Micromanipulator for Inverted-Design Microscope

The invention relates to a micromanipulator for an inverted-design microscope, or more particularly, to a micromanipulator associated with an inverted design microscope for performing a variety of minute operations upon an object to be observed.

A typical micromanipulator for inverted-design microscope of the prior art is illustrated in FIG. 1. As shown, the microscope includes a stage 1 on which is placed a culture vessel 2 which contains a specimen such as cells 2a. An illuminion optical system comprising a condenser lens 3 having a ring slit 3a internally is disposed above the stage 1 for illuminating the specimen. On the other hand, a revolver 4 is disposed below the stage 1, and an objective lens 5 internally including a phase plate 5a is mounted on the revolver for observation of the specimen. The manipulator shown is mounted with respect to the stage 1 of such microscope. Specifically, a fixture 6 is secured to the lower surface of the stage 1, and is coupled to a micromanipulator 7 through a vertical motion mechanism, a back-and-forth motion mechanism and a lateral motion mechanism, each of which may comprise a rack and a pinion, so that a glass needle 7a disposed at the extremity of the micromanipulator extends toward the cell 2a in the culture vessel 2 or toward the focal position of the microscope.

The purpose of the micromanipulator is to effect a pricking technique which causes the glass needle 7a of the micromanipulator 7 to pierce through one of the cells 2a in the vessel 2 to permit an enzyme contained in the culture solution to permeate into the cell. When the glass needle 7a is to pierce one of the cells 2a, resulting damage to the particular cell must be minimized, by directing the extremity of the manipulator in the exact direction toward the particular cell 2a and enabling a minute movement of the manipulator which is free from oscillations or meandering movement. However, with a conventional arrangement, this must be achieved by directly operating the operating members 7b of the manipulator 7 manually, and hence any trembling motion which occurs in the finger or fingers of the hand is likely to be transmitted to the glass needle 7a through the manipulator 7. In addition, an increased length from the mounting location of the fixture 6 on the stage 1 to the extremity of the glass needle 7a tends to magnify any oscillating motion transmitted to the glass needle 7a, and thus any external oscillation or trembling motion of the finger may be magnified when it is transmitted to the glass needle 7a. For this reason, when the pricking operation is to be performed, the manipulator 7 must be operated gently while suppressing any trembling motion of the finger to the maximum possible degree and while waiting for the influence of any external oscillation to settle down. Thus, the operation of the manipulator requires extreme care and is quite difficult, and thus requires a high level of skill and time.

Another problem of the prior manipulator 7 relates to the plurality of operating members 7b thereof which are mechanically interlocked with each other. This results in a complex construction of the mechanically interlocked mechanism, resulting in a reduced level of reliability and durability of the apparatus. Also, the location of the individual operating members 7b is limited, presenting another difficulty relating to the degraded ease of operation. The bottom surface of the culture vessel 2 or the surface of a cell being cultured are generally not uniform, so that when the free end of the glass needle 7a is lowered to the focal position, the free end of the glass needle 7a may inadvertently be brought into abutment against the bottom surface of the vessel 2, thus causing a fracture of the glass needle 7a. As a consequence, the efficiency of the pricking operation is degraded.

It is a primary object of the invention to overcome the described difficulties of the prior art, by providing a micromanipulator for an inverted-design microscope which enables a greatly facilitated and rapid operation without the need for suppressing any trembling motion of the finger and while minimizing the influence of any external oscillations.

It is another object of the invention to provide a micromanipulator for an inverted-design microscope which achieves a high level of reliability and durability of the manipulator while drastically improving the operational efficiency of the pricking technique, by avoiding a fracture of the glass needle during the pricking technique.

In accordance with the invention, the micromanipulator includes a needle holder which is remotely operated under hydraulic drive. This technique prevents any trembling motion of the finger from being transmitted to a glass needle. The needle holder is located above a stage while the hydraulic drive mechanism is mounted on the upper surface of the stage, thus reducing the length from the location of the mechanism to the free end of the glass needle. This arrangement reduces the susceptibility of the glass needle to oscillations. Thus, it is unnecessary to suppress any trembling motion of the finger intentionally during the operation, and the influence of any external oscillation is minimized, allowing the manipulator to be operated rapidly and in a facilitated manner. By way of example, when the pricking operation is to be conducted, it has been an upper limit to treat about five hundred cells per hour in the prior art practice, while with the manipulator of the invention, an increased number of cells between one to two thousand can be treated. A hydraulic operator which causes a vertical motion of the glass needle is connected to the hydraulic drive mechanism merely through a hydraulic transmission tube, allowing the hydraulic operator to be freely located relative to the microscope, thus improving the ease of operation. Since the manipulator is mounted on the upper surface of the stage, mere replacement of the stage allows a conversion from an inverted-design microscope with manipulator to a normal inverted-design microscope. The glass needle is carried by an annular needle holder in the direction of the optical axis through a hollow or transparent member, thus preventing it from causing an interference with a phase contrast operation.

A plurality of hydraulic drive mechanisms can be connected together through hydraulic transmission tubes for coupling them together in a simple manner, thus greatly simplifying the construction of interlocked mechanisms, and with consequent increase in the level of reliability and durability of the apparatus.

In the event the bottom surface of a culture vessel or the surface of a cell being cultured is not a flat plane, the lower limit for the position of the free end of the glass needle may be established at a focal position, whereby it is assured that the free end will come to a stop at the focal position whenever the glass needle is lowered. If the focal position is changed, the free end is again stopped at the changed focal position. This avoids a fracture of the glass needle during the pricking operation, thus drastically improving the operational efficiency thereof.

FIG. 1 is a front view of one exemplary micromanipulator for inverted-design microscope of the prior art;

FIGS. 2 and 3 are a side elevation and a front view of a micromanipulator for inverted-design microscope according to one embodiment of the invention;

FIGS. 4 and 5 are a plan veiw and a longitudinal section, partly broken away, of the needle holder of the embodiment shown in FIGS. 2 and 3;

FIGS. 6 and 7 are a plan view and a longitudinal section, partly broken away, of an hydraulic drive mechanism associated with a holder which is used in the embodiment shown in FIGS. 2 and 3;

FIG. 8 is a transverse section of an hydraulic drive mechanism which is mechanically associated with a focussing operation used in the embodiment of FIGS. 2 and 3;

FIG. 9 is a longitudinal section of a source of hydraulic pressure and an associated operating unit which are used in the embodiment shown in FIGS. 2 and 3;

FIG. 10 is a diagrammatic view of the overall arrangement of the hydraulic drive system used in the embodiment of FIGS. 2 and 3;

FIGS. 11 and 12 are a plan view and a longitudinal section, partly broken away, of another form of needle holder;

FIG. 13 is a transverse section of another form of source of hydraulic pressure and its associated operating unit; and

FIG. 14 is a plan view of a fine motion knob shown in FIG. 13.

An inverted design microscope which is adapted to have a micromanipulator according to the invention mounted thereon is illustrated in FIGS. 2 and 3. As shown, the microscope includes a body 10, from the rear portion of which extends an integral stanchion 11 in an upright manner which is used for mounting an illumination system thereon. Intermediate the length of the stanchion 11, a stage 12 is detachably mounted relative to the body 10 through a translational structure including a dovetail 12a and a dovetail groove 10a. A condenser lens 14 is carried by a condenser holder 13 which is mounted on the stanchion 11 at a location above the stage 12, thus projecting light from a source of illumination, which is secured to the top of the stanchion 11, over the stage.

An objective lens 21 is detachably mounted on a revolver 20 disposed below the stage 12, and allows a specimen which is placed on the stage 12 to be observed through the objective lens and through a pair of binocular lens barrels 9a, 9b. The revolver 20 is laterally disposed and extends through the bottom portion of the body 10 and includes a pair of externally exposed ends, which are coupled through a combination of a rack 20a and a pinion 22b to a coarse/fine motion assembly of a known construction which includes a drive shaft carrying a pair of handles including a coarse motion handle 22 and a fine motion handle 22a for the focussing operation. As the handles 22 and 22a are turned, the revolver 20 can be moved vertically up and down relative to the body 10 to achieve a focussing of the objective lens 21.

The micromanipulator according to the invention essentially comprises a needle carrier 15 which carries a glass needle 16 of the manipulator in the direction of an optical axis of observation, an hydraulic drive mechanism 17 for providing a vertical motion of the carrier 15 hydraulically, an hydraulic operator 26 for operating the hydraulic drive mechanism 17, and another hydraulic drive mechanism 23 for detecting the stroke of movement of the objective lens 21 and for transmitting the stroke to the hydraulic drive mechanism 17 associated with the carrier. The manipulator is mounted on the microscope in a manner such that the needle carrier 15 is disposed intermediate the stage 12 and the condenser lens 14 while allowing the carrier 15 to maintain the glass needles 16 in a vertically depending manner. The needle carrier 15 includes a centering mechanism for the glass needle 16.

As shown in FIGS. 4 and 5, the needle carrier 15 essentially comprises an annular support frame 28 which is substantially integrally mounted on the hydraulic drive mechanism 17 associated with the holder, an annular centering frame 30 which is disposed within the support frame 28 so as to be movable in a plane orthogonal to the direction of the optical axis, a holder frame 33 fitted in and detachably secured to the feed frame 30, and an L-shaped holder 35 having a horizontal portion which extends through the holder frame 33 and its free end extending downwardly at the center of the holder frame 33 and having a clamp screw 36 which is used to secure the glass needle 16, the central portion of the holder being formed of a transparent material and its one end opposite from the free end being connected to a hydraulic pressure transmitting tube 19.

The holder frame 33 comprises an annular body fitted inside the centering frame 30 and having a flange integrally formed around its periphery at its top, which flange is adapted to be placed on the support frame 28. By tightening a lock screw 34 which extends through the support frame 28 and threadably engaged with the centering frame 30, the holder frame is secured to the centering frame 30. The holder frame can be separated from the centering frame 30 by loosening the lock screw 34. The centering mechanism comprises a pair of spring sets 31 which are threadably engaged with the support frame 28 in a radial direction and a pair of threaded shafts 32 each having a centering knob 32a, both of which sets are urged against a V-shaped groove 30a formed in the peripheral surface of the centering frame 30. Each spring set 31 comprises a spring 31a and an abutment pin 31b which are received within a hollow screw, with the hollow screw being threadably engaged with the support frame 28 and the abutment pin 31b disposed in abutment against the peripheral groove 30a. The threaded shaft 32 which carries the centering knob 32a is disposed in opposing relationship with one of the spring sets 31 and is threadably engaged with the support frame 28 so that its free end presses against the peripheral groove 30a. It will be noted that the pair of spring sets 31 and the pair of threaded shafts 32 are equi-angularly spaced apart. Accordingly, by adjusting the spring sets and the threaded shafts to move the centering frame 30, the movement of the latter can be transmitted through the holder frame 33 to control the centering operation which positions the glass needle 26 on the optical axis of the objective lens.

The needle carrier 15 thus constructed includes a dovetail 28a extending vertically and secured to one lateral side of the support frame 28 by means of a mounting member 28b. The dovetail 28a fits in a dovetail groove 46a (see FIGS. 6 and 7) formed in the hydraulic drive mechanism 17 associated with the holder, and by tightening a clamp screw 29 which is threadably engaged with the mounting member 28b, it can be integrally coupled to a slide 46 (see FIGS. 6 and 7), having the dovetail groove 46a formed therein, of the hydraulic drive mechanism 17. Accordingly, the needle carrier 15 mvoes up and down together with the slide 46. The L-shaped holder 35 which is carried by the needle carrier 15 is connected to an injector 18 which is disposed externally of the body 10 of the microscope, through the hydraulic pressure transmitting tube 19, as shown in FIG. 2, whereby by filling the tube 19 with a silicone oil or the like, pressure can be applied to a liquid chemical contained within the glass needle 16 to displace it from the needle tip.

As shown in FIGS. 6 and 7, the hydraulic drive mechanism 17 associated with the holder comprises a mounting base 37 having a laterally reversed L-configuration in cross section and having its horizontal limb secured to the upper surface of the stage 12 by means of a screw 12b, a pinion shaft 38 disposed horizontally to extend through the vertical limb of the mounting base 37 in a rotatable manner and carrying a coarse motion handle 40 mounted on one end thereof for rotation, a first hydraulic cylinder 41 integrally mounted on a rack 43 which meshes with a pinion 38a formed on the pinion shaft 38 intermediate its length, a piston rod 45 fitted into the first hydraulic cylinder 41 and is actuated by oil under pressure within the cylinder 41, and the slide 46 mentioned above which is integrally secured to the piston rod 45 by a set screw and having the dovetail groove 46a formed therein in which the support frame 28 of the needle carrier 15 is mounted.

The pinion shaft 38 extends through and is rotatably carried in the mounting base 37, and is formed with a head 38b at its one end which prevents its withdrawal and has a Belleville spring 39 fitted on its other end, which is followed by the coarse motion handle 40 which is threadably engaged thereon. When the pinion shaft 38 is maintained fixed while the coarse motion handle 40 is rotated in a direction to screw it on, the Belleville spring 39 is compressed to produce a reaction of an increased magnitude, which can be utilized to adjust an operating force to move the pinion shaft 38 and the coarse motion handle 40 in an integral manner. The force of friction acting between the pinion shaft 38 and the coarse motion handle 40 is chosen to be sufficiently great, due to the force of friction produced by the Belleville spring 39, to prevent the coarse motion handle 40 alone from rotating relative to the pinion shaft 38, thus causing its loosening.

A longitudinally extending dovetail groove 37a is formed in the inside of the vertical limb of the mounting base 37, and is engaged by a dovetail 42 which carries the rack 43 meshing with the pinion 38a. The cylinder 41 is secured to the dovetail 42. Accordingly, the cylinder 41 may be moved up and down relative the mounting base 37 by utilizing the coarse motion handle 40. The interior of the cylinder 41 is partitioned by a rolling diaphragm 44 into an oil chamber 41a and a vacant chamber 41b, with the top end of the piston rod 45 being secured to the rolling diaphragm 44. The slide 46 is channel-shaped in transverse section so as to surround the cylinder 41, and thus is mounted on the outside of the cylinder 41 so as to be movable in the vertical direction with roller guides 47 interposed therebetween. At its lower end, the slide 46 includes a horizontally extending limb, to which the lower end of the piston rod 45 is secured. The dovetail groove 46a is formed in the backside of the vertical wall of the slide 46 and is engaged by the dovetail 28a on the support frame 28.

As shown in FIG. 8, the hydraulic drive mechanism 23 associated with the focussing operation is disposed around one of the focussing handles, 22. The drive mechanism 23 comprises a cylindrical frame 48 secured to the body 10 of the microscope in coaxial relationshp with the focussing handle 22, a transmission drum 52 disposed within the frame 48 and urged into abutment against the fine motion handle 22a for integral rotation therewith, a threaded shaft 55 disposed for axial movement back and forth through a transmission shaft 51 which is coupled integrally with the transmission drum 52, and a piston rod 58 disposed for movement within an hydraulic cylinder 54 by the action of the threaded shaft 55, the oil pressure within the cylinder 54 being changed in response to movement of the piston rod 58.

The frame 48 is mounted on a mounting ring 49, which is secured to the body 10 of the microscope in coaxial relationship with the focussing handle 22, in a detachable manner by means of a lock screw 24, with the open end of frame 48 having a head 50 secured thereto. The transmission drum 52 is formed of an elastic material such as rubber which is fitted around and urged against the fine motion handle 22a, and is secured to one end of the transmission shaft 51 by a mounting plate 52a. The transmission shaft 51 extends through an intermediate wall 48a of the frame 48 and is rotatably disposed in coaxial relationship with the focussing handle 22. The other end of the transmission shaft is integrally formed with a transmission drum 51b having a notched groove 51a which is formed to extend in a direction parallel to the transmission shaft 51. A pin 56 fixedly mounted on the periphery of the threaded shaft 55 is fitted into the groove 51a, and the threaded shaft 55 carries the piston rod 58 fitted therein, which is disposed in the cylinder 54 which is secured so as to extend through the head 50 as by threadable engagement therewith. The threaded shaft 55 is threadably engaged with the hydraulic cylinder 54 which is threadably engaged with the head 50, and the piston rod 58 is fitted into a central axial bore therein which is coaxial with the transmission shaft 51.

The hydraulic cylinder 54 has its interior also partitioned into an oil chamber 54a and a vacant chamber 54b by means of a rolling diaphragm 57, to which is secured the free end of the piston rod 58. The oil chamber 54a in the cylinder 54 communicates with the oil chamber 41a in the first hydraulic cylinder 41 within the hydraulic drive mechanism 17 associated with the holder through hydraulic transmission tube 25 (see FIG. 2).

The hydraulic drive mechanism 23 responsive to a focussing operation is constructed such that when one revolution of the fine motion handle 22a causes the objective lens 21 to move though a distance of 200 .mu.m, for example, such rotation of the handle 22a is transmitted through the transmission drum 52, the transmission shaft 51 and the threaded shaft 55 to be converted into an axial movement of the piston rod 58 to cause a corresponding movement of the rolling diaphragm 57, so that an amount of oil which corresponds to the stroke of the objective lens 21 is fed through the hydraulic transmission tube 25 to the cylinder 41 of the hydraulic drive mechanism 17 associated with the holder, thereby moving the needle carrier 15 through the same distance as the distance through which the object lens 21 has moved.

It will be appreciated that the above description applies when the space between the objective lens 21 and the glass needle 16 comprises air. However, a cell which is to be operated upon is frequently situated in a liquid such as a culture solution, water or the like, and when a focal position is displaced within the liquid, the length of the optical path changes, with a consequent change in the distance through which the objective lens must be moved, thus causing an offset in the focussed position of the glass needle 16 which has initially been attained. representing a variation in the length of optical path within the liquid by .DELTA.l, there results a change in the distance through which the objective lens 21 must be operated which is equal to .DELTA.l/n where n is the refractive index of the liquid. To compensate for this error, the pitch of the threads on the threaded shaft 55 is suitably chosen so that the ratio of the stroke of the objective lens 21 to the distance of movement of the needle carrier 15 in response to an adjustment of the fine motion handle 22a be equal to or close to the value of the refractive index n of the liquid, thus overcoming the problem.

As shown in FIG. 9, the source of hydraulic pressure and its associated operator 26 which are used to operate the hydraulic drive mechanism 17 associated with the holder comprise a hand rest 60 mounted on a base 59 by means of stanchions 61 and centrally formed with an elongate slot 60a which determines the extent of oscillating movement of a joystick 70, a cylindrical frame 62 secured to the base 59, a second hydraulic cylinder 63 secured in the sidewall of the frame 62, a piston rod 65 operated by the hydraulic pressure in the second hydraulic cylinder 63, a slider 66 disposed inside the frame 62 on the base 59 so as to be slidable in the horizontal direction and carrying a ball 66a disposed for movement and to which the piston rod 65 is connected, a support frame 67 threadably engaged with the top of the frame 62, an operating shaft 68 rotatably carried within the support frame 67 and having a spherical recess 68a therein in which the ball 66a is fitted to cause a movement thereof, a third hydraulic cylinder 69 mounted on the top of the operating shaft 68, a fine motion joystick 70 extending through the slot 60a and having a piston rod 73, which causes the hydraulic pressure within the third hydraluic cylinder 69 to change, extending therein, and a coarse motion knob 74 rotatably mounted around the joystick 70 and connected with the piston rod 73.

The interior of the second hydraulic cylinder 63 is partitioned into an oil chamber 63a and a vacant chamber 63b by a rolling diaphragm 64, to which diaphragm is secured one end of the piston rod 65. The other end of the piston rod 65 is connected to the slider 66 which is slidable on the base 59 in the horizontal direction, as indicated by a double-ended arrow A. The ball 66a is secured centrally on the top surface of the slider 66 as by a pivot.

The operating shaft 68 includes a spherical articulation member which is rotatably fitted in the support frame 67 at its bottom, the support frame 67 being threadably engaged with the top of the frame 62 so as to be movable up and down. The top portion of the operating shaft 68 includes the third hydraulic cylinder 69. It will be seen that the spherical articulation member is formed with a spherical recess 68a in its bottom, in which the ball 66a is fitted. The lower end of the joystick 70 which extends downwardly through the slot 60a formed in the hand rest 60 is secured around the top of the cylinder 69. Accordingly, it is possible to oscillate the joystick 70 within the slot 60a in a direction indicated by a double-ended arrow B about the articulation member, thus causing the slider 66 to slide in either of the directions indicated by the arrow A, acting through the ball 66a.

The interior of the third hydraulic cylinder 69 is also partitioned into an oil chamber 69a and a vacant chamber 69b by a rolling diaphragm 71, to which diaphragm the lower end of the piston rod 73 is secured. The coarse motion knob 74 is mounted on the top end of the joystick 70 so as to be rotatable about the center axis of the joystick 70, as indicated by a double-ended arrow C, and is also connected to the upper end of the piston rod 73 through a threaded shaft, not shown, so that the rotation of the knob 74 causes an axial movement of the piston rod 73.

The oil chamber 63a of the second hydraulic cylinder 63 is connected to the oil chamber 69a of the third hydraulic cylinder 69 through an hydraulic transmission tube 72, thereby transmitting a displacement of the piston rod 73 in response to rotation of the coarse motion knob 74 to the second hydraliuc cylinder 63. The oil chamber 63a of the second hydraulic cylinder 63 is also connected to the oil chamber 54a of the hydraulic cylinder 54 in the hydraulic drive mechanism 23 which is responsive to the focussing operation, through the hydraulic transmission tube 27. It will be seen that the opposite edges of the elongate slot 60a formed in the hand rest 60 serve as stops which determine the extent of oscillating motion of the joystick 70.

FIG. 10 is a diagrammatic view illustrating the general arrangement of the hydraulic drive system. The hydraulic drive mechanism 17 which drives the needle carrier 15 has a cylinder capacity corresponding to a stroke of 12 mm, for example, and accordingly stops are provided on the hydraulic drive mechanism 23 and the hydraulic operator 26 so that a combined stroke of the mechanism 23 and the operator 26 does not exceed 12 mm, thus preventing the individual rolling diaphragms from being damaged. In the present embodiment, the hydraulic drive mechanism 23 and the hydraulic operator 26 are connected in tandem to the hydraulic drive mechanism 17, but they can be connected in parallel relationship as well. In addition, it will be seen that any desired number of hydraulic drive mechanisms may be used, provided a combined stroke of the respective hydraulic drives does not exceed the stroke of the hydraulic drive mechanism 17. In the present embodiment, the hydraulic drive mechanism 23 which is responsive to the focussing operation is connected to the fine motion handle 22a of the focussing handle 22, but alternatively, it may be connected to a vertical drive mechanism associated with the objective lens 21 or to revolver 20 directly.

As a result of the described construction of the manipulator according to the invention, the focussing handle 22 is used to perform a focussing operation upon a specimen initially placed on the stage 12. The hydraulic drive mechanism 23 responsive to the focussing operation is then connected to the fine motion handle 22a of the focussing handle 22.

The glass needle 16 is then set up. Initially, the condenser lens 14 is sprung back or removed since it stands in the way of adjusting the glass needle 16. The coarse motion handle 40 of the hydraulic drive mechanism 17 is operated to raise the support frame 28 of the needle carrier 15 sufficiently upward. A suitable length of glass needle 16 is attached to the holder 35 on the holder frame 33, which is then fitted into the feed frame 30 and secured in place by the lock screw 34. By utilizing the coarse motion handle 40 and the coarse motion knob 74 of the hydraulic operator 26 in combination, the needle carrier 15 is lowered to bring the tip of the glass needle 16 to a position slightly above the focussed position. If the objective lens having a magnification of 4.times.to 10.times.is used, the tip of the glass needle 16 will appear as a white spot in the field of view. The centering knobs 32a are then operated to move the threaded shafts 32 back and forth to adjust the location of the centering frame 30, thus bringing the glass needle 16 to the center of the field of view. Preferably the glass needle is brought to the exact center by utilizing a cross scale of an eyepiece. The joystick 70 associated with the hydraulic operator 26 is then slowly oscillated to tilt it to its lower limit stop position. While maintaining the joystick 70 at this position, the coarse motion knob 74 is turned to lower the glass needle 26 until the tip thereof coincides with the focussed position, whereupon the entire set-up operation is completed.

A pricking operation upon a cell being cultured is then performed. As the joystick 70 is oscillated, it is possible to cause the glass needle 16 to move up and down in an extent from 100 to 500 .mu.m with the focussed position as the lower limit. When the fine motion handle 22a is turned in order to modify the focussed position, such movement is transmitted from the hydraulic drive mechanism 23 to the hydraulic drive mechanism 17, thus changing the lower limit position for the needle carrier 15. Accordingly, if the bottom surface of the culture vessel or the surface of a cell being cultured does not have a uniform plane, the tip of the glass needle 16 is maintained at a focussed position for the cell, thus automatically preventing the occurrence that the tip of the needle may be moved against the bottom surface of the vessel to cause a fracture thereof. In this manner, a pricking operation can be successively performed on sequential cells. Since the glass needle 16 is carried by the annular needle carrier 15 with the holder 35 interposed therebetween, there is no interference with the phase contrast operation which takes place between the condenser lens 14 and the specimen.

FIGS. 11 and 12 show another form of needle carrier 15A. Instead of being carried by the holder frame 33 as shown in FIGS. 4 and 5, in this instance, a transparent disc 75 is fitted in the feed frame 30, and the base of the holder 35 is carried by the central portion of the transparent disc 75. In this manner, the length as measured from the base end of the holder 35 to the tip of the glass needle 16 is reduced. Since the holder 35 is carried by the phase contrast operation which takes place between the condenser lens 14 and the specimen.

FIGS. 13 and 14 show another form of hydraulic operator 26A. In the hydraulic operator 26A, the joystick 70 shown in FIG. 9 is replaced by a combination of a fine motion knob 76 and a threaded shaft 77, and the coarse motion knob 74 is replaced by a coarse motion knob 78 having a threaded shaft 79. Specifically, the piston rod 65 associated with the cylinder 64 is urged for outward movement by a coiled spring 81, and a connecting rod 65a is connected to the threaded shaft 77 so that the end of the rod 65a may be displaced by the threaded shaft 77 which is threadably engaged with a stationary member 82. The threaded shaft 77 is turned by the fine motion knob 76 in which an elongate slot 76a is formed in which a control pin 80 fixedly mounted on the stationary member 82 is fitted to restrict the extent of angular movement. The piston 73 associated with the cylinder 69 is also urged to move outwardly by a coiled spring 83, and a connecting rod 73a couples the piston 73 with the threaded shaft 79 so that the end of the rod 73a may be displaced by the threaded shaft 79 which is threadably engaged with a stationary member 84 and turned by the coarse motion knob 78.

Such construction again achieves the similar effect and function as described in connection with the first mentioned embodiments.