WO2015101785A1 - Non-invasive fluorescence-based eye lens diagnostics - Google Patents

Non-invasive fluorescence-based eye lens diagnostics Download PDF

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Publication number
WO2015101785A1
WO2015101785A1 PCT/GB2014/053854 GB2014053854W WO2015101785A1 WO 2015101785 A1 WO2015101785 A1 WO 2015101785A1 GB 2014053854 W GB2014053854 W GB 2014053854W WO 2015101785 A1 WO2015101785 A1 WO 2015101785A1
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Prior art keywords
tryptophan
photo
fluorescent
products
lens
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PCT/GB2014/053854
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French (fr)
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Dmitry Gakamsky
Desmond Smith
Rory DUNCAN
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Heriot-Watt University
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Priority to EP14821265.7A priority Critical patent/EP3090250A1/en
Publication of WO2015101785A1 publication Critical patent/WO2015101785A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/117—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for examining the anterior chamber or the anterior chamber angle, e.g. gonioscopes
    • A61B3/1173—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for examining the anterior chamber or the anterior chamber angle, e.g. gonioscopes for examining the eye lens
    • A61B3/1176—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for examining the anterior chamber or the anterior chamber angle, e.g. gonioscopes for examining the eye lens for determining lens opacity, e.g. cataract
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission

Definitions

  • the present invention relates to a non-invasive system and method for detecting changes indicative of cataract or cataract formation using intrinsic fluorescence reporters of the eye lens proteins.
  • the invention can be used in vivo to detect early changes in the eye lens structure and allows quantitative categorisation of cataract types and stage of its formation. It can be also used for in vitro or ex vivo drug screening applications.
  • the ocular lens grows continuously throughout life.
  • the major components of the lens are highly stable and water soluble structural protein encoded as alpha-, beta- and gamma-crystallins.
  • the high concentration of these proteins and the presence of a substantial fraction (-35%) of alpha-crystallin, which has a chaperon-like function in the protein composition, plays two major roles: maintaining high refractive index and binding of misfolded (damaged) proteins to the chaperon molecules to prevent their irreversible aggregation (Borkman, Knight and Obi, The molecular chaperon alpha- crystallin inhibits UV-induced protein aggregation. Exp. Eye Res. 62, 141 -148 (1996)).
  • PTMs Post-translational modifications in the lens proteins caused by chemical, photochemical and environmental factors accumulate over the life span of patients. PTMs lead to protein misfolding, denaturation and aggregation and hence structural defects. Accumulation of PTMs in the lens structure can be also caused by aging (Age- related changes in human crystallins determined from comparative analysis of post- translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility? Wilmarth at al. J Proteome Res. 2006 Oct;5(10):2554-66) or concomitant diseases, e.g. diabetes, (Truscott, R.J.W. Protein misfolding, aggregation, and conformational diseases, Uverski VN and Fink A (eds.), pp. 435-447, Springer, 2007).
  • cataract detection is based on measurements of lens opacity by light scattering. This can be done using a conventional slit lamp-based technique or other more advanced methods, such as Optical Coherence Tomography etc, see KonstantopoulosA, Hossain,P. & Anderson, D.F. Recent advances in ophthalmic anterior segment imaging: a new era for ophthalmic diagnosis? Br. J. Ophthalmol. 91 , 551 -557 (2007).
  • a limitation of these scattering techniques is that the sizes of structural defects must be comparable with the wavelength of light used, i.e. ca 0.4 - 0.6 micrometer or greater. Therefore, methods based on light scattering only allow detection of relatively large-scale changes in the eye lens.
  • US 5203328 describes a method for noninvasively diagnosing diabetes mellitus and cataracts using a fluorescent response of eye lens. Diagnoses are made by illuminating eye lens tissue with a narrow-band light source at a selected wavelength, detecting the back-scattered radiation intensity at the peak of a non-tryptophan fluoresce, and normalizing the detected value with the intensity of its Rayleigh component.
  • This method uses auto-fluorescence of the lens, i.e. emission of fluorescent products accumulated in the lens over time.
  • the eye lens exhibits UV emission in the 300 - 450nm range caused by tryptophan and in the 350 - 750nm range by its photo-products and other fluorescent PTMs.
  • WO2010097582 describes a tryptophan-based method for detecting changes in a human or animal eye. The method involves illuminating the eye or at least a part thereof, in particular the lens and/or cornea, using light at a tryptophan red edge excitation wavelength and detecting tryptophan fluorescence. The detected tryptophan fluorescence is used to detect or identify structural defects or changes in the eye, such as the formation of cataracts.
  • PTMs such as phosphorylation, methylation, acethylation, glycation etc
  • ROS reactive oxygen species
  • UV light UV light
  • gamma-irradiation Oxidation_of Free Tryptophan and Tryptophan Residues in Peptides and Proteins. Simat and Steinhart, Agric Food Chem. 46(2):490-498 (1998)).
  • the human molecular chaperons alpha crystallin A and B contain one or two tryptophan residues respectively.
  • Human beta- and gamma-crystallins contain four to eight tryptophan residues.
  • Susceptibility of a tryptophan residue side chain to photo- oxidation is a function of the protein structure, which protects it from exposure to an aqueous environment where it can interact with ROS.
  • Some tryptophan photo- products such as 5-OH-tryptophan (5-OH-trp), N-formylkynurenine (NFK) and kynutrenine (Kyn), have substantial extinction coefficients in the 320 - 450nm spectral range (3000 - 4000 M "1 cm “1 ) and emit in the UV-Visible range (Impact of UVR-A on whole human lenses, supernatants of buffered human lens homogenates, and purified argpyrimidine and 3-OH-kynurenine. Kessel et al. Acta Ophthalmol Scand. 83(2):221 -7 (2005)).
  • tryptophan photo-products can inhibit the chaperone-like function of alpha-crystallins (Preferential and Specific Binding of Human aB-Crystallin to a Cataract-Related Variant of 7S-Crystallin. Kingsley et al. Structure 21 (12):2221 -7 (2013)) and critically affect the lens homeostasis.
  • Some tryptophan modifications together with modifications of other side chains can cause misfolding of beta- and gamma-crystallins which together with the impaired chaperone-like function of alpha- crystallins leads to irreversible aggregation of the lens proteins which in turn affects the homogeneity of the lens refractive index which causes light scattering.
  • relatively large extinction coefficients of tryptophan photo-products and other PTMs cause the lens optical absorbance in the UV-Visible range, which attenuates its light transmission and impairs vision.
  • a method for detecting structural changes in a human or animal eye lens comprising: illuminating the eye with radiation that causes fluorescence of tryptophan, its photo-products and/or at least one other fluorescent post-translational modification (PTM); and detecting features in the fluorescence.
  • Red-edge excitation enables excitation of tryptophan fluorescence in the lens interior together with the excitation of its photo-products and/or at least one other fluorescent PTM.
  • the features detected in the fluorescence may be at least one of the total intensity of the fluorescence emission, the spectral shape of the fluorescence emission and the position of at least one peak. Values of relative fractions of the individual spectral components caused by tryptophan, its photo-products and other fluorescent PTMs can be calculated. One or more of the total intensity of the fluorescence emission, the spectral shape of the fluorescence emission, the position of at least one peak and the values of relative fractions can be used to quantify the level of the lens protein photo-oxidation and structural defects accumulated in the body of the lens.
  • the structural defects may be associated with protein damage, such as protein post-translational modifications, misfolding, denaturation and aggregation. Hence, such data can be used for grading cataracts.
  • the method may involve decomposing the emission stimulated by excitation of the tryptophan, its photo-products and other fluorescent PTMs into individual spectral components each associated with one of the tryptophan and the at least one photo- product or fluorescent PTM.
  • the total spectrum S tota i (A) may be given by a sum of its individual components Sj(A) weighted by fractional factors f, proportional to concentrations of the individual components.
  • the spectrum is decomposed into at least two components or at least three components or at least four components or at least five components, or at least six components.
  • the method may involve predicting one or more conditions based on the detected features, for example cataracts.
  • the method may involve using 3D fluorescence mapping of the eye lens for identification of a cataract type and grade.
  • the excitation wavelength may be in the range 305nm to 320nm, for example 317nm.
  • the method may involve illuminating the eye lens with radiation that causes fluorescence of tryptophan, its photo-products and other fluorescent PTMs at multiple different positions within the lens.
  • an apparatus for detecting changes in a human or animal eye comprising: an illuminator for illuminating the eye lens or at least a part thereof using a wavelength suitable for excitation of tryptophan and at least one of its photo-products or other fluorescent post-translational modification (PTM) ; a detector for detecting features in the fluorescence associated with an emission stimulated by excitation of the tryptophan and at least one of its photo-products or other PTMs, and processor for using the detected features to detect modifications and/or structural defects in the lens.
  • PTM fluorescent post-translational modification
  • the processor may be operable to use the detected features to determine relative concentrations of individual components of the mix of tryptophan and at least one of its photo-products or another fluorescent PTM.
  • the processor may be operable to decompose the emission stimulated by excitation of the tryptophan and its photo-products into individual components each associated with one of the tryptophan and the at least one photo-product or another fluorescent PTM.
  • the total spectrum S tota i (A) is given by a sum of its individual components Sj(A) weighted by fractional factors f, proportional to concentrations of the individual components.
  • the processor may be operable to predict one or more conditions based on the detected features, for example cataracts.
  • the illuminator may be operable to emit an excitation wavelength in the range 305nm to 320nm, for example 317nm.
  • a system for detecting changes in a human or non-human eye lens comprising means for illuminating the eye lens using tryptophan red-edge excitation to cause fluorescence of its intrinsic fluorphores, for example tryptophan or/and its derivatives; a detector for detecting features in the eye lens fluorescence associated with crystalline proteins and means for using the detected features associated with the eye lens fluorescence to detect structural defects in the eye lens.
  • the structural defects may be associated with protein damage, such as protein post-translational modifications, misfolding, denaturation and aggregation.
  • the system may be configured to predict one or more conditions based detected features in the proteins, for example cataracts or diabetes.
  • the tryptophan photo- products may include at least one of: 5-Hydroxytryptophan (50H-Trp), N- Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H-Kyn).
  • the tryptophan photo-products may include all of: 50H-Trp, NFK, Kyn and 30H-Kyn.
  • Figure 1 is a schematic layout of an experimental setup for in vitro tryptophan and non-tryptophan fluorescence measurements in the eye lens;
  • Figure 2 shows UV-Visible spectrally corrected emission spectra of a normal
  • Figure 3 shows chemical structures of tryptophan, its photo-products and arginine fluorescent PTMs, argpyrimidine and pentosidine;
  • Figure 4 shows absorption spectra of N-acetyl-tryptophanamide (NATA) and tryptophan photo-products: 5-Hydroxytryptophan (50H-Trp), N-Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H-Kyn) in PBS measured in a Shimadzu UV2550 spectrophotometer;
  • N-acetyl-tryptophanamide tryptophan photo-products: 5-Hydroxytryptophan (50H-Trp), N-Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H-Kyn) in PBS measured in a Shimadzu UV2550 spectrophotometer;
  • Figure 5 shows corrected fluorescence spectra of NATA and tryptophan photo- products: 50H-Trp, NFK, Kyn and 30H-Kyn in PBS measured in FLS980 spectrometer (Edinburgh Instruments);
  • Figure 6 shows a spectral decomposition of a total fluorescence spectrum of a mixture of NATA, 50H-Trp, NFK, Kyn and 30H-Kyn into the individual spectral components
  • Figure 7 shows a table of results of mass spectrometric analysis of a solubilised in PBS pig's eye lens proteins
  • Figure 8 shows absorption (upper panel) and emission spectra (lower panel) of eye lens fluorophores, 50H-Trp, NATA, Pentosidine, Argpyrimidine, NFK, Kyn and 30H-Kyn, which can be excited in the 310-320nm spectral range;
  • Figure 9 is a schematic diagram of a confocal instrument for non-invasive measurement of tryptophan and non-tryptophan fluorescence from the eye lens;
  • Figure 10 shows fluorescence spectra of an insoluble fraction of protein samples of control (dashed) and cataractous human eye lenses (solid lines) dissolved in PBS/8M urea and measured at 310nm excitation. Fluorescence spectrum of normal pig's eye lens protein in PNS/8M urea solution measured at the same excitation wavelength (dashed-dot line);
  • Figure 1 1 compares the intensity of non-tryptophan emission of the control (grey bar) and cataractous samples (black bars);
  • Figure 12 shows spectral decomposition of fluorescence spectra for an insoluble fraction of a lens protein sample at 310nm excitation (dashed line) and a fit-function (solid line), and Figure 12 (lower panel) shows residuals;
  • Figure 13 is a plot of normalised on tryptophan fractional coefficient (f2) coefficients of spectral decomposition of spectra of the human donor eye lens samples shown in Figure 1 1 : (f4+f5)/f2 (Argpyrimidine + NFK) - filled circles, f1/f2 (50H-Trp) - squares, f3/f2 (Pentosidine) - open circles, f6/f2 (Kynurenine) - crosses;
  • Figure 14 shows fluorescence spectra of a donor eye lens measured in a
  • Figure 15 is a plot of intensity of non-tryptophan fluorescence calculated by integration of the emission spectra shown in Figure 14 in the 375-420nm spectral range as a function of age (open circles), together with a linear fit of the experimental data (straight line).
  • the present invention detects structural changes in a human or animal eye lens by detecting fluorescence of tryptophan, its photo-products and other fluorescent PTMs, wherein the fluorescence is caused by simultaneous excitation at the same wavelength.
  • tryptophan photo-products are 5-Hydroxytryptophan (50H- Trp), N-Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H- Kyn).
  • Examples of other fluorescent PTMs are Argpyrimidine and Pentosidine.
  • the method of the invention is based on quantification of the concentration in the lens of tryptophan, its photo-products and other fluorescent PTMs, in which tryptophan emission, emission of its photo-products and other fluorescent PTMs is excited by the same excitation wavelength.
  • the method uses total emission spectrum measured in different points of the eye lens for spectral decomposition into individual spectral components, which are associated with emission of tryptopan, its photo-products and other fluorescent PTMs.
  • the method requires simultaneous excitation of tryptophan, its products and other fluorescent PTMs in the excitation wavelength range of 305-320nm. This is at the red edge of the tryptophan absorption band.
  • Fluorescence is a relative characteristic and its intensity is a function of experimental parameters, such as intensity of excitation light, excitation and emission wavelength, geometrical and other experimental factors. Simultaneously monitoring emission of individual spectral components of tryptophan photo-products and other fluorescent PTMs associated with eye lens proteins using the same wavelength and intensity of excitation light in the same instrument, upon the same experimental geometry, and a normalization of the spectrum on intensity of tryptophan emission allows quantitative determination of concentrations of tryptophan photoproducts and other fluorescent PTMs in eye lens proteins.
  • Concentration of florescent products of tryptophan and other amino acids are related to the total score of PTMs in the eye lens proteins and hence can be used for categorisation and quantification of structural changes in the eye lens caused by e.g. cataractogenesis.
  • the eye lens contains high concentration of protein and hence of tryptophan amino residues, which strongly absorbs excitation light and prevents penetration into the lens at wavelengths shorter than 300nm.
  • the decrease in tryptophan extinction at the red edge of its absorption spectrum allows the lens interior to be excited by light in the 305- 320nm range. Together with tryptophan several of its fluorescent photo-products and other fluorescent PTMs are also excited by these wavelengths.
  • the optimal excitation wavelength at which both tryptophan and its photo-products will be excited in comparable proportions is around 315 - 317nm. Conversion of tryptophan side chains to the photo-products decreases tryptophan concentration and increases concentration of its derivatives. When tryptophan concentration depletes and its photo-products accumulate, the excitation should be taken at shorter wavelength at around 305nm in order to provide comparable levels of excitation of all fluorophores. Thus, variations in the concentration of tryptophan and its photo-products in the lens may require optimisation of the excitation wavelength.
  • Figure 1 shows a schematic layout of an experimental setup for in vitro fluorescence measurements in the eye lens.
  • This has a light source, for example a Xenon (450W) lamp.
  • a light source for example a Xenon (450W) lamp.
  • Light passes into an excitation monochromator, where the excitation wavelength is selected by a diffraction grating, before exiting via variable slits.
  • the spectrally selected light is focused using a lens along an excitation path onto the eye lens.
  • Fluorescence stimulated by the excitation is conveyed along an emission path to an emission monochromator.
  • the emission monochromator wavelength is scanned between 330-700 nm, and the emitted light is detected by a detector, for example a photomultiplier (PMT) operated in a single photon counting mode to detect light.
  • PMT photomultiplier
  • the photomultiplier signal is analysed by a computer processor and displayed on a computer screen (not shown).
  • the computer processing of the system of Figure 1 includes analysis software arranged to analyse the emission spectra to provide a measure of the relative fractions of eye lens tryptophan, tryptophan photo-products and other fluorescent PTMs. The analysis will be described in more detail later.
  • Figure 2 shows corrected fluorescence spectra of a normal (left) and UVB-irradiated (damaged) pig's eye lens (right) as function of excitation wavelength (300nm (1 ), 305nm (2), 310nm (3), 4317nm (4), 320nm (5)) measured in the setup shown in Figure 1 . From this, it can be clearly seen that the spectra of the normal eye and the spectra of the irradiated, damaged eye are easily distinguishable.
  • Figure 3 shows chemical structures of tryptophan, its photo-products 50H-Trp, Kyn, 30H-Kyn, NFK, 30H-Kyn and two other fluorescent PMTs which can be excited in the 310-320nm range: Argpyrimidine and Pentosidine.
  • the total emission spectrum of the eye lens varies depending on the level of its damage.
  • By measuring a fluorescence spectrum of the eye lens relative proportions of each of tryptophan, its photo-products and other fluorescent PTM can be deduced and used to identify the level of damage to the lens.
  • Figure 4 shows absorption spectra of NATA and tryptophan photo-products: 50H-Trp, NFK, Kyn and 30H-Kyn in PBS measured in a Shimadzu UV2550 spectrophotometer.
  • the vertical lines show the spectral range suitable for excitation of tryptophan and its photo-products.
  • Figure 5 shows corrected fluorescence spectra of NATA and tryptophan photo- products: 50H-Trp NFK, Kynurenine and 30H-Kyn in PBS measured using the setup shown in Figure 1 .
  • Figure 6 shows a spectral decomposition of the total spectra of a mixture of NATA and 50H-Trp, NFK, Kynurenine and 30H-Kyn in PBS into the individual spectral components using Nelder-Mead list squared minimisation algorithm (Matlab).
  • the total spectrum S tota i (A) is given by a sum of its individual components Sj(A) weighted by the fractional factors f,. which are proportional to concentrations of the individual components of the mixture of these fluorophores:
  • S tatal (A) frSifi) + f 2 S 2 (A) + f 3 sS 3 (A) + f 4 S 4 (A)
  • the spectral decomposition yields the relative fractions 32%, 8%, 43% 15% and 2% of tryptophan, 50H-Trp, NFK, Kyn and 30H-Kyn respectively in the mixture.
  • Figure 7 shows a table of results of mass spectrometric analysis of a solubilised in PBS pig's eye lens proteins.
  • Figure 8 shows absorption (upper panel) and emission spectra (lower panel) of human eye lens fluorophores emitting in the 320-650nm range, which can be excited in the 310-320mn range.
  • Figure 9 shows an optical scheme of an instrument for measuring fluorescence spectra from the eye lens in vivo.
  • Excitation light is reflected by a Dichroic beam splitter and directed to the eye by a X-Y and Z scan L2 deflection system.
  • the beam is focused on the eye lens by an objective lens L1 .
  • Fluorescence from the eye lens is collected by the objective L1 and collimated by the lens L2, and separated from the excitation light by the dichroic beam splitter.
  • the fluorescence is then filtered by a spatial filter (L3/pinhole/L4) and spectrally filtered by a tunable interference filter. Fluorescence is measured by an optical detector.
  • Figure 10 shows fluorescence spectra of an insoluble fraction of human eye lens proteins dissolved in PBS/8M urea and measured in a FLS980 spectrometer (Edinburgh Instruments, UK) at an excitation wavelength of 310nm (solid lines).
  • the dashed line shows a spectrum from a control sample (46 years old man)
  • a fluorescence spectrum of a normal pig's eye lens proteins (which have substantially lower post translational modification score) dissolved in PBS/8M urea (dashed-dot line). The latter was taken as a spectrum of tryptophan.
  • Subtracting the emission spectrum of tryptophan from the fluorescence spectra of the insoluble fraction of human eye lens protein samples and integrating the resulting spectra allows the non- tryptophan emission intensity of the above samples to be calculated.
  • Figure 1 1 shows the fluorescence intensity of non-tryptophan emission of the control (grey bar) and cataractous samples (black bars) calculated from the data shown in Figure 10 by subtracting the tryptophan fluorescence from the total emission spectra. This shows that the intensity of the non-tryptophan fluorescence is significantly higher in the cataractous samples than in the control sample.
  • Figure 12 shows an example of decomposition of fluorescence spectra of the insoluble fraction of human donor eye lens proteins dissolved in PBS/8M urea with the use of the known spectral functions of the individual components shown in Figure 8. The spectrum was measured using 310nm excitation (dashed line).
  • Figure 13 is a plot of fractional coefficients representing relative concentrations of the fluorescent PTMs normalised using the fractional coefficient of tryptophan emission (f2): (f4+f5)/f2 (Argpyrimidine + NFK) - filled circles, f1/f2 (50H-Trp) - squares, f3/f2 (Pentosidine) - open circles, f6/f2 (Kyn) - crosses
  • the fractional coefficients were calculated by spectral decomposition of the fluorescence spectra shown in Figure 10.
  • Figure 13 shows that the (Argpyrimidine + NFK) fraction emission dominates in the non-tryptophan emission of the insoluble fraction of the samples shown in Figure 10. This suggests that the formation of Argpyrimidine and NFK play an important role in cataractogenesis.
  • Figure 14 shows fluorescence spectra of whole donor eye lenses measured in a FLS980 spectrometer at 315nm excitation. Sixteen eye lens samples of donors in the age range of 30 to 83 years were measured 36 to 48 hours after death. Measurements were carried out using a 315nm excitation wavelength. The spectral functions were integrated in the 375-420nm range where Argpyrimidine and NFK predominantly emit. The emission intensity values are plotted as a function of age in Figure 15. This shows that the intensity of non-tryptophan (NFK and Agrpyrimidine) emission in eye lens proteins consistently increases with aging of the eye lens.
  • NFK and Agrpyrimidine non-tryptophan
  • the method of the invention can be used in drug screening applications to monitor changes in the eye lens structure induced by application of various chemical compounds. For this, identical extracted animal eye lenses are plated into a multi-well plate and different compounds of a chemical library are added to the plate's wells. Changes in fluorescence emission properties can be monitored in real-time or in an end-point format.
  • the method of the invention can also be used for monitoring results of therapeutic treatment of the eye lens.
  • An instrument for fluorescence measurements of emission from eye lenses in vivo will be used to monitor changes in the lens fluorescence features caused by administration of a medication to the patient's eyes.

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Abstract

A method for detecting changes in a human or animal eye comprising: illuminating the eye lens or at least a part thereof using a wavelength suitable for excitation of tryptophan and at least one of its photo- products and/or at least one other fluorescent post-translational modification (PTM); detecting features in the fluorescence associated with an emission stimulated by excitation of the tryptophan and at least one of its photo-products and/or the other fluorescent PTM, and using the detected features to detect modifications and/or structural defects in the lens. A correspomding system for detecting changes in a human or non-human eye is also disclosed.

Description

Non-Invasive Fluorescence- Based Eye Lens Diagnostics
Field of the Invention
The present invention relates to a non-invasive system and method for detecting changes indicative of cataract or cataract formation using intrinsic fluorescence reporters of the eye lens proteins. The invention can be used in vivo to detect early changes in the eye lens structure and allows quantitative categorisation of cataract types and stage of its formation. It can be also used for in vitro or ex vivo drug screening applications.
Background of the Invention
The ocular lens grows continuously throughout life. The major components of the lens are highly stable and water soluble structural protein encoded as alpha-, beta- and gamma-crystallins. The high concentration of these proteins and the presence of a substantial fraction (-35%) of alpha-crystallin, which has a chaperon-like function in the protein composition, plays two major roles: maintaining high refractive index and binding of misfolded (damaged) proteins to the chaperon molecules to prevent their irreversible aggregation (Borkman, Knight and Obi, The molecular chaperon alpha- crystallin inhibits UV-induced protein aggregation. Exp. Eye Res. 62, 141 -148 (1996)).
Post-translational modifications (PTMs) in the lens proteins caused by chemical, photochemical and environmental factors accumulate over the life span of patients. PTMs lead to protein misfolding, denaturation and aggregation and hence structural defects. Accumulation of PTMs in the lens structure can be also caused by aging (Age- related changes in human crystallins determined from comparative analysis of post- translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility? Wilmarth at al. J Proteome Res. 2006 Oct;5(10):2554-66) or concomitant diseases, e.g. diabetes, (Truscott, R.J.W. Protein misfolding, aggregation, and conformational diseases, Uverski VN and Fink A (eds.), pp. 435-447, Springer, 2007).
Currently cataract detection is based on measurements of lens opacity by light scattering. This can be done using a conventional slit lamp-based technique or other more advanced methods, such as Optical Coherence Tomography etc, see KonstantopoulosA, Hossain,P. & Anderson, D.F. Recent advances in ophthalmic anterior segment imaging: a new era for ophthalmic diagnosis? Br. J. Ophthalmol. 91 , 551 -557 (2007). A limitation of these scattering techniques is that the sizes of structural defects must be comparable with the wavelength of light used, i.e. ca 0.4 - 0.6 micrometer or greater. Therefore, methods based on light scattering only allow detection of relatively large-scale changes in the eye lens.
US 5203328 describes a method for noninvasively diagnosing diabetes mellitus and cataracts using a fluorescent response of eye lens. Diagnoses are made by illuminating eye lens tissue with a narrow-band light source at a selected wavelength, detecting the back-scattered radiation intensity at the peak of a non-tryptophan fluoresce, and normalizing the detected value with the intensity of its Rayleigh component. This method uses auto-fluorescence of the lens, i.e. emission of fluorescent products accumulated in the lens over time. The eye lens exhibits UV emission in the 300 - 450nm range caused by tryptophan and in the 350 - 750nm range by its photo-products and other fluorescent PTMs. Various fluorescence techniques have been proposed to investigate the eye, for example as described by Van den Berg, T.J. in the article "Quantal and visual efficiency of fluorescence in the lens of the human eye", Invest Ophthalmol. Vis. Sci. 34, 3566-3573 (1993) and by Bessems,G.J., et al in the article "Non-tryptophan fluorescence of crystallins from normal and cataractous human lenses" Invest Ophthalmol. Vis. Sci. 28, 1 157-1 163 (1987).
WO2010097582 describes a tryptophan-based method for detecting changes in a human or animal eye. The method involves illuminating the eye or at least a part thereof, in particular the lens and/or cornea, using light at a tryptophan red edge excitation wavelength and detecting tryptophan fluorescence. The detected tryptophan fluorescence is used to detect or identify structural defects or changes in the eye, such as the formation of cataracts. Among various types of PTMs, such as phosphorylation, methylation, acethylation, glycation etc, there are several modifications of the tryptophan side chain, triggered by the interaction with reactive oxygen species (ROS), UV light or gamma-irradiation (Oxidation_of Free Tryptophan and Tryptophan Residues in Peptides and Proteins. Simat and Steinhart, Agric Food Chem. 46(2):490-498 (1998)). The human molecular chaperons alpha crystallin A and B contain one or two tryptophan residues respectively. Human beta- and gamma-crystallins contain four to eight tryptophan residues. Susceptibility of a tryptophan residue side chain to photo- oxidation is a function of the protein structure, which protects it from exposure to an aqueous environment where it can interact with ROS. Some tryptophan photo- products, such as 5-OH-tryptophan (5-OH-trp), N-formylkynurenine (NFK) and kynutrenine (Kyn), have substantial extinction coefficients in the 320 - 450nm spectral range (3000 - 4000 M"1 cm"1) and emit in the UV-Visible range (Impact of UVR-A on whole human lenses, supernatants of buffered human lens homogenates, and purified argpyrimidine and 3-OH-kynurenine. Kessel et al. Acta Ophthalmol Scand. 83(2):221 -7 (2005)).
The formation of tryptophan photo-products can inhibit the chaperone-like function of alpha-crystallins (Preferential and Specific Binding of Human aB-Crystallin to a Cataract-Related Variant of 7S-Crystallin. Kingsley et al. Structure 21 (12):2221 -7 (2013)) and critically affect the lens homeostasis. Some tryptophan modifications together with modifications of other side chains can cause misfolding of beta- and gamma-crystallins which together with the impaired chaperone-like function of alpha- crystallins leads to irreversible aggregation of the lens proteins which in turn affects the homogeneity of the lens refractive index which causes light scattering. In addition, relatively large extinction coefficients of tryptophan photo-products and other PTMs cause the lens optical absorbance in the UV-Visible range, which attenuates its light transmission and impairs vision. Summary of the invention
According to one aspect of the present invention, there is provided a method for detecting structural changes in a human or animal eye lens comprising: illuminating the eye with radiation that causes fluorescence of tryptophan, its photo-products and/or at least one other fluorescent post-translational modification (PTM); and detecting features in the fluorescence. Red-edge excitation enables excitation of tryptophan fluorescence in the lens interior together with the excitation of its photo-products and/or at least one other fluorescent PTM.
The features detected in the fluorescence may be at least one of the total intensity of the fluorescence emission, the spectral shape of the fluorescence emission and the position of at least one peak. Values of relative fractions of the individual spectral components caused by tryptophan, its photo-products and other fluorescent PTMs can be calculated. One or more of the total intensity of the fluorescence emission, the spectral shape of the fluorescence emission, the position of at least one peak and the values of relative fractions can be used to quantify the level of the lens protein photo-oxidation and structural defects accumulated in the body of the lens. The structural defects may be associated with protein damage, such as protein post-translational modifications, misfolding, denaturation and aggregation. Hence, such data can be used for grading cataracts.
The method may involve decomposing the emission stimulated by excitation of the tryptophan, its photo-products and other fluorescent PTMs into individual spectral components each associated with one of the tryptophan and the at least one photo- product or fluorescent PTM. The total spectrum Stotai (A) may be given by a sum of its individual components Sj(A) weighted by fractional factors f, proportional to concentrations of the individual components. Preferably, the spectrum is decomposed into at least two components or at least three components or at least four components or at least five components, or at least six components.
The method may involve predicting one or more conditions based on the detected features, for example cataracts. The method may involve using 3D fluorescence mapping of the eye lens for identification of a cataract type and grade.
The excitation wavelength may be in the range 305nm to 320nm, for example 317nm. The method may involve illuminating the eye lens with radiation that causes fluorescence of tryptophan, its photo-products and other fluorescent PTMs at multiple different positions within the lens.
According to another aspect of the invention, there is provided an apparatus for detecting changes in a human or animal eye comprising: an illuminator for illuminating the eye lens or at least a part thereof using a wavelength suitable for excitation of tryptophan and at least one of its photo-products or other fluorescent post-translational modification (PTM) ; a detector for detecting features in the fluorescence associated with an emission stimulated by excitation of the tryptophan and at least one of its photo-products or other PTMs, and processor for using the detected features to detect modifications and/or structural defects in the lens.
The processor may be operable to use the detected features to determine relative concentrations of individual components of the mix of tryptophan and at least one of its photo-products or another fluorescent PTM.
The processor may be operable to decompose the emission stimulated by excitation of the tryptophan and its photo-products into individual components each associated with one of the tryptophan and the at least one photo-product or another fluorescent PTM. The total spectrum Stotai (A) is given by a sum of its individual components Sj(A) weighted by fractional factors f, proportional to concentrations of the individual components.
The processor may be operable to predict one or more conditions based on the detected features, for example cataracts.
The illuminator may be operable to emit an excitation wavelength in the range 305nm to 320nm, for example 317nm. According to yet another aspect of the invention, there is provided a system for detecting changes in a human or non-human eye lens comprising means for illuminating the eye lens using tryptophan red-edge excitation to cause fluorescence of its intrinsic fluorphores, for example tryptophan or/and its derivatives; a detector for detecting features in the eye lens fluorescence associated with crystalline proteins and means for using the detected features associated with the eye lens fluorescence to detect structural defects in the eye lens. The structural defects may be associated with protein damage, such as protein post-translational modifications, misfolding, denaturation and aggregation. The system may be configured to predict one or more conditions based detected features in the proteins, for example cataracts or diabetes.
In any method, system and apparatus of the present invention, the tryptophan photo- products may include at least one of: 5-Hydroxytryptophan (50H-Trp), N- Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H-Kyn). In some cases, the tryptophan photo-products may include all of: 50H-Trp, NFK, Kyn and 30H-Kyn. Brief description of the drawings
Various aspects of the invention will now be described by way of example only, with reference to the accompanying drawings, of which:
Figure 1 is a schematic layout of an experimental setup for in vitro tryptophan and non-tryptophan fluorescence measurements in the eye lens;
Figure 2 shows UV-Visible spectrally corrected emission spectra of a normal
(left) and UVB-irradiated (right) pig's eye lens as function of excitation wavelength;
Figure 3 shows chemical structures of tryptophan, its photo-products and arginine fluorescent PTMs, argpyrimidine and pentosidine;
Figure 4 shows absorption spectra of N-acetyl-tryptophanamide (NATA) and tryptophan photo-products: 5-Hydroxytryptophan (50H-Trp), N-Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H-Kyn) in PBS measured in a Shimadzu UV2550 spectrophotometer;
Figure 5 shows corrected fluorescence spectra of NATA and tryptophan photo- products: 50H-Trp, NFK, Kyn and 30H-Kyn in PBS measured in FLS980 spectrometer (Edinburgh Instruments);
Figure 6 shows a spectral decomposition of a total fluorescence spectrum of a mixture of NATA, 50H-Trp, NFK, Kyn and 30H-Kyn into the individual spectral components, and
Figure 7 shows a table of results of mass spectrometric analysis of a solubilised in PBS pig's eye lens proteins;
Figure 8 shows absorption (upper panel) and emission spectra (lower panel) of eye lens fluorophores, 50H-Trp, NATA, Pentosidine, Argpyrimidine, NFK, Kyn and 30H-Kyn, which can be excited in the 310-320nm spectral range;
Figure 9 is a schematic diagram of a confocal instrument for non-invasive measurement of tryptophan and non-tryptophan fluorescence from the eye lens; Figure 10 shows fluorescence spectra of an insoluble fraction of protein samples of control (dashed) and cataractous human eye lenses (solid lines) dissolved in PBS/8M urea and measured at 310nm excitation. Fluorescence spectrum of normal pig's eye lens protein in PNS/8M urea solution measured at the same excitation wavelength (dashed-dot line);
Figure 1 1 compares the intensity of non-tryptophan emission of the control (grey bar) and cataractous samples (black bars);
Figure 12 (upper panel) shows spectral decomposition of fluorescence spectra for an insoluble fraction of a lens protein sample at 310nm excitation (dashed line) and a fit-function (solid line), and Figure 12 (lower panel) shows residuals;
Figure 13 is a plot of normalised on tryptophan fractional coefficient (f2) coefficients of spectral decomposition of spectra of the human donor eye lens samples shown in Figure 1 1 : (f4+f5)/f2 (Argpyrimidine + NFK) - filled circles, f1/f2 (50H-Trp) - squares, f3/f2 (Pentosidine) - open circles, f6/f2 (Kynurenine) - crosses;
Figure 14 shows fluorescence spectra of a donor eye lens measured in a
FLS980 spectrometer at an excitation wavelength of 315nm, and
Figure 15 is a plot of intensity of non-tryptophan fluorescence calculated by integration of the emission spectra shown in Figure 14 in the 375-420nm spectral range as a function of age (open circles), together with a linear fit of the experimental data (straight line).
Detailed description of the drawings
The present invention detects structural changes in a human or animal eye lens by detecting fluorescence of tryptophan, its photo-products and other fluorescent PTMs, wherein the fluorescence is caused by simultaneous excitation at the same wavelength. Examples of tryptophan photo-products are 5-Hydroxytryptophan (50H- Trp), N-Formylkynurenine (NFK), Kynurenine (Kyn) and 3-Hydroxykynurenine (30H- Kyn). Examples of other fluorescent PTMs are Argpyrimidine and Pentosidine. The method of the invention is based on quantification of the concentration in the lens of tryptophan, its photo-products and other fluorescent PTMs, in which tryptophan emission, emission of its photo-products and other fluorescent PTMs is excited by the same excitation wavelength. The method uses total emission spectrum measured in different points of the eye lens for spectral decomposition into individual spectral components, which are associated with emission of tryptopan, its photo-products and other fluorescent PTMs. The method requires simultaneous excitation of tryptophan, its products and other fluorescent PTMs in the excitation wavelength range of 305-320nm. This is at the red edge of the tryptophan absorption band. By detecting features in the fluorescence of tryptophan, its photo-products and other fluorescent PTMs, the level of the lens protein's photo-oxidation and structural defects can be quantified and used to identify and grade cataract formation.
Fluorescence is a relative characteristic and its intensity is a function of experimental parameters, such as intensity of excitation light, excitation and emission wavelength, geometrical and other experimental factors. Simultaneously monitoring emission of individual spectral components of tryptophan photo-products and other fluorescent PTMs associated with eye lens proteins using the same wavelength and intensity of excitation light in the same instrument, upon the same experimental geometry, and a normalization of the spectrum on intensity of tryptophan emission allows quantitative determination of concentrations of tryptophan photoproducts and other fluorescent PTMs in eye lens proteins.
Fluorescence spectra of tryptophan, its photo-products and other fluorescent PTMs overlap and the total emission spectrum extends from 300nm to 750nm. The identification by mass spectroscopy of all major photo-products of tryptophan and other fluorescent PTMs and their spectral characterisation substantially simplifies the decomposition of the total emission spectrum into its individual components. This allows the emission spectra of these fluorophores as known spectral functions to be used to calculate relative concentrations of individual species associated with these spectra.
Concentration of florescent products of tryptophan and other amino acids are related to the total score of PTMs in the eye lens proteins and hence can be used for categorisation and quantification of structural changes in the eye lens caused by e.g. cataractogenesis.
The eye lens contains high concentration of protein and hence of tryptophan amino residues, which strongly absorbs excitation light and prevents penetration into the lens at wavelengths shorter than 300nm. The decrease in tryptophan extinction at the red edge of its absorption spectrum allows the lens interior to be excited by light in the 305- 320nm range. Together with tryptophan several of its fluorescent photo-products and other fluorescent PTMs are also excited by these wavelengths.
At an early stage of lens protein modification when tryptophan concentration is high and concentrations of fluorescent PTMs are small, the optimal excitation wavelength at which both tryptophan and its photo-products will be excited in comparable proportions is around 315 - 317nm. Conversion of tryptophan side chains to the photo-products decreases tryptophan concentration and increases concentration of its derivatives. When tryptophan concentration depletes and its photo-products accumulate, the excitation should be taken at shorter wavelength at around 305nm in order to provide comparable levels of excitation of all fluorophores. Thus, variations in the concentration of tryptophan and its photo-products in the lens may require optimisation of the excitation wavelength. Figure 1 shows a schematic layout of an experimental setup for in vitro fluorescence measurements in the eye lens. This has a light source, for example a Xenon (450W) lamp. Light passes into an excitation monochromator, where the excitation wavelength is selected by a diffraction grating, before exiting via variable slits. The spectrally selected light is focused using a lens along an excitation path onto the eye lens. Fluorescence stimulated by the excitation is conveyed along an emission path to an emission monochromator. The emission monochromator wavelength is scanned between 330-700 nm, and the emitted light is detected by a detector, for example a photomultiplier (PMT) operated in a single photon counting mode to detect light. The photomultiplier signal is analysed by a computer processor and displayed on a computer screen (not shown). The computer processing of the system of Figure 1 includes analysis software arranged to analyse the emission spectra to provide a measure of the relative fractions of eye lens tryptophan, tryptophan photo-products and other fluorescent PTMs. The analysis will be described in more detail later.
Figure 2 shows corrected fluorescence spectra of a normal (left) and UVB-irradiated (damaged) pig's eye lens (right) as function of excitation wavelength (300nm (1 ), 305nm (2), 310nm (3), 4317nm (4), 320nm (5)) measured in the setup shown in Figure 1 . From this, it can be clearly seen that the spectra of the normal eye and the spectra of the irradiated, damaged eye are easily distinguishable. Figure 3 shows chemical structures of tryptophan, its photo-products 50H-Trp, Kyn, 30H-Kyn, NFK, 30H-Kyn and two other fluorescent PMTs which can be excited in the 310-320nm range: Argpyrimidine and Pentosidine. As noted above, the total emission spectrum of the eye lens varies depending on the level of its damage. By measuring a fluorescence spectrum of the eye lens relative proportions of each of tryptophan, its photo-products and other fluorescent PTM can be deduced and used to identify the level of damage to the lens.
Figure 4 shows absorption spectra of NATA and tryptophan photo-products: 50H-Trp, NFK, Kyn and 30H-Kyn in PBS measured in a Shimadzu UV2550 spectrophotometer. The vertical lines show the spectral range suitable for excitation of tryptophan and its photo-products.
Figure 5 shows corrected fluorescence spectra of NATA and tryptophan photo- products: 50H-Trp NFK, Kynurenine and 30H-Kyn in PBS measured using the setup shown in Figure 1 .
Figure 6 shows a spectral decomposition of the total spectra of a mixture of NATA and 50H-Trp, NFK, Kynurenine and 30H-Kyn in PBS into the individual spectral components using Nelder-Mead list squared minimisation algorithm (Matlab). The total spectrum Stotai (A) is given by a sum of its individual components Sj(A) weighted by the fractional factors f,. which are proportional to concentrations of the individual components of the mixture of these fluorophores: Statal (A) = frSifi) + f2S2(A) + f3sS3(A) + f4S4(A)
From the data of Figure 6, the spectral decomposition yields the relative fractions 32%, 8%, 43% 15% and 2% of tryptophan, 50H-Trp, NFK, Kyn and 30H-Kyn respectively in the mixture.
Figure 7 shows a table of results of mass spectrometric analysis of a solubilised in PBS pig's eye lens proteins. The data shows that 7 mW UVB (310 +/- 10nm) irradiation decreases by 26% the concentration of alpha-crystallin A in the soluble fraction and increases ~3-fold the rate of post translational modifications of tryptophan amino residues (Tryptophan => 50H-Trp, NFK, Kyn). Hence, knowledge of the fractional components of tryptophan and its photo-products can give a direct indication of the level of damage to a lens.
In further tests, human eye lens protein samples have been analysed by tandem electrospray ionization mass spectrometry. In addition to the UV-emitting tryptophan derivatives such as 50H-Trp, tryptophan, NATA, NFK, Kyn and 30H-Kyn, another fluorescent PTM, Argpyrimidine, was identified. The literature also suggests the formation of a cross-link between Lysine and Arginine in the eye lens proteins called Pentosidine, which emits in the 350-450nm range (Kessel et al. Photochem. Photobiol. (2002) 76(5):549-554). Argpyrimidine and Pentosidine do not belong to the tryptophan degradation pathway, but their concentrations can be determined from evaluation of eye lens UV-Vis spectra.
Figure 8 shows absorption (upper panel) and emission spectra (lower panel) of human eye lens fluorophores emitting in the 320-650nm range, which can be excited in the 310-320mn range.
Figure 9 shows an optical scheme of an instrument for measuring fluorescence spectra from the eye lens in vivo. Excitation light is reflected by a Dichroic beam splitter and directed to the eye by a X-Y and Z scan L2 deflection system. The beam is focused on the eye lens by an objective lens L1 . Fluorescence from the eye lens is collected by the objective L1 and collimated by the lens L2, and separated from the excitation light by the dichroic beam splitter. The fluorescence is then filtered by a spatial filter (L3/pinhole/L4) and spectrally filtered by a tunable interference filter. Fluorescence is measured by an optical detector.
Figure 10 shows fluorescence spectra of an insoluble fraction of human eye lens proteins dissolved in PBS/8M urea and measured in a FLS980 spectrometer (Edinburgh Instruments, UK) at an excitation wavelength of 310nm (solid lines). The dashed line shows a spectrum from a control sample (46 years old man) Also shown is a fluorescence spectrum of a normal pig's eye lens proteins (which have substantially lower post translational modification score) dissolved in PBS/8M urea (dashed-dot line). The latter was taken as a spectrum of tryptophan. Subtracting the emission spectrum of tryptophan from the fluorescence spectra of the insoluble fraction of human eye lens protein samples and integrating the resulting spectra allows the non- tryptophan emission intensity of the above samples to be calculated.
Figure 1 1 shows the fluorescence intensity of non-tryptophan emission of the control (grey bar) and cataractous samples (black bars) calculated from the data shown in Figure 10 by subtracting the tryptophan fluorescence from the total emission spectra. This shows that the intensity of the non-tryptophan fluorescence is significantly higher in the cataractous samples than in the control sample. Figure 12 shows an example of decomposition of fluorescence spectra of the insoluble fraction of human donor eye lens proteins dissolved in PBS/8M urea with the use of the known spectral functions of the individual components shown in Figure 8. The spectrum was measured using 310nm excitation (dashed line). The spectral evaluation was carried out using proprietary software based on Nelder-Mead list squared minimisation algorithm (Matlab) using the following model:
Figure imgf000013_0001
where S\(k) are spectra of 50H-Trp (i=1 ), NATA (i=2), Pensotidine (i=3), Argpyrimidine (i=4), NFK (i=5) and Kyn (i=6) and f, are fractional coefficients (Upper panel). Residuals are shown in lower panel.
Figure 13 is a plot of fractional coefficients representing relative concentrations of the fluorescent PTMs normalised using the fractional coefficient of tryptophan emission (f2): (f4+f5)/f2 (Argpyrimidine + NFK) - filled circles, f1/f2 (50H-Trp) - squares, f3/f2 (Pentosidine) - open circles, f6/f2 (Kyn) - crosses The fractional coefficients were calculated by spectral decomposition of the fluorescence spectra shown in Figure 10. Figure 13 shows that the (Argpyrimidine + NFK) fraction emission dominates in the non-tryptophan emission of the insoluble fraction of the samples shown in Figure 10. This suggests that the formation of Argpyrimidine and NFK play an important role in cataractogenesis.
Figure 14 shows fluorescence spectra of whole donor eye lenses measured in a FLS980 spectrometer at 315nm excitation. Sixteen eye lens samples of donors in the age range of 30 to 83 years were measured 36 to 48 hours after death. Measurements were carried out using a 315nm excitation wavelength. The spectral functions were integrated in the 375-420nm range where Argpyrimidine and NFK predominantly emit. The emission intensity values are plotted as a function of age in Figure 15. This shows that the intensity of non-tryptophan (NFK and Agrpyrimidine) emission in eye lens proteins consistently increases with aging of the eye lens.
The method of the invention can be used in drug screening applications to monitor changes in the eye lens structure induced by application of various chemical compounds. For this, identical extracted animal eye lenses are plated into a multi-well plate and different compounds of a chemical library are added to the plate's wells. Changes in fluorescence emission properties can be monitored in real-time or in an end-point format.
The method of the invention can also be used for monitoring results of therapeutic treatment of the eye lens. An instrument for fluorescence measurements of emission from eye lenses in vivo will be used to monitor changes in the lens fluorescence features caused by administration of a medication to the patient's eyes.
A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the scope of the invention. Accordingly, the above description of the specific embodiment is made by way of example only and not for the purposes of limitation. It will be clear to the skilled person that minor modifications may be made without significant changes to the operation described.

Claims

A method for detecting changes in a human or animal eye comprising:
illuminating the eye lens or at least a part thereof using a wavelength suitable for excitation of tryptophan and at least one of its photo-products and/or at least one other fluorescent post-translational modification (PTM);
detecting features in the fluorescence associated with an emission stimulated by excitation of the tryptophan and at least one of its photo-products and/or the other fluorescent PTM, and
using the detected features to detect modifications and/or structural defects in the lens.
A method as claimed in claim 1 wherein the structural defects are associated with protein damage, such as protein post-translational modifications, misfolding, denaturation and aggregation.
A method as claimed in claim 1 or claim 2 comprising using the detected features to determine relative concentrations of individual components of the tryptophan and at least one of its photo-products and/or at least other fluorescent PTM.
A method as claimed in any of the preceding claims comprising decomposing the emission stimulated by excitation of the tryptophan and its photo-products and/or the other fluorescent PTM into separate components each associated with one of the tryptophan and the at least one photo-product or the at least one other fluorescent PTM.
A method as claimed in claim 4 wherein the total spectrum Statai ( ) is given by a sum of its individual components Sj(A) weighted by fractional factors f, proportional to concentrations of the individual components.
A method as claimed in any of the preceding claims comprising predicting or more conditions based on the detected features, for example cataracts.
7. A method as claimed in any of the preceding claims comprising using an excitation wavelength in the range 305nm to 320nm, for example 317nm.
8. A method as claimed in any of the preceding claims comprising illuminating the eye in vivo.
9. A screening method that uses a method according to any of the preceding claims further.
10. A method for monitoring results of therapeutic treatment of the eye lens comprising the method of any of claims 1 to 8.
1 1 . A system for detecting changes in a human or non-human eye lens comprising a light source for illuminating the eye using tryptophan red edge excitation to cause fluorescence of intrinsic fluorphores, for example tryptophan or/and its derivatives / photo-products and/or at least one other fluorescent post- translational modification PTM associated with eye lens proteins; a detector for detecting features in the lens fluorescence associated crystalline proteins and means for using the detected features associated with the lens fluorescence to detect structural defects in the eye lens.
12. A system as claimed in claim 1 1 wherein the structural defects are associated with protein modifications, such as protein post-translational modifications causing misfolding, denaturation and aggregation.
13. A system as claimed in claim 1 1 or claim 12 adapted to predict one or more conditions based on the detected features in the proteins, for example cataracts or diabetes.
14. A system as claimed in any of claims 1 1 to 13 arranged to use the detected features to determine relative concentrations of individual components of the mix of tryptophan and at least one of its photo-products or another fluorescent PTM.
15. A system as claimed in any of claims 1 1 to 14 arranged to decompose the emission stimulated by excitation of the tryptophan and its derivatives / photo- products and/or other fluorescent PTMs into separate components each associated with one of the tryptophan and the at least one derivative / photo- product and/or other fluorescent PTMs.
16. A system as claimed in any of claims 1 1 to 15 arranged to determine the relative proportions of the tryptophan and the at least one photo-product and/or another fluorescent PTM.
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