JPH0697303B2 - Time-sharing optical adjustment method for live cell laser perforator - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for adjusting a focal position of an optical system of a live cell laser perforation apparatus.

(Prior Art) A method for transferring a substance into a living cell is disclosed in which a laser pulse is projected onto a living cell to modify a part of the living cell to take in a foreign substance such as a DNA fragment in the surroundings.
No. 60-83584) "was proposed by the present applicant, and a" live cell laser perforation device (Japanese Patent Laid-Open No. 60-83583) "was similarly proposed as a specific device therefor. This device is equipped with a monitor TV and is configured to project laser pulses while confirming the position of living cells.
In this case, if the highest optical adjustment on the monitor and the highest focus adjustment position for laser light irradiation match, the state at the projection depth position of the laser pulse is considered to be convenient.

(Problems to be solved by the invention) However, a clear image can be obtained only when the living cell surface is in focus, and the objective optical system of the monitor optical system and the laser pulse projection optical system is commonly used. Therefore, it has been found that there is a problem in that if the inside of the cell, which is suitable as the laser projection position, is focused, the image becomes unclear and monitor observation cannot be performed satisfactorily.

(Means for Solving Problems) The above-mentioned problems are objectives shared by the first optical system for obtaining a clear image of the sample and the second optical system for irradiating the laser under optimum conditions. This can be solved by using the time-division adjusting method of the present invention in which the distance between the sample and the sample is changed in a time-division manner while using the optical system.

In the present specification, the "objective optical system" means an optical system that is closest to the sample object, and includes not only the objective lens of the imaging system but also the condensing lens of the light projection system.

The distance between the objective optical system and the sample is changed by moving the objective optical system or the sample.

(Operation) In the present invention, the focal depth positions of the first optical system and the second optical system with respect to the sample are set to desired positions of the sample in a time division manner.

(Effect of the Invention) According to the present invention, the focal position of the first optical system is set to a certain depth position of the sample for a certain period, and the focal position of the second optical system is set to another certain depth position of the sample for another period. Can be set to position. In the above-mentioned live cell perforation device, the position of the objective lens of the microscope is adjusted so that the clearest monitor image can be obtained during cell observation, and the laser pulse is applied only when projecting the laser pulse on the live cells. The objective lens can be moved to a suitable position so that is projected at a desired depth position of the sample.

(Example) Hereinafter, an example of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a schematic view of the first embodiment of the present invention, showing an example of a laser perforation apparatus. FIG. 2 is a timing chart of signals in this example. Laser punching device body 1
Is a function of a microscope for enlarging the image of the sample 2 to the TV camera 3 and a laser beam 5 emitted from the laser oscillator 4.
Performs the two functions of a light collecting device that collects light and projects it onto the sample 2. The focus of the laser light 5 and the focus position of the imaging surface change depending on the vertical movement of the objective lens 1a. This vertical movement of the electrostrictive element 6 provided between the objective lens 1a and the lens barrel 1b of the apparatus main body 1. It is performed by distortion deformation. The projection timing of the laser light 5 is controlled by the master signal generated by the master signal generator 7. This master signal is
The signal is once input to the delay trigger circuit 8 and delayed for a predetermined time (the time required for the electrostrictive element to deform), and then input to the laser oscillator 4 as a laser light emission instruction signal. The master signal is also input to the electrostrictive element drive device 9 to generate an electrostrictive element drive voltage for controlling the drive of the electrostrictive element 6.
Therefore, at the time of laser projection, the objective lens 1a is positioned so that the laser light 5 focuses on a desired and suitable position of the sample 2, and in other cases, the objective lens 1a is set to a position suitable for monitor observation. Can be positioned. Specifically, in the state where no voltage is applied to the electrostrictive element, the objective lens 1a is adjusted so that the monitor image is projected most clearly as a whole, and then the object that is going to project the laser light (for example, The voltage applied to the electrostrictive element is set in advance so that only the (cell nucleus) is clearly displayed, and after that, when the laser beam is projected, the voltage of this set value is applied to the electrostrictive element. do it. If the objective lens 1a is located at a position other than the position suitable for monitor observation, the image projected on the monitor TV 10 via the TV camera 3 becomes unclear, so the shutter is adjusted in time with the signal from the master signal generator 7.
It is preferable that the opening / closing of 11 is controlled by the shutter drive circuit 12. In the apparatus thus configured, while the sample 2 is moved by the moving stage 13 and pulse signals are generated from the master signal generator 7 at predetermined intervals, the laser is repeatedly and continuously provided at a predetermined depth position of the sample 2. As the light is projected, a clear monitor image can be continuously observed.

In order to prevent the sample image acquisition, in addition to using the shutter 11, the sample certification may be turned off. The shutter may be an electromagnetically driven mechanical shutter or an optical shutter using a filter or a polarizer.

FIG. 3 is a schematic view of the second embodiment of the present invention, which is an example of the laser perforation apparatus similar to the first embodiment. FIG. 4 is a timing chart of signals in this example. In this embodiment, the laser light is projected at the position indicated by the light pen 15. Light pen 15 on monitor TV10
When the instruction is given, the position control signal generator 16 calculates the indicated position based on the pulse signal output from the light pen 15, and generates a control signal based on the calculated value. The beam deflecting device 17 including a galvanometer mirror is provided with the deflecting device driving means 18 that receives this control.
The laser light 5 is projected by the light pen 15 at the position designated by the light pen 15. The beam deflecting device 17 receives the control signal and drives a galvanometer mirror or the like so that the laser beam 5 is projected at the position indicated by the light pen 15. The pulse signal from the light pen 15 is simultaneously input to the time control signal generator 19. The time control signal generator 19 converts a pulse signal generated in each scanning cycle of the TV camera 3 into a time control signal at a desired interval.
This time control signal is input to the driving device 9 for the electrostrictive element 6 in the same manner as in the first embodiment to generate an electrostrictive element drive voltage for controlling the drive of the electrostrictive element 6. Further, the time control signal controls opening / closing of the laser optical path shutter 14 provided between the microscope body 1 and the laser oscillator 4. As shown in FIG. 4, the laser optical path shutter 14 is opened during the voltage application period of the electrostrictive element 6 excluding the transient period, and the laser light 5 is continuously projected onto the sample during that period (pulse form). . The focus of the laser beam for punching may be set so that a predetermined voltage value is applied to the electrostrictive element 6 when the laser beam 5 is projected, as in the case of the first embodiment. However, when the laser light 5 is always projected onto the predetermined focal plane as described above, the laser light 5 is not always projected onto a suitable position of the sample. In this embodiment, the focal depth position of the laser beam 5 is automatically set to the object pointed by the light pen 15. As described in JP-A-60-83583, the reference laser beam 21 is constantly projected onto the sample. The light reflected from the sample 2 by this projection is extracted from the microscope body 1 by the semitransparent mirror 22. The extracted light is the lens 2
After passing through the iris diaphragm 3 and 24, it is picked up by the photoelectric conversion element 26. An oscillating voltage of a predetermined cycle is constantly generated from the oscillator 17, and this oscillating voltage is applied to the electrostrictive element 6 while the switch 28 is closed. The phase sensitive detector 29 outputs the phase difference between the output of the photoelectric converter 26 and the output of the oscillator 27 as a voltage value. The relationship between the voltage V applied to the electrostrictive element 6, that is, the position of the focal plane, and the output I of the photoelectric converter is as shown in FIG. 5, and the output V'of the phase sensitive detector is the electrostrictive element. FIG. 6 shows the relationship between the voltage applied to and. Therefore, the focal plane of the laser projection optical system is set at the depth position where the reflected light becomes maximum with respect to the (two-dimensional) pointing position of the light pen 15, and the laser beam 5 for perforation is projected on the sample 2. . During the driving period of the electrostrictive element 6 including the transition period, in order to prevent an inappropriate screen from being displayed on the monitor TV, the opening / closing of the gate switch 20 provided between the TV camera 3 and the monitor TV10 is time-controlled. It is better to carry out based on the signal. It is more preferable that the image immediately before the gate switch 20 is closed is displayed on the monitor TV 10 when the gate switch 20 is closed. When configured as in this example, while accurately grasping the position of the living cells, it is possible to project a laser beam in the vicinity of the center of the living cells, the living cells described in JP-A-60-83584 It is considered to be extremely preferable for the method of substance transfer into the interior. Since the projection of the laser light is controlled by cutting off the laser light itself as in this embodiment, either pulsed laser light or continuous laser light can be used.

FIG. 7 is a schematic view of a third embodiment of the present invention, which is an example of a laser perforation apparatus similar to each of the above embodiments. In this embodiment as well, the laser beam is projected to the designated position of the light pen as in the second embodiment, but the focal depth position of the laser light 5 with respect to the sample 2 is changed according to the pressing force of the light pen. ing. As shown in FIG. 8, the light pen used in this embodiment is a light guide unit 1 movably provided in front of and behind a cavity 15b at the tip of the light pen body 15a.
It has a 5c, monitor TV10 through this light guide 15c
Light is guided to the photodetector 15d. A pressure detecting element 15e is provided behind the light guide portion 15c. The pressure detecting element 15e is sandwiched between the light guide portion 15e and the spring 15d, and generates a voltage value between the facing surfaces according to the pressure applied between the light pen body 15a and the light guide portion 15c. That is, a signal having a value corresponding to the pressing force on the monitor TV10 when the monitor TV10 is instructed by the light pen 15 is emitted together with the detection signal of the fluorescence emission during the electron beam scanning. The voltage signal emitted from the light pen 15 corresponding to the pressing force is sent to the focal depth position control signal generator 30. The signal from the focal depth position control signal generator 30 controls the electrostrictive element driving voltage generated from the electrostrictive element driving device 9. Therefore, a laser beam waist is formed at the focal depth position with respect to the sample 2 according to the pressing force of the light pen 15 on the monitor TV 10.

In the present embodiment, the "depth of the laser focus" corresponding to the "depth of the scalpel" is added to enable three-dimensional control, and the processing accuracy can be dramatically improved. It is possible to perform highly precise cell surgery such as laser irradiation aimed at a specific depth inside a cell, for example, a cell nucleus at a specified depth, or perforation aimed at the inner surface of a biological membrane.

[Brief description of drawings]

1 is a schematic diagram of the first embodiment of the present invention, FIG. 2 is a timing diagram of signals in the first embodiment, FIG. 3 is a schematic diagram of the second embodiment of the present invention, and FIG. FIG. 5 is a timing chart of signals in the embodiment, FIG. 5 is a diagram showing the relationship between the voltage applied to the electrostrictive element and the output of the photoelectric converter, and FIG. 6 is the output of the phase sensitive detector and the electrostrictive element. FIG. 7 is a diagram showing the relationship with applied voltage, FIG. 7 is a schematic diagram of a third embodiment of the present invention, and FIG. 8 is a sectional view of an embodiment of a light pen used in the third embodiment. 1 ... microscope body, 1a ... objective lens 1b ... lens barrel, 2 ... sample, 3 ... TV camera 4 ... laser oscillator 5 ... laser light 6 ... electrostrictive element, 7 ... master signal generation Device 8 ...... Delay trigger circuit 9 ...... Electrostrictive element drive device, 10 ...... Monitor TV 11 ...... Shutter 12 ...... Shutter drive circuit 13 ...... Moving stage 14 ...... Laser optical path shutter, 15 ...... Light pen 16 ... ... Position control signal generator 17 ... Beam deflector 18 ... Deflection device control means 19 ... Time control signal generator 20 ... Gate switch 21 ... Reference laser light 26 ... Photoelectric converter, 27 ... Oscillator 29 …… Phase sensitive detector 30 …… Depth of focus position control signal generator

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G02B 21/36 7625-2K

Claims (1)

[Claims] 1. A first method for obtaining a clear image of a living cell sample.
And a second optical system for irradiating a desired position of a living cell sample with a laser under an optimum condition.
Is an optical adjustment method of a live cell laser perforation apparatus in which the optical system of 1) and the second optical system share only the objective optical system, and the distance between the objective optical system and the live cell sample is According to the alternative use of the first optical system and the second optical system, the first distance and the laser for obtaining a clear image of the live cell sample are set to the desired position of the live cell sample under optimum conditions. A time-division optical adjustment method for a live-cell laser perforation apparatus, characterized in that it is changed in a time-division manner with respect to a second distance for irradiating the cells.