May 2005
Volume 46, Issue 5
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Noncontact Optical Measurement of Lens Capsule Thickness in Human, Monkey, and Rabbit Postmortem Eyes
Author Affiliations
  • Noël M. Ziebarth
    From the Ophthalmic Biophysics Center and the
    Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida; the
  • Fabrice Manns
    From the Ophthalmic Biophysics Center and the
    Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida; the
  • Stephen R. Uhlhorn
    From the Ophthalmic Biophysics Center and the
    Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida; the
  • Anna S. Venkatraman
    Biostatistics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; the
  • Jean-Marie Parel
    From the Ophthalmic Biophysics Center and the
    Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida; the
    Department of Ophthalmology, University of Liege, Centre Hospitalier Universitaire Sart-Tillman, Liege, Belgium; and the
    University of Paris Hotel-Dieu Hospital, Paris, France.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1690-1697. doi:10.1167/iovs.05-0039
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      Noël M. Ziebarth, Fabrice Manns, Stephen R. Uhlhorn, Anna S. Venkatraman, Jean-Marie Parel; Noncontact Optical Measurement of Lens Capsule Thickness in Human, Monkey, and Rabbit Postmortem Eyes. Invest. Ophthalmol. Vis. Sci. 2005;46(5):1690-1697. doi: 10.1167/iovs.05-0039.

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

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Abstract

purpose. To measure interspecies thickness differences in the central anterior and posterior capsules of postmortem crystalline lenses, by a technique that maintains the anatomic integrity of the lens.

methods. Central capsule thickness was measured with a custom-built, noncontact optical system, using a focus detection technique. Anterior and posterior lens capsule thickness measurements were performed on 22 human, 29 monkey, and 34 New Zealand White rabbit intact postmortem lenses in situ. Eyes were prepared for optical measurements by bonding a PMMA ring to the sclera in the region of the ciliary body after the conjunctiva, adipose, and muscle tissues were removed. The posterior pole was removed by making a circumferential incision through the sclera approximately 7 mm posterior to the limbus. Excess vitreous was removed to expose the posterior capsule surface, and the eye assembly was placed on a Teflon slide. The cornea and iris were sectioned to expose the anterior capsule surface. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Lens capsule thickness was measured from the histologic slides and compared to the optical results.

results. Central anterior lens capsule thickness was 8.2 ± 5.5 (human), 7.5 ± 4.4 (monkey), and 10.7 ± 4.2 (rabbit) μm optically and 12.4 ± 2.5 (human), 10.7 ± 3.7 (monkey), and 10.4 ± 2.0 (rabbit) μm histologically. Central posterior capsule thickness was 6.3 ± 2.2 (human), 5.9 ± 1.7 (monkey), and 7.8 ± 2.3 (rabbit) μm optically and 4.1 ± 1.5 (human), 3.5 ± 1.6 (monkey), and 4.7 ± 2.5 (rabbit) μm histologically.

conclusions. The central anterior and posterior lens capsule thicknesses do not appear to vary considerably among human, rabbit, and monkey eyes. There were significant differences between optical in situ measurements and histology, which indicates that histologic preparation may affect lens capsule thickness.

The lens capsule is an acellular membrane that maintains the shape of the lens. It is thicker anteriorly, and the anterior and posterior portions become thicker toward the periphery. 1 Studies have shown that the thickness of the human anterior lens capsule increases until the middle of the eighth decade and then begins to decrease. 2 The human posterior lens capsule does not exhibit this age dependence. 3  
According to Fincham, 1 the nonuniform thickness distribution of the anterior lens capsule could affect the lens shape changes during accommodation. He also found greater accommodative amplitudes in those species with a nonuniform anterior lens capsule thickness. His work suggests that the nonuniform thickness distribution affects the shape of the lens in the accommodated form. 4  
In extracapsular cataract surgery, a 3- to 6-mm diameter capsulorrhexis is made manually in the anterior lens capsule. During the removal of the lens contents and the placement of the intraocular lens, the capsulorrhexis is stretched and manipulated. The maximum force that can be applied to the capsule without rupture can be influenced by the capsular thickness. Calculation of the ultimate stress sustainable by the capsule cannot be determined without information on the thickness. 2 A full characterization of the lens capsule’s mechanical properties, incorporating the effects of thickness, is therefore important for improved cataract surgery outcomes. This information can help optimize the size, method, and location of the capsulorrhexis, especially in experimental cataract surgery techniques, involving accommodating intraocular lenses or lens refilling, which require a smaller capsulorrhexis. 
The thickness of the lens capsule has been measured in the past by several researchers. The techniques were invasive, requiring the lens capsule to be removed from the eye before the measurements. Seland, 5 Streeten et al., 6 and Kato et al. 7 measured the thickness of fixed, sectioned human lens capsules by electron microscopy. Fincham, 1 Ruotsalainen and Tarkkanen, 8 Straatsma et al., 9 and Schneider et al. 10 measured the thickness of fixed human lens capsules by light microscopy. Fisher 11 and Krag and Andreassen 2 3 12 13 measured excised, nonfixed human lens capsules with a technique involving the use of microspherules. The thickness measured by these researchers was between 4 and 30 μm for the human central anterior lens capsule and between 2 and 9 μm for the human central posterior lens capsule. A change in lens capsule thickness with age could explain the variability in the thickness measurements. 2 However, the manipulation of such a thin sample could have affected the measurements as well. Because the thickness of the lens capsule has never been noninvasively measured, its true thickness is not known. 
To the best of our knowledge, Fincham 1 is the only researcher who has investigated the lens capsule thickness in monkeys and rabbits. His published data contain only information on the thickness of the anterior lens capsule. The thickness of the rabbit central anterior lens capsule is 20 μm and that of the monkey central anterior lens capsule is 5 to 6 μm (depending on the monkey species). These animals are commonly used as models for experimental cataract surgery techniques 14 15 and accommodation. 16 Accurate measurements of the lens capsule thickness in monkeys and rabbits are needed for the development of experimental cataract surgery procedures and to help determine whether it plays an important role in accommodation in these species. 
The purpose of this study was to measure the thickness of the lens capsule in human, monkey, and rabbit postmortem eyes, using a noncontact optical technique that minimizes tissue manipulation and does not require excision of the capsule. The thickness of the lens capsule was also measured by using histology, to compare with those results obtained optically and determine whether histologic preparation affects capsule thickness. 
Materials and Methods
Optical System
The noncontact optical system constructed to measure the thickness of the lens capsule is based on a focus detection scheme, in which light from a laser source is focused at various depths in the sample. The focused beam is reflected from the surface of the sample and from each internal boundary (Fig. 1) . As the point of focus of the incident light is scanned through the depth of the sample and meets the successive internal boundaries, various intensities of reflected light are observed. The intensity of the reflected light is at its maximum when the incident light focuses on the surface of the sample or at an interface between structures with different refractive indices. The difference in position between two successive maxima is directly proportional to the thickness of the corresponding layer, according to the following relationship  
\[t_{\mathrm{sample}}{=}\ \frac{n_{\mathrm{sample}}}{n_{0}}{\Delta}z\]
, where t sample is the thickness of the sample being measured, n sample is the sample refractive index, n 0 is the incident medium refractive index, and Δz is the displacement of the focusing lens. 
The optical system designed for measurement of lens capsule thickness is shown in Figure 2 . A laser diode emitting 1 mW at 670 nm was butt-coupled into a 2 × 2 bidirectional fiber coupler (F-CPL-2 × 2-OPT-50-20-55, 50/50, 633-nm design wavelength, Newport Corp., Irvine, CA). The fiber coupler split the incident radiation equally between the two output arms. One of the output arms was used as the sample arm, and the other was not used. The light exiting the fiber coupler from the sample arm was collimated with a 10× microscope objective (numerical aperture [NA] = 0.25). The light was focused onto the surface of the lens capsule by a high-NA aspheric lens (NA = 0.68, working distance = 3.1 mm, 350330-B; ThorLabs, Newton, NJ). The aspheric lens was mounted on a translation stage (DM-13L; Newport Corp.) with a motorized actuator with a resolution of 0.05 μm (CMA-12CCL; Newport Corp.). The power delivered to the sample was 320 μW. Light reflected from the surface of the sample was collected by the fiber coupler and sent to a silicon photodiode detector (818-SL/CM; Newport Corp.) connected to a power meter (1825-C-CAL; Newport Corp.). The power meter was connected to a data acquisition card (DAQ-6024E; National Instruments, Austin, TX) located in a laptop computer. The acquisition from the power meter was controlled by the computer running commercial software (LabView; National Instruments), and the motorization of the actuator was controlled with a separate program provided by the manufacturer (ESP100, Newport Corp.). 
Calibration of the Optical System
A cell consisting of a PMMA plate bonded to a glass slide was specially designed for the calibration experiments. An excimer laser was used to make ablations in the PMMA plate at depths of 20, 15, 10, and 5 μm. High-resolution, low-coherence interferometry was then used to determine the actual depths of the ablations in the PMMA plate, because the excimer laser was calibrated for treatment of human corneas. The optical system was used to measure the excimer laser ablation depth in the PMMA plate, with each measurement repeated five times. The measurements obtained with low-coherence interferometry and the optical system were compared by the Bland-Altman technique, with the thickness obtained with low-coherence interferometry on the x-axis and the difference between the low-coherence interferometry and the optical system on the y-axis. This analysis showed that the error of the optical system (defined as twice the standard deviation of the differences) was ±0.5 μm (Fig. 3)
Experimental Protocol
Optical thickness measurements of the anterior and posterior lens capsule of postmortem eyes of 34 New Zealand White (NZW) rabbits (weight average, 3.68 ± 0.36 kg; range, 3.18–4.63), 19 cynomolgus (Macaca fascicularis) and 10 rhesus (Macaca mulatta) monkeys (average age, 8 ± 3 years; range, 2–14), and 22 humans (average age, 74 ± 13 years; range, 40–92) were obtained using the focus detection system. After enucleation, all eyes were placed in sealed containers with gauze soaked with physiologic saline (BSS; Alcon Laboratories, Fort Worth, TX) to prevent dehydration of the globe. Experiments were performed on rabbit eyes immediately after enucleation (less than 1 hour after death). Experiments were performed on monkey eyes less than 2 days after death (0.7 ± 0.6 days) and on human eyes less than 4 days after death (2.3 ± 0.9 days). The monkey and human eyes were stored in the refrigerator before they were used. The animal eyes were obtained after enucleation according to approved institutional animal care guidelines. The human donor eyes were obtained and used in compliance with guidelines of the Declaration of Helsinki for research involving the use of human tissue. 
The protocol was as follows. A custom-made circular PMMA ring (Fig. 4)was bonded onto the sclera in the region of the ciliary body approximately 2 mm posterior to the limbus with cyanoacrylate adhesive (Duro Quick Gel super glue; Loctite Corp., Rocky Hill, CT) after the conjunctiva, adipose, and muscle tissues were removed. The ring’s inner radius of curvature was machined to fit the globe (12 mm for human, 9.5 mm for monkey, and 8 mm for rabbit), enabling dissection of the globe with minimal deformation while keeping the ciliary body–zonule–lens framework intact. Dissection was not initiated until the glue had dried, to ensure that the fumes from the glue did not cause any dehydration of the lens. The posterior pole was removed by making a circumferential incision through the sclera approximately 7 mm posterior to the limbus. Excess vitreous was removed, and the eye was placed on a Teflon slide. The cornea and iris were then sectioned. The clinical appearance of all lenses and lens capsules was examined under an operation microscope. All monkey and rabbit lenses were noted to be intact and clear. All human lenses were intact, but some of the older human lenses (>70 years old) had signs of cataract. All lens capsules were intact and transparent, with no signs of dehydration. The mounted tissue specimen was then placed in a well filled with balanced saline solution (Fig. 5 , left) under the focusing lens of the optical system (Fig. 5 , right). The well was 33 mm in diameter and 6 mm deep, to enable placement of the PMMA ring. A circular groove 14 mm in diameter and 2 mm deep was made in the center of the well to ensure that the lens did not touch the bottom. The well was filled so that the entire capsule except for a region approximately 3 mm in diameter at the central pole of the lens was covered in balanced saline during the measurements. The lens was adjusted in the xy plane until the incident laser beam was located in the center of the lens. The determination of the center of the lens was subjective. However, as a test of the precision of the xy alignment technique, in one separate experiment optical measurements were repeated five times on one anterior lens capsule. In this experiment the sample was removed from the system between each successive measurement and placed under the focusing objective again, and the position was adjusted until the beam was in the center of the lens. The measurement difference was found to be only 0.5 μm in these successive measurements. The sample was also on a platform with tilt adjustment. The tilt was adjusted until maximum reflected light was sent to the photodetector. This confirmed perpendicularity of the sample, since the maximum light detected at the photodetector signifies that the sample is aligned perpendicularly to the coupling system. The lens objective was translated toward the sample at 0.1 mm/s as the detected signal was acquired at a rate of 1000 Hz (1 sample every 0.1 μm). Except for the alignment experiment (described earlier) in which five measurements were taken, optical measurements were performed three times on each sample, with the sample alignment adjusted before each measurement. The mounted specimen was flipped, and the same procedure was used to measure the posterior lens capsule. Repeatability for the optical technique was defined as the difference between the largest and smallest measurement obtained for the same eye (Table 1) . The dissection process took an average of 15 minutes per eye, and measurements took an average of 5 minutes per eye. Both dissection and measurements were performed at room temperature. The lens capsule was kept hydrated during the dissection with the saline. In a time-related study on the saline solution (BSS; Alcon Laboratories) as a preservative medium for the crystalline lens, it caused swelling of <3% over a testing period of 4 to 5 hours. 17 Because the surgical preparation and measurements took only ∼20 minutes, the saline solution (BSS; Alcon Laboratories) physiologically maintained the shape of the entire lens. Only the central pole of the capsule was exposed to air during the measurements (∼5 minutes), and dehydration was therefore unlikely. This was demonstrated by the repeatability of the measurements. Successive measurements did not show a trend toward decreasing thickness. The clinical appearance of the lens capsules was normal throughout the experiments, with no clouding of the lens capsule observed. Intensity maxima obtained during the measurements corresponded to the anterior and posterior surfaces of the lens capsule. The intensity maxima were detected with a peak detection algorithm from the graphing software (Origin; Microcal, Northampton, MA), and the validity of the peaks was verified manually. The physical thickness of the lens capsule was calculated by multiplying the distance between successive maxima by the refractive index (1.4 for the human lens capsule 2 ). Because there are no published data on the refractive index of the rabbit or monkey lens capsule, the value was assumed to be 1.4 as well. Slight variations in refractive index of the capsule were found to have a negligible effect on the calculated thickness, assuming that a refractive index in the range of 1.37 to 1.47 produces only a 1.5 μm measurement difference for a 20-μm thick sample. Figure 6shows typical graphs obtained during the experiments. 
Histology Measurements
All lenses were excised after the experiments and placed in 10% buffered formalin. Sections were taken through the lens, and slides were prepared with these sections. Digital micrographs were taken with a digital camera (Optronics, Goleta, CA) connected to the light microscope (Nikon, Tokyo, Japan) at a magnification of 40× of the central anterior and posterior lens capsule (Fig. 7) . One digital micrograph was taken of the histologic section from two separate histology slides. The thickness of the lens capsule was measured by drawing a line across the capsule on the micrograph, using image-editing software (Canvas 8; ACD Systems, Inc., Miami, FL). The length of this line was divided by a calibration factor that was calculated by using the same technique with a 40× micrograph of an objective microruler (Olympus, Melville, NY) with 10-μm divisions. The pixel size was 0.15 μm. This technique was used on the two micrographs taken. The two thicknesses obtained were averaged to obtain the value reported as the capsule thickness obtained using histology. Repeatability of the histologic technique was defined as the difference between the two measurements obtained for the same eye (Table 1)
Statistical Analysis
Bland-Altman plots 18 were created to find the strength of agreement between the optical and histologic measurements. The paired Student’s t-test was used to compare optical versus histologic and anterior versus posterior measurements. Interspecies comparisons were performed with Student’s t-test. The criterion for statistical significance for the Student’s t-tests was P < 0.05. 
Results
Optical versus Histologic Results
Histologic results were obtained for all eyes, but only the histology measurements from eyes with measurable optical data were included. Measurable results for the anterior lens capsule thickness according to the optical system were obtained from 21 of 22 human, 25 of 29 monkey, and 28 of 34 NZW rabbit eyes (Table 2) . Measurable results for the posterior lens capsule thickness were obtained from 21 of 22 human, 25 of 29 monkey, and 22 of 34 NZW rabbit eyes (Table 3) . Some of the data were not measurable because the peaks corresponding to the capsule were not detectable, because the gain setting on the power meter had not been properly adjusted before the measurements were taken. Other data were not measurable because the resolution of the optical system was not sufficient to distinguish separate peaks. The optical results were plotted against the histologic results (Fig. 8) . Agreement between the two techniques was investigated with the Bland-Altman technique, 18 which plots the difference between the optical and histologic measurements against the average of the two methods (Fig. 9) . This analysis showed that the error between the two techniques (twice the standard deviation of the differences between measurements obtained using the optical system and histology for the same sample) was 10.9 μm for the anterior and 5.8 μm for the posterior lens capsule. Analysis of the optical and histologic measurements using a paired Student’s t-test showed that there was a significant difference in all measurements, except in the case of the NZW rabbit anterior lens capsule (Tables 2 3)
Anterior versus Posterior Lens Capsule Thickness
The thickness measurements obtained for the anterior and posterior lens capsule were compared for statistical significance with a paired Student’s t-test (Table 4) . The measurements obtained by histology showed that the anterior and posterior thicknesses were significantly different in humans, monkeys, and rabbits (P < 0.001 in all three cases), but the optical measurements were only significantly different in rabbits (P = 0.012). 
Interspecies Correlation
The thickness measurements obtained optically and histologically for the anterior and posterior lens capsule of the different species were compared by using the Student’s t-test for statistical significance (Table 5) . All optical and histologic data were included in this analysis. There were no significant differences between human and monkey lens capsule thicknesses. There were significant differences between thickness in humans and rabbits (anterior with histology, posterior with optical system) and in monkeys and rabbits (anterior with optical system, posterior with histology and optical system). 
Discussion
The human lens capsule thickness measurements obtained with the optical system and histology correspond to those found by other researchers. Previous experiments had shown thicknesses between 4 and 30 μm in the anterior lens capsule. 1 2 5 6 8 9 10 11 13 The measurements using the optical system (range, 1.8–24.9 μm) and histology (6.5–15.2 μm) are within this window. The human posterior lens capsule has been found to be between 2 and 9 μm thick. 1 3 5 8 The optical system–measured thicknesses were slightly above this range (range, 2.0–11.7 μm). Histology, however, provided values closer to those found in the past (range, 2.4–8.0 μm). 
For individual measurements, the optical system and histology produced different lens capsule thickness measurements (Fig. 8) . The Bland-Altman technique for method comparison 18 showed that the 95% confidence interval lays between ±10.9 μm for the anterior and ±5.8 μm for the posterior lens capsule (Fig. 9) , which indicates that there is not good agreement between the two techniques. We anticipated that the optical system would produce thickness measurements greater than those found with histology, as histologic preparation causes sample dehydration, producing a decrease in thickness. The optical system did not always produce greater thickness measurements than histology, however. The thickness of 48 of 74 anterior lens capsules and 10 of 68 posterior lens capsules was greater histologically than optically. Some of the differences could be because the lens capsule’s thickness depends on position. Measurements at different positions when using histology or the optical system yield different values. Alignment of the lens capsule at the anterior and posterior poles to measure it using both the optical system and histology was subjective. The histologic sections may not always have been taken perpendicularly, which would have caused increased thickness measurements. In addition, in its anatomic position, the lens capsule is under tension. The cutting of the lens during histologic preparation releases this tension and may also cause an increase in thickness. 19 Although the optical system produced different measurements than did histology, this does not indicate that the measurements are incorrect. The calibration of the system shows that the precision of the optical system was ±0.5 μm. The differences between the optical and histologic measurements are most likely due to changes in the tissue resulting from preparation techniques. The optical system measured lens capsular tissue that had not been manipulated, whereas histology measured excised, fixed tissue samples. Because the state of the lens capsule during optical measurements is closer to the normal anatomic and physiological state, we believe that optical measurements are a better estimate of the true capsule thickness in situ. 
The optical system produced measurements with a higher variability than histology. This may be because the optical system measures tissue in situ under different stress and hydration conditions between eyes. Variations in the state of stress or hydration between samples introduce an additional variable into the optical measurements that may have contributed to the higher variability. 
This study found that the thicknesses of the anterior and posterior lens capsule of monkeys and humans are not different (Table 5) . There was a significant difference between the anterior lens capsule of rabbits and humans when measured histologically, the anterior lens capsule of monkeys and rabbits and the posterior lens capsule of humans and rabbits measured optically, and the posterior lens capsule of rabbits and monkeys measured both histologically and optically. If additional interspecies differences in central anterior and posterior lens capsule thickness exist, they are smaller than the resolution of both the histologic and the optical techniques. Fincham 1 measured the anterior lens capsule of two monkeys and one rabbit. He found a thickness of 20 μm in the rabbit, which is at the high end of the range for the two techniques (4.0–22.3 μm optically and 6.7–15.5 μm histologically). He measured the monkey anterior lens capsule as 5 to 6 μm, which is at the low end of the range for the two techniques (3.5–22.7 μm optically and 5.4–19.7 μm histologically). The few samples measured by Fincham fit within the experimental results of this study. 
Because the thickness of the central anterior and posterior lens capsule of monkeys is not significantly different from that of humans (Table 5) , these animals can be used as experimental models when the thickness of the lens capsule is important. The central anterior lens capsule of rabbits and humans was only different histologically, indicating that this animal is probably a good experimental model. 
Conclusion
This study shows that the optical system produces lens capsule thickness measurements that are within the range obtained by previous researchers using established techniques. Within the precision of the optical system and histology, the central thickness of the lens capsule does not appear to vary considerably among humans, monkeys, and NZW rabbits. There were significant differences between optical in situ measurements and histology, which indicates that histologic preparation may affect lens capsule thickness. 
 
Figure 1.
 
Representation of the path of light rays in a focus detection system, illustrating the relationship between change in position (Δz) and thickness (t sample). The front surface of the sample bends the incident light rays, due to the difference in refractive index. At the second intensity maximum, after a displacement of Δz, the beam appears to be focused at a point located in front of the posterior surface of the sample. This point is the virtual image created by the plane-parallel plate of the point located on the optical axis at the posterior surface.
Figure 1.
 
Representation of the path of light rays in a focus detection system, illustrating the relationship between change in position (Δz) and thickness (t sample). The front surface of the sample bends the incident light rays, due to the difference in refractive index. At the second intensity maximum, after a displacement of Δz, the beam appears to be focused at a point located in front of the posterior surface of the sample. This point is the virtual image created by the plane-parallel plate of the point located on the optical axis at the posterior surface.
Figure 2.
 
Focus detection system for measurement of ocular tissues. A laser diode emitting at 670 nm was butt-coupled to a 2 × 2 bidirectional fiber coupler. The fiber coupler split the incident radiation equally between the two output arms. One of the output arms was used as the sample arm, and the other was not used. The light exiting the fiber coupler from the sample arm was collimated with a 10× microscope objective (NA = 0.25). The light was focused onto the surface of the lens capsule using a high-NA aspheric lens (NA = 0.68), mounted on a translation stage with a motorized actuator. Light reflected from the surface of the sample was collected by the fiber coupler and sent to a photodetector connected to a power meter.
Figure 2.
 
Focus detection system for measurement of ocular tissues. A laser diode emitting at 670 nm was butt-coupled to a 2 × 2 bidirectional fiber coupler. The fiber coupler split the incident radiation equally between the two output arms. One of the output arms was used as the sample arm, and the other was not used. The light exiting the fiber coupler from the sample arm was collimated with a 10× microscope objective (NA = 0.25). The light was focused onto the surface of the lens capsule using a high-NA aspheric lens (NA = 0.68), mounted on a translation stage with a motorized actuator. Light reflected from the surface of the sample was collected by the fiber coupler and sent to a photodetector connected to a power meter.
Figure 3.
 
The thickness of the calibration cell ablations found using the optical system was subtracted from the thickness found using low-coherence interferometry and plotted against the thickness found using low-coherence interferometry (Bland-Altman analysis). The dotted lines indicate the 95% confidence intervals, or twice the SD of the difference. The error of the optical system was ±0.5 μm.
Figure 3.
 
The thickness of the calibration cell ablations found using the optical system was subtracted from the thickness found using low-coherence interferometry and plotted against the thickness found using low-coherence interferometry (Bland-Altman analysis). The dotted lines indicate the 95% confidence intervals, or twice the SD of the difference. The error of the optical system was ±0.5 μm.
Figure 4.
 
A cross section of the lens attached to the custom-made PMMA ring. The ring’s inner radius of curvature (R) was machined to fit the globe (12 mm for human, 9.5 mm for monkey, and 8 mm for rabbit), enabling dissection of the globe with minimal deformation while keeping the ciliary body–zonule–lens framework intact.
Figure 4.
 
A cross section of the lens attached to the custom-made PMMA ring. The ring’s inner radius of curvature (R) was machined to fit the globe (12 mm for human, 9.5 mm for monkey, and 8 mm for rabbit), enabling dissection of the globe with minimal deformation while keeping the ciliary body–zonule–lens framework intact.
Figure 5.
 
Left: Top view of lens in specially designed holder mounted to a tilt platform. Right: side view, showing placement in the optical system.
Figure 5.
 
Left: Top view of lens in specially designed holder mounted to a tilt platform. Right: side view, showing placement in the optical system.
Table 1.
 
Repeatability of the Optical Method and Histology
Table 1.
 
Repeatability of the Optical Method and Histology
Species Eyes Anterior Repeatability (μm) Posterior Repeatability (μm)
Anterior Posterior Optical Histology Optical Histology
Human 22 22 3.4 ± 3.7 1.4 ± 1.1 1.0 ± 0.4 1.4 ± 1.7
(1.4–8.4) (0.4–3.6) (0.7–1.4) (0.1–5.3)
Monkey 29 29 3.0 ± 4.1 2.2 ± 2.2 1.2 ± 1.0 2.4 ± 1.4
(0–10.1) (0.9–6.1) (0.3–2.7) (0.9–4.2)
NZW rabbit 34 34 1.3 ± 0.9 1.7 ± 1.3 1.6 ± 1.2 1.7 ± 1.5
(0.1–3.4) (0.1–5.5) (0.1–5.2) (0.1–5.5)
Figure 6.
 
Typical results obtained for the anterior (left) and posterior (right) lens capsules. Δz, the displacement of the objective between the two peaks. To obtain the true thickness, the thickness was Δz multiplied by the refractive index (1.4) of the capsule.
Figure 6.
 
Typical results obtained for the anterior (left) and posterior (right) lens capsules. Δz, the displacement of the objective between the two peaks. To obtain the true thickness, the thickness was Δz multiplied by the refractive index (1.4) of the capsule.
Figure 7.
 
Histology images of the central anterior (left) and posterior (right) lens capsules of a human eye (74 years of age). Thickness was measured manually by magnifying the image until the pixels were visible. A line was then drawn through the thickness of the sample. The edges of the capsule were detected within ±5 pixels, corresponding to 0.75 μm. Magnification, ×40.
Figure 7.
 
Histology images of the central anterior (left) and posterior (right) lens capsules of a human eye (74 years of age). Thickness was measured manually by magnifying the image until the pixels were visible. A line was then drawn through the thickness of the sample. The edges of the capsule were detected within ±5 pixels, corresponding to 0.75 μm. Magnification, ×40.
Table 2.
 
Anterior Lens Capsule Thickness Results Obtained by the Optical Method and Histology
Table 2.
 
Anterior Lens Capsule Thickness Results Obtained by the Optical Method and Histology
Species Eyes (n) Optical Results (μm) Histology Results (μm) Average Difference (μm) P
Human 22 8.2 ± 5.5 12.4 ± 2.5 −3.9 ± 6.3 0.012*
(1.8–24.9) (6.5–15.2) (−11.9–14.8)
Monkey 29 7.5 ± 4.4 10.7 ± 3.7 −2.9 ± 4.3 0.002*
(4.1–22.7) (5.4–19.7) (−10.3–7.0)
NZW rabbit 34 10.7 ± 4.2 10.4 ± 2.0 0.5 ± 5.1 0.635
(4.0–22.3) (6.7–15.5) (−9.3–9.6)
Table 3.
 
Posterior Lens Capsule Thickness Results from the Optical Method and Histology
Table 3.
 
Posterior Lens Capsule Thickness Results from the Optical Method and Histology
Species Eyes (n) Optical Results (μm) Histology Results (μm) Average Difference (μm) P
Human 22 6.3 ± 2.2 4.2 ± 1.5 2.3 ± 2.3 <0.001*
(2.0–11.7) (2.4–8.0) (−1.3–6.6)
Monkey 29 5.9 ± 1.7 3.5 ± 1.6 2.5 ± 2.4 <0.001*
(3.6–9.2) (1.6–6.7) (−1.9–6.8)
NZW rabbit 34 7.8 ± 2.3 4.7 ± 2.5 3.0 ± 3.8 0.001*
(3.8–15.1) (2.5–12.9) (−4.9–12.1)
Figure 8.
 
Thickness measured using histology versus the thickness measured using the optical system for anterior (left) and posterior (right) lens capsules. Diagonal line: where the measurements would lie with perfect 1:1 correlation.
Figure 8.
 
Thickness measured using histology versus the thickness measured using the optical system for anterior (left) and posterior (right) lens capsules. Diagonal line: where the measurements would lie with perfect 1:1 correlation.
Figure 9.
 
Thickness found using the optical system subtracted from the histologic results plotted against the average of the two techniques (Bland-Altman technique) for the anterior (left) and posterior (right) lens capsule. Dashed lines: 95% CI (±2 SD).
Figure 9.
 
Thickness found using the optical system subtracted from the histologic results plotted against the average of the two techniques (Bland-Altman technique) for the anterior (left) and posterior (right) lens capsule. Dashed lines: 95% CI (±2 SD).
Table 4.
 
Probabilities Obtained for the Anterior Versus Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Table 4.
 
Probabilities Obtained for the Anterior Versus Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Species P (Optical System) P (Histology)
Human 0.077 <0.001*
Monkey 0.095 <0.001*
NZW rabbit 0.012* <0.001*
Table 5.
 
Probabilities Obtained for Interspecies Differences in Anterior and Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Table 5.
 
Probabilities Obtained for Interspecies Differences in Anterior and Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Species P (Optical System) P (Histology)
Human Monkey NZW Rabbit Human Monkey NZW Rabbit
Anterior
 Human 0.641 0.077 0.075 0.002*
 Monkey 0.641 0.009* 0.075 0.691
 NZW Rabbit 0.077 0.009* 0.002* 0.691
Posterior
 Human 0.506 0.029* 0.174 0.260
 Monkey 0.506 0.002* 0.174 0.030*
 NZW Rabbit 0.029* 0.002* 0.260 0.030*
The authors thank Bobby Collins, DVM (Division of Veterinary Resources, University of Miami, Miami, FL), and Philip McCabe, PhD (Department of Psychology, University of Miami), for donating several NZW rabbits; Patricia Gullett, DVM, and Daniel Rothen, DVM (Division of Veterinary Resources), and Norma Kenyon, PhD (Diabetic Research Institute, University of Miami), for donating nonhuman primate eyes; the Florida Lions Eye Bank for donating human eyes; Sonia Yoo, MD (Bascom Palmer Eye Institute, University of Miami), for assistance with the excimer laser; and Magda Celdran (Bascom Palmer Eye Institute, University of Miami) for preparation of the histology slides. 
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Figure 1.
 
Representation of the path of light rays in a focus detection system, illustrating the relationship between change in position (Δz) and thickness (t sample). The front surface of the sample bends the incident light rays, due to the difference in refractive index. At the second intensity maximum, after a displacement of Δz, the beam appears to be focused at a point located in front of the posterior surface of the sample. This point is the virtual image created by the plane-parallel plate of the point located on the optical axis at the posterior surface.
Figure 1.
 
Representation of the path of light rays in a focus detection system, illustrating the relationship between change in position (Δz) and thickness (t sample). The front surface of the sample bends the incident light rays, due to the difference in refractive index. At the second intensity maximum, after a displacement of Δz, the beam appears to be focused at a point located in front of the posterior surface of the sample. This point is the virtual image created by the plane-parallel plate of the point located on the optical axis at the posterior surface.
Figure 2.
 
Focus detection system for measurement of ocular tissues. A laser diode emitting at 670 nm was butt-coupled to a 2 × 2 bidirectional fiber coupler. The fiber coupler split the incident radiation equally between the two output arms. One of the output arms was used as the sample arm, and the other was not used. The light exiting the fiber coupler from the sample arm was collimated with a 10× microscope objective (NA = 0.25). The light was focused onto the surface of the lens capsule using a high-NA aspheric lens (NA = 0.68), mounted on a translation stage with a motorized actuator. Light reflected from the surface of the sample was collected by the fiber coupler and sent to a photodetector connected to a power meter.
Figure 2.
 
Focus detection system for measurement of ocular tissues. A laser diode emitting at 670 nm was butt-coupled to a 2 × 2 bidirectional fiber coupler. The fiber coupler split the incident radiation equally between the two output arms. One of the output arms was used as the sample arm, and the other was not used. The light exiting the fiber coupler from the sample arm was collimated with a 10× microscope objective (NA = 0.25). The light was focused onto the surface of the lens capsule using a high-NA aspheric lens (NA = 0.68), mounted on a translation stage with a motorized actuator. Light reflected from the surface of the sample was collected by the fiber coupler and sent to a photodetector connected to a power meter.
Figure 3.
 
The thickness of the calibration cell ablations found using the optical system was subtracted from the thickness found using low-coherence interferometry and plotted against the thickness found using low-coherence interferometry (Bland-Altman analysis). The dotted lines indicate the 95% confidence intervals, or twice the SD of the difference. The error of the optical system was ±0.5 μm.
Figure 3.
 
The thickness of the calibration cell ablations found using the optical system was subtracted from the thickness found using low-coherence interferometry and plotted against the thickness found using low-coherence interferometry (Bland-Altman analysis). The dotted lines indicate the 95% confidence intervals, or twice the SD of the difference. The error of the optical system was ±0.5 μm.
Figure 4.
 
A cross section of the lens attached to the custom-made PMMA ring. The ring’s inner radius of curvature (R) was machined to fit the globe (12 mm for human, 9.5 mm for monkey, and 8 mm for rabbit), enabling dissection of the globe with minimal deformation while keeping the ciliary body–zonule–lens framework intact.
Figure 4.
 
A cross section of the lens attached to the custom-made PMMA ring. The ring’s inner radius of curvature (R) was machined to fit the globe (12 mm for human, 9.5 mm for monkey, and 8 mm for rabbit), enabling dissection of the globe with minimal deformation while keeping the ciliary body–zonule–lens framework intact.
Figure 5.
 
Left: Top view of lens in specially designed holder mounted to a tilt platform. Right: side view, showing placement in the optical system.
Figure 5.
 
Left: Top view of lens in specially designed holder mounted to a tilt platform. Right: side view, showing placement in the optical system.
Figure 6.
 
Typical results obtained for the anterior (left) and posterior (right) lens capsules. Δz, the displacement of the objective between the two peaks. To obtain the true thickness, the thickness was Δz multiplied by the refractive index (1.4) of the capsule.
Figure 6.
 
Typical results obtained for the anterior (left) and posterior (right) lens capsules. Δz, the displacement of the objective between the two peaks. To obtain the true thickness, the thickness was Δz multiplied by the refractive index (1.4) of the capsule.
Figure 7.
 
Histology images of the central anterior (left) and posterior (right) lens capsules of a human eye (74 years of age). Thickness was measured manually by magnifying the image until the pixels were visible. A line was then drawn through the thickness of the sample. The edges of the capsule were detected within ±5 pixels, corresponding to 0.75 μm. Magnification, ×40.
Figure 7.
 
Histology images of the central anterior (left) and posterior (right) lens capsules of a human eye (74 years of age). Thickness was measured manually by magnifying the image until the pixels were visible. A line was then drawn through the thickness of the sample. The edges of the capsule were detected within ±5 pixels, corresponding to 0.75 μm. Magnification, ×40.
Figure 8.
 
Thickness measured using histology versus the thickness measured using the optical system for anterior (left) and posterior (right) lens capsules. Diagonal line: where the measurements would lie with perfect 1:1 correlation.
Figure 8.
 
Thickness measured using histology versus the thickness measured using the optical system for anterior (left) and posterior (right) lens capsules. Diagonal line: where the measurements would lie with perfect 1:1 correlation.
Figure 9.
 
Thickness found using the optical system subtracted from the histologic results plotted against the average of the two techniques (Bland-Altman technique) for the anterior (left) and posterior (right) lens capsule. Dashed lines: 95% CI (±2 SD).
Figure 9.
 
Thickness found using the optical system subtracted from the histologic results plotted against the average of the two techniques (Bland-Altman technique) for the anterior (left) and posterior (right) lens capsule. Dashed lines: 95% CI (±2 SD).
Table 1.
 
Repeatability of the Optical Method and Histology
Table 1.
 
Repeatability of the Optical Method and Histology
Species Eyes Anterior Repeatability (μm) Posterior Repeatability (μm)
Anterior Posterior Optical Histology Optical Histology
Human 22 22 3.4 ± 3.7 1.4 ± 1.1 1.0 ± 0.4 1.4 ± 1.7
(1.4–8.4) (0.4–3.6) (0.7–1.4) (0.1–5.3)
Monkey 29 29 3.0 ± 4.1 2.2 ± 2.2 1.2 ± 1.0 2.4 ± 1.4
(0–10.1) (0.9–6.1) (0.3–2.7) (0.9–4.2)
NZW rabbit 34 34 1.3 ± 0.9 1.7 ± 1.3 1.6 ± 1.2 1.7 ± 1.5
(0.1–3.4) (0.1–5.5) (0.1–5.2) (0.1–5.5)
Table 2.
 
Anterior Lens Capsule Thickness Results Obtained by the Optical Method and Histology
Table 2.
 
Anterior Lens Capsule Thickness Results Obtained by the Optical Method and Histology
Species Eyes (n) Optical Results (μm) Histology Results (μm) Average Difference (μm) P
Human 22 8.2 ± 5.5 12.4 ± 2.5 −3.9 ± 6.3 0.012*
(1.8–24.9) (6.5–15.2) (−11.9–14.8)
Monkey 29 7.5 ± 4.4 10.7 ± 3.7 −2.9 ± 4.3 0.002*
(4.1–22.7) (5.4–19.7) (−10.3–7.0)
NZW rabbit 34 10.7 ± 4.2 10.4 ± 2.0 0.5 ± 5.1 0.635
(4.0–22.3) (6.7–15.5) (−9.3–9.6)
Table 3.
 
Posterior Lens Capsule Thickness Results from the Optical Method and Histology
Table 3.
 
Posterior Lens Capsule Thickness Results from the Optical Method and Histology
Species Eyes (n) Optical Results (μm) Histology Results (μm) Average Difference (μm) P
Human 22 6.3 ± 2.2 4.2 ± 1.5 2.3 ± 2.3 <0.001*
(2.0–11.7) (2.4–8.0) (−1.3–6.6)
Monkey 29 5.9 ± 1.7 3.5 ± 1.6 2.5 ± 2.4 <0.001*
(3.6–9.2) (1.6–6.7) (−1.9–6.8)
NZW rabbit 34 7.8 ± 2.3 4.7 ± 2.5 3.0 ± 3.8 0.001*
(3.8–15.1) (2.5–12.9) (−4.9–12.1)
Table 4.
 
Probabilities Obtained for the Anterior Versus Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Table 4.
 
Probabilities Obtained for the Anterior Versus Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Species P (Optical System) P (Histology)
Human 0.077 <0.001*
Monkey 0.095 <0.001*
NZW rabbit 0.012* <0.001*
Table 5.
 
Probabilities Obtained for Interspecies Differences in Anterior and Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Table 5.
 
Probabilities Obtained for Interspecies Differences in Anterior and Posterior Lens Capsule Thickness Found Using the Optical System and Histology
Species P (Optical System) P (Histology)
Human Monkey NZW Rabbit Human Monkey NZW Rabbit
Anterior
 Human 0.641 0.077 0.075 0.002*
 Monkey 0.641 0.009* 0.075 0.691
 NZW Rabbit 0.077 0.009* 0.002* 0.691
Posterior
 Human 0.506 0.029* 0.174 0.260
 Monkey 0.506 0.002* 0.174 0.030*
 NZW Rabbit 0.029* 0.002* 0.260 0.030*
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