(Investigative Ophthalmology and Visual Science. 2001;42:885-894.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
High-Resolution Ultrasonic Imaging and Characterization of the Ciliary Body
Ronald H. Silverman1,
Frederic L. Lizzi2,
Bogdan G. Ursea1,
Mark J. Rondeau1,
Nashwa Badr Eldeen1,
Andy Kaliscz2,
Harriet O. Lloyd1 and
D. Jackson Coleman1
1 From the Weill Medical College of Cornell University, New York; and
2 Riverside Research Institute, New York.
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Abstract
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PURPOSE. To develop a means for noninvasive in vivo visualization of the ciliary
processes using very-high-frequency (50 MHz) ultrasound and to develop
quantitative morphologic descriptors that may relate to physiologic
function.
METHODS. The region of the ciliary body was scanned with very-high-frequency
ultrasound, both in rabbits and in normal human subjects. Data were
acquired in a series of planes so that the spacing between them was
less than the beam width of the transducer in its focal plane.
Three-dimensional perspective images were constructed, representing the
anatomy of the angle region, including the ciliary processes. The
automatically detected boundaries of the ciliary processes were
analyzed to compute their periphery, area, shape factor, and fractal
dimension. These measures were compared between the human and the
rabbit eye and analyzed for periodicities related to the spacing of
successive processes.
RESULTS. Three-dimensional images allowed visualization of the radial
arrangement of the processes. All biometric descriptors were
significantly different between the rabbit and human eye and showed
periodicities consistent with spacing between processes.
CONCLUSIONS. The methods described in this report are sensitive descriptors of the
state of the ciliary processes. These techniques may be of value in
measurement of changes in the ciliary body associated with disease,
medical therapy, and aging.
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Introduction
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The ciliary processes, the site of aqueous fluid
production, are largely inaccessible to direct visualization because of
their location posterior to the optically opaque iris and sclera.
Intraoperative viewing of the ciliary processes is now possible
(invasively) using fine endoscopic systems. In this report we describe
noninvasive visualization of the ciliary body with very-high-frequency
ultrasound (VHFU). We also demonstrate quantitative morphologic
descriptors of the ciliary processes that may relate to function.
Lizzi et al.1
and Pavlin et al.2
3
introduced VHFU and ultrasound biomicroscopy (UBM) in the early 1990s
for ocular studies. These techniques, although differing in certain
aspects of signal processing, both use 50-MHz polyvinylidene fluoride
(PVDF) transducers. Ultrasound allows imaging of tissues that are
located behind optically opaque structures. At the high frequencies
used in these techniques, ultrasound is necessarily limited to study of
the anterior segment of the eye because of the exponential increase in
acoustic attenuation that occurs with frequency. With resolution on the
order of 35 µm axially by 65 µm laterally, visualization of the
ciliary processes can be accomplished. By scanning in a series of
parallel planes, high-resolution, three-dimensional (3-D) perspective
images can be produced as well.4
However, unless scan
planes (and pulse–echo vectors within planes) are within a beam’s
width of each other, 3-D images will not obtain the resolution to which
they are entitled. The major factor that tends to militate against
increasing scan plane density is time: The longer the acquisition
process, the more likely motion-induced blurring or distortion will
occur. We have recently demonstrated, however, that this problem can be
superseded both in animal and human subjects.5
Since their inception, VHFU and UBM systems have been used in many
clinical and preclinical studies related to glaucoma. As the site of
aqueous fluid production, the ciliary body is also the site of medical
intervention intended to reduce inflow. Until now, study of the effects
of medical therapies have been largely focused on the effect of drugs
on intraocular pressure. The ability to study drug action on the
morphology of the ciliary body itself may offer useful insights
regarding mechanisms of action.
A number of UBM studies of ciliary body morphometrics have been
conducted. Several reports have described the effects of pharmacologic
agents on ciliary body thickness.6
7
8
Gentile et
al.9
measured ciliary body cross-sectional area in a
series of patients with uveitis. Frieling and Dembinsky10
measured ciliary body length and thickness in relation to axial length.
Gohdo et al.11
examined ciliary body thickness in normal
eyes with narrow angles. Several studies have been made of ciliary body
anatomic changes that occur during accommodation.12
13
14
In these studies, the ciliary body was treated as a unit—that is, the
epithelial and muscular tissues were not distinguished for biometric
purposes. In general, overall ciliary body thickness was treated as the
measure of interest. None of these reports attempted to describe 3-D
anatomy of the ciliary body or to distinguish the ciliary processes
from muscle. In this report we describe the 3-D anatomy of the ciliary
processes in rabbits and in normal human subjects. We describe
quantitative methods for characterizing the processes, including
surface area, volume, and fractal dimension. To validate these
measures, we demonstrate that their values differ between human and
rabbit ciliary processes and that in each species, these parameters
show periodicities that correspond to the spacing between successive
processes.
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Methods
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The transducer used in these studies consists of a spherical
PVDF section with an aperture of 6 mm and focal length of 12 mm. The
reflectance spectrum from a glass plate aligned perpendicular to the
beam axis in the focal plane reveals a 38-MHz center frequency and a
-15-dB bandwidth extending from 10 to 55 MHz. The scanning system
consisted of two orthogonal linear stages with computer-controlled
stepper motors providing a positional resolution of 10 µm. We used a
digitizing oscilloscope to store echo data, which were subsequently
transferred to the computer hard drive.
Each scan sequence consisted of series of parallel scan planes spaced
at 40-µm intervals, and each plane consisted of 128 pulse–echo
vectors spaced 40 µm apart (less than the 65-µm lateral
resolution). Vectors consisted of 2048 samples of radio frequency echo
data, acquired at a sample rate of 250 MHz. Thus, the 3-D data
comprised a block 5.1 mm in length by 6.4 mm in depth by 3 to 4 mm in
width (depending on the number of planes acquired). Once acquired, we
determined the envelope of the echo data and generated a series of
B-mode images.
The experiments were performed in compliance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research. Dutchbelt
rabbits were used in these studies. To scan rabbits, we first induced
general anesthesia with intramuscular injection of xylazine (5 mg/kg)
and ketamine (35 mg/kg). The eye was then gently proptosed and placed
through a hole in a rubber membrane. This allowed formation of a normal
saline water bath that provided acoustic coupling between the
transducer and the eye. We obtained four sets of scans on two rabbit
eyes.
Experiments on human subjects were performed in accordance with the
Declaration of Helsinki after the purpose and the risks of the protocol
had been explained and written consent obtained from the
subjects. In human subjects, we formed a reservoir around the
eye using a sterile drape (1020 Steridrape; 3M Health Care, St. Paul,
MN), which provides an adhesive ring around a central aperture. After
administering a few drops of topical proparacaine HCl (0.5%), we
inserted a lid speculum to prevent blinking. We then filled the
reservoir to a depth of roughly 2 cm. We provided a fixation target
visible to the subject’s other eye. The subject’s task was to
maintain constant gaze (with the other eye) on the target during the 1
to 2 minutes required to acquire the many scan planes. We twice scanned
one eye on each of two healthy human subjects, a 49-year-old man and a
30-year-old woman. Neither subject had a history of glaucoma or used
glaucoma medications.
The 3-D volume-rendered images were generated from the B-mode image
series using a computer workstation (VoxelView software; Vital Images,
Fairfield, IA). The 3-D images could be oriented for viewing from a
variety of perspectives, and we could examine individual planes cut
either orthogonally to the original scan plane or at arbitrary
orientations through the data set. This provided a qualitative
evaluation of the ciliary body and adjacent structures.
Biometric analysis of the ciliary processes was accomplished in a
semiautomated manner using image analysis software (PhotoShop, ver.
5.0; Adobe Systems, San Jose, CA) as illustrated in Figure 1 . We analyzed one complete scan set each of a human eye (90 planes) and
a rabbit eye (80 planes). In both cases, planes were oriented parallel
to the processes. In each scan plane, we manually delineated and
selected the region of the ciliary processes. This was easily
accomplished in the rabbit eye, where the ciliary muscles are quite
diminutive in comparison with the prominent processes. In the human
eye, we carefully delineated the boundary between the muscle and
epithelium, which appeared as a thin anechoic line between the two
tissues in the VHFU images (Fig. 2)
. We reduced speckle by use of a median filter (span, 1 pixel) and used
a thresholding function to form a binary image consisting of a black
background corresponding to regions with little or no echo signal
(vitreous, aqueous, or other) versus echogenic solid tissues (the
ciliary processes) in white. We then used an edge filter to outline the
borders of the detected region. The only manual step in this process
was the initial circling of the area of interest.
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Figure 1. Stages of processing of rabbit HFU image. The ciliary processes are
manually selected (A) and isolated (B). Noise is
reduced using a median filter (C) and thresholding applied
to create a binary image (D) in which pixel count is
proportional to area. Edge detection is then used to determine the
boundary of the processes (E) from which peripheral
dimension can be computed. The fractal dimension of the edge-detected
image is then determined by counting the number of boxes of increasing
size required to cover the boundary (F).
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Figure 2. (A) Photomicrograph of typical human ciliary body region.
Hematoxylin-eosin; original magnification, x20. (B)
Comparative HFU image of ciliary body region. Note the thin, dark
line at the interface of the ciliary muscle and epithelium.
(C) Manually drawn boundary for isolation of ciliary
processes. (D) Automatically detected region.
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We determined the area and perimeter of the ciliary processes by
counting the number of pixels (and using the appropriate scaling
factor) in the filled binary and edge-detected images, respectively. We
divided the periphery by the area to obtain a ratio that we term the
shape factor. A circle has the smallest shape factor for an object of a
given size. For a circle, the shape factor is defined as
2
r/r2 =
2/r. Note that this ratio of circumference to area decreases
as the size of a circle increases. Finally, we converted periphery and
area measurements to surface area and volume units by multiplying by
the interplane interval.
Fractal dimension denotes a concept related to certain shapes in the
natural world and in mathematics. Such shapes exhibit a property called
self-similarity, in which form is invariant over changes in scale.
Examples of this are coastlines, snowflakes, and river networks. The
measured length of these objects depends on the length of the yardstick
used for making measurements. As the yardstick gets smaller, we are
able to measure finer and finer features, and the measured overall
length increases. Fractal dimension is a function of the relationship
between length and yardstick size. A straight line is one-dimensional,
and fractal objects have a dimension greater than 1.0 but less than
2.0. We determined the fractal dimension of the ciliary process
boundary directly from the edge-detected image using specially designed
software (Fractal Dimension Calculator, ver. 1.5 (Paul Bourke, Auckland
University School of Architecture, New Zealand; Shareware available for
the Macintosh at
http://www.swin.edu.au/astronomy/pbourke/software). This
software tool calculates the Hausdorff–Besicovitch dimension by
superimposing meshes of various sizes over the edge-detected image and
counting the number of mesh boxes containing part of the boundary (see
Fig. 1F
).15
The fractal dimension is determined from the
linear best-fit slope of a plot of log(Ns)
versus log(1/s), where s is box size, and
Ns is the number of boxes of size
s containing edge regions.
We compared the mean values of each parameter in the human versus the
rabbit eye using an unpaired, two-tailed t-test. We examined
the parameters for periodicities by ordering the values by plane
number, centering the data in a 128-point array, and computing the
spectrum using a fast Fourier transform. Lastly, we used a continuous
wavelet transform (Morlet with 6 df) to analyze the
data.16
This method determines the presence of
periodicities in a spatially localized manner. (Wavelet software is
provided in the public domain by Christopher Torrence and Gilbert
Compo, University of Colorado, Boulder, and is available at
http://paos.colorado.edu/research/wavelets.)
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Results
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Figures 3
(top) is an example of a 3-D volume-rendered image of the scanned area
in the rabbit eye. Below the 3-D rendering is a series of coronal plane
cuts through the above volume that demonstrate the radial arrangement
of processes. Figure 4
provides a series of false-color 3-D renderings of the rabbit eye. With
the separately color encoded cornea and sclera, iris, and ciliary body,
anatomic structure is more readily appreciated. In the rabbit eye, we
can see the ciliary web and its attachments to the posterior surface of
the iris. Figure 5
provides a series of edge-on 3-D false-color renderings of the ciliary
processes of a normal human subject (the 49-year-old man). Individual
processes are apparent, as is a small occult and asymptomatic cyst.
Figure 6 provides a gray-scale 3-D volume-rendered image of the scanned region
in the same person, viewed from a posterior perspective.
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Figure 3. Top: A 3-D volume-rendered image of rabbit eye. The
cornea (C) shows reduced reflectivity because of its oblique angle of
presentation and because it is largely outside the focal plane. The
iris plane (I) is evident, as are the prominent ciliary processes (P)
typical of the rabbit eye. Bottom: A series of coronal
planes cut through the volume shown above reveal the radial arrangement
of processes around the pupil.
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Figure 4. False-color 3-D volume renderings of a rabbit eye as seen from behind,
derived from HFU data. Gray tones were used to depict
the cornea-sclera, blue for the iris, and
red for the ciliary processes. Center and
right images: Progressive cuts were made through the
volume to reveal the complex structure of the processes and their
attachments to the iris and each other.
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Figure 5. The 3-D renderings of the human ciliary processes in which the
processes are depicted in red tones. The three images
show the same region with progressively more tissue removed.
Arrow: occult cyst.
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Figure 6. A 3-D rendering of a scanned region in a normal human subject, seen
from a perspective posterior to the eye. Several ciliary processes (P)
are seen posterior to the iris (I) plane. S, sclera.
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Table 1
provides a statistical comparison of the average values of each
biometric factor for human and rabbit eyes. Averaged over the scan
sets, the rabbit ciliary processes have both a significantly larger
periphery and area than the human ciliary processes. It was interesting
to note that the rabbit’s shape factor was smaller than that of the
human, whereas the rabbit fractal dimension was larger. Taken together,
these indicate that although the rabbit ciliary processes were more
convoluted than those of the human, the actual ratio of surface area to
mass was lower in the rabbit.
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Table 1. Statistical Comparison of Mean Values of Four Biometric Parameters for
Rabbit and Human Ciliary Processes
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If we assume a diameter of 10 mm for the ciliary band, then we get a
circumference of 31.4 mm. We therefore scanned approximately one tenth
of the circumference of the eye. Given the spacing of 0.04 mm between
successive planes, we can compute the mean surface area of the rabbit
ciliary processes per plane to be 16.80 mm x 0.04 mm = 0.67
mm2, and the total surface area to be 0.67
mm2 x (31.4/0.04) = 528
mm2. We can similarly calculate the total surface
area of the processes for the human subject to be 222
mm2. Ciliary body volumes can be calculated in
the same manner. The volume of ciliary processes for the rabbit and
human eye was found to be 59.6 mm3 and 19.2
mm3, respectively.
Figure 7
provides plots of periphery, area, shape factor, and fractal dimension
as a function of scan plane position for the rabbit eye. The
corresponding plots for the human eye are provided in Figure 8
. In some instances, periodicity of the biometric parameters is quite
evident, such as for the ciliary process area and shape factor of the
rabbit. The human data seem noisier overall, which may well be due to
inevitable small eye movements that occurred during scanning.
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Figure 7. Plots of peripheral dimension (A), area (B),
shape factor (C), and fractal dimension (D) of
successive scan planes in a rabbit eye.
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Figure 8. Plots of peripheral dimension (A), area (B),
shape factor (C), and fractal dimension (D) of
successive scan planes in a human eye.
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Spectral plots for each factor are provided in Figures 9
and 10 for rabbit and human eyes, respectively. The spectral plots for all
parameters in the rabbit eye show a peak at a frequency of 10,
indicating that 10 cycles occurred over 5.1 mm (128 x 40 µm).
This suggests a spacing of ciliary processes at approximately 0.5-mm
intervals. In human subjects, a 0.5-mm periodicity was also observed
for all parameters except fractal dimension, for which no prominent
peak was evident.
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Figure 9. Plots of spectral amplitude of peripheral dimension (A),
area (B), shape factor (C), and fractal dimension
(D) for the rabbit eye. The prominent peak at a frequency of
10 seen for all factors corresponds to a spatial frequency of 0.5 mm,
the expected frequency of ciliary processes.
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Figure 10. Plots of spectral amplitude of peripheral dimension (A),
area (B), shape factor (C), and fractal dimension
(D) for the human eye. Peaks corresponding to a 0.5 mm
spacing between processes appear, but less prominently than in the
rabbit eye.
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Figure 11
provides comparative wavelet transform maps of the peripheral dimension
of the rabbit and human ciliary processes. The abscissas for these maps
are directly comparable to those of the plots of peripheral dimension
in the upper left of Figures 7 (rabbit) and 8
(human). The ordinates
indicate detected periodicities in mm. The wavelet map of the rabbit
shows a dominant band extending from approximately 0.5 to 0.75 mm. For
the human data, a similar but less regular pattern is seen but at
somewhat higher spatial frequency, approximately 0.3 to 0.6 mm.
Comparable results were obtained for the other three factors.
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Figure 11. Wavelet transform maps of ciliary body peripheral dimension in rabbit
(top) and human (bottom) data. The
abscissa represents spatial position in the data series and the
ordinate represents periodicity. Dark
areas: Regions of high wavelet spectral density;
superimposed semicircle: region within which valid
results are expected. The rabbit shows a dominant band with a period
0.5 to 0.75 mm across the entire series. In the human, periodicities
are more intermittent, appearing in the region from 0.3 to 0.6 mm.
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Fourier and wavelet findings was consistent with the spacing of
processes as seen in the 3-D images shown in Figures 4
and 6 . In Figure 4
(rabbit), six processes are seen over the 3.2-mm width of the scan
series. The image also shows that the spacing between successive
iridial processes can be somewhat irregular. In Figure 6
(human) seven
to eight processes are seen over a 3.6-mm range.
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Discussion
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In this study we demonstrated that not only can the ciliary
processes of human and animal eyes be visualized three dimensionally,
but that quantitative descriptors can be defined that might be useful
for clinical studies related to ciliary body function. These
descriptors relate to the surface area, volume, and degree of
convolution of the ciliary processes. They differed significantly
between rabbit and human eyes and also showed periodicities that
corresponded to the spacing of successive processes.
The significance of differences between the values of the above
quantitative descriptors between human and rabbit eyes and between scan
planes encompassing and not encompassing individual processes is not
that we can prove that these anatomic differences exist. This is
evident by casual inspection of the 3-D images. Rather, the
significance is that these descriptors can be used in experimental
studies as quantitative measures of anatomic morphology.
Our goal is to develop improved systems that allow scanning at higher
frame rates with consequent reduction of the time necessary to acquire
high-resolution 3-D data sets. The methods described in this report
will allow study of ciliary body functional anatomy in glaucoma,
hypotony, aging, and other conditions in the clinical population.
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Footnotes
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Supported by National Institutes of Health Grant EY01212, the Dyson
Foundation, and Research to Prevent Blindness.
Submitted for publication August 14, 2000; accepted November 3, 2000.
Commercial relationships policy: N.
Corresponding author: Ronald H. Silverman, Department of Ophthalmology,
Weill Medical College of Cornell University, 1300 York Avenue, Room
A855, New York, NY 10021.
prof{at}boodle.med.cornell.edu
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References
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Top
Abstract
Introduction
