INFRARED LASER DAMAGE TO CILIARY MOTION IN PHRAGMATOPOMA

Transcription

1 J. Cell Sci. M, (i977) 361 Printed in Great Britain INFRARED LASER DAMAGE TO CILIARY MOTION IN PHRAGMATOPOMA ROBERT RIKMENSPOEL, SANDRA E. ORRIS AND PETER O'DAY Department of Biological Sciences, State University of New York, Albany, New York 12222, U.S.A. SUMMARY A 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 is sufficiently absorbed by water to work without the additions of dyes. Ciliary arrest in Phragmatopoma gills was produced by an amount of energy, sufficient to cause a rise in temperature of 150 C in an area of 2 fim 3. At these low doses the effect was fully reversible. With higher doses of laser energy the cilia stopped permanently, probably because of structural damage of the irradiated cells. INTRODUCTION Laser microsurgery has been used in recent years to investigate the control processes of the motility of cilia (Motokawa & Satir, 1975), sperm flagella (Goldstein, 1969) and cellular flagella (Goldstein, Holwill & Silvester, 1970). In all cases a ruby laser was used with a wavelength of the irradiating light of nm. Since biological materials have no absorption at that wavelength a dye has to be added externally to the preparation. This presents difficulties for precise microsurgical experiments inside intact cells. In this paper experiments are reported in which microsurgery was performed on the control of ciliary motion in Phragmatopoma, using a glass neodymium laser. The absorption 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 quantitatively known 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 from the Pacific Bio-Marine Co. (Venice, California). Gills of the organisms were excised and suspended in artificial sea water for experimentation. Normal motion was maintained by the cilia on the gills for several hours. The cilia are arranged on the conically shaped gills of Phragmatopoma in rows as shown in Fig. 1. In most experiments the gills were positioned in such a way that the optical axis was along a row of cilia. The plane of beating of the cilia was then in the plane of focus of the microscope. For a few separately mentioned experiments the gills were oriented so that a row of cilia was obliquely viewed. Since this latter orientation was difficult to produce and to maintain, only a limited amount of data could be obtained this way. All experiments were carried out at a room temperature of 22 ± 2 C.

2 362 R. Rikmenspoel, S. E. Orris and P. O'Day Normal direction of viewing Oblique direction of viewing' Fig. 1. Fragment of a gill oiphragmatopoma, with rows of cilia. The normal direction I 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 Universal microscope (Carl Zeiss Inc., New York, N.Y.). Nomarski differential interference optics were used with a Zeiss 40 x water-immersion objective. By rotating the polarizer the depth of focus could be adjusted so that just one or two cilia of a row were sharply visible, or so that a greater depth of focus was obtained with reduced contrast. The sliding prism that deflects the light beam in the microscope to the viewing tube or the photography tube was replaced by a permanent partially reflecting mirror (MARC 45-g of Bausch 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., New Rochelle, 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 1 part in io 8. Laser set-up A TRG model 513 Biolaser (Hadron Inc., Westbury, N.Y.), equipped with a glass neodymium rod, was mounted on the photography tube of the Universal microscope. Fig. 2 gives the general outlay of the optical train. In the first experiments a target was used consisting of a slide on which a thin layer of feltpen ink, had been spread. The area in which the ink layer was removed by a laser pulse from the slide 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 of the laser light. Improvement of the focusing of the laser beam was obtained by replacing the lens L, in Fig. 2 by a 10 x Zeiss objective. The parallel laser beam emerging from this objective had a diameter of approximately 1 mm. The dichroic mirror in Fig. 2, which directs the laser beam into the microscope eyepiece, was originally positioned about 2 cm above the top of the eyepiece. By removing part of the material of the eyepiece (including cutting through the top lens), it was possible to lower the laser assembly. In the final adjustment the dichroic mirror intersected the optical axis of the microscope at the point where the cone of the microscope imaging light converged (about 8 mm above the top of the eyepiece). This ensures that the laser light is paraxial as it passes through the microscope. After these modifications holes of 1-2 fim diameter could be produced in the felt-pen ink target.

3 Infrared laser damage to ciliary motion in Phragmatopoma 363 Filter-== : Slide Objective Laser head Fig. 2. Schematic diagram of the laser apparatus. In the experiments the filter was a Schott UG2, of 2 mm thickness. -,3 10 E o Z Input energy, J Fig. 3. Energy of the laser beam emerging from a 40 x Zeiss microscope objective as a function of the energy input to the laser. The scale at the right shows the values applicable to the experiments in this paper, where a UG2 glass filter and the Nomarski Wollaston prism were present in the laser light path. Arrow indicates threshold value. 250

4 364 R. Rikmenspoel, S. E. Orris and P. O'Day The energy of the beam after passage through the microscope was measured with a model TRG Substage Energy Monitor (Hadron Inc., Westbury, N.Y.). Fig. 3 shows the calibration of the energy output on to the target as a function of the input energy, using the 40 x Zeiss objective. The values for a 10 x Zeiss objective were within 10 % of those for the 40 x objective. In the actual experiments reported in this paper, the Nomarski Wollaston prism was inserted in the light path between the objective and the eyepiece of the microscope. The transmission of this Wollaston prism at A = 1-06 fim was measured with the Energy Monitor to be 86%. It was found convenient to have a 2-mm Schott UG2 filter, with a measured transmission at A = 1-06 fim of 28 %, present in the laser beam as indicated in Fig. 2. The reduction of the laser output energy by the UG2 filter eliminated the need to work near the laser threshold (at 125 J input), where the calibration of the output energy is not accurate. The calibration of the energy incident on the target in the experiments reported in this paper is given on the right side of Fig. 3. The duration of the laser output pulse, measured with a photocell, was <^ 1 ms L ' Distance S, Fig. 4. Percentage of sperm which ceased motion upon irradiation by a laser pulse of 180 J input, as a function of the distance S. The diagram at the left illustrates the position of the sperm during the experiments in which the laser was aimed at the centre of the indicated cross hairs. Distance between small arrows is the full width at half maximum of the dose-effect curve. The effective size of the laser beam at the focus of the microscope is given by the area which is heated by the absorbed light, not necessarily by its optical diameter. To measure the effective beam size the following procedure was used. Bull spermatozoa were washed repeatedly. Most of the spermatozoa after washing became stuck to the microscope slide by the heads and midpieces, while the fiagella continued waving (Lindemann & Rikmenspoel, 1972a, b). When the midpiece area was hit by a laser pulse with sufficient energy the flagellar motion stopped. The damage due to the laser pulse appeared to be to the membrane of the sperm, not to the contractile system, since the flagellar motility was always maintained in the presence of external ATP 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 of the distance between the midpiece of the sperm and the target point. In this way the laser beam 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 shows that 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 /im 1. At a numerical aperture of 0-75, as with the 40 x Zeiss objective, the height of the area in which the beam is focused 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 fraction of energy absorbed is 11 x io~* per/tm pathlength along the optical axis. Therefore a pulse

5 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 cal per /(m pathlength in a 1-5 fim* cross-section. The local rise in temperature is thus 170 C per o-i mj of beam energy. It appears, therefore, that approximately o-i mj of beam energy is just sufficient to cause a small vapour bubble (w 1-2 /tm B ) to be produced at the focus. It should be noted that the temperature rise is independent of the height of the area in which the beam is focused. Table 1. Effective diameter of beam of laser pulse as a function of the pulse energy Beam energy, mj o No niters were Input energy, J present in the Full width at half maximum of scan curve (Fig. 3), /»m Beam diameter, /im i-8 light path during these measurements. Cinemicrography 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 the Milliken camera was replaced by a triplet lens of/= 40 mm and a diameter of 30mm (Bausch and Lomb, Rochester, N.Y.) to ensure that the imagefilledthe wholefilmframe. The entrance pupil of a normal 16-mm cine objective, when placed above the dichroic mirror (cf. Fig. 2), is too small to intercept the full light cone emerging from the microscope eyepiece. For analysis the films were projected at afinalmagnification of 1000 x in a Vanguard Motion Analyser (Vanguard Instrument Corp., Melville, N.Y.). When desired, the ciliary motion was traced 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 motion stopped in approximately 20 % of the cases when the input energy was just above lasing threshold. Increasing the energy input led to a greater percentage of stoppage of the ciliary motion. In a fraction of the cases the cilia, after having been stopped by a laser pulse, recovered apparently normal activity. The delay between the laser pulse and the resumption 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 from films. Using the 40 x Zeiss water-immersion objective in Nomarski illumination, the depth 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 a ciliary cycle as it appears on the film. The frequency of the ciliary motion varied in the different preparations from 8 to 14 Hz. After firing of the laser the ciliary motion continued for 3 to 8 cycles before it came to a stop. The cilia invariably stopped at the end of a recovery stroke. This is illustrated in Fig. 5B, where it can be seen that the two or three cilia imaged on the film have all stopped in approximately the same position.

6 Fig. 5. A, 5 film frames representing stages of a normal cycle of ciliary motion of Phragmatopoma. Two cilia of a row are sharply in focus. B, z cases of reversible stoppage of ciliary motion after a laser pulse at the end of the recovery stroke. c, frayed cilia after permanent stoppage of motion by a 2-mJ laser pulse. Blisters of cytoplasmic extrusion are indicated by arrows.

7 Infrared laser damage to ciliary motion in Phragmatopoma 367 In the period between the laser firing and the halting of the ciliary motion, the shape of the ciliary beat was largely unchanged. Fig. 6 shows tracings of 2 cilia before the laser 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 the final cycle, but also that the motion is not or only slightly slowed down. Normal cycle V/, 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 last cycle before stopping after the laser pulse. In A the duration of the final cycle is equal to that of a normal one; in B the final cycle appears ro be slightly slower. The numbers at the various ciliary positions give the time in ms. The cilia which resumed motion, did so after a delay time varying between 8 and 30 s. The spread and unpredictability of this delay time made it impractical to document the starting-up process on film. From visual observation it appeared that the motion began with a few slow partial beat cycles, which over a period of 1-2 s speeded up until normal motion was again present. When the ciliary motion was permanently stopped by a laser pulse the cilia frayed into a disorganized mass of axonemes, as illustrated in Fig. 5 c. Usually an extrusion of cytoplasm would develop over a period of several seconds following the laser pulse in these cases. Both of these observations indicate that permanent damage to the integrity 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 a length of fim along a row. Recovery of the motion proceeded from the sides inwards to the centre of the affected area. Since in this skew orientation the location of

8 368 R. Rikmenspoel, S. E. Orris and P. O'Day the lesion produced by the laser was not well known and since the limited depth of focus made filming of the entire affected section impossible, we have not tried to quantitate this observation. Dose response curves All data on dose-response curves were gathered by observing the ciliary motion before and after the firing of the laser. The orientation of the gills of the Phragmatopoma was such that viewing was along the ' normal' direction indicated in Fig. i. 100 y 0 s * 50 /I :opped inently, % in E V Q. 100 r- 50 o. B / / ^ / 1 A y i, i, 150 Input energy, J Target : Basal body, Fig. 7. Dose-response curve for laser-induced ciliary arrest, with the laser beam aimed 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, which represents 5 firings. Vertical dotted line indicates E 0. b value. Fig. 7 A shows the dose-response curve when the laser beam was aimed at the basal body 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 the cilia and cytoplasmic extrusions, the dose-response curve of Fig. 7B gives a value for E 0. 6 of 0-16 mj. Lesions were produced at distances of 16 and 32 fim from the basal body in a direction perpendicular to the cell surface and also in a direction almost parallel to the cell surface. Fig. 8 shows the values for E 0. 5 for all laser-induced stoppage, and for the permanent stoppage only as a function of the distance of the lesion to the basal

9 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 the ciliary motion is not much dependent on the location of the lesion. This shows that the laser effect is not damage of a contractile structure required for the ciliary motion. The laser energy required to produce permanent cessation of motion increases according to Fig. 8 with the distance to the basal body. This supports the notion that permanent damage, probably to the cell surface, was the cause of that effect. 1-5f 10 OS I I d, ftm /, 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 the basal body. The diagram on the right defines the direction of location of the target spots. DISCUSSION The experimental results presented in this paper show that quantitative microsurgery 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 ciliary motion indicates that the effect was brought about by the creation of a microvapour bubble of 1-2 fim 3 volume and a temperature of around 150 C. The neodymium glass laser appears, in this respect, to have the capability to produce more precise microlesions than the ruby laser. With the latter the induced lesions reported were larger and were always accompanied by damage to the cells (Goldstein, 1972; Motokawa & Satir, 1975). 24 CEL 24

10 370 R. Rikmenspoel, S. E. Orris and P. O'Day The permanent cessation of ciliary motion reported in this paper was, as explained in 'Results', most probably caused by cellular damage due to the laser pulse. This phenomenon probably is not of much investigative interest. The discussion that follows will therefore be restricted to the reversible stoppage induced by the low doess of laser energy. The energy needed to cause (reversible) stoppage of ciliary motion appears from Fig. 8 to be largely independent of the location of the produced lesion. The cessation of motion over a large segment of a row of cilia was observed also in the ruby laser experiments in Mytilus by Motokawa & Satir (1975). Together these results indicate that the laser irradiation affects a cellular control mechanism of the ciliary motion. Ca 2+ has been shown (Eckert, 1972; Naitoh & Eckert, 1974) to control the reversal of ciliary motion in Paramecium. The frequency of ciliary motion was reported to be increased by an increased internal Ca 2+ concentration in Necturus maculosus by Murakami & Eckert (1972), but to be decreased in freshwater mussel by Satir (1975). Even though the role of Ca 2+ in the control of ciliary motion is apparently not a simple one, it would be desirable to investigate whether this ion is involved in the effects produced by the laser. We have found, however, that it was not possible to maintain Phragmatopoma gill preparations even for limited periods in Ca 2 +-free or low Ca 2+ media. This instability in Ca 2+ -free medium was reported also for Mytilus by Motokawa & Satir (1975). These observations might indicate that the role of Ca 2 " 1 " in the control of ciliary motion could be investigated more profitably in freshwater organisms. In the experiments reported in this paper the cilia of Phragmatopoma always stopped at 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 forthcoming. That the effective stroke of cilia is triggered by a signal, with the recovery stroke following autonomously, was also found from theoretical analysis of the contractile events in ciliary motion by Rikmenspoel & Rudd (1973). The rather abrupt cessation of motion after several cycles of almost normal motion following a laser pulse suggests that the signal depends on a threshold level of a substance which is either released or sequestered by the cell. The present experiments do not give information as to whether metachronal coordination is mechanical or not (Machemer, 1972, 1974). To investigate the coordination of cilia and the role of ions in the control of ciliary motion, freshwater organisms with the direction of the metachronal coordination in the plane of the ciliary beat (synplectic metachronism) appear to be better candidates. The experiments should include kinetic studies on the spread of ciliary arrest (Motokawa & Satir, 1975). This investigation was supported in part by the National Institute for Child Health and Human Development through grant HD-6445.

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