues [31].

2.4.1.2 Flow Cytometry

An important use of fluorescence in the study of biological cells is in fluorescence activated cell sorting (FACS) [32]. FACS is a specialised form of flow cytometry [33] that is used to sort and analyse a fluorescently labelled cell population. Fluorescent markers can be used to target cells with a specific trait of interest within a general heterogeneous population. A solution of the cells is then placed into the flow cytometer where they are flowed past a bank of lasers and detectors to excite and detect any fluorescence emissions from the cells. According to their fluorescence the cells can be sorted into different chambers thus allowing the isolation and counting of the cell population of interest. This is a very high throughput technique that can sort analyse up to ten thousand samples per second. As well as sorting populations of cells FACS has been used in cancer diagnostics in a derivative technique known as DNA flow cytometry or DNA-FCM [34]. In this technique the nuclei of cells, taken from a biopsy, are stained with a fluorescent marker and passed through the flow cytometer which measures the level of fluorescence and hence the DNA content present in the sample. As cancer is often associated with an increase in cellular DNA content DNA- FCM can potentially give a diagnosis. However the technique requires a large amount of material to give an accurate diagnosis and may not be able to detect the onset of cancer at a very early stage.

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2.4.1.3 Forster Resonance Energy Transfer (FRET)

FRET is a fluorescence based technique that allows the examination of protein-protein [35] or protein-DNA [36] interactions with resolutions of a few hundred angstroms. The process of FRET requires the two interacting molecules to be tagged with fluorescent proteins, a donor and an acceptor fluorophore, where the emission band of one fluorophore overlaps with the excitation band of the other. The technique operates by observing the transfer of energy between the two fluorescent molecules. The transfer of energy, from the donor in an excited state to the acceptor, is not mediated by photon emission and absorption, rather the energy is transfered via, as the name suggests, a non-radiative fashion through long range dipole interactions. The acceptor then manifests the occurrence of a transition by fluorescing at a different wavelength in comparison to the donor emission. Throughout an entire sample the interactions are manifested, when the sample is exposed to the exciting wavelength of the donor, as a drop in fluorescence from the donor molecule and an increase in fluorescence from the acceptor molecule. Furthermore FRET is extremely sensitive to separation of the interacting fluorphores thus can be used a molecular ruler [36].

FRET is a very sensitive technique that has opened up the study of molecule interactions in the cellular environment with unprecedented precision, however only one specific interaction can be studied at one particular time and is dependant on suitable fluorophores to tag the molecules of interest. FRET is best used in the study of individual protein interactions rather than ensembles.

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2.4.1.4 Total Internal Reflectance Fluorescence Microscopy (TIRFM)

TIRFM is a microscopy based technique that allows the excitation of only a very fine layer close to a refractive index border, normally a microscope slide and the solution in which the sample under study is immersed, via an evanescent field caused by total internal reflection. The objects for study are normally tagged with fluorescent markers so that their presence or position within the solution or a cell may be observed. Normally a laser beam is introduced through a specialised microscope objective at an angle such as when the laser strikes the glass solution boundary it is totally internally reflected. This creates a short range evanescent field at the boundary that is capable of exciting the fluorophores close to the boundary. The use of this method to excite the fluorophores results in an increase of signal to noise ratio by a factor of approximately twentyfive. It should be noted that this technique can be performed with prisms rather than microscopes, however microscopy is the preferred option due to its added advantages. TIRFM can be used to study single biologically important molecules [37] all the way up to full cell imaging [38].

TIRFM is an extremely useful technique in studying cellular and single molecule function giving excellent signal to noise ratios; however it remains dependant on fluorophores to monitor its desired target thus cannot engage an entire ensemble, such as complete cell behaviour, simultaneously.

Fluorescence imaging has had a massive impact on the world of biology opening up many research aspects of molecular biology, it has also been investigated as a possible diagnostic technique for cancer with promising results. However fluorescence imaging using tags is molecule specific and finds difficulty engaging large ensembles such as whole cells and is best exploited to study the function of

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single molecules in systems. When studying whole systems with natural fluorescence, such as tissues, the retrieved signal is an amalgamation of many molecules and contributions from individual groups of molecules is difficult to discern, thus it may be difficult to pick up subtle variations such as those involved in the earliest stages of neoplastic development.