Ophthalmic Surgery Lasers and Imaging 39, 485-490

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e x p e r i m e n t a l
S c i e n c e
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Femtosecond Laser Photodisruption of Porcine
Anterior Chamber Angle: An Ex Vivo Study
Yaoming Liu, PhD; Hiroshi Nakamura, MD, PhD; Tana Elizabeth Witt; Deepak P. Edward, MD;
Robert J. Gordon, PhD
n BACKGROUND AND OBJECTIVE: The
femtosecond laser is a promising tool for the micromachining of biological tissues. The current study
investigated the feasibility of noninvasive glaucoma
therapy by femtosecond laser ablation in enucleated
porcine eyes by delivering the laser energy through a
gonioscopic lens.
n MATERIALS AND METHODS: Freshly enucleated porcine eyes were kept in Optisol GS solution
(Bausch & Lomb, Rochester, NY) at 4°C for up to 48
hours. Photodisruption was attempted at the peripheral inner surface of the cornea using a gonioscopic lens
and a custom femtosecond laser ablation system. Femtosecond laser ablation was performed using a Ti:Sapphire laser (800-nm wavelength) focused with a 0.15
numerical aperture lens. The laser treatment process
was recorded by real time video and the lesions were
examined histologically.
From the Department of Chemistry (YL, TEW, RJG) and the Department
of Ophthalmology and Visual Sciences (HN, DPE), University of Illinois at
Chicago College of Medicine, Chicago, Illinois.
Accepted for publication October 3, 2007.
Supported by grants from NSF CHE-0640306 and American Health Assistance Foundation, Clarksburg, Maryland; and Research to Prevent Blindness,
Inc., New York, New York.
The authors thank Sima Singha for her assistance in setting up the optical
delivery system and Xin Li for assistance in histopathologic examination.
Address correspondence to Robert J. Gordon, PhD, Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, Room 4500,
Chicago, IL 60607-7061.
Femtosecond Laser Photodisruption · Liu et al.
n RESULTS: During laser surgery, bubble formation
was observed and appeared to be positively related to
laser energy and exposure time. When examined histologically, areas of ablation were consistently observed
at the peripheral inner surface of the cornea. The extent of the lesion increased nonlinearly with both pulse
energy and exposure time, showing saturation at high
energy and long times. For a given energy dose, fewer
pulses of high energy were more effective than many
low energy pulses.
n CONCLUSION: The current study demonstrates
that laser ablation at the anterior chamber angle is feasible by a femtosecond laser ablation system using a
gonioscopic lens in ex vivo eyes. The results indicate
the potential of a novel noninvasive glaucoma laser
therapy.
[Ophthalmic Surg Lasers Imaging 2008;39:485-490.]
Introduction
In glaucoma surgical therapy, there is a great need
for a simple, effective, and noninvasive surgical procedure. As a noninvasive surgical therapy, laser trabecular
ablation with Nd:YAG1,2 and Er:YAG3,4 lasers has been
attempted previously. These treatments were ineffective
or were not widely accepted.4 The primary reason for
the failure of laser trabecular ablation is related to the
thermal effects of the lasers used for treatment.3-7 For
485
Figure 1. Schematic diagram of the
optical delivery system for ex vivo laser surgery. M = mirror; BS = beam
splitter, L = lens; D = detector; H =
half-wave plate; GL = gonioscopic
lens. Inset: Sketch showing the path
of the laser beam in the eye.
pulse durations of several nanoseconds or longer, thermal and mechanical relaxation occurs during the laser
pulse and competes with material ablation, resulting in
thermal damage of tissues and scar formation.8
The use of an ultrashort, sub-picosecond pulsed
laser confines the thermal and mechanical stresses to
a small volume. Depending on the properties of the
tissue, a significant amount of material may be ablated before thermal diffusion, shock propagation, and
cavitation occur and thermal and mechanical collateral damage are reduced.7 Previously, our laboratory
showed that photodisruption by a femtosecond laser
can be employed to create lesions in the human trabecular meshwork from corneoscleral rim tissues of donor
eyes without damaging the surrounding tissues.9 The
current study was undertaken to extend our previous
efforts to ex vivo eyes. We investigated the feasibility of
a noninvasive glaucoma therapy by delivering femtosecond laser energy to enucleated porcine eyes through
a gonioscopic lens.
Materials and Methods
Laser and Delivery System
The optical delivery system is shown schematically
in Figure 1. Briefly, a mode-locked Ti:Sapphire Oscillator (Tsunami; Spectra-Physics, Mountain View, CA) 486
pumped by a diode laser (Millennia V; Spectra-Physics) generates 82 MHz, 45 femtosecond pulses. The oscillator output is amplified by a regenerative amplifier
(Spitfire; Spectra-Physics) to produce 800 nm, 45 femtosecond pulses with energies of 2 mJ/pulse at a 1 KHz
repetition rate with a beam diameter of 9 mm. A halfwave plate and a polarizer placed between mirror M3
and beam splitter M5 are used to attenuate the pulse
energy to a desirable level. To obtain a high numerical
aperture (NA), the beam is expanded to a diameter of
30 mm by lenses L3 and L4, producing an NA of 0.15
at lens L5.
We used a He-Ne (632.8 nm) laser as a guiding
beam because the surgical femtosecond laser beam is
barely visible at 800 nm. To match the diameter of the
surgical beam, the He-Ne laser beam was expanded by
lenses L1 and L2. Both the guiding He-Ne and the surgical femtosecond laser beams were combined at the
beam splitter BS2 to produce coaxially propagating
beams. Both beams were then focused by lens L5 (f =
100 mm) and directed by the beam splitter BS3 to the
enucleated eye using a gonioscopic lens as described
below.
The illumination beam from the light bulb of a
surgical microscope (OPMI-6; Carl Zeiss Meditec
Vertriebsgesellschaft mbH, Jena, Germany) was transmitted through the same beam splitter and focused on
Ophthalmic Surgery, Lasers & Imaging · November/December 2008 · Vol 39, No 6
the target by the gonioscopic lens. The procedure was
viewed by an operator through the eyepiece of the microscope or via a television monitor. The focal point
of both laser beams was finely adjusted by lens L5,
which was mounted on a motorized computer-controlled three-dimensional translation stage. The laser
treatment time was precisely controlled by a computer,
which triggered the Pockell’s cell in the amplifier.
Laser Procedure in Porcine Eyes
Freshly enucleated porcine eyes were kept in Optisol GS solution (Bausch & Lomb, New York, NY)
at 4°C for up to 48 hours before use. Photodisruption was conducted at the peripheral inner surface of
the cornea using a gonioscopic lens (OMVGL Magna
View Gonio Laser Lens; Ocular Instruments, Inc., Bellevue, WA) and the custom femtosecond laser ablation
system described above.
A two-beam focusing technique was developed because precise focusing of the laser beam to the target
is critical to obtain consistent photodisruption. The
He-Ne guiding beam, which is coaxial with the surgical
femtosecond laser beam, was split into two beams at lens
L2. When the guide beam focused onto the target, the
two visible beams coalesced. A single spot of the guiding
beam indicated precise focusing on the target because
these beams overlap each other only when the target is at
a focal point. Variable settings of laser energy and exposure times (energy = 40 to 240 µJ; exposure time = 0.02
to 0.50 second) were used to investigate the relationship
between lesion size and laser parameters. The laser treatment process was recorded by a real time video system
(DXC-760MD; Sony, Minato, Tokyo, Japan).
Histopathologic Examination
After laser surgery, porcine eyes were fixed with
10% buffered formalin for 24 hours. After the eyes
were dissected, processed, and embedded in paraffin,
5-micron thick serial sections of the anterior segment
were prepared. The sections were stained with hematoxylin and eosin to assess the laser lesion at the peripheral inner surface of the cornea. To evaluate the
relationship between properties of the lesions and the
laser parameters, the section with the largest lesion was
selected from the serial sections prepared from each
photodisruption. A straight-line measurement was
made between each edge of the corneal inner surface
of the lesion. Hereafter, this distance will be referred
Femtosecond Laser Photodisruption · Liu et al.
to as the lesion length. Photographs were taken using
Zeiss Axiovision software 4.2 and Axioscope 2 (Carl
Zeiss MicroImaging, Thornwood, NY). The lesion
length was measured using ImageJ (National Institutes
of Health, Bethesda, MD).
Results
By using the split-focus technique described
above, a precise focus of the guide laser on the target
was obtained with relative ease. Bubble formation was
observed during laser surgery, as has been previously
described,10 and such bubble formation appeared to be
positively related to laser energy and exposure time (a
video of this phenomenon is available from the authors
upon request).
When examined histologically, areas of ablation
were consistently observed at the peripheral inner surface of the cornea of the porcine eyes. The ablated lesions were observed to have a trough-like shape. The
edges of the lesions were sharp, and there was no evidence of a coagulative effect in any of the photodisruptive lesions examined (Fig. 2). The extent of the lesion
correlated significantly to both laser pulse energy and
total exposure time. The lesion length increased nonlinearly with both energy and time, showing saturation
at high energy and long time.
The dose response is shown graphically in Figure 3.
The variation of lesion length with pulse energy for a
fixed exposure time of 0.5 second is fitted in Figure 3A
to the functional form
L = A(1 – ea(E - E0))
(1).
The fitted result is A = 141 ± 17 µm, a = 0.031 ± 0.021
µJ-1, and E0 = 31.2 ± 9.0 µJ, with R2 = 0.949. The variation of lesion length with exposure time for a fixed
pulse energy of 240 µJ is fitted in Figure 3B to the
functional form
L = B (1 – ebt)
(2).
The fitted result is B = 181 ± 7 µm and b = 17.8 ±
2.5 s-1, with R2 = 0.992. Figure 3C displays the same
data of panels (A) and (B) plotted against the total energy dose (pulse energy times number of pulses). It is
apparent that for a given dose, fewer pulses at higher
pulse energy are more effective than more pulses of
487
A
A
B
B
C
C
Figure 2. Photomicrographs of laser lesions. Eyes ablated with
the femtosecond laser were stained with hematoxylin and eosin to
assess laser lesions. (A) Pulse energy = 240 µJ; exposure time =
0.02 s. (B) Pulse energy = 240 µJ; exposure time = 0.50 second.
(C) pulse energy = 60 µJ; exposure time = 0.50 second. Arrow =
lesion; Double headed arrow = lesion length; Arrow head = Descemet’s membrane; S = corneal stroma; I = iris; TM = trabecular
meshwork; black bar = 50 µm.
488
Figure 3. (A) Lesion length versus pulse energy at a fixed exposure time of 0.50 second. The curve is a least-squares fit to Eq. (1).
(B) Lesion length versus exposure time at a fixed pulse energy of
240 µJ. The curve is a least-squares fit to Eq. (2). (C) Same data
as in panels (A) and (B) plotted as a function of total energy dose
(pulse energy times number of pulses).
Ophthalmic Surgery, Lasers & Imaging · November/December 2008 · Vol 39, No 6
Table 1
Table 2
Lesions Created With Different Energies at a
Fixed Exposure Time of 0.5 Second
Lesions Created With Different Exposure
Times at Fixed Energy of 240 µJ/pulse
Total
Energy
(mJ)
Average
Lesion
Length (µm)
Standard
Deviation
(µm)
39.6
19.8
29.4
2.9
0.02
4.80
59.90
2.60
60.0
30.0
92.4
4.1
0.10
24.00
145.90
13.50
120.0
60.0
117.3
4.7
0.50
120.00
183.50
13.10
240.0
120.0
150.2
8.3
Energy/
Pulse (µJ)
lower pulse energy. The error bars indicate a single
standard deviation corresponding to a sample size of 2
to 4 lesions for each point.
The quantitative results are summarized in Tables
1 and 2. Table 1 displays the length of the lesions obtained with different pulse energies at a fixed exposure
time of 0.5 second. Table 2 displays the lesions obtained
with different exposure times at a fixed pulse energy of
240 µJ. We also list the total energy dose for each set of
conditions in column 2 of both tables.
Discussion
We reported the ablation of the peripheral inner
surface of the cornea in ex vivo eyes using a femtosecond laser with a gonioscopic lens. The femtosecond
laser is a promising tool for micromachining biological
tissue11 because of the fundamental advantages in using ultrashort Ti:Sapphire lasers for ablation compared
with other types of lasers. First, the 800-nm wavelength
of the Ti:Sapphire laser falls within the absorption window of most tissues, allowing radiation to be focused
to a point within the target (eg, the cornea) without
damaging the outer layers.7
Second, the use of sub-picosecond pulses allows
the radiant energy to be absorbed on a time scale that is
much shorter than both the thermal diffusion and shock
wave propagation times. This property leads to thermal
and stress confinement, thereby reducing the region of
material damage to the vicinity of the laser focus.7
Third, the lower fluence threshold for femtosecond
laser ablation reduces the overall thermal and mechanical load on the tissue.7 Therefore, the femtosecond laser appears to be useful for noninvasive trabecular ablation. The laser lesions after ablation were consistently
observed, the edges of lesions were sharp, and there
Femtosecond Laser Photodisruption · Liu et al.
Exposure
Time (sec)
Total
Energy
(mJ)
Average
Lesion
Length (µm)
Standard
Deviation
(µm)
was no evidence of a coagulative effect, indicating that
a noninvasive laser trabecular ablation is feasible by a
femtosecond laser ablation system.
Porcine eyes were used in this feasibility study because of their availability and because the volume and
size of the porcine eye (its anterior chamber particularly) are closer to those of the human eye than any
other species reported.12 In the porcine eye, however,
the ciliary cleft and trabecular meshwork are bridged
anteriorly by robust pectinate ligaments that precede
fine uveal cords, both of which form the uveal or iridocorneal meshwork, a pattern common in other nonprimates.12,13
In our preliminary experiments, it was revealed that
consistent photodisruption on the uveal or iridocorneal meshwork in the porcine eye was difficult to achieve
because of the nonprimate–mammalian structure of
the outflow system (data not shown). In addition, laser
ablation of the pectinate iris ligaments was clearly observed during the procedure (data not shown). It was
also discovered that it was difficult to evaluate lesions
in ligaments quantitatively by histologic or other approaches. Therefore, the peripheral cornea in the porcine eye at the anterior chamber angle was chosen for
the site of ablation.
Precise focusing of the laser was revealed preliminarily to be critical in ex vivo experiments because
the laser procedure was performed under unfavorable
conditions (ie, blurred observation through a manually supported gonioscopic lens and somewhat edematous cornea in the enucleated eye) compared with the
in vitro experiments.9 Split-beam focusing of the guide
laser alleviated this problem and allowed us to consistently obtain lesions with the femtosecond laser ablation system. Using a femtosecond laser, an earlier study
using in vivo rabbit and monkey eyes failed to produce
489
deep retinal lesions, and that failure was attributed to
bubble formation.10 We observed bubble formation in
our study but were able to produce consistent lesions.
From the current study, we conclude that precise focusing is the most critical factor and can compensate for
bubble interference.
Furthermore, the lesions displayed trough-shaped
craters compared with the cylindrical shape observed in
our previous in vitro study.9 To ablate the cornea in this
ex vivo study, higher doses with a total energy ranging
from 4.8 to 120 mJ were used. In addition, the laser
beam was focused on the target by the gonioscopic lens
at a more grazing angle. Higher doses or a grazing angle
of incidence resulted in trough-shaped lesions.
Quantitative measurements of the lesion length revealed a positive, nonlinear correlation with pulse energy and exposure time. At a fixed exposure time of 0.5
second (ie, 500 laser shots), a threshold energy of 31 ±
9.0 µJ/pulse was observed, with a saturation depth of
141 ± 17 µm occurring at pulse energies greater than
approximately 150 µJ. At a fixed pulse energy of 240
µJ, no time threshold was observed and a saturation
depth of 181 ± 7 µm was obtained at exposure times
greater than approximately 0.2 second. Although we
expect these results to be qualitatively general, the specific numerical values are likely sensitive to the NA of
the focusing lens, which was 0.15 in the current study.
Of particular interest is the comparative effectiveness of pulse energy versus exposure time for a fixed
total energy dose. Figure 3C shows that fewer pulses of
higher energy are more effective for a fixed dose than
many low-energy pulses. This effect may be explained
by the possibility that interference by bubble formation
increases with the number of pulses. This observation
may prove to be a useful guide for selecting the most effective laser parameters for future clinical applications
of femtosecond laser surgery.
The current study demonstrates laser ablation at
the anterior chamber angle produced by a femtosecond
laser ablation system using a gonioscopic lens in ex vivo
eyes. Our results underscore the potential effectiveness
of a novel noninvasive trabecular glaucoma laser therapy in the form of laser trabecular ablation. This approach will be tested in the trabecular meshwork with
primate eyes.
490
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