October 2010
Volume 51, Issue 10
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Physiology and Pharmacology  |   October 2010
Two-Photon Fluorescence Excitation Microscopy to Assess Transscleral Diffusional Pathways in an Isolated Perfused Bovine Eye Model
Author Affiliations & Notes
  • Wee Kuan Kek
    From the Singapore Eye Research Institute, Singapore;
    the Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, Scotland, United Kingdom;
  • Wallace S. Foulds
    From the Singapore Eye Research Institute, Singapore;
  • Gail McConnell
    the Centre of Biophotonics and
  • Amanda J. Wright
    the Institute of Photonics, SUPA (Scottish Universities Physics Alliance), University of Strathclyde, Glasgow, Scotland, United Kingdom; and
  • John M. Girkin
    the Centre for Advanced Instrumentation, Department of Physics, University of Durham, Durham, United Kingdom.
  • Clive G. Wilson
    the Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, Scotland, United Kingdom;
  • Corresponding author: Wee Kuan Kek, Abbott Laboratories, Global Formulation Sciences, Department R4P7, Building AP31-4-264, 200 Abbott Park Road, Abbott Park, IL 60064-6199; wee.kek@abbott.com
Investigative Ophthalmology & Visual Science October 2010, Vol.51, 5182-5189. doi:10.1167/iovs.09-3854
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      Wee Kuan Kek, Wallace S. Foulds, Gail McConnell, Amanda J. Wright, John M. Girkin, Clive G. Wilson; Two-Photon Fluorescence Excitation Microscopy to Assess Transscleral Diffusional Pathways in an Isolated Perfused Bovine Eye Model. Invest. Ophthalmol. Vis. Sci. 2010;51(10):5182-5189. doi: 10.1167/iovs.09-3854.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).

Methods.: Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.

Results.: TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.

Conclusions.: TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.

Topical administration is by far the most common route chosen to treat the eye with therapeutic agents, and the efficacy of delivery is restricted by several factors. A significant proportion of the topically applied drug is lost through the nasolacrimal duct or through the conjunctiva, whereas the penetration of drugs across the cornea is governed by the physicochemical properties of the active moiety and a corneal barrier that has both hydrophilic and hydrophobic characteristics. Restricted diffusion between the anterior and posterior segments of the eye also prevents adequate therapeutic drug concentrations from being attained in the posterior segment of the eye (retina, choroid, and vitreous), when drugs are applied topically to the surface of the eye. Intravitreal injection of a drug depot or an intravitreal drug delivery device carries a small risk of intraocular hemorrhage and damage to the retina and rarely, intraocular infection. 
Sustained transscleral drug delivery to the posterior segment of the eye has received some attention, primarily because it may avoid many of the disadvantages associated with direct intravitreal delivery. 15 Subconjunctival administration may fail to achieve sustained retinal delivery of therapeutic drugs, mainly because of removal by blood vessels and lymphatics of the conjunctiva. 6,7 The sclera provides a potentially useful route for delivery of drugs to the vitreoretinal tissues 8,9 ; however, an understanding of the route by which drugs travel through the sclera is necessary if this drug delivery route is to be fully exploited. 
The sclera consists of three layers: episclera, stroma, and lamina fusca. All three layers are composed of loosely arranged collagen fibrils within a proteoglycan matrix. This loose arrangement of collagen fibers allows easy diffusion across the sclera. 4 The scleral stroma is fibrous with a high water content and, as a consequence, the sclera is highly permeable for hydrophilic drugs. 10 The classic experiments by Ahmed and Patton 1 showed that the intact sclera provides much less resistance to the entry of hydrophilic drugs than does the cornea, whereas there is little difference between the corneal and scleral penetration of hydrophobic drugs, such as timolol, propranolol, or penbutolol. Schoenwald et al. 11 noted that certain drugs, when applied topically to the eye, may be preferentially absorbed through the sclera and enter the iris-ciliary body without first entering the aqueous humor. These data suggest that the scleral pathway is a potential route for drug delivery and is worthy of investigation. 
We report here the results of studies undertaken to determine the feasibility of using two-photon fluorescence excitation microscopy to study the lateral diffusion and penetrance into the sclera of a test drug, tazarotenic acid (TA), a vitamin A analogue. 
Materials and Methods
In summary, polyvinyl alcohol-tazarotenic acid (PVA-TA) films were applied to the surface of the sclera of perfused bovine eyes, and a neighboring area of sclera (5 or 25 mm from the edge of the PVA-TA film was examined by two-photon microscopy (TPM) to determine the time course of the appearance of the test drug in the superficial layers of the sclera and its subsequent diffusion into the deeper scleral layers as indicated by the intensity of fluorescence stimulated by two-photon absorption by the drug. PVA films were used for delivery of TA because they form a gel after the addition of water, reducing lateral diffusion on the surface. 12  
Preparation of PVA Film
PVA-TA films were prepared by using a solvent casting technique. Granules of PVA containing 87% to 89% hydrolyzed PVA with an average molecular weight of 85,000 to 146,000 daltons (Sigma-Aldrich, Paisley, UK) were added to distilled water (15% wt/wt) that was heated to ∼90°C with constant stirring until all granules were dissolved. The PVA solution was then cooled to 60°C to increase its viscosity. TA (1% wt/wt) was added under constant stirring to maintain a uniform dispersion of the drug. 
The stirring was stopped when a satisfactory dispersion was achieved as judged by a uniform color of the mixture and homogeneity of the dispersed particles. This mixture was then sonicated for 30 minutes. 
The PVA-TA mixture were left overnight to allow complete de-aeration to prevent the formation of air bubbles in the cast films. The mixture was then cast onto a Perspex plate with a hand spreader. The films were left to dry in the dark at room temperature for 2 days until the dried film readily peeled off the Perspex plate. The films were then stored in the dark, in a cool room in an amber desiccator for future use. With a hole punch, 1% wt/wt PVA-TA films were produced with dimensions of 6.5-mm diameter and 250-μm thickness. 
Two-Photon Microscopy
In TPM an infrared (IR) femtosecond-pulsed laser is used to excite fluorescence at visible wavelengths. 12 The IR laser provides light at approximately twice the single-photon absorption wavelength and excites dye molecules via a two-photon excitation process. This process is a nonlinear one; only at the focal plane is the peak intensity sufficiently high to instigate simultaneous absorption of two-photons, leading to subsequent fluorescent emission. Thus, the method has an inherent optical sectioning capability. An optical section can be obtained by scanning the focused spot in the x–y plane. With its inherent three-dimensional capability TPM can image living tissue with high resolution. 13 Furthermore, this technique is able to image living tissues for longer periods than with the ultraviolet radiation used in conventional confocal microscopy, 13 as infrared radiation reduces phototoxicity and photobleaching that may occur with ultraviolet radiation. The TPM technique can thus be regarded as minimally invasive to living tissues. 14,15  
The TPM system consisted of a (multiphoton) scanning system (1024 MP; Bio-Rad, Hemel Hempstead, UK) coupled to the upright, fixed stage of a microscope (Eclipse E600FN; Nikon Tokyo, Japan). A mode-locked femtosecond Ti:Sapphire laser (Coherent Mira, Palo Alto, CA) which was tuneable from 700 to 920 nm was set to a wavelength of 745 nm as the excitation source. This was approximately twice the single-photon absorption wavelength of TA at 338 nm. The peak emission wavelengths of TA with single-photon absorption are 450 and 678 nm. The peak emission with two-photon absorption, however, is broad, 12 so that the use of a photomultiplier tube (PMT) as a detector, together with a high-quality optical band pass filter 525 ± 25 nm (Chroma, Rockingham, VT) (midway between the peak emission wavelengths of single-photon excitation) not only provides an adequate signal from the fluorescing TA, but allows the rejection of stray excitation radiation that would contribute to noise. Images were obtained with a 60×/1.0-numerical-aperture, water-immersion lens with a working distance of 2.0 mm and were captured with image-acquisition software (LaserSharp, ver. 4; Bio-Rad Ltd.). 
Calibration Study for Sclera
To determine the fluorescence yield at different drug concentrations, we used four concentrations of TA in PBS solution and a control solution of PBS without TA. Samples of sclera 10 × 10 mm and approximately 1 mm thick were cut from bovine eyes. The samples were placed in test tubes, and 5 mL of each concentration of TA (0, 3.125, 6.25, 12.5, and 25 μg/mL) was added to quadruplicate samples that were then incubated in the various concentrations of TA or PBS alone for 24 hours. The incubated samples were then imaged by TPM using 20%, 40%, and 100% laser power. Image analysis software (LaserPix software; Bio-Rad, Ltd.) was used to measure the average intensity of each image 10 μm below the surface where the images showed uniform fluorescence. 
Scleral Binding
Forty samples of sclera with weights in the range of 0.1 to 1 g from fresh bovine eyes obtained from a local abattoir were used. The samples distributed within the weight range were each immersed for 24 hours in 5 mL of a TA solution of 70, 7.0 or 0.7 μg/mL, or a phosphate-buffered solution as a control. After immersion for 24 hours, 1 mL of the supernatant was withdrawn and centrifuged for 10 minutes at 5000 rpm and then transferred into HPLC vials and analyzed by HPLC. 16  
Protein and Collagen Binding
TA solutions in six concentrations ranging from 0.01 to 100 μg/mL were prepared, and each concentration was verified with HPLC. Protein and collagen binding were determined by using plasma protein (Blood Transfusion Centre, Gartnavel General Hospital, Glasgow) and collagen (Sigma-Aldrich) in similar concentrations of approximately 72 mg/mL for each so that their relative affinities for TA could be compared. 
Two hundred microliters of plasma were transferred to a tube (Eppendorf, Fremont, CA) equilibrated in a water bath for 5 minutes at 37°C. One milliliter of TA solution (of various concentrations) was added to the tube and shaken vigorously for 30 seconds before incubation for 5 minutes. This mixture was then centrifuged for 5 minutes and 400 μL transferred into a centrifugal filter unit (Ultra-free tube; Millipore, Billerica, MA; molecular weight cutoff point, 5000 daltons) and further centrifuged for 40 min at 10,000 rpm. The ultrafiltrate was then transferred into HPLC vials for analysis. The procedure was repeated for various concentrations of TA in the collagen suspension. 
Arterially Perfused Bovine Eye for TPM
Bovine eyes were obtained from the local abattoir and were used within 1 to 2 hours of death. After the eyes were enucleated, excess adnexal tissue was trimmed away, and a long posterior ciliary artery was cleared for cannulation. The tip of an extruded plastic cannula was inserted into the artery and secured with a circumferentially placed surgical suture before perfusion commenced. Heat exchangers and jacket-heated beakers were used to maintain the perfusate and the perfused eye at a constant temperature of ∼37°C, as previously described. 16,17 A flow rate of 0.2 mL/min was used initially and slowly increased to 1.0 mL/min over 20 minutes. Arterial perfusion pressure was maintained at no more that 50 mm Hg, with a drift of no more than 2 mm Hg/h. Eyes were rejected if there was failure to achieve a stable intraocular pressure. 
Once the arterially perfused bovine eye was prepared, it was placed under the TPM (Fig. 1) and carefully positioned to enable TPM measurements to be made, with the TPM centered 10 mm posterior to the corneoscleral limbus between neighboring extraocular muscles. As a control, an XYZ series of scans was made without the presence of a PVA film. 
Figure 1.
 
Perfusion setup for transscleral delivery study.
Figure 1.
 
Perfusion setup for transscleral delivery study.
A PVA-TA film was then placed on the sclera near the equator with its edge at either 5 or 25 mm from the optical axis of the objective lens. TPM images of an x-y-z-series were taken at selected time intervals based on initial trial experiments. Images were then analyzed (LaserPix software; Bio-Rad, Ltd.). 
Results
Scleral Binding
The results of scleral binding of TA (Fig. 2A) showed that, at concentrations of 7 or 70 μg/mL, sclera binding of TA was similar and on the order of 20%. Even at the lowest concentration of TA (0.7 μg/mL), binding to the sclera was no greater than 70%, indicating that TA has a relatively weak binding affinity to the sclera. This finding was supported by the observation (Fig. 2B) that binding of TA to plasma protein was stronger than to collagen. More than 95% of TA was bound to plasma protein, whereas less than 55% was bound to collagen in TA concentrations ranging from 0.01 to 100 μg/mL. 
Figure 2.
 
(A) Unbound TA concentrations as a percentage of control values, with a range of concentrations of TA in which samples of sclera of various weights were immersed for 24 hours. Each point represents the result of an individual sample of sclera. Not unexpectedly, scleral binding increased with increasing sample weight. For most weights of sclera, the values of unbound TA were identical for 7 and 70 μg/mL TA, so that the graphs for each are largely coincident. (B) Binding affinities for TA to plasma and collagen. Binding to plasma was high, with <10% of unbound TA present at any concentration of TA tested. Binding to collagen was much less, with some 50% to 60% of TA remaining unbound. Each point represents the mean of two determinations.
Figure 2.
 
(A) Unbound TA concentrations as a percentage of control values, with a range of concentrations of TA in which samples of sclera of various weights were immersed for 24 hours. Each point represents the result of an individual sample of sclera. Not unexpectedly, scleral binding increased with increasing sample weight. For most weights of sclera, the values of unbound TA were identical for 7 and 70 μg/mL TA, so that the graphs for each are largely coincident. (B) Binding affinities for TA to plasma and collagen. Binding to plasma was high, with <10% of unbound TA present at any concentration of TA tested. Binding to collagen was much less, with some 50% to 60% of TA remaining unbound. Each point represents the mean of two determinations.
Fluorescence Intensity Calibration Study of Impregnated Sclera
For each of the laser intensities used, an initial image was taken to ensure that there were no saturated pixels in the image, so as to allow the full dynamic range of the 8-bit digitization to be used. Care was also taken to ensure that the black level of the system was correctly set according to the Bio-Rad-prescribed method. As expected (Fig. 3, Table 1), the fluorescence yield rose with increasing drug concentration and increasing laser power, but with similar slopes for each laser power used. The similar responses at each laser power indicated that there was little photobleaching of the fluorophore. At even the highest powers and lowest concentrations, the excitation rate of the fluorophores was not exceeded. 
Figure 3.
 
Fluorescence intensities of TPA images of bovine sclera after immersion in differing TA concentrations using 20%, 40%, or 100% laser power. As expected, there was increasing intensity of fluorescence with increasing concentrations of TA (R 2 = 0.9684, 0.961, and 0.9884, respectively, for increasing laser power). Each point represents the mean of four measurements. The actual numerical results are detailed in Table 1.
Figure 3.
 
Fluorescence intensities of TPA images of bovine sclera after immersion in differing TA concentrations using 20%, 40%, or 100% laser power. As expected, there was increasing intensity of fluorescence with increasing concentrations of TA (R 2 = 0.9684, 0.961, and 0.9884, respectively, for increasing laser power). Each point represents the mean of four measurements. The actual numerical results are detailed in Table 1.
Table 1.
 
Mean TPM Fluorescence Intensities and SDs Imaged at a Depth of 10 μm in Scleral Samples Incubated in Different Concentrations of TA in PBS
Table 1.
 
Mean TPM Fluorescence Intensities and SDs Imaged at a Depth of 10 μm in Scleral Samples Incubated in Different Concentrations of TA in PBS
Concentration TA (μg/mL) 20% Laser 40% Laser 100% Laser
Mean Intensity SD Mean Intensity SD Mean Intensity SD
0.000 43.67 0.76 48.67 1.26 64.33 2.89
3.125 69.33 14.74 97.33 20.53 94.00 35.21
6.25 113.75 24.14 146 33.98 114.50 13.70
12.5 119 42.03 134 16.49 116.50 20.42
25 177 60.23 185 77.78 160.00 56.40
TPM of TA in Sclera of Isolated Perfused Bovine Eyes
In these experiments two-photon–induced fluorescence was recorded at different depths in the sclera at various time intervals after application of a PVA-TA film placed either 5 or 25 mm away from the area of sclera being imaged by TPM. In the former case 40% laser power was used and in the latter 100%. 
In control experiments, no fluorescence was detected at any time interval or scleral depth from 10 to 210 μm. After application of the drug PVA-TA 5 mm from the imaged area of sclera, induced fluorescence of collagen bundles was detected from 15 minutes onward (Fig. 4), with a peak intensity at 50 minutes. At time intervals of 15–100 minutes induced fluorescence was detected at increasingly greater depths in the sclera at 15 minutes, being at its maximum at 130 μm, increasing after 100 minutes to a maximum at 300 μm (Figs. 5, 6, 7). The images obtained indicate that the drug diffused both laterally and deeper into the scleral tissues over time. At image depths between 250 and 340 μm, an increase in visible collagen fiber bundles accompanied an increase in fluorescence. 
Figure 4.
 
Two-photon images of sclera selected from a z-series obtained 15 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 90- to 130-μm depth. (The thickness of the bovine sclera is approximately 1 mm at the locations illustrated in Figs. 4 56).
Figure 4.
 
Two-photon images of sclera selected from a z-series obtained 15 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 90- to 130-μm depth. (The thickness of the bovine sclera is approximately 1 mm at the locations illustrated in Figs. 4 56).
Figure 5.
 
Two-photon images selected from a z-series obtained 50 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 210- to 250-μm depth and appeared to be associated with scleral collagen bundles.
Figure 5.
 
Two-photon images selected from a z-series obtained 50 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 210- to 250-μm depth and appeared to be associated with scleral collagen bundles.
Figure 6.
 
Two-photon images selected from a z-series obtained 85 minutes after application of drug 5 mm from imaged area. Maximum fluorescence was at a 250- to 290-μm depth.
Figure 6.
 
Two-photon images selected from a z-series obtained 85 minutes after application of drug 5 mm from imaged area. Maximum fluorescence was at a 250- to 290-μm depth.
Figure 7.
 
Diffusion of drug into the sclera when a PVA-TA film was placed 5 mm away from the area imaged by TPM. Maximum fluorescence intensity (i.e., concentration of TA) occurred at a depth of 180 μm at 50 minutes after application of TA. At increasing times up to 100 minutes, fluorescence occurred at increasing depths up to 350 μm but with decreased intensity, probably due to a combination of decreasing concentration of TA and physical factors related to TPM imaging at depth within convex tissue.
Figure 7.
 
Diffusion of drug into the sclera when a PVA-TA film was placed 5 mm away from the area imaged by TPM. Maximum fluorescence intensity (i.e., concentration of TA) occurred at a depth of 180 μm at 50 minutes after application of TA. At increasing times up to 100 minutes, fluorescence occurred at increasing depths up to 350 μm but with decreased intensity, probably due to a combination of decreasing concentration of TA and physical factors related to TPM imaging at depth within convex tissue.
When the PVA-TA film was placed 25 mm rather than 5 mm away from the TPM observation area, initial fluorescence from the tracer drug was detected after 45 minutes. The intensity of induced fluorescence at different depths in the sclera over periods of 45 to 200 minutes showed that fluorescence peaked at a 110 μm depth at 65 minutes and thereafter was distributed fairly uniformly throughout the depth of the sclera (Fig. 8). This pattern no doubt reflected the distance between the PVA-TA film and the measurement site and the combined effects of lateral diffusion and vertical diffusion through the scleral thickness. 
Figure 8.
 
Diffusion of drug with TA explants 25 mm from areas imaged by TPM. The pattern is somewhat similar to that in Figure 7 but the depth of penetration of TA achieved and the intensity of fluorescence were both less than when the explants were closer to the areas imaged. Loss of TA from the imaged areas as a result of lateral diffusion within the sclera is a likely explanation.
Figure 8.
 
Diffusion of drug with TA explants 25 mm from areas imaged by TPM. The pattern is somewhat similar to that in Figure 7 but the depth of penetration of TA achieved and the intensity of fluorescence were both less than when the explants were closer to the areas imaged. Loss of TA from the imaged areas as a result of lateral diffusion within the sclera is a likely explanation.
Discussion
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. 
Footnotes
 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.
Footnotes
 Disclosure: W.K. Kek, None; W.S. Foulds, None; G. McConnell, None; A.J. Wright, None; J.M. Girkin, None; C.G. Wilson, None
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Figure 1.
 
Perfusion setup for transscleral delivery study.
Figure 1.
 
Perfusion setup for transscleral delivery study.
Figure 2.
 
(A) Unbound TA concentrations as a percentage of control values, with a range of concentrations of TA in which samples of sclera of various weights were immersed for 24 hours. Each point represents the result of an individual sample of sclera. Not unexpectedly, scleral binding increased with increasing sample weight. For most weights of sclera, the values of unbound TA were identical for 7 and 70 μg/mL TA, so that the graphs for each are largely coincident. (B) Binding affinities for TA to plasma and collagen. Binding to plasma was high, with <10% of unbound TA present at any concentration of TA tested. Binding to collagen was much less, with some 50% to 60% of TA remaining unbound. Each point represents the mean of two determinations.
Figure 2.
 
(A) Unbound TA concentrations as a percentage of control values, with a range of concentrations of TA in which samples of sclera of various weights were immersed for 24 hours. Each point represents the result of an individual sample of sclera. Not unexpectedly, scleral binding increased with increasing sample weight. For most weights of sclera, the values of unbound TA were identical for 7 and 70 μg/mL TA, so that the graphs for each are largely coincident. (B) Binding affinities for TA to plasma and collagen. Binding to plasma was high, with <10% of unbound TA present at any concentration of TA tested. Binding to collagen was much less, with some 50% to 60% of TA remaining unbound. Each point represents the mean of two determinations.
Figure 3.
 
Fluorescence intensities of TPA images of bovine sclera after immersion in differing TA concentrations using 20%, 40%, or 100% laser power. As expected, there was increasing intensity of fluorescence with increasing concentrations of TA (R 2 = 0.9684, 0.961, and 0.9884, respectively, for increasing laser power). Each point represents the mean of four measurements. The actual numerical results are detailed in Table 1.
Figure 3.
 
Fluorescence intensities of TPA images of bovine sclera after immersion in differing TA concentrations using 20%, 40%, or 100% laser power. As expected, there was increasing intensity of fluorescence with increasing concentrations of TA (R 2 = 0.9684, 0.961, and 0.9884, respectively, for increasing laser power). Each point represents the mean of four measurements. The actual numerical results are detailed in Table 1.
Figure 4.
 
Two-photon images of sclera selected from a z-series obtained 15 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 90- to 130-μm depth. (The thickness of the bovine sclera is approximately 1 mm at the locations illustrated in Figs. 4 56).
Figure 4.
 
Two-photon images of sclera selected from a z-series obtained 15 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 90- to 130-μm depth. (The thickness of the bovine sclera is approximately 1 mm at the locations illustrated in Figs. 4 56).
Figure 5.
 
Two-photon images selected from a z-series obtained 50 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 210- to 250-μm depth and appeared to be associated with scleral collagen bundles.
Figure 5.
 
Two-photon images selected from a z-series obtained 50 minutes after application of drug 5 mm from the imaged area. Maximum fluorescence was at a 210- to 250-μm depth and appeared to be associated with scleral collagen bundles.
Figure 6.
 
Two-photon images selected from a z-series obtained 85 minutes after application of drug 5 mm from imaged area. Maximum fluorescence was at a 250- to 290-μm depth.
Figure 6.
 
Two-photon images selected from a z-series obtained 85 minutes after application of drug 5 mm from imaged area. Maximum fluorescence was at a 250- to 290-μm depth.
Figure 7.
 
Diffusion of drug into the sclera when a PVA-TA film was placed 5 mm away from the area imaged by TPM. Maximum fluorescence intensity (i.e., concentration of TA) occurred at a depth of 180 μm at 50 minutes after application of TA. At increasing times up to 100 minutes, fluorescence occurred at increasing depths up to 350 μm but with decreased intensity, probably due to a combination of decreasing concentration of TA and physical factors related to TPM imaging at depth within convex tissue.
Figure 7.
 
Diffusion of drug into the sclera when a PVA-TA film was placed 5 mm away from the area imaged by TPM. Maximum fluorescence intensity (i.e., concentration of TA) occurred at a depth of 180 μm at 50 minutes after application of TA. At increasing times up to 100 minutes, fluorescence occurred at increasing depths up to 350 μm but with decreased intensity, probably due to a combination of decreasing concentration of TA and physical factors related to TPM imaging at depth within convex tissue.
Figure 8.
 
Diffusion of drug with TA explants 25 mm from areas imaged by TPM. The pattern is somewhat similar to that in Figure 7 but the depth of penetration of TA achieved and the intensity of fluorescence were both less than when the explants were closer to the areas imaged. Loss of TA from the imaged areas as a result of lateral diffusion within the sclera is a likely explanation.
Figure 8.
 
Diffusion of drug with TA explants 25 mm from areas imaged by TPM. The pattern is somewhat similar to that in Figure 7 but the depth of penetration of TA achieved and the intensity of fluorescence were both less than when the explants were closer to the areas imaged. Loss of TA from the imaged areas as a result of lateral diffusion within the sclera is a likely explanation.
Table 1.
 
Mean TPM Fluorescence Intensities and SDs Imaged at a Depth of 10 μm in Scleral Samples Incubated in Different Concentrations of TA in PBS
Table 1.
 
Mean TPM Fluorescence Intensities and SDs Imaged at a Depth of 10 μm in Scleral Samples Incubated in Different Concentrations of TA in PBS
Concentration TA (μg/mL) 20% Laser 40% Laser 100% Laser
Mean Intensity SD Mean Intensity SD Mean Intensity SD
0.000 43.67 0.76 48.67 1.26 64.33 2.89
3.125 69.33 14.74 97.33 20.53 94.00 35.21
6.25 113.75 24.14 146 33.98 114.50 13.70
12.5 119 42.03 134 16.49 116.50 20.42
25 177 60.23 185 77.78 160.00 56.40
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