Lasers - Douglas hamilton, conia - 2001 - thermal effects in laser-assisted pre-embryo zona drilling

Journal of Biomedical Optics 6(2), 205–213 (April 2001)

Thermal effects in laser-assisted pre-embryo zonadrilling

Diarmaid H. Douglas-HamiltonHamilton Thorne Research102c Cummings CenterBeverly, Massachusetts 01915

Jerome ConiaCell Robotics Inc.2715 Broadbent Parkway NEAlbuquerque, New Mexico 87107

Abstract. Diode lasers @l51480 nm# are used with in vitro fertiliza-tion to dissect the zona pellucida (shell) of pre-embryos. A focusedlaser beam is applied in vitro to form a channel or trench in the zonapellucida. The procedure is used to facilitate biopsy or as a promoterof embryo hatching. We present examples and measurements of zonapellucida ablation using animal models. In using the laser it is vital notto damage pre-embryo cells, e.g., by overheating. In order to definesafe regimes we have derived some thermal side effects of zona pel-lucida removal. The temperature profile in the beam and vicinity ispredicted as function of laser pulse duration and power. In a crossed-beam experiment a HeNe laser probe is used to detect thetemperature-induced change in the refractive index of an aqueoussolution, and estimate local thermal gradient. We find that the diodelaser beam produces superheated water approaching 200°C on thebeam axis. Thermal histories during and following the laser pulse aregiven for regions in the neighborhood of the beam. We conclude thatan optimum regime exists with pulse duration <5 ms and laser power;100 mW. © 2001 Society of Photo-Optical Instrumentation Engineers.[DOI: 10.1117/1.1353796]

Keywords: embryo; laser; temperature; zona pellucida; infrared; microscope.

Paper 20019 received May 23, 2000; revised manuscript received Nov. 21, 2000;accepted for publication Nov. 21, 2000.

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1 IntroductionA growing number of patients treated for infertility are under-going in vitro fertilization ~IVF! with assisted reproductiontechnologies. Some apparently healthy pre-embryos are impaired in their ability to hatch from their protective shell, thezona pellucida~ZP!, after in vitro fertilization. Fortunately, itis possible to initiate hatching of the pre-embryos in the laboratory, before reimplantation in the reproductive tract of apatient.1,2 In the laboratory, assistance is provided to thesepre-embryos by creating a notch or channel—a region ostress release—in the zona for the purpose of improving thchances of successful implantation. After transfer into theuterus the embryo hatches: it extrudes itself through the channel and implants into the uterine wall. The ZP may be pre-pared using a focused beam of infrared~IR! light, and laser-assisted hatching can result in improving implantation andpregnancy success rates.3 In addition, preimplantation genetictesting may also improve the success rate of IVF procedureA panel of tests has been reported that can identify chromosomal abnormalities in the developing embryo before implantation, thus reducing the rate of spontaneous miscarriage4

Here again, IR lasers may be usefully applied to facilitatebiopsy and the collection of important genetic clues from thepre-embryo in the laboratory.5,6

Precise channeling of the zona pellucida may be accomplished efficiently under microscopy using the ablative prop-erties of pulsed laser radiation. A laser beam is introduced inthe optical path of an inverted microscope and then tightly

Address all correspondence to Diarmaid H. Douglas-Hamilton. Tel: 978-921-2050; Fax: 978-921-0250; E-mail: [email protected]

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focused on the target zona pellucida using the same objeclens utilized to view the specimen. Since the pre-embryosfragile and valuable, the procedure must be well controland very reliable. The wavelength of light, the duration of tpulse, the laser spot size, and the light energy delivered arfactors that must be taken into consideration when evaluathe safety of the procedure. Ultraviolet wavelengthsavoided since the potential for mutagenic effects exists.contrast, these concerns are minimized with a near-infralaser source. A laser operating at wavelengthl51480 nmispreferred because water molecules absorb strongly atwavelength@attenuation coefficienta521 cm21 ~Ref. 7!#, anddestruction of tissue bonds is due mainly to heat transfer frwater. Microscopic dissection of the zona pellucida at 14nm was first reported for animal models, in particular wspecimens of murine origin. The well-established moumodel is advantageous because it is informative and thecome of laser treatment can be readily studied after culturthe pre-embryosin vitro for only a few days.

In experiments using a 1480 nm diode laser with a rangelaser pulse duration and intensity,8,9 channels from 5 to 20mmin diameter were drilled in the 7.5-mm-thick zona pellucida ofmouse pre-embryos. The reported laser pulse durationt wasin the range3,t,100 ms,and laser powerP in the range22,P,70 mW. Laser spot size was 2–8mm and laser en-ergy was 0.5–1.3 mJ. The safety of the procedure was evated by the absence of visible damage at the ultrastructand biological levels.9,10 No laser-induced structural alterations of the neighboring cytoplasm were observed after sc

1083-3668/2001/$15.00 © 2001 SPIE

Journal of Biomedical Optics d April 2001 d Vol. 6 No. 2 205

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ning and transmission electron microscopy. Improved implantation rate was also observed after transfer of the treated prembryos in surrogate mothers.3 After birth, the developmentof the mice was followed and no morphological anomaly wasdetected; the reproductive capacity of the mice was confirmeamong subsequent generations.

Laser-mediated zona ablation with the 1480 nm diode lasewas further applied to human pre-embryos. Laser-assistehatching in pre-embryos recently resulted in successful clinical pregnancies and healthy babies.11 In particular, assistedhatching of frozen–thawed pre-embryos with the 1480 nmlaser enhanced pregnancy outcome in patients who had seeral previous implantation failures.12 Opening of the zona pel-lucida and removal of degenerated pre-embryonic cells ofrozen–thawed pre-embryos also increased implantation anpregnancy rates.13

As more successful pregnancy results are published, inteest in laser applications with pre-embryos is likely to growamong both clinics and patients. However, as with any clini-cal application, the introduction of a new technology inevita-bly raises questions and/or fears of unexpected consequencIn particular, with all its promises of improvements, the ap-plication of laser technology to human pre-embryos must beprevented from being unintentionally harmful, for example,by overheating the blastomeres adjacent to the site of lasedelivery. In previous experiments, measurements of laseheating in beams focused in medium with heat-sensitive molecules introduced into cells were made atl51064 ~used foroptical tweezers!, by Liu et al.14 At 100 mW incident power,with l51064 nm(a50.1 cm21), they obtained steady statetemperature rise near the focus ofDT51.15°C. RecentlyCellier and Conia15 made interferometric longitudinal phase-change measurements, using 100 mW atl5985 nm (a50.42), and obtainedDT57.3°C. We can estimate the tem-perature rise that would have been obtained in these experments atl51480 nm.Since in a thermal-diffusion controlledsituation the temperature will scale asaP, at l51480 theabove temperature increases would scale toDT;242 and366°C, respectively. Such high values will cause significanlocal heating. A laser instrument for use in clinical practicemust therefore be designed with extensive safety consideations in mind. In this paper, we present the evaluation osome thermal side effects of zona pellucida removal by laseas a function of various laser parameters. Limits to local heating are estimated. The temperature profile in the focused lasebeam and in the nearby region as function of time is used tminimize potentially dangerous regimes of pulse duration andlaser intensity.

2 Preliminary Experiments2.1 Laser Module, Microscope Specifications andDesignWe used a Nikon TE-300 laser-equipped inverted microscopewhich has the IR laser beneath the stage. Laser light proceeup through the objective and is focused on the target. Thlaser module~ZLTS, Hamilton Thorne Research, Beverly,MA ! has maximum power 200 mW atl51480 nm,and isutilized in pulsed mode. Peak transmitted laser power througthe objective is;140 mW. Pulse duration is adjustable in halfmillisecond increments from 0.5 to 25 ms. In addition to the

206 Journal of Biomedical Optics d April 2001 d Vol. 6 No. 2

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laser diode itself, the laser module includes control boaadjustable collimating lens and dichroic mirror, and is intelocked to prevent potentially hazardous operation.

The laser module fits into the microscope filter cube sunder the stage, and the beam is deflected by a dichroic malong the optic axis of the microscope. The nearly collimabeam is directed toward the back aperture of a 403, 0.60numerical aperature~NA! objective lens, designed to maxmize transmission at 1480 nm. The long working distan(WD53 mm) objective lens is used to focus the laser beaon to the target specimen; it has high transmission ratio ininfrared range~typically ;71% at 1480 nm!. The collimationof the beam is adjusted to give parfocality of IR and visiblight. Biological specimens, e.g., bovine oocytes or moupre-embryos, are maintained in an aqueous culture mediuclear polystyrene Petrie culture dishes~Falcon, Lincoln Park,NJ!. The incident focused laser beam travels sequentithrough air, plastic, and then the aqueous solution bereaching the target area of the zona pellucida.

Delivered laser energy at the objective was measured ua thermal detector head~Ophir Optronics, Peabody, MA!placed immediately above the stage with objective removTransmission of the objective was determined by placingidentical opposed objective above the focal point to recomate the beam, which passed into the detector. Usingderived transmission, 71% atl51480 nm,maximum beampower at the focal point was estimated at 103 mW, allowfor reflection at the polystyrene Petrie dish and absorptthrough 75mm H2O. Pulse duration was checked using aoscilloscope~Tektronix TDS 360! monitoring laser radiationscattered on to a Ge photodiode detector~B1918-01, Ham-mamatsu, Bridgewater, NJ!. In a preliminary measurementhe focal beam radius in air was determined by interceptthe beam at the focal point with a knife edge, which wmoved across the beam at known rate while the IR intenwas monitored with the Ge detector. At the focus, the radto the 1/e2 intensity level wasR53.160.5mm. The focalcone half angle in the medium is 26.7°.

2.2 Examples: Laser-Assisted Zona Dissection withBovine EggsBovine eggs are readily available. They constitute a useand cost effective model for testing laser-assisted zona ding protocols. The Nikon TE300 inverted microscope is uswith illumination source and condenser above specimenobjective below. The Petrie dish specimen chamber~wallthickness 1 mm, diameter 50 mm! is on a motorized stageThe eggs settle at the bottom of the dish, and the objectivfocused on the edge of the ZP, about 75mm into the medium.

A typical zona drilling is shown in Figure 1 for a bovinoocyte. The egg diameter is;150 mm and the thickness othe zona pellucida, the translucent shell surrounding the eis ;12 mm. The laser power was set to 100 mW deliveredthe focal point, and the three holes depicted in Fig. 1 cospond to pulse duration 1.5, 3, and 4.5 ms. Beam diamete1/e2 was 6.2mm, independent of beam power. The hole dameters are 4.3, 7.8, and 10.5mm; the hole diameter dependmore on pulse duration than on laser beam size.

In Figure 2~a! an electron photomicrograph of severchannels in a bovine egg is shown. Repeated firing at cons

Thermal Effects in Laser-Assisted Pre-embryo

Fig. 1 Laser ablation of ZP of bovine egg at 100 mW, pulse duration1.5, 3, 4.5 ms.

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laser pulse length 25 ms and laser power 50 mW producechannels of consistent size.

The close-up in Figure 2~b! emphasizes the sharply drilledwall of each channel. The channels have clear cylindricacross sections of width;12 mm, with no significant beambroadening at either end. The channels reflect a thermal dissolution ‘‘melting’’ temperature, and its locus is not cotermi-nous with the beam edge. Although no evidence of thermadamage can be seen surrounding the target area, the transithermal excursion may cause damage invisible under the mcroscope to nearby living cells. The thermal history will bederived in the next section.

3 Thermal Predictions3.1 Standard ConditionsThe objective~NA50.6, 403, infinity corrected! used trans-mits 71% of the incident energy atl51480 nm.We will takethe typical laser beam power arriving at the vicinity of the ZP,following transmissive, reflective, and attenuation loss, as 10mW. We take the typical beam focal radius asa53 mm, andassume the beam is passing up through an inverted microscope into medium of refractive indexn51.333.The thermalhistory expected along the path of the laser beam, and espcially in the vicinity of the pre-embryo, will be derived.

3.2 Thermal Constants and Source FunctionGeometryThe medium is almost pure water, and the cellular material is;80% water. While the infrared absorptances in the mediumand cell atl51480 nmhave not been measured, they areunlikely to be significantly above that ofH2O, and cannot besignificantly lower. Similarly, the thermal conductivity of thematerial will not be higher than that of water. By taking theabsorptance as identical to that ofH2O, a521 cm21 ~Ref. 7!we will obtain a lower limit to the temperature produced. Onthe time scales of interest~1–25 ms!, the only significantmode of heat loss from the heated liquid will be thermal con-duction, since convection does not have time to develop anradiation may be neglected. The thermal conductivity of wateis 631023 w/cm/° at room temperature, but since the ther-

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mal excursion during the laser pulse is typically 100–200we take the conductivity of the medium asK56.831023,corresponding toH2O at 100°C.16

The laser path is approximated as three regions:~1! a con-verging cone of semiangleu, ~2! the waist region of the beamand ~3! a diverging cone with semiangleu. The beam hashigher net power at the converging than at the diverging codue to attenuation. The cone semiangle isu5arcsin(NA/n)526.7°. For the thermal calculation we approximate thwaist region as a cylinder of radiusa53 mm and length2a•cotu.

The laser beam converges through the medium to a fopoint 75 mm above the floor of the Petrie dish. Absorptiowill only be important in the medium and may be neglectedthe Petrie dish. Self-focusing of the IR beam will not signicantly affect the thermal distribution outside the beam andignored. The radiation intensity is symmetric in azimuthal cordinate, varying in intensity only with axial distance anradius. Ignoring angular variation, the heat diffusion equatin axisymmetric cylindrical coordinates may be written

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Fig. 2 (a) Bovine egg in which several channels have been cut bylaser. (b) Close-up view of channels in bovine zona pellucida. Thechannels are ;25 mm long, almost constant-radius cylindrical inter-cepts with sharp edges and do not show effects of beam convergenceor divergence.


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where the heat diffusion coefficient isk5K/rCp , and rCp

54.18 j/cm3, so k51.631023 cm2/s. Variation of the diffu-sion coefficient with temperature is small and will be ignored.The origin is taken as the focal point, withr andz as the radialand axial distances. The source functionS represents the laserheating power per unit volume, and varies across the entirdomain.

The source functions in the three regions of the laser patare normalized to the laser powerP reaching the focal plane,so that the converging beam is more intense below the waisand attenuates as it progresses upward, diverging above twaist. The central cylinder length is short(2a.cotu512mm), and attenuation is ignored in region 2.

The source functions for attenuated uniform beam in thethree regions are1. Converging

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constant, and forS2 , we use the factorAe22(r /a)2.

3.3 Boundaries for Solution of the Heat DiffusionEquationEquation ~1! was solved by finite element analysis~FEA!,using the codes COSMOS~Structural Research Co, Los An-geles, CA! and Mathlab PDE Toolbox~Mathworks Inc, Nat-ick, MA!, with source functions from Eqs.~2!–~4!. The beamtravels parallel to the axis of a cylinder of radius 100mm andlength 200mm. For short pulses(t,6 ms) these dimensionsare sufficient to make edge effects negligible, since the thermal diffusion distance in timet is x,30mm. The axis is aNeumann boundary with zero radial temperature gradientSince the beam is attenuated we cannot take the focal planea Neumann boundary but have to solve the equation in thentire upper half plane. The cylinder end planes atz56100mm, and cylinder wall atr 5100mm, are both Di-richlet boundaries with temperature held atT0537°C. Theinitial temperature of the entire domain is taken asT0 .

Of practical interest is the thermal history on the focalplane itself, at various radial distances from the axis, whichwill give information on the thermal excursion experienced bythe nearest blastomeres.

3.4 Preliminary VerificationFor comparison with an analytic case, the FEA was run witha uniformly heated cylinder of radius 3mm in a 200-mm-diam


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right cylinder containingH2O at T0537, and with sourcefunction as in Eq.~2!, P5100 mWanda521 cm21.

The analytic steady-state solution for the temperatureference between edge and center of an infinite cylindricalgion of radiusRmax heated on axis by a uniform beam oradiusR is

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The FEA steady-state result was within 0.5% of the tempeture difference from Eq.~5! and we conclude that the systeis integrating correctly.

3.5 Standard CaseWe take initial temperature in the medium at the physiolocal T0537°C, in a cylinder of diameter 200mm and length200 mm, with attenuated converging beam ofNA50.6. Thefield is divided into1.133105 elements. The same temperture T0 is held at the end planes and at the radiusRmax5100mm. Beam power is 100 mW at the focal waist, witpulse duration 3 ms and radius 3mm. The problem is solvedin the half plane and has been converted to the full planeFigures 3~a! and 3~b!, where the beam direction is bottom ttop, shown for times 0.1 and 3 ms, respectively. At 0.1 mscentral temperature is 98.8°C. In Figure 3~b!, at 3 ms thecentral temperature is 189°C. If the beam is flattop insteadGaussian, central temperature is reduced;15%. In either casethese high temperatures imply the potential presence of suheated medium. The temperature change from the initial vaat any time depends on the pulse duration and is direproportional to the beam power@see Eqs.~1!–~5!#. Predic-tions for any beam power may be scaled from the resreported.

The effect of attenuation is apparent in the slightly nonvtical slope of the isotherms as the beam progresses fromlow to above the waist in Figures 3~a! and 3~b!. Apart fromthis effect the converging and diverging parts of the beaway from the end planes result in almost cylindrical istherms. For the present purpose the focal plane is the oregion of interest in questions of cell heating, and we thefore examine whether the focal plane temperature heatingbe approximated by a simpler~and faster! one-dimensionalsolution.

To test the effect of geometry the FEA system was runthe Gaussian beam in the following three cases, all wpower 100 mW, pulse 3 ms and radius 3mm:

1. Attenuated beam,NA50.6 ~converging cone, centracylinder radius 3mm, converging/diverging cone!;

2. Unattenuated beam,NA50.6 ~same as above, exponential terms in Eqs.~2! and ~4! set to unity! and

3. Unattenuated beam,NA50 ~uniform central cylinderradius 3mm, exp. terms set to unity!.

The latter case corresponds to a constant cylindrical beand has one-dimensional symmetry, whereas the first twoquire two dimensions.

For all three cases, predicted temperatures on the fplane for radii 5<radius<50mm as function of time are

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Fig. 3 (a) FEA analysis of 3 ms pulse, 100 mW, converging Gaussianattenuated beam solved in half plane. The isotherms at 0.1 ms into thepulse are indicated. Isotherm interval is 2.5°C. Central temperature is98.8°C. (b) FEA analysis of same case as 5a, at 3 ms. Isotherm intervalis 2.5°C. Central temperature is 189°C.

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within 0.5°C over 10 ms integration time. At least on the focalplane, in the present case the converging and diverging beaproduces heating very similar to that from a long heated cylinder, and if the power is normalized to its value at the focalplane the effect of beam attenuation may be ignored. Henc

the mathematically simpler case of the cylinder can be ufor predicting the temperature history at points on the foplane. In the following we have therefore used the ondimensional Gaussian elimination solution outlined below.

4 Solution in Cylindrical Symmetry4.1 Gaussian EliminationThe FEA solution shows that radial temperature historyalmost cylindrical, with isotherms close to parallel to the aover the region of interest. If the heating effect of the beamapproximated as a cylindrical region of constant radiusaequal to its focal radius, Eq.~1! can be written as a onedimensional difference equation, which can be rapidly solvby Gaussian elimination,17,18 to give the thermal time historyin the region near the laser focus. The advantage of this issolution is much faster than FEA, and the case of pure cydrical geometry without converging and diverging beamgives a good approximation to the focal plane temperatuWe have written a Gaussian elimination~GE! code to modelthe thermal behavior in the radial dimension.

We cover the area0<r<Rmax, and takeRmax5100mm,chosen because at the laser pulse lengths considered, themal excursion does not reachRmax before the end of the pulseWe divide the cylindrical area intoN shells, each separated bDr 5Rmax/N. Accurate results are obtained withDr 51 mm.As boundary conditions, the initial temperature and the teperature atr 5Rmax, are set toT0537°C. The temperaturegradient at beam center is set to zero. In the GE analysiswill conservatively assume the beam is flattop rather thGaussian: this reduces central temperature by;15% but doesnot affect temperature history outside the beam. We use~5! to check the result and find agreement within 0.1% btween the GE code and Eq.~5! for very long~200 ms! pulses,so the GE code gives correct results for the analytic case

4.2 Thermal Histories on Focal PlaneThe GE solutions for temperature at various radial positioon the focal plane are shown as function of time in FigureThe standard case of beam power 100 mW, pulse duratioms, in a 200mm right cylinder initially at 37°C is used. Atbeam center the peak temperature reaches over 170°C. Tperature falls off sharply as the pulse terminates due toshort diffusion time over 3mm. As expected the thermal excursion seen near the beam has approximately the sametion as the laser pulse.

4.3 SuperheatingThe calculated~GE! peak central temperatures at typical beapowers with beam radius 3mm in water are given for flattopbeam focal spots of various pulse durations in Table 1, wsteady-state values from Eq.~5! for reference. Again we takethe region of the analysis as a cylinder of length 200mm andradius 100mm. The calculated temperature of water exposto the beam is very high, with peak central beam temperaranges up to above 200°C, corresponding to highly supheated water. The question is whether this temperature isor whether a phase change occurs, which would reducecentral temperature and form a column of vapor bubbalong the beam axis near the focal point. Miotello and Kell19

have examined the formation rate of homogeneous nuclei


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subsequent explosive phase change in superheated liquids.the absence of a surface for heterogeneous nucleation, homgeneous nucleation is necessary for rapid phase change, aits rate depends strongly on how close the liquid is to thecritical temperature~Tc5647 K for H2O!. The homogeneousnucleation rate becomes significant near 300°C. However, aand below 250°C the rate of homogeneous nuclei formation inwater is negligible, and in the absence of sites for heterogeneous nucleation we would not expect boiling within a 25 mspulse. If nucleation sites are present in the liquid, remote fromthe walls, boiling could occur. While it is possible that the ZPor local suspended particles may provide sites for nucleationit is unlikely since sharp solid edges are generally more favorable.

Thermal calculations indicate that highly superheated water is briefly formed in the focal waist of the IR beam. Heat isconducted mainly radially away from the waist, and regions

Table 1 Peak central temperatures.

PowermW

Pulse durationms

At end pulse°C

Steady state°C

100 1.5 155.4 233.9

100 3.0 172.3 233.9

100 4.5 182.3 233.9

100 6 189.3 233.9

100 10 201.9 233.9

100 15.0 211.7 233.9

100 25 223.0 233.9

80 15.0 176.8 194.5

40 15.0 106.9 115.8


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near the beam will be heated by conduction. Superheatedter is an excellent solvent, and it is not surprising that tregion of ZP within the focal waist rapidly disappears.

After the beam is turned off any vapor bubbles formwould be expected to collapse in less than 1 ms, releasingto the liquid. If a significant degree of phase change hadcurred the temperature at the beam core would be heldclose to 100°C, which would result in lower radial tempertures in the focal plane. We could therefore test the preseof superheating by measuring local temperature excursion

5 Temperature Check From Probe BeamSteering5.1 Beam SteeringIn order to confirm the predicted temperature excursion,decided to estimate the change in local refractive indaround the IR focal waist caused by the temperature excsion. We used the steering effect caused by the therminduced refractive index gradient on an orthogonal probeam. Estimates show that a 2° change in ray direction wobe possible for light passing near the IR heated water colu

5.2 Optical SystemThe setup is shown in Figure 5. The IR beam passes verticinto a 131 mm glass-walled rectangular tube~Vitrocom,Mountain View, NJ! containing distilledH2O. The laser is setto pulse at 4 Hz, while beam power and pulse durationvaried. The IR beam focus is set at approximately 200mmabove the bottom wall of the rectangular tube. The IR befocal diameter has already been measured at close to2a56 mm. A horizontal probe continuous wave~cw! HeNe la-ser beam is focused with anf 5120 mmlens into the regionof the IR beam focus, with the HeNe optic axis orthogonalthe IR beam axis. The HeNe probe beam radius to the1/e2

points at its focal point was measured by knife intercept22.361.2mm. The probe covers the region of interest nethe IR focus. The HeNe beam is then focused with a cylindcal lens on to a 1 mmaperture in front of a Si detector. Byscanning theaperture1detectorin the direction orthogonal toboth laser axes, the angle by which the probe beam isfracted~or scattered! by the thermal field around the IR beamis determined. The system is used to measure the maxim

Fig. 5 Measurement of beam steering. Horizontal view.

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Fig. 6 Bending of ray with impact factor b near a central refractivefield. Vertical view.

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angle through which the probe beam is turned at given pulslength/power. This is then compared with the angle derivedfrom the predicted thermal field.

5.3 Probe Ray SteeringThe curvature of the probe ray is proportional to~and in thedirection of! the normalized refractive index gradient¹n/n.In the IR beam focal plane the temperature field, and consequently the refractive index, in the specimen must be radiallysymmetric around the IR axis. The refractive index will de-crease toward the IR axis during the IR pulse, and a probe ratraveling in the focal plane of the IR beam will be steeredaway from the axis~see Figure 6!. The steering angleC in a

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centrally symmetric refractive field20 is given by the expres-sion

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whereb is the impact factor,r is radial distance from the IRbeam axis,n(r ) is the refractive index, andR5b/n(R) is thedistance of closest approach.

The refractive indexn(T) of superheated water is given bIAPWS21 from 0 to 150°C. This covers the required ranexcept for the beam center. For higher temperatures we hextrapolated from the IAPWS data. We use the GE solutifor the thermal field to calculaten(r ), and derive the ex-pected probe ray steering by the changed refractive indexing the laser pulse, as a function of IR laser power and pulength. The impact factor covers the range0,b,22mm dueto the large probe beam diameter.

The maximum beam bending is a measure of the mamum temperature gradient, which occurs adjacent to the bradius and corresponds to the specific temperature field.predicted values for pulse duration 25 and 2.5 ms are shoand maximum measured steering angles are given in Figufor IR power 20–100 mW and pulse duration 1–25 ms. Tlaser power shown in Figure 7 is that delivered at the fopoint, allowing for absorption after 200mm H2O .

After the beam is turned off any vapor bubbles formwould be expected to collapse in less than 1 ms, releasingto the liquid. We will neglect possible phase change in

Fig. 7 Probe beam steering angle in the IR laser thermal field, vs delivered IR power. Measurements are denoted by symbols and predictions bysolid lines. The dashed line corresponds to the prediction when maximum temperature cannot exceed 100°C.


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Fig. 8 Measured channel diameter vs pulse duration averaged overthree bovine eggs and two murine embryos. Predicted maximum tem-perature in focal plane at given radius is shown vs pulse duration andradial distance from axis.

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time scales of interest. Consequently, we can use predictionderived from Eq.~1! to compute temperatures in the beamfocal plane.

For higher laser delivered power, the central temperaturrises and the thermal gradient becomes steeper. The predictvalues therefore increase. The measured values show a tedency to reach a limiting value at high laser powers, in theregion where the predicted temperature is.200°C, implyingthat the gradient has maximized. This is unlikely to reflectphase transition: once phase change nucleates at these supheated temperatures, it would be expected to continue untthe medium reachesT5100.

We note that, if the medium can reach a maximum tem-perature of only 100°C due to phase change, the predicterefractive index gradient never rises high enough to cause thobserved beam bending, but flattens out atu50.6°. Since theobserved maximum ray bending angle isu51.8°, the resultsare consistent with central beam temperature.100°C.

5.4 ‘‘Melting Point’’ of Zona PellucidaWe have measured the diameter of the channels cut in the Zof three bovine oocytes by pulses of power 100 mW andduration 1.5–25 ms, using a 3mm radius beam under standardconditions. We have also measured channels cut in the ZP omurine two-cell pre-embryos by the same beam. The channediameter versus pulse duration is shown for both species iFigure 8. The GE model is applied to determine temperatur


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history on the focal plane at various distances from the beaxis, and the peak temperature calculated at each positioshown in Figure 8.

The bovine ZP material appears to undergo dissolut~melt! between 140 and 150°C, the temperature decreawith longer pulse time. The murine ZP appears to be measily removed and vanishes at;5–10°C lower temperaturethan the bovine.

Descloux and Delacre´taz22 measured the thermal dissolution of mouse ZP at temperatures 61–73°C over times up1000 s, and derived a rate constant. We extrapolate thiestimate the mouse ZP dissolution temperature for 10pulse as 111°C, lower than our experimental estimate aaged over the ZP from mouse pre-embryos. The minimtemperature that must be attained in the beam center in oto ablate human ZP material in the pulse times used is likto be between 130 and 150°C.

6 Conditions for Minimizing Collateral DamageConservatively, we will assume that the beam central teperature should be maintained at or above;150°C in order topenetrate the ZP within pulse time of 10 ms. At the same tithe temperature at the surface of the nearest cell shouldminimized. We take typical distance to nearest cell as 20mm,and use the thermal model above to derive temperaturethe focal plane at 20mm from the beam axis: in Figure 9graph is shown of the peak temperature reached duringpulse as function of pulse duration, for various beam poweIn the present case a typical thermal excursion would lastthe order of the pulse duration, and have peak amplitshown. The cells therefore undergo rapid heating and cooof amplitude tens of degrees in a few milliseconds.

We are not aware of published work on the deleterioeffects of brief thermal pulses on cells. If we assume therm

Fig. 9 The predicted maximum of the temperature pulse 20 mm frombeam axis in the focal plane is given vs laser pulse duration and beampower. Also shown are limit lines corresponding to beam central tem-perature 150 and 110°C.

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damage scales as a chemical reaction, then damage wouldproportional tot•exp(2DQ/RT), wheret is pulse duration,Tis the mean pulse temperature, andDQ is reaction energy.This would imply that the pulse time must be kept extremelyshort if the temperature rise is significant.

It is clearly advisable to minimize the thermal excursionamplitude and duration, both of which imply keeping pulsetime low. Short pulse duration and high beam power give themost efficient system for maximizing beam cutting power andminimizing local heating. We have indicated in Figure 9 lim-iting lines corresponding to beam center peak temperatures150 and 110°C. The peak central temperature should exceethe limiting lines for rapid ZP ablation, while maintainingminimum peak temperature at 20mm. It is evident that thelaser pulse duration should be as short as possible consistewith effective ZP dissolution, preferably below 5 ms.

7 Summary and ConclusionIR laser beams introduced into the optical path of an invertedmicroscope are now routinely used to perform delicate operations on human pre-embryosin vivo. The ablative propertiesof a pulsed beam ofl51480 nmlaser light are of consider-able interest for penetration of the shell surrounding the preembryo. Using the laser, highly predictable and reproducibleresults are obtained and the amount of time necessary to peform a treatment on pre-embryos is kept very short. The introduction of laser technology into the clinical IVF laboratoryhas allowed the development of more standardized procedurwhich are not so dependent on individual operator skill.

The action of the laser must be strictly limited to the tar-geted region of the ZP, since focused laser irradiation on ancell would be damaging and probably fatal to that cell. Fol-lowing irradiation the heat is conducted away from the targeonto which the laser focal spot is directed, and is dissipateinto the surrounding medium. Our interest is to determine thetemperature in the medium exposed to the beam and its suroundings, and set limits to beam pulse and beam power. Whave examined the thermal regime associated with pulsed laser ablation of pre-embryo zona pellucida, and found that aone-dimensional model gives adequate accuracy to preditemperature. Only conductive heat transfer is important, anthe result that the aqueous medium in fact reaches supeheated temperatures in the laser beam is obtained. Experimeconfirms the presence of the high thermal gradients predictein the medium. We estimate the temperature of zona pellucidablation in bovine oocytes between 140 and 160°C, and murine embryos 130–150°C. Potentially damaging thermal excursions at the embryo blastomeres are minimized by usinpulse durations 1–5 ms.

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Lasers - Douglas hamilton, conia - 2001 - thermal effects in laser-assisted pre-embryo zona drilling

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