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A simpler, gentler eye test based on Raman spectroscopy could spot ocular infection and other problems without irritating patients, although they may be required to yawn during the procedure. Whichever way you look at them, whether through the emotional blur of crying or as lachrymal secretions ripe for analysis, tears are complex. Now, researchers in the UK, have taken a close look at this aqueous solution of proteins, metabolites, electrolytes and lipids using Raman spectroscopy and obtained results that would make any ocular enthusiast cry with joy. Jacob Filik and Nick Stone of the Biophotonics Research Unit, Gloucester Royal Hospital, Gloucester, point out how powerful Raman spectroscopy can be in the analysis of biological samples. To demonstrate its prowess they analysed 3 microlitre samples of human tear fluid from three healthy volunteers using two different methods to obtain Raman spectra. The first, solution-phase Raman spectroscopy, and the second, drop-coating deposition Raman spectroscopy (DCDRS). Tears not only create a smooth surface through which light can pass, but also lubricate the eye and provide it with nutrients, waste disposal services and protect the eye from injury and infection. The composition of tears is critical to these functions and so the good health of the eye and even subtle changes can cause problems with the surface that can affect the transparent cornea. Variations may also be symptomatic of underlying disease in the eye and elsewhere in the body and so might provide a novel approach to diagnostics. The researchers have turned to Raman to study the complexities of tears because even highly sensitive techniques such as immunoassays, electrophoresis and mass spectrometry are often time consuming and impractical. "To be truly a fast and simple aid to the diagnosis of ocular disease, an analytical technique needs to be able to be performed near patient, with minimal specialist knowledge, little sample processing and yet be sensitive enough to yield diagnostic information," the researchers explain, "These requirements can be met by using Raman spectroscopy." The researchers confess that the Raman signals were very weak, whether the tears were "basal" (non-reflex) fluid or yawn reflex secretions. Nevertheless, they were able to obtain protein concentrations and observe urea levels, for instance. DCDRS gave them the best signal-to-noise ratio even with short acquisition times and the small sample volumes. However, it was the processing of the Raman data using principal components analysis (PCA) that allowed them to obtain protein, urea, bicarbonate and lipid values that could be correlated with the characteristic, so-called "coffee-ring" drying pattern - used in sample pre-concentration - and so provide the means to calibrate tear protein concentrations as well as fingerprinting the tear protein and lipid composition. The researchers add that the results of the DCDRS studies showed a clear difference in the lipid content of tears collected by the two different methods. However, they decided that basal tear collection was the better method not only because it sampled the lipid component of the volunteers' tears, but moreover because it did not require them to yawn in order to carry on with the experiments. On a more serious note, the team adds that, although their study is a preliminary one, it does demonstrate that it should be possible using the two Raman techniques together with visual analysis of the way the drying tear sample produces fern-like patterns in diagnosing ocular infection. The main advantage, aside from speed of analysis and potential simplicity of the instrumentation used to carry out a test, is that only minute volumes of tear fluid, just a couple of microlitres, are required. This latter point is important from the point of view of patient comfort. Tears are often collected using glass capillary tubes, polyester rods, Schirmer strips and eye washes, the team points out. An adequate sample size is usually elicited by irritating the eye with onion or ammonia vapour. Moreover, tear composition is affected by flow rate and irritation, so samples collected using those methods may not accurately reflect the base-line composition required for a definitive diagnosis. If larger samples are required then, the yawn reflex is a less extreme way to increase the production of tears, than ammonia and glass tubes, the researchers add. Related links:
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Tears are enough, for Raman-based ocular diagnostics |
