The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

We report on the development of a non-invasive instrument based on scanning confocal microscopy for tracking inherently fluorescent drugs and measuring spatial features in the anterior chamber of the eye. The new instrument incorporates all features of the initial instrument 1 with the addition of fluorescence detection from within the anterior chamber of the eye. We have measured the diffusion of Fluorescein with high time resolution within a cuvette, an artificial eye and ex vivo porcine eyes. Results are be presented that demonstrate the capability of the instrument to accurately measure the concentration and the location of the fluorescent drug over a given period of time along the optical axis of the eye with an axial resolution of under 200 μm and temporal resolution of < 1s. We show that the instrument has high sensitivity and can measure concentrations of < 1µM/L of compounds having a quantum yield as low as 0.01 with high specificity for the compound of interest over competing background signals. The role of the instrument in assessing the efficiency of any inherently fluorescent ophthalmic drug as well as monitoring other medication that might produce fluorescent compounds in the eye will be discussed. We furthermore believe that the instrument might also be capable of monitoring certain bodily processes which have an impact on the compounds present in the eye.

We report on the development of a novel, low cost instrument that is capable of accurately measuring small, short and long term changes in the thickness of the cornea and tear film at high speed. The performance of the instrument was tested by measuring the influence of Allergan's OPTIVE™ lubricating eye drops on the thickness of the cornea and tear film. Comparative measurements to quantify the performance were taken using Haag-Streit's LenStar. It was found that the newly developed instrument accurately measured a change in thickness of around 9 µm with an accuracy comparable to the LenStar, and with a standard deviation of less than 1 micrometer. Since the new instrument was not configured to resolve the tear film from the cornea, we are not yet able to distinguish the cause of the thickening.

We report on the development of a non-contact, low cost instrument based upon confocal microscopy that is capable of rapidly and accurately measuring changes in the axial dimensions of components in the anterior section of the eye. The performance of the instrument was tested by applying an oil film to a vertically mounted artificial cornea to simulate a breaking up tear film. By measuring the influence of lubricating eye drops on the thickness of the human cornea and tear film in vivo with a commercial, interferometer based instrument, we show the need for higher resolution instruments. It was found that the new instrument measured a change in thickness as small as 5 µm in the artificial system with a standard deviation of less than 1 µm. The change of thickness in the in vivo cornea was measured accurately with the interferometer based instrument, but no distinction between the thickness of the cornea and that of the tear film was possible. We therefore believe that the new confocal instrument and its further developments will play an important role in the fast and accurate measurement of tear film and corneal thickness and also in relation to glaucoma screening with applanation tonometry.