For imaging at depth, TPM has advantages over conventional confocal microscopy, as the use of long-IR wavelengths for TPM reduces the effect of Rayleigh scattering in tissue media.
18 Therefore, with TPM, images can be obtained routinely at depths in excess of 250 μm,
19,20 compared with confocal microscopy, which typically loses contrast at depths of ∼75 μm.
21 TPM imaging in the neocortex is capable of penetration depths greater than 600 μm,
22 and it has been reported that imaging up to a 2 mm depth is possible in brain tissue by using gradient index lenses together with in vivo TPM.
23 It should be noted, however, that the imaging depth is dependent on whether tissue structures (such as thick cell body layers or surface blood vessels) contribute to optical aberrations.
24 TPM therefore allows imaging at greater tissue depths than does conventional confocal microscopy.
The results obtained in this study of TPM imaging of the diffusion of TA through the sclera of isolated perfused bovine eyes are qualitative rather than quantitative in terms of actual concentrations of TA at various scleral depths. The feasibility of detecting the diffusional movement of TA through the bovine sclera by TPM presents several problems that have to be overcome before exact quantification based on fluorescence intensity can be achieved.
The technique is limited to drugs that are inherently fluorescent, with an absorption maximum that is within the range of excitatory wavelengths available from the IR laser used. We chose to use TA for our experiments, because not only is this a drug that is under investigation for its potential use in ocular therapy, but because, fortuitously, it has the required fluorescence characteristics to allow its visualization by TPM. In the case of nonfluorescent drugs, the incorporation of a fluorescent marker may be needed to allow their tracking by TPM.
From calibration studies, we have demonstrated that fluorescence intensity in the isolated sclera can be related to the drug concentration present when imaged at a depth of 10 μm, but when imaging more deeply in the sclera, optical aberrations induced by the structure of the tissue alter the focal spot size within the sample.
25
In multiphoton microscopy, fluorescence intensity is a function of the square of the laser intensity so that a small increase in spot size leads to a large decrease in signal. As the spot size increases with greater depth due to sample-induced aberrations,
24 a correspondingly lower signal is detected for a given concentration. This variation was expected but validation studies are needed to compare TPM images against the actual concentration of TA at different depths in the sclera at specific time points after drug application to the scleral surface. As the sclera varies considerably in its composition (e.g., size and number of collagen bundles) in different areas and at various depths, there could also be increased variance in measurements made at depths in the sclera.
A further disadvantage in using TPM to obtain images at different depths in the sclera of the intact eye is that the images obtained are scanned under the curved surface of the preparation. Thus, the curved surface of the sclera acts as a lens, meaning that the excitation light travels different distances at different points within the optical section, thus altering the relative intensity of fluorescence across the section. With adaptive optics, however (i.e., flexible membrane mirror), in combination with TPM, it has been shown that imaging at depth with enhanced resolution is possible.
26,27 We thus believe that this article demonstrates that the technique of two-photon excitation and imaging within the eye for drug tracking is a viable method and that the complications that have been listed can be overcome in the longer term.
Thus, factors pertaining to the optical characteristics of the laser beam, structural differences in the sclera at different depths, and locations and the curvature of the eye wall all must be taken into account, and the technology must be refined if TPM is to be used in quantifying drug penetration into and across the sclera.
In our experiments, the release of TA from the PVA-TA films was primarily dependent on the rate of diffusion from the films rather than the rate of PVA dissolution. There was a measured linear relationship between drug release from the PVA films and the square root of elapsed time, indicating that diffusion was the predominant process of drug release. This conclusion was based on the Higuchi kinetic equation.
28
In the experiments reported herein, the induced fluorescence of TA over time at various depths in the sclera was imaged at some distance (5 or 25 mm) from the source of TA in the PVA-TA films. The intensity of the fluorescence was influenced by the various routes along which TA diffused from the PVA-TA film, some undoubtedly diffusing directly inward from the deep aspect of the PVA-TA film and some not only diffusing laterally in the superficial layers of the sclera but penetrating progressively into the deeper sclera layers at a rate dependent on the decreasing concentration of TA as it diffused laterally from the PVA-TA film. Thus, both lateral diffusion and diffusion more deeply into the sclera have to be considered when assessing the penetration into the sclera of a drug delivered to the scleral surface.
Diffusion through the sclera would obviously be affected by the nature of the scleral constituents in different locations in the tissue and by the route through which diffusion primarily occurred. Factors of relevance would include the density and spacing of sclera collagen bundles, the resistance to diffusion posed by the proteoglycan ground substance, the presence of perivascular tissue spaces, the effect of protein or collagen binding, and the thickness of the sclera. The sclera is mainly composed of collagenous and elastic tissues that vary substantially in shape and thickness.
29 These collagenous and elastic fibers have interdependent bonds. The elastic tissue is not present at birth, but increases with growth and then dies out on ageing. The collagenous fibers adopt a wavy configuration under normal conditions and straighten when the elastic fibers are stretched. The collagen fiber bands in the most anterior part of the sclera are arranged in a circular direction, building a firm platform for the attachment of the extraocular muscles.
30
Scanning electron microscopy (SEM) reveals clear differences in the organization of the collagen bundles at different depths in the sclera. On the surface, fine fibers are interwoven and form a dense reticulated mat, which, at greater depths, changes into a criss-crossing layer of fibers described as a rhombic lattice.
30 Measurements from SEM images show that, on the surface, the fibers have a diameter of 80 to 140 nm, whereas deeper in the tissue, the fiber bundles vary from 0.5 to 6 μm in diameter. These latter fibers scatter light strongly and may be responsible for the relative opacity of the scleral tissue. The thickness and distribution of collagen fibers within the sclera and the nature of the intervening ground substance affects the diffusion of drugs into and across the sclera.
In SEM cross-section, bovine sclera
31 shows an architecture that is remarkably similar to observations made using the two-photon technique. The TPM
x-y sections shown in
Figures 4 to
6, as expected, revealed limited information on collagen orientation. The fine surface fibrils evident on SEM
30 are not easily resolved by TPM, although examination of some the images taken during experiments in which the PVA-TA film was only 5 mm from the imaged area show the presence of fine fluorescent fibrils. These collagen fibers that are too fine to be visualized at the magnification used are visible only on TPM secondary to the fluorescence of the TA.
Maurice and Mishima
32 have defined three potential pathways for diffusion of drugs across the sclera: through perivascular tissue spaces, through the aqueous phase of the gel-like glycosaminoglycans between collagen bundles, or along scleral collagen fibrils.
Perivascular tissue spaces are scant in the sclera, being largely confined to the posterior sclera around the optic nerve head where posterior ciliary arteries traverse the posterior sclera to supply the choroid and optic nerve head and to the retroequatorial sclera where the sclera is traversed by the four vortex veins. As a result, there is a reduced potential for drugs to diffuse in any significant amount through scleral perivascular spaces.
As regards whether diffusion through the proteoglycans and glycosaminoglycans that fill the spaces between collagen bundles or through the collagen bundles themselves constitute routes by which drugs may diffuse across the sclera, the pattern of fluorescence seen on TPM of the sclera delineates the collagen bundles with no visible fluorescence between these bundles. The diffusion of TA into the sclera may be along the collagen bundles. In many of the images we obtained, entire bundles were uniformly fluorescent, and therefore the diffusion of TA may actually be within these bundles themselves.
We found that TA bound more avidly to plasma than to sclera or collagen. Binding to plasma proteins would undoubtedly affect the molecular size of bound TA and slow its diffusional movement through the sclera, although the relatively avascular nature of the sclera would make binding to plasma proteins less important than in the case of more vascularized tissues.
Although the sclera is highly permeable to many substances, the extent of drug loss into the systemic circulation by way of the vascular choroid would have to be considered in the development of transscleral drug delivery. Binding of administered drug to plasma protein in the choroidal blood or melanin in the choroid and retinal pigment epithelium also appears to be important in influencing drug deposition and elimination.
17 Ambati and Adamis,
2 however, showed that a target concentration of IgG antibody, administered transsclerally, was achievable in the choroid and retina without significant systemic absorption.
As regards the effects of scleral thickness on the diffusion of drugs across the sclera, the equatorial region, which is the thinnest,
33 would be the preferred location for transscleral drug delivery. The purpose of the present investigation was to determine whether two-photon excitation fluorescence microscopy would allow the real-time visualization of the passage of a test drug into and across the tissues of the bovine sclera. The results indicate that not only is this possible but fluorescence of the test drug TA could be imaged to a depth of around 350 μm, which is approximately the thickness of the average human equatorial sclera, so that TPM appears capable of imaging drug penetrance across almost all if not all the full thickness of the human equatorial sclera. This would certainly be possible in vitro and perhaps also in vivo.
Although TPM has provided an opportunity to image drug dispersion in the sclera, further improvement in the resolution and contrast of images at depth is necessary. Although TPM shows promise of being able to determine the real-time pattern of diffusion of therapeutic drugs across the sclera, validation of the method necessitates verification by alternative methods of determining drug concentration at various depths in the sclera. The use of a radioactive tag and autoradiography of histologic specimens would be one possibility, although numerous serial sections would be necessary to simulate the real-time information provided by TPM. In quantifying actual drug concentrations at different depths in the sclera, parallel frozen sections to correlate with the TPM images would be of value.
We have demonstrated the first real-time images of the diffusion of a tracer drug through excised and intact bovine sclera by using a two-photon technique. We have also shown that the disappearance of the drug from the superficial layers of the sclera and the movement of TA through the sclera can be followed at increasing depth at increasing time intervals after application of a PVA-TA film to the scleral surface. We have shown that TA appears to follow the fibrous structural pathway of the scleral collagen bundles, which are observed to be of different thicknesses. Although currently the technique of TPM shows promise in the assessment of the scleral penetrance of drugs designed for transscleral delivery to the posterior segment of the eye, the differences in the structure and thickness of the bovine sclera compared with those of the human eye indicate a need for the method to be assessed in the human eye or in an eye more closely resembling that of the human before any firm conclusions can be drawn in relation to its use in the assessment of therapeutic drugs designed for transscleral delivery to treat disorders affecting the posterior segment of the human eye.
Supported by grants from Allergan Inc., Irvine, CA, and the Medical Research Council of the United Kingdom. AJW received funding under the Basic Technology program from Research Councils, UK, and GMcC received a Fellowship from the Royal Society of Edinburgh.