British Journal of Ophthalmology, 1985, 69, 86-91

In vitro studies with a pulsed neodymium/YAG laserS VENKATESH,' S GUTHRIE,2 W S FOULDS,' W R LEE,'F R CRUICKSHANK,2 AND R T BAILEY2

From the ' Tennent Institute of Ophthalmology, University of Glasgow; and the 2Department ofPureand Applied Chemistry, University ofStrathclyde, Glasgow

SUMMARY The relationships between the destructive effects of Q-switched Nd/YAG laser pulsesand a number of experimental parameters were studied for various target materials including inparticular excised, fixed samples of human trabecular meshwork. The laser parameters alteredwere the pulse energy, the convergence angle of the focused beam, and the position of the focus ofthe beam relative to the target's axial position. The main finding was that it was possible to makedeep holes, of a diameter less than 100 ,um, in virtually transparent samples of trabecular meshworkwith a laser delivery system of 6° convergence and pulse energies of 14 mJ or more. The relevanceof this and the other experimental results to the development of a reliable system for performinginternal trabeculotomies for the treatment of open-angle glaucoma is presented.

Over the past few years interest has grown in thepossible applications of high power pulsedneodymium/YAG lasers in ophthalmology, particu-larly in the destruction of transparent tissues. Mode-locked Nd/YAG lasers, which give extremely highpower and low energy pulses, have been used success-fully clinically to open the anterior lens capsulebefore extracapsular extraction, to pierce secondarymembranes, to treat pupillary block and updrawnpupil, and to cut vitreous bands.' 2 Q-switched neo-dymium/YAG lasers, which give higher pulseenergies. but lower peak powers, have been usedexperimentally on tissues of the anterior chamber,3and more recently clinical success had been reportedin a range of treatments-iridectomy, membran-otomy in pseudophakia, membranotomy and dissec-tion of vitreous bands,4 coreplasty, and the division ofsynechiae.5Our long term objective is to develop a system

based on a Q-switched Nd/YAG laser to performinternal trabeculotomies as a possible treatment foropen-angle glaucoma. Previous attempts by others touse Q-switched pulses to treat open-angle glaucomahave not been successful possibly because of thecollapse of Schlemm's canal around the site of thelaser hole.6 Cases where new channels into thesuprachoroidal space have been made have also failedto produce a lasting reduction in intraocular

Correspondcnce to Dr S Vcnkatcsh, Tcnnent Institutc of Oph-thalmology, University of Glasgow, Glasgow GIl 6NT.

pressure .7We would hope to avoid the risk ofinducingSchlemm's canal to collapse by making laser holes ofvery small diameter, say 50 ,um, spaced around theband, each penetrating the meshwork through to thecanal. The demands of such a technique on precisionof focusing, reproducibility of the laser output, andconfidence in its predicted effect would be great, and,as a first step towards our objective, a series ofsystematic studies was carried out to investigate therelationships between the observed destructiveeffects of Q-switched pulses and experimentalparameters such as pulse energy, convergence angleof the focused beam, and the nature of the target.

Materials and methods

The laser system used in these studies was the Hyper-YAG 2000 produced by JK Lasers Ltd. It wasoperated at its fundamental wavelength of 1 06 ,um.Pulse durations were compressed to between 30 and40 ns by Q-switching, so that high powers could beachieved with relatively low energies. A fast photo-diode detector (Centronic APD 05 4R) was used tocheck the pulse duration occasionally. One of itstypical output traces, proportional to pulse powerversus time, is shown in Fig. 1.The laser pumping voltage could be altered to vary

the pulse energy. The actual pulse energy output wasmeasured before and after each experiment with aphotodiode based energy monitor (JK Lasers Ltd).In these experiments the pulse energies used were in

86

In vitro studies with apulsedneodymiumlYAG laser

TIME INTENSITY

)J . m1.4 I.

DISTANCE

Fig. 1 Temporal andspatial intensity profiles ofQ-switchedNdlYAG pulses.

the range from 3 mJ to 30 mJ corresponding to pulsepowers in the range from 105 to 106 W.The laser output was carefully channelled into its

fundamental transverse mode, which offered twoadvantages-the theoretical limit for the focusedbeam diameter is smaller than for other modes, andthe variability in the pattern of intensity from onepulse to another is minimised. The mode pattern ofthe unfocused Nd/YAG beam was measured period-ically with a photodiode array detector (EG and GReticon RL 512G), and adjustments were made tothe laser cavity optics if necessary to regain thefundamental mode. Fig. 1 shows a typical outputtrace proportional to pulse power versus distance.From such spatial intensity profiles the beam diameterwas found to be 1-5 mm. (The diameter is measuredacross the beam at the points where the signal intensityis l/e (that is, 1/2*718) of its peak value.)

Fig. 2 shows the experimental arrangement usedfor these studies. A low power continuous wavehelium neon laser beam was directed coaxially intothe invisible Nd/YAG beam to mark its path andallow visual focusing. The delivery system suppliedwith the laser was removed, and the two beams were

delivered to the target through a beam expandingtelescope and a single focusing lens. Different lensescould be used in this position to converge theNd/YAGbeam at different angles.

Coaxial Nd/YAG Targetand tissueHe/Ne lserbeams

-_________

BSem expandig Focusing lenstelescope

The target was mounted on a micropositioningstage, so that its position along the axis of the beamscould be altered and set with precision relative to thepoint at which the HeNe beam could be seen to befocused. The viewing system used to observe thisfocusing, and so position the target, was quiteseparate from the laser focusing optics and con-sisted of a binocular microscope with fibre opticillumination.

Glutaraldehyde-fixed human tissue, obtained atnecropsy was used in this study. As the eventualobjective was to make holes through the outflowsystem, many of the experiments used excised piecesof anterior chamber angle tissue with the uvealmeshwork surface facing the NdIYAG beam. Insome cases a suspension of saccharated iron oxidewas injected into Schlemm's canal in an attempt tomimic the in vivo situation when blood might bepresent in the canal and, if so, might affect the tissueresponse to the laser radiation. Corneal tissue wasalso used as a target, because in the clinical applica-tion ofthis work the convergingNd/YAG beam wouldpass through the cornea on its way to the meshwork.It was therefore necessary to knowwhether the corneawould be likely to suffer damage when situated only afew mm away from the focal spot of the Nd/YAGbeam.A smooth, transparent plastic material (Mylar)

was also chosen to act as a relatively homogeneousand reproducible non-biological target.The morphology of the destructive effects of the

Nd/YAG laser pulses on the various targets wasstudied primarily by scanning electron microscopy(Jeol Ltd JSM-T200), for which the tissue sampleswere prepared by critical point drying. Image analysis(Micro Measurements Ltd, Optomax) was carriedout on the scanning electron micrographs to givemeasurements of hole areas, and a stereoscope with aparallax measurement unit (Cartographic Engin-eering Ltd) was used on tilted pairs of micrographs togive hole depth measurements. Some of the scannedtissue specimens were reprocessed for Aralditeembedding by a technique to be described elsewhere,and 1-0 ,gm toluidine blue stained sections were

Binocular

microscope

J fibre-optic

illumination

Fig. 2 Schematic diagram ofexperimental delivery system.

Stage, mobile in 3Dby micrometeradjustments

87

S Venkatesh, S Guthrie, WS Foulds, WR Lee, FR Cruickshank, andR TBailey

Fig. 3 Laser hole made by a single30mJpulse converging at 2° on totissuepositioned at the HeNefocus.M= Uveal meshwork; R=Redblood cells.

studied by light microscopy (Leitz Dialux 20/WildPhotoautomat MPS 45).

Procedure and results

Six sequential sets of experiments were carried out asfollows:Meshwork tissue at low convergence angle. A lens

of focal length 52mm was used to focus the NdIYAGbeam on to the uveal surface of meshwork samples.This would give a beam convergence angle of only 20,much lower than we expected to use in our in vivoapplication, but it did offer a long working distance,which was very useful in this preliminary work. Singlepulses of 30 mJ each were fired into tissue positionedat the point of focus of the HeNe beam. Determiningthis focal point with a precision of even 100 gm wasfound to be difficult.

Scanning electron microscopy of the tissue samples

subsequently showed well defined holes at 13 out of22 target sites. Holes that were clearly seen topenetrate through to Schlemm's canal were made infive cases. Fig. 3 shows one of these holes.Measurements of hole area and depth yielded

coefficients of variation of over 70% indicating greatvariability in the dimensions of holes made intrabecular tissue under apparently identical con-ditions. In nine cases no hole was made at all. Asimpler target material was therefore chosen for thenext set of experiments.Mylar at low convergence angle. Single pulses of 10

mJ were fired into plastic placed at a series ofpositionsbetween 0*5 mm in front of the HeNe focus and 3-5mm beyond it. The plastic was then coated with goldand observed by scanning electron microscopy.

Large variations were again found in hole area anddepth at a given target position shown by coefficientsof variation of approximately 50%. Possibly because

11.0 mJ - +

6.0 mJ

CORNEAL ENDOTHELIUM at 30.0 mJ

CORNEAL ENDOTHELIUM at 13.0 mJ

CORNEAL EPITHELIUM at 11.0 mJ

-1 .0

+

+ + + + + + -

- _-++ + + ++ -

- + ++ ++ -

_-++ +--

0

TARGET DISPLACEMENT FROM HeNe FOCUS (mm)Fig. 4 Effects ofNd/YAG pulses at a convergence angle of6° targeted on Mylarandhuman cornea. + =Hole made; -=Nohole made.

MYLAR at

MYLAR at

+ ++ + + + + ++ -

+1 .0

88

In vitro studies with apulsed neodymiumlYAG laser

NORMAL MESHWORK at 30.O mJ - + ++

20.0 mJ

16.0 mJ

+ * + * + * + +

+

+

COLOURED MESHWORK at 30.0 mJ

of 9.6 mJ

6.0 mJ

of 3. 0 mJ

-1 .0

4+ + + +

0 +1 .0

TARGET DISPLACEMENT FROM HeNe FOCUS (mm)

Fig. 5 Effects ofNdlYAG pulses ata convergence angle of6° targeted on normaland artificially colouredhuman meshwork.+ =Hole made; * =Hole extending through to Schlemm 's canal made; - =No hole made.

of this no systematic change in area or depth withtarget position was observed. The main finding wasthat all 75 target sites fired at did show laser damage,though the target position had varied over a range of4 mm. The effective depth of focus of this deliverysystem was at least 4mm then, obviously too large forthe intended clinical application. The focusing lenswas therefore replaced with one ofshorter focal lengthfor the next set of experiments.Mylar at high convergence angle. A lens of focal

length 14-6 mm was used to focus NdIYAG pulses at

a convergence angle of 6° on to pieces of Mylar heldat a series of positions between 1.2 mm in front of theHeNe focus and 1-4 mm beyond it. Single pulses ofeither 6 mJ or 11 mJ were used.The results are indicated in the upper section of

Fig. 4. They show that 11 mJ made holes over a targetposition range of approximately 2 mm, so the depthof focus of the delivery system had indeed beenreduced. It can also be seen that the depth of focuswas reduced from 2 mm to approximately 1 mm bylowering the pulse energy to 6 mJ. While carrying out

Fig. 6 A toluidine blue stained

lItu section oftissue previouslypositionedO-5mm beyond the

HeNefocus and exposed to a single30 mJpulse at 60 cohvergence.

S=Schlemm's canal; L=Laser

hole; M= Uveal meshwork.

~~~~~(x298).

" 14.0 mJ

of 11.0 mJ

of 9.6 mJ

7.2 mJ

6.0 mJ

89

- - -

S Venkatesh, S Guthrie, WS Foulds, WR Lee, FR Cruickshank, and R TBailey

this set of pulse firings we found it possible to locatethe HeNe focus with a precision of approximately 10g,m with this high convergence system. These resultswere encouraging enough to warrant tissue experi-ments with the same system.Cornea athigh convergence angle. Nd/YAG pulses.

converged at an angle of 60, were fired into cornealtissue held at positions between 300 gim in front of theHeNe focus and 400 ,um beyond it. Single pulses of 30mJ, 13 mJ, or 11 mJ were fired into each target site. Insome cases the corneal tissue was positioned with itsepithelial surface facing the Nd/YAG beam. In othersthe endothelial surface was exposed instead.The results are indicated in the lower section of

Fig. 4. It was found that even at 30 mJ, Nd/YAG

Figs. 7A and B Scanning electronmicrographs oftissue exposed toNdl/YA G pulses at 60 convergence.

A: EffectofoneM3Opulsefiredinto tissue position at 0-2 mmbeyond the HeNefocus. M= Uvealmeshwork.

Fig. 7B:_Effectsoftwo14m pulsesfired side by side into tissuepositionedfirst at the HeNefocusthen 0-4mm beyond it. M= Uvealmeshwork; S=Schlemm's canal;C= Collector channel.

pulses caused damage only if the corneal surface waspositioned between 100 gim in front of the HeNefocus and approximately 300 ,um beyond it. Sucha small depth of focus for damage in the corneasuggested that the 60 convergence angle systemwould be acceptable in that context for our eventualin vivo application.Normal meshwork at high convergence angle.

Excised samples of meshwork were held at a series ofpositions between 1-5 mm in front of the HeNe focusand 1-6mm beyond it. Eight different NdIYAG pulseenergies in the range 6 mJ to 30 mJ were tried, firing asingle pulse into each target site as before.The results are indicated in the upper section of

Fig. 5. There were four main points of interest in

90

In vitro studies with apulsed neodymiuml/YAG laser

them. The effective depth of focus for making holesin meshwork with 30 mJ pulses was approximately2-4 mm centred at the point of focus for the HeNebeam. This depth of focus was reduced as pulse-energy was lowered. The threshold for meshworkdamage was about 7 mJ. The deeper holes were madewhen the target was closer to the HeNe focal point.

It was found that holes as small as 50 ,um indiameter but extending into Schlemm's canal couldbe made. Fig. 6 is a photomicrograph of a toluidineblue stained section cut through one of the laserholes. Fig. 7 shows scanning electron micrographs ofsome others.

Stained meshwork at high convergence angle. Fourvalues of pulse energy in the range 3 mJ to 30 mJ were

tried with the artificially coloured meshworksamples. In other respects the experimental protocolwas as for the previous set of experiments.The results are shown in the lower section of Fig. 5.

It was found that 30 mJ pulses made holes over

approximately the same range as for unstained tissue,but the threshold of damage was much lower. Eventhe lowest Q-switched pulse energy available withour system, 3 mJ, resulted in a hole, and one pulse ofonly 6 mJ made a hole through to Schlemm's canal.These results can be explained as follows. Laser

damage by Q-switched pulses can occur by non-linearprocesses of fundamental structural breakdown,8 butonly at very high power densities, of the order of 1010watt/cm2. These processes would be expected toapply to transparent and coloured matter equally,and our results at a pulse energy of 30 mJ confirmthis. At lower power densities damage can occur bynormal, linear absorption processes. The effective-ness of these processes would be expected to dependon the properties of the target matter, in particular itscolour. Our results below 7 mJ fit this reasoning.

Discussion

This series of in vitro experiments has yielded in-formation of interest and value in furthering ourdevelopment of a noedymium/YAG laser system forperforming internal trabeculotomies. The initialwork, done with a small YAG convergence angle,showed the feasibility of making discrete holesthrough meshwork but also showed problems of poorreproducibility of the effects in terms of the dimen-sions of the holes made. This variability was found todepend partly on the irregularity of the tissue surface,as it improved slightly if a smooth plastic surface wasused instead, but also on the difficulty found in

accurately determining the focal point of the HeNemarker beam.When the convergence angle of the two laser

beams was increased by a factor of three, the focalpoint of the HeNe beam could be found more reliably,and at the same time the effective depth of focus fortheYAG pulses was significantly reduced resulting ina more useful delivery system.

This system enabled the effects of displacing thetarget from the geometric focus, of lowering pulseenergy, and of changing the nature of the target to beobserved. Although these effects could have beenpredicted qualitatively, our system allowed precisemeasurements of the changes and correspondingeffects to be made. It also showed that holes of therequired morphology-diameter 100 gim and depthsufficient to reach Schlemm's canal-could be createdquite easily.

Similar in vitro studies are currently being pursuedwith fresh tissue rather than fixed being used, an evenlarger beam convergence angle, and a speciallydesigned gonioglass, to approach the in vivo situationmore closely. In this way we hope to gather system-atically the data on which a clinical neodymium/YAGlaser system and operating protocol can be soundlybased.

This study was supported by the Rank Prize Fund (Grant No 7269).

References

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2 Aron-Rosa D, Cohn HC, Aron T. Use of a pulsed neodymium-YAG laser (picosecond) in endocular surgery. In: Birngruber R,Gabel V-P, eds. Laser treatment and photocoagulation of the eye.Amsterdam: Junk, 1984.

3 Van der Zypen E, Bebie H, Fankhauser F. Morphological studiesabout the efficiency of laser beams upon the structures of the angleof the anterior chamber. Int Ophthalmol 1979; 1: 109-22.

4 Fankhauser F, Lortscher H, Van der Zypen E. Clinical studies onhigh and lower power laser radiation upon some structures of theanterior and posterior segments of the eye. Int Ophthalmol 1982;5:15-32.

5 Fankhauser F, Roussel P, Steffen J, et al. Clinical studies uponsome structures of the anterior segment of the eye. Int Ophthalmol1981;3: 129-39.

6 Van der Zypen E, Fankhauser F. The ultrastructural features oflaser trabeculopuncture and cyclodialysis. Ophthalmnologica 1979;179: 189-20().

7 Van der Zypen E, Fankhauser F. Lasers in the treatment ofchronic simple glaucoma. Trans Ophthalmol Soc UK 1982; 102:147-53.

8 Bloembergen N. Laser-induced electric breakdown in solids.IEEEJ Quant Electr 1974; QEIO: 375-86.

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