J. Cell Sci. M, 361-371 (i977) 361Printed in Great Britain

INFRARED LASER DAMAGE TO CILIARY

MOTION IN PHRAGMATOPOMA

ROBERT RIKMENSPOEL, SANDRA E. ORRISAND PETER O'DAYDepartment of Biological Sciences, State University of New York, Albany,New York 12222, U.S.A.

SUMMARYA glass neodymium laser was modified to make it possible to produce small lesions of 1-2 fim

size with a quantitatively known amount of energy. The i-o6-/tm radiation of this laser issufficiently absorbed by water to work without the additions of dyes. Ciliary arrest in Phrag-matopoma gills was produced by an amount of energy, sufficient to cause a rise in temperatureof 150 °C in an area of 2 fim3. At these low doses the effect was fully reversible. With higherdoses of laser energy the cilia stopped permanently, probably because of structural damage ofthe irradiated cells.

INTRODUCTION

Laser microsurgery has been used in recent years to investigate the control processesof the motility of cilia (Motokawa & Satir, 1975), sperm flagella (Goldstein, 1969) andcellular flagella (Goldstein, Holwill & Silvester, 1970). In all cases a ruby laser wasused with a wavelength of the irradiating light of 694-3 nm. Since biological materialshave no absorption at that wavelength a dye has to be added externally to the prepara-tion. This presents difficulties for precise microsurgical experiments inside intact cells.

In this paper experiments are reported in which microsurgery was performed onthe control of ciliary motion in Phragmatopoma, using a glass neodymium laser. Theabsorption of water at the i-o6-/tm wavelength of this laser is approximately 11 %/cm.This makes it possible to produce lesions inside an intact cell with a quantitativelyknown amount of energy, without the addition of a dye.

EXPERIMENTAL METHODS

Organisms and materials

Phragmatopoma, an organism quite similar to the more familiar Sabellaria, was obtained fromthe Pacific Bio-Marine Co. (Venice, California). Gills of the organisms were excised andsuspended in artificial sea water for experimentation. Normal motion was maintained by thecilia on the gills for several hours.

The cilia are arranged on the conically shaped gills of Phragmatopoma in rows as shown inFig. 1. In most experiments the gills were positioned in such a way that the optical axis wasalong a row of cilia. The plane of beating of the cilia was then in the plane of focus of themicroscope. For a few separately mentioned experiments the gills were oriented so that a rowof cilia was obliquely viewed. Since this latter orientation was difficult to produce and tomaintain, only a limited amount of data could be obtained this way.

All experiments were carried out at a room temperature of 22 ± 2 °C.

362 R. Rikmenspoel, S. E. Orris and P. O'Day

Normal directionof viewing

Oblique directionof viewing'

Fig. 1. Fragment of a gill oiPhragmatopoma, with rows of cilia. The normal directionI of viewing, along a row of cilia, and the skew orientation are indicated by arrows.

Microscopy

During the experiments the gill fragments were under observation on a Zeiss Universalmicroscope (Carl Zeiss Inc., New York, N.Y.). Nomarski differential interference optics wereused with a Zeiss 40 x water-immersion objective. By rotating the polarizer the depth of focuscould be adjusted so that just one or two cilia of a row were sharply visible, or so that a greaterdepth of focus was obtained with reduced contrast.

The sliding prism that deflects the light beam in the microscope to the viewing tube or thephotography tube was replaced by a permanent partially reflecting mirror (MARC 45-g ofBausch and Lomb, Rochester, N.Y.), which deflected 35 % of the intensity to the viewing tube.For protection against eye damage a 2-mm Schott BG18 filter (Fish-Schurman Corp., NewRochelle, N.Y.) was inserted in the viewing tube. At the wavelength of the laser light used,106/tm, the BG18 filter has, according to the catalogue value, a transmission of less than 1part in io8.

Laser set-up

A TRG model 513 Biolaser (Hadron Inc., Westbury, N.Y.), equipped with a glass neodymiumrod, was mounted on the photography tube of the Universal microscope. Fig. 2 gives the generaloutlay of the optical train.

In the first experiments a target was used consisting of a slide on which a thin layer of felt-pen ink, had been spread. The area in which the ink layer was removed by a laser pulse from theslide showed a central hole of ~io/tm diameter surrounded by a halo of K2O/im diameter.This indicates the presence of spherical aberration in the optics at the wavelength of 106 /im ofthe laser light.

Improvement of the focusing of the laser beam was obtained by replacing the lens L, in Fig. 2by a 10 x Zeiss objective. The parallel laser beam emerging from this objective had a diameterof approximately 1 mm. The dichroic mirror in Fig. 2, which directs the laser beam into themicroscope eyepiece, was originally positioned about 2 cm above the top of the eyepiece. Byremoving part of the material of the eyepiece (including cutting through the top lens), it waspossible to lower the laser assembly. In the final adjustment the dichroic mirror intersected theoptical axis of the microscope at the point where the cone of the microscope imaging light con-verged (about 8 mm above the top of the eyepiece). This ensures that the laser light is paraxialas it passes through the microscope. After these modifications holes of 1-2 fim diameter couldbe produced in the felt-pen ink target.

Infrared laser damage to ciliary motion in Phragmatopoma 363

F i l t e r -== :

ObjectiveSlide Laser

head

Fig. 2. Schematic diagram of the laser apparatus. In the experiments the filter was aSchott UG2, of 2 mm thickness.

10

-,3

EoZ

100 150 200

Input energy, J

250

Fig. 3. Energy of the laser beam emerging from a 40 x Zeiss microscope objectiveas a function of the energy input to the laser. The scale at the right shows the valuesapplicable to the experiments in this paper, where a UG2 glass filter and the NomarskiWollaston prism were present in the laser light path. Arrow indicates threshold value.

364 R. Rikmenspoel, S. E. Orris and P. O'Day

The energy of the beam after passage through the microscope was measured with a modelTRG 513-5 Substage Energy Monitor (Hadron Inc., Westbury, N.Y.). Fig. 3 shows the cali-bration of the energy output on to the target as a function of the input energy, using the 40 xZeiss objective. The values for a 10 x Zeiss objective were within 10 % of those for the 40 xobjective.

In the actual experiments reported in this paper, the Nomarski Wollaston prism was insertedin the light path between the objective and the eyepiece of the microscope. The transmission ofthis Wollaston prism at A = 1-06 fim was measured with the Energy Monitor to be 86%. Itwas found convenient to have a 2-mm Schott UG2 filter, with a measured transmission atA = 1-06 fim of 28 %, present in the laser beam as indicated in Fig. 2. The reduction of thelaser output energy by the UG2 filter eliminated the need to work near the laser threshold (at125 J input), where the calibration of the output energy is not accurate. The calibration of theenergy incident on the target in the experiments reported in this paper is given on the right sideof Fig. 3. The duration of the laser output pulse, measured with a photocell, was <̂ 1 ms.

0100

50

0 L

- 2 - 1 0'Distance S,

Fig. 4. Percentage of sperm which ceased motion upon irradiation by a laser pulse of180 J input, as a function of the distance S. The diagram at the left illustrates theposition of the sperm during the experiments in which the laser was aimed at thecentre of the indicated cross hairs. Distance between small arrows is the full width athalf maximum of the dose-effect curve.

The effective size of the laser beam at the focus of the microscope is given by the area whichis heated by the absorbed light, not necessarily by its optical diameter. To measure the effectivebeam size the following procedure was used. Bull spermatozoa were washed repeatedly. Most ofthe spermatozoa after washing became stuck to the microscope slide by the heads and mid-pieces, while the fiagella continued waving (Lindemann & Rikmenspoel, 1972a, b). When themidpiece area was hit by a laser pulse with sufficient energy the flagellar motion stopped. Thedamage due to the laser pulse appeared to be to the membrane of the sperm, not to the con-tractile system, since the flagellar motility was always maintained in the presence of externalATP or ADP, even at the highest doses of laser energy. In the absence of external ATP or ADP,the percentage of sperm that ceased motion after one laser pulse was measured as a function ofthe distance between the midpiece of the sperm and the target point. In this way the laserbeam was 'scanned' with a slit represented by the midpiece.

Fig. 4 shows the results at an input energy of 180 J. The full width at half maximum (FWHM)of the dose—effect curve in Fig. 4 is 1 -9 fim. The width of the midpiece of bull sperm being 0-4 fim(Rothschild, 1962), this gives an effective diameter of the laser beam of 1-5 fim. Table 1 showsthat the beam size increases slightly with the energy of the laser pulse.

At pulse energies below 1 mj the effective beam cross-section is close to 1 5 /im1. At a numeri-cal aperture of 0-75, as with the 40 x Zeiss objective, the height of the area in which the beam isfocused is 1-2 fim, dependent on whether or not a remnant of spherical aberration is present.At an absorption of water at A = 1-06 fim of 11 % per cm (Curcio & Petty, 1951), the fractionof energy absorbed is 11 x io~* per/tm pathlength along the optical axis. Therefore a pulse

Infrared laser damage to ciliary motion in Phragmatopoma 365

of o-i mj = 2-4 x io~* cal deposits at the focus of the microscope an energy of 26 x io"10 calper /(m pathlength in a 1-5 fim* cross-section. The local rise in temperature is thus 170 °C pero-i mj of beam energy. It appears, therefore, that approximately o-i mj of beam energy is justsufficient to cause a small vapour bubble (w 1-2 /tmB) to be produced at the focus. It should benoted that the temperature rise is independent of the height of the area in which the beam isfocused.

Table 1. Effective diameter of beam of laser pulse as a function of the pulse energy

Beam energy,mj

o-63670

No niters were

Cinemicrography

Inputenergy,

J

140180220

present in the

Full width athalf maximum of

scan curve (Fig. 3),/»m

Beamdiameter,

/im

17 1-31-9 1-52 2 i-8

light path during these measurements.

Cinemicrographs at 400 or 200 frames/s were made with a Milliken DBM 5 C Camera(Teledyne Corp., Arcadia, California) on Kodak Plus X negative film. The objective lens of theMilliken camera was replaced by a triplet lens of/= 40 mm and a diameter of 30mm (Bausch andLomb, Rochester, N.Y.) to ensure that the image filled the whole film frame. The entrance pupilof a normal 16-mm cine objective, when placed above the dichroic mirror (cf. Fig. 2), is toosmall to intercept the full light cone emerging from the microscope eyepiece.

For analysis the films were projected at a final magnification of 1000 x in a Vanguard MotionAnalyser (Vanguard Instrument Corp., Melville, N.Y.). When desired, the ciliary motion wastraced from successive frames on tracing paper.

RESULTS

Description of laser effects

With the laser beam aimed at the basal body of the cilium in focus, the ciliary motionstopped in approximately 20 % of the cases when the input energy was just abovelasing threshold. Increasing the energy input led to a greater percentage of stoppage ofthe ciliary motion.

In a fraction of the cases the cilia, after having been stopped by a laser pulse, re-covered apparently normal activity. The delay between the laser pulse and theresumption of motion was from 8 to 30 s.

During the firing of the laser the preparation cannot be viewed for safety reasons.Information on the course of the process of laser-induced stoppage was obtained fromfilms. Using the 40 x Zeiss water-immersion objective in Nomarski illumination, thedepth of focus was such that usually two (sometimes three) cilia of a row along the'normal' viewing direction (cf. Fig. 1) were visible on the film. Fig. 5A shows aciliary cycle as it appears on the film. The frequency of the ciliary motion varied in thedifferent preparations from 8 to 14 Hz.

After firing of the laser the ciliary motion continued for 3 to 8 cycles before it cameto a stop. The cilia invariably stopped at the end of a recovery stroke. This is illustratedin Fig. 5B, where it can be seen that the two or three cilia imaged on the film have allstopped in approximately the same position.

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Infrared laser damage to ciliary motion in Phragmatopoma 367

In the period between the laser firing and the halting of the ciliary motion, the shapeof the ciliary beat was largely unchanged. Fig. 6 shows tracings of 2 cilia before thelaser firing and in each case a tracing of the last cycle before the motion had stopped.It can be seen in Fig. 6 not only that the shape of the ciliary beat is maintained in thefinal cycle, but also that the motion is not or only slightly slowed down.

Normal cycleV/, V/,

Last cycle of motion

-40

Normal cycle Last cycle of motion

Fig. 6. Normal cycle of ciliary motion before laser irradiation (left) and the lastcycle before stopping after the laser pulse. In A the duration of the final cycle is equalto that of a normal one; in B the final cycle appears ro be slightly slower. The numbersat the various ciliary positions give the time in ms.

The cilia which resumed motion, did so after a delay time varying between 8 and30 s. The spread and unpredictability of this delay time made it impractical todocument the starting-up process on film. From visual observation it appeared thatthe motion began with a few slow partial beat cycles, which over a period of 1-2 sspeeded up until normal motion was again present.

When the ciliary motion was permanently stopped by a laser pulse the cilia frayedinto a disorganized mass of axonemes, as illustrated in Fig. 5 c. Usually an extrusionof cytoplasm would develop over a period of several seconds following the laserpulse in these cases. Both of these observations indicate that permanent damage to theintegrity of the cell was the cause of the irreversible cessation of ciliary motion.

With Phragmatopoma gills oriented in an oblique fashion, as indicated in Fig. 1,p. 362, it was possible to view the effects of the laser irradiation along a row of cilia.When ciliary motion stopped following the firing of the laser, it always did so over alength of 40—60 fim along a row. Recovery of the motion proceeded from the sides in-wards to the centre of the affected area. Since in this skew orientation the location of

368 R. Rikmenspoel, S. E. Orris and P. O'Day

the lesion produced by the laser was not well known and since the limited depth offocus made filming of the entire affected section impossible, we have not tried toquantitate this observation.

Dose—response curves

All data on dose-response curves were gathered by observing the ciliary motionbefore and after the firing of the laser. The orientation of the gills of the Phragmato-poma was such that viewing was along the ' normal' direction indicated in Fig. i.

100

0 s *

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y

150 200

100 r -

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in EVQ.

50

o

. B

-

100

/

/

^ / 1

Ay

i , i ,

150

InputTarget

energy , J: Basal body

, 1200

Fig. 7. Dose-response curve for laser-induced ciliary arrest, with the laser beamaimed at the basal body. A, total fraction, of stoppage; B, permanent stoppage only.Each point represents 7-10 laser firings, except at the highest input energy, whichrepresents 5 firings. Vertical dotted line indicates E0.b value.

Fig. 7 A shows the dose-response curve when the laser beam was aimed at the basalbody of the cilium under observation in the microscope. The half value dose, EQ.6,for laser-induced stoppage according to Fig. 7 A, using the calibration curve of Fig. 2,is 0-08 mj. For permanent laser-induced stoppage, accompanied by fraying of thecilia and cytoplasmic extrusions, the dose-response curve of Fig. 7B gives a value forE0.6 of 0-16 mj.

Lesions were produced at distances of 16 and 32 fim from the basal body in adirection perpendicular to the cell surface and also in a direction almost parallel to thecell surface. Fig. 8 shows the values for E0.5 for all laser-induced stoppage, and forthe permanent stoppage only as a function of the distance of the lesion to the basal

Infrared laser damage to ciliary motion in Phragmatopoma 369

body. It can be seen in Fig. 8 that the energy needed to produce stoppage of theciliary motion is not much dependent on the location of the lesion. This shows thatthe laser effect is not damage of a contractile structure required for the ciliary motion.

The laser energy required to produce permanent cessation of motion increasesaccording to Fig. 8 with the distance to the basal body. This supports the notion thatpermanent damage, probably to the cell surface, was the cause of that effect.

1-5f—

10

OS

II

10 20 30d, ftm

•40

1 0

0-5

10 20 30/,

Fig. 8. Laser beam energy needed to produce ciliary arrest (•) or irreversible arrest( x ) in 50 % of the laser firings, as a function of the distance between the target and thebasal body. The diagram on the right defines the direction of location of the targetspots.

DISCUSSIONThe experimental results presented in this paper show that quantitative micro-

surgery is possible with the use of a neodymium glass laser, without the use of dyes.The characteristic dose E ^ = 0-08 mj for the reversible stoppage of the ciliarymotion indicates that the effect was brought about by the creation of a microvapourbubble of 1-2 fim3 volume and a temperature of around 150 °C. The neodymiumglass laser appears, in this respect, to have the capability to produce more precisemicrolesions than the ruby laser. With the latter the induced lesions reported werelarger and were always accompanied by damage to the cells (Goldstein, 1972; Moto-kawa & Satir, 1975).

24 CEL 24

370 R. Rikmenspoel, S. E. Orris and P. O'Day

The permanent cessation of ciliary motion reported in this paper was, as explainedin 'Results', most probably caused by cellular damage due to the laser pulse. Thisphenomenon probably is not of much investigative interest. The discussion that fol-lows will therefore be restricted to the reversible stoppage induced by the low doess oflaser energy.

The energy needed to cause (reversible) stoppage of ciliary motion appears fromFig. 8 to be largely independent of the location of the produced lesion. The cessationof motion over a large segment of a row of cilia was observed also in the ruby laserexperiments in Mytilus by Motokawa & Satir (1975). Together these results indicatethat the laser irradiation affects a cellular control mechanism of the ciliary motion.Ca2+ has been shown (Eckert, 1972; Naitoh & Eckert, 1974) to control the reversal ofciliary motion in Paramecium. The frequency of ciliary motion was reported to beincreased by an increased internal Ca2+ concentration in Necturus maculosus byMurakami & Eckert (1972), but to be decreased in freshwater mussel by Satir (1975).Even though the role of Ca2+ in the control of ciliary motion is apparently not a simpleone, it would be desirable to investigate whether this ion is involved in the effectsproduced by the laser. We have found, however, that it was not possible to maintainPhragmatopoma gill preparations even for limited periods in Ca2+-free or lowCa2+ media. This instability in Ca2+-free medium was reported also for Mytilus byMotokawa & Satir (1975). These observations might indicate that the role of Ca2"1"in the control of ciliary motion could be investigated more profitably in freshwaterorganisms.

In the experiments reported in this paper the cilia of Phragmatopoma always stoppedat the end of a recovery stroke, just before a new effective stroke was to have started.This indicates that a cellular ' signal' to trigger the effective stroke was not forthcom-ing. That the effective stroke of cilia is triggered by a signal, with the recovery strokefollowing autonomously, was also found from theoretical analysis of the contractileevents in ciliary motion by Rikmenspoel & Rudd (1973). The rather abrupt cessation ofmotion after several cycles of almost normal motion following a laser pulse suggeststhat the signal depends on a threshold level of a substance which is either released orsequestered by the cell.

The present experiments do not give information as to whether metachronal co-ordination is mechanical or not (Machemer, 1972, 1974). To investigate the coordina-tion of cilia and the role of ions in the control of ciliary motion, freshwater organismswith the direction of the metachronal coordination in the plane of the ciliary beat(synplectic metachronism) appear to be better candidates. The experiments shouldinclude kinetic studies on the spread of ciliary arrest (Motokawa & Satir, 1975).

This investigation was supported in part by the National Institute for Child Health andHuman Development through grant HD-6445.

Infrared laser damage to ciliary motion in Phragmatopoma 371

REFERENCES

CURCIO, J. A. & PETTY, C. C. (1951). The near infrared absorption spectrum of liquid water.J. Opt. Soc. Am. 41, 302-304.

ECKERT, R. (1972). Bioelectric control of ciliary activity. Science, N.Y. 176, 473-481.GOLDSTEIN, S. F. (1969). Irradiation of sperm tails by laser microbeam. J. exp. Biol. 51,

431-441.GOLDSTEIN, S. F. (1972). Effects of laser irradiation on the structure and function of cilia and

flagella. Acta protozool. 11, 259-264.GOLDSTEIN, S. F., HOLWILL, M. E. J. & SILVESTER, N. R. (1970). The effects of laser micro-

beam irradiation on the flagellum of Crithidia (Strigomonas) oconpelti. J. exp. Biol. 53,401-409.

LINDEMANN, C. B. & RIKMENSPOEL, R. (1972a). Sperm flagella; autonomous oscillations of thecontractile system. Science, N. Y. 176, 337-338.

LINDEMANN, C. B. & RIKMENSPOEL, R. (19726). Sperm flagellar motion maintained by ADP.Expl Cell Res. 73, 255-259.

MACHEMER, H. (1972). Properties of polarized ciliary beat in Paramecium. Acta protozool. 11,295-300.

MACHEMER, H. (1974). Ciliary activity and metachronism in cilia. In Cilia and Flagella (ed.M. A. Sleigh), pp. 199-286. London & New York: Academic Press.

MOTOKAWA, T. & SATIR, P. (1975). Laser induced spreading arrest of Mytilus gill cilia. J. CellBiol. 66, 377-39I-

MURAKAMI, A. & ECKERT, R. (1972). Cilia; activation coupled to mechanical stimulation bycalcium influx. Science, N.Y. 175, 1375-1377.

NAITOH, Y. & ECKERT, R. (1974). The control of ciliary activity in protozoa. In Cilia and Flagella(ed. M. A. Sleigh), pp. 305-352. London & New York: Academic Press.

RIKMENSPOEL, R. & RUDD, W. G. (1973). The contractile mechanism in cilia. Biophys. J. 13,955-995-

ROTHSCHILD, LORD (1962). Sperm movement; problems and observations. In SpermatozoonMotility (ed. D. W. Bishop), pp. 13-29. Washington, D.C.: American Association for theAdvancement of Science.

SATIR, P. (1975). Ionophore mediated calcium entry induces mussel gill ciliary arrest. Science,N. Y. 190, 586-587.

{Received 6 August 1976)