TWO-PHOTON EXCITED FLUORESCENCE OF THE LENS FOR THE DIAGNOSIS OF PRESBYOPIA

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1 ОПТИКА И СПЕКТРОСКОПИЯ, 29, том 17, 3, с УДК :375 НАНОФОТОНИКА И БИОМЕДИЦИНА TWO-PHOTON EXCITED FLUORESCENCE OF THE LENS FOR THE DIAGNOSIS OF PRESBYOPIA 29 г. R. Steiner, M. Kessler, O. Fugger, F. Dolp, and D. Russ Institüt fur Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm, D-91 Ulm, Germany Received January 22, 29 Abstract Presbyopia is a wide spread phenomenon in elder people and is caused by the hardening of the lens in human eyes. Research is performed to make such lenses again more flexible by application of geometrically optimised cuts through the lens with a femtosecond-laser. Different protein agglomerations are responsible for the flexibility reduction of the lens. Two-photon excited fluorescence of the lens can be used as a diagnostic tool to localise such protein accumulations. In in-vitro experiments with human cataract lenses and also lenses of the Philly-mouse it could be demonstrated that with age the fluorescence increases as presbyopia proceeds. The distribution of the fluorescing compounds are not homogeneous but rather cloudy. Discrimination of the compounds by fluorescence lifetime measurements in relation of the depth in the lens is possible. PACS: Be INTRODUCTION Humans suffer from presbyopia with increasing age due to a loss of accommodation of the lens. This is caused by metabolism of the proteins in the lens and oxidative stress. Most of these proteins fluoresce and cannot be degraded in the lens. Therefore the concentration of the fluorophores are a measure of the life span index of the metabolism and the oxidative stress. The fluorescence of the human lens increases with the enrichment of advanced glycation end-products (AG- Es) produced by non enzymatic glycolysis of proteins in the lens [1]. Fluorescence is therefore not only an indicator of presbyopia but its intensity will be raised significantly in patients with diabetes or in patients suffering from ischemic heart diseases. The biophysical consequence of the change of the proteins and fibres [3, 5] is a hardening of the lens, increase of viscosity of up to 5% and loss of accommodation up to 9%. Endogen fluorophores (α, β and γ) in the crystalline lens are tryptophan (λ em = 329 nm) hydroxykynurenine glucoside (λ em = 55 nm) and other fluorophores except tryptophan (λ em = nm) which are not present in the lenses of newborns. Protein aggregation increases with age of the lens and also the water insoluble part of the proteins increases until 4% as a result of photooxidative processes. When diseases like diabetes or the development of cataract is superimposed to presbyopia then the fluorescence of the lens increases due to glycolysis of collagen. The aim of this study is to demonstrate the localisation of the fluorescence [2] in the lens and the fluorescence increase with age as a measure of presbyopia. By fluorescence lifetime imaging it is possible to differentiate between fluorophores with similar emission spectra but which differ in structural condition or chemical environment. MATERIAL AND METHODS Lenses For experimental reasons extracted eye lenses from pigs and mice have been used within a short time after they have been killed. The lenses were kept in a buffer solution and placed in a flat open cuvette for microscopy. Thin slices of lens tissue have been prepared after freezing with a microtome. Philly-mouse lenses, a strain with inbred cataract and abnormal βb2-cristallin, were obtained from GSF-National Research Center for Environment & Health, Institute of Developmental Genetics (Prof. Dr. Jochen Graw). Philly-mice develop cataracts with age. Therefore it is possible to study the fluorescence increase with aging until months. Human lenses have been provided after star operations from an eye-centre in Africa and from the HE- LIOS clinic in Erfurt, Germany, by Priv.-Doz. Dr. med. Marcus Blum. These lenses are opaque and stiff. The fluorescence excitation by two photons with 1 fs laser pulses was possible only superficially down to 1 µm depth. Microscopy Laser scanning microscopes (LSM) were used to get 2D and 3D fluorescence images of the lenses. For single photon fluorescence excitation a Zeiss LSM 51META (objectives 63 and 2 ) has been used. For two photon fluorescence excitation a Zeiss LSM

2 496 STEINER и др. Intensity λ cm, nm Fig. 1. Fluorescence spectra of a cut human cataract lens. The positions where the spectra are measured are marked by a cross in the picture. The fluorescence intensity of the nucleus is higher than of the surrounding cortex. Z-plane Z-plane Fig. 2. Scanning into the depth of the cataract lens by LSM51META (Carl Zeiss). Stack of layers each µm thick and 6 µm apart. The fluorescence intensity is expressed in false colour. 5 was equipped with a fs-laser (Spectra Physics, Tsunami 1 fs, 2 MHz endowed with spitfire and OPAC). Fluorescence detection occurred by single photon counting (Becker & Hickl, parallel 16 channel spectrometer, PML-Spec, and time correlation spectroscopy for fluorescence lifetime images, PML-16-C). OCT Optical coherence tomography images of structural changes in the lenses were taken with the instrument from ISIS optronics GmbH, SkinDex3. RESULTS Spectra Fluorescence spectra of a human cataract lens are shown in Fig. 1. The spectra were taken with the Carl Zeiss LSM51META. The cut through the lens demonstrate the strong cloudiness which is caused by precipitation and agglomeration of protein structures. Within the nucleus of the lens (green cross and green curve) the fluorescence intensity, excited with 45 nm, is increased. It shows three peaks at 5, 52, and 55 nm. The two peaks at 5 and 52 nm correspond to non-tryptophan fluorescence and should increase with age. The intensity in the nucleus is slightly higher than in the cortex. To image the fluorescence of the lens in three dimensions, a stack of 13 layers in the z-direction has been measured. Each layer had a thickness of µm ОПТИКА И СПЕКТРОСКОПИЯ том

3 TWO-PHOTON EXCITED FLUORESCENCE OF THE LENS 497 Relative fluorescence, ( nm)/55 nm kat-527 nm 16 kat-514 nm kat-51 nm kat-4 nm kat-475 nm 4 Intensity, a.u. Fluorescence spectra/age of mouse lens 7 3Mo 4Mo 6 5Mo 6Mo 7Mo 5 Mo 1Mo 4 Mo 14Mo λ, nm 6 Age of mouse lens, month 1 Fig. 3. Increase of the fluorescence of mouse lenses with age (Philly-mouse). The plot shows the relations of fluorescence intensities ( nm) after two-photon excitation to that of 55 nm. The inset are the corresponding spectra. Y-coordinates X-coordinates Z-coordinates Fig. 4. Demonstration of the three dimensional distribution of the fluorescence intensity. It shows a cloudy behaviour. and was 6 µm apart from the next layer in z-direction. Figure 2 represents in false colours the fluorescence intensity in the measured layers. This was performed still by single photon excitation with the LSM51META. As it is nearly impossible to experimentally study the fluorescence of the lens excited by two photons with fs-laser pulses live in human eyes with age, an appropriate model was needed. Here, the Philly-mouse seems to be the right animal which develops a cataract during the life-span. Therefore, one can follow the fluorescence intensity with age, preparing the lenses starting at three months up to months. According to the literature [2] the non-tryptophan fluorescence ( nm) should increase with age and can be an indication of developing presbyopia. Calibration of the measurements were done by calculating the relation between the fluorescence of the detector channels 1 ОПТИКА И СПЕКТРОСКОПИЯ том

4 49 STEINER и др. Fig. 5. OCT image of the mouse lens with lines marking the depth where the lifetimes are measured in Fig. 6. The visible structures are similar to those measured by fluorescence. Also three dimensional plots of the fluorescence of the mouse lens can be generated showing the inhomogeneous distribution of the fluorescence. This represents the cloudy distribution of the proteins leading to an increased stiffness of the lens. Therapeutically, it is planned to cut with a high repetitive fs-laser planes and figures into the lens to make it more flexible and prevent the eye from presbyopia at least for several years. Figure 4 shows the three-dimensional distribution of the fluorescence intensity. When it originates from protein accumulations in the lens then these structures should be seen also in OCT-pictures. Optical coherence tomography (OCT) is an ideal method to image structures which have different refractive indices and therefore reflecting/scattering back photons within the biological tissue. Such an OCT-picture of a mouse lens demonstrates Fig. 5. Clearly one can see the cloudy distribution of the scattering structures, similar to the fluorescence images. Also the nucleus of the lens is well separated from the cortex. The lines in the picture mark the depths where fluorescence lifetime has been measured. corresponding to 475 nm until 527 to 55 nm which is always present, in young as well as in old lenses. The plot of this intensity relation is given in Fig. 3. After an age of months the fluorescence intensity of the mouse lens is steadily increasing for all channels corresponding to 475 nm up to 527 nm, relative to the 55 nm fluorescence. Fluorescence lifetime Measurement of the fluorescence lifetime of different compounds allows to distinguish between them, even if the spectral information is the same. Different chemical surrounding, attachment to other structures or agglutination of molecules may be the cause for fluorescence lifetime changes. In the lens it is supposed Fig. 6. Fluorescence lifetime measurements at different depths of the mouse lens (25, 115, and 225 µm). Experiments were performed with a Becker&Hickl detector and imaged by Becker&Hickl software. ОПТИКА И СПЕКТРОСКОПИЯ том

5 TWO-PHOTON EXCITED FLUORESCENCE OF THE LENS 499 that in the nucleus protein agglomeration starts resulting in more dense structures than in the cortex. If so, then differences in the fluorescence lifetime should occur. With the Becker&Hickl setup and the evaluation software, such lifetime measurements of mouse lenses have been performed. Figure 6 gives examples of lifetime measurements of three different depths (25, 115, and 225 µm) in the lens of a months old Phillymouse. The pictures represent the lifetimes in ps until 6 ps in false colours. Superficially, the lifetime is rather short with a maximum marked by a cross at 14 ps. Lifetime in deeper layers starts to spread and much longer lifetimes occur. At 225 µm depth, a second well separated peak arises with a lifetime of 2727 ps. The whole spectrum fills the range until 6 ps. The total number of measured photons in the lifetime plots is nearly the same, but because of the larger distribution the photon counts per channel become smaller with depth in the lens. Whether this is a physical effect or the cross-linking of the fluorescing compounds might change with depth of the lens is not quite clear. This has to be investigated more intensively. DISCUSSION The advantage to use femtosecond-lasers in the near infrared wavelength region to excite fluorophores by two photons is the possibility to penetrate deeper into scattering biological tissue. But intense fs-laser pulses can also produce second harmonic generation (SHG) which might mix up with the fluorescence emission [4]. To discriminate between these two phenomena, lifetime measurements have been performed which demonstrate that the structures of the eye lens does not produce SHG, at least not measurable with the setup used by single photon counting. Presbyopia is a common phenomenon with age leading to a reduction of the flexibility of the human eye lens. It is accompanied by increasing protein content and protein agglomeration in the lens. Therefore, it should be possible to follow the process of presbyopia by measuring the increase of fluorescence intensity with age. This has been shown on an integral scale [1] and we could demonstrate the localisation and distribution of the fluorescence over the lens by two-photon excitation. This diagnostic information could be used for therapeutic treatment of the lens with fs-lasers, a research being performed by our project partners (Institute of Applied Physics, University of Jena). Studying the fluorescence increase in human lenses in vivo is not yet possible, therefore, a mouse model, the Phillymouse, was selected. It is a strain which develops inbred cataract with age. With this model we were able to demonstrate the increase of the fluorescence up to twelve months. The fluorescence distribution is very cloudy which has also been proved by OCT imaging of the same lenses. Fluorescence lifetime measurements revealed that lifetime increases with depth in the lens. Even separate peaks appear. Fluorescence lifetime depends on the chemical surrounding of the chromophore or the attachment to other structures or is due to agglutination. The origin of the lifetime spread is not yet clear und must be further evaluated. ACKNOWLEDGMENTS The assistance of Andrea Böhmler is gratefully acknowledged. This project was supported by the German Federal Ministry of Education and Research (BMBF), grant 13N32. REFERENCES 1. L. Kessel, Lens Fluorescence as a Marker of Aging in Ralation to Heritability, Diabetes Mellitus, and Ischaemic Heart Disease. PhD-Thesis, University of Copenhagen, L. Rovati, F. Docchio, J. Biomed. Optics 9 (1), 9 (24). 3. J. R. Kuszak, R. K. Zoltoski, and C. E. Tiedemann, Int. J. Dev. Biol., 4, 9 (24). 4. J. Eichler, B.-M. Kim, Proceedings Int. Conf. Lasers 21 (USA). 5. S. J. McGinty, R. J. W. Truscott, Ophthalmic Research, 3, 137 (26). ОПТИКА И СПЕКТРОСКОПИЯ том *