JPS62234113A - Time-division optical adjusting method - Google Patents

Abstract

PURPOSE:To obtain a clear image by time-dividedly changing a distance between an objective optical system to be shared by the 1st and 2nd optical systems and a sample. CONSTITUTION:The distance between the objective optical system to be shared by the 1st optical system and the 2nd optical system for projecting a laser beam under an optimum condition and the sample is time-dividedly changed. Consequently, the focal position of the 1st optical system can be set up on a certain depth position in a certain period and the focal position of the 2nd optical system can be set up on a certain depth position in another period. In addition, light beams having different wavelengths each other can be focused on the same position by using the two focal positions.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for adjusting the focal position of an optical device.

(Prior art) A method for transferring substances into living cells (Japanese Unexamined Patent Application Publication No. 1989-1999) involves projecting a laser pulse onto a living cell and thereby modifying a part of the living cell to take in surrounding foreign substances such as DNA fragments. -83584)'' was proposed by the present applicant, and as a specific device for this purpose, the ``Live Cell Laser Perforation Apparatus (Japanese Unexamined Patent Publication No. 60-83583)'' was similarly proposed.

This device is equipped with a monitor TV and is configured to project laser pulses while confirming the location of living cells. In this case, it is considered advantageous if the highest optical adjustment and the highest focus adjustment position for laser beam irradiation match on the monitor, since the state at the laser pulse projection depth position can be monitored.

(Problem to be solved by the invention) However, a clear image can only be obtained when the surface of living cells is in focus, and the objective optical system of the monitor optical system and the laser pulse projection optical system are commonly used. It has been found that there is a problem in that when the laser is focused on a position inside the cell that is suitable for the laser projection position, the image becomes unclear and it becomes impossible to carry out satisfactory monitoring observations.

(Means for solving the problem) The problem mentioned above is that the objective shared by the first optical system for obtaining a clear image of the sample and the second optical system for irradiating the laser under optimal conditions. This problem can be solved by using the time-division adjustment method of the present invention, which uses an optical system but changes the distance between it and the sample in a time-division manner.

In this specification, the term "objective optical system" refers to the optical system 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.

(Function) 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 on the sample in a time-sharing manner.

(Effects of the Invention) According to the present invention, the focal position of the first optical system is set at a certain depth of the sample for a certain period, and the focal position of the second optical system is set at another certain depth of the sample for another period. Can be set to any position. Also,
Using these two focal position settings, it is possible to focus lights of different wavelengths onto the same location on the sample. Therefore, for example, in the above-mentioned living cell perforation device, the position of the objective lens of the microscope is adjusted to obtain the clearest monitor image during cell observation, and when projecting a laser pulse onto the living cells, The objective lens can only be moved to a suitable position so that the laser pulse is projected to the 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 diagram of a first embodiment of the present invention, showing an example of a laser drilling device. FIG. 2 is a timing diagram of signals in this example. The laser drilling device main body 1 functions as a microscope that magnifies an image of a sample 2 and guides it to a TV camera 3, and a laser beam 5 emitted from a laser oscillator 4.
It performs the two functions of a light condensing device that condenses light and projects it onto the sample 2. The focal point of the laser beam 5 and the focal position of the image plane change due to the vertical movement of the objective lens 1a. This is done by strain deformation. The projection timing of the laser beam 5 is controlled by a master signal generated from a master signal generator 7. This master signal is once manually input to the delay trigger circuit 8, delayed for a predetermined time (the time required for the electrostrictive element to deform), and then inputted to the laser oscillator 4 as a laser beam emission instruction signal. The master signal is also input to the electrostrictive element driving device 9 to generate an electrostrictive element driving voltage for controlling the driving of the electrostrictive element 6. Therefore, during laser projection, the objective lens 1a is positioned so that the laser beam 5 is focused on a suitable position on the beam of the sample 2; otherwise, the objective lens 1a
a can be positioned at a position suitable for monitor observation.

Specifically, with no voltage applied to the electrostrictive element, the objective lens la is adjusted so that the monitor image as a whole is displayed most clearly, and then the target object (for example, a cell nucleus) is ) is projected clearly, the voltage applied to the electrostrictive element is set in advance, and then the voltage of this set value is applied to the electrostrictive element when laser light is projected. Bye. If the objective lens la is located at a position other than the one suitable for monitor observation, the image projected on the monitor television 10 via the TV camera 3 will be unclear, so the shutter must be It is preferable that the opening and closing of the shutter 11 be controlled by the shutter drive circuit 12. In the apparatus configured in this way, when the master signal generator 7 generates pulse signals at predetermined intervals while the sample 2 is moved by the moving stage 13, the laser beam is repeatedly and continuously placed at a predetermined depth position on the sample 2. Light is projected and a clear monitor image can be continuously observed.

In order to prevent sample image collection, in addition to using the shutter 11, the illumination of the sample may be turned off. The shutter may be a mechanical shutter with electromagnetic island movement, or an optical shutter using a filter or polarizer.

FIG. 3 is a schematic diagram of a second embodiment of the present invention, and is an example of a laser drilling device similar to the first embodiment. FIG. 4 is a timing chart of signals in this example. In this embodiment, the laser beam is projected onto the position indicated by the light pen 15. When the monitor TVIO is instructed to use the light pen 15, the position control signal generator 16 calculates the indicated position based on the pulse signal output from the light pen 15, and also generates a control signal based on the calculated value. occurs. The beam deflection device 17 consisting of a galvanometer mirror or the like is driven by the deflection device driving means 18 under this control, so that the laser beam 5 is projected onto the position specified by the light pen 15. The beam deflector 17 receives this control signal and drives a galvanometer mirror, etc., so that the laser beam 5 is projected onto 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 zero time control signal generator 19 converts the pulse signal generated every scanning period of the TV camera 3 into a time control signal at a desired interval. Convert.

This time control signal is input to the driving device 9 for the electrostrictive element 6 to generate an electrostrictive element driving voltage for controlling the driving of the electrostrictive element 6 in the same manner as in the first embodiment. Further, the time control signal controls opening and closing of a laser beam path shutter 14 provided between the microscope main body l and the laser oscillator 4. As shown in FIG. 4, the laser beam path shutter 14 is opened during the voltage application period of the electrostrictive element 6, excluding the transient period.
During that period, the (pulsed) laser light 5 is continuously projected onto the sample. The focus of the laser beam that performs perforation 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 first embodiment. However, when the laser beam 5 is always projected onto a predetermined focal plane in this way, the laser beam 5 is not necessarily projected onto a suitable position on the sample. In this embodiment, the focal depth position of the laser beam 5 is automatically set to the object indicated by the light pen 15. As described in Japanese Patent Application Laid-Open No. 60-83583, a reference laser beam 21 is constantly projected onto the sample.

The light reflected from the sample 2 by this projection is transmitted to the semi-transparent mirror 22.
It is taken out from the microscope body 1 by. The extracted light passes through the lenses 23, 24 and the iris diaphragm 15, and is then picked up by the photoelectric conversion element 26. Oscillator 17 constantly generates an oscillating voltage with a predetermined period.
This oscillating voltage is applied to the electrostrictive element 6 while the switch 28 is closed. The phase sensitive detector 29 is a photoelectric converter 23
The phase difference between the output of oscillator 26 and the output of oscillator 27 is output as a voltage value. A voltage V applied to the electrostrictive element 6,
That is, the relationship between the position of the focal plane and the output of the photoelectric converter is as shown in Figure 5, and the output of the phase sensitive detector is as shown in Figure 6 with respect to the voltage applied to the electrostrictive element. It becomes like this.

Therefore, with respect to the (two-dimensional) indicated position of the light pen 15, the focal plane of the laser projection optical system is set at the depth position where the reflecting mirror is at its maximum, and the drilling laser beam 5 is projected onto the sample 2. . During the driving period of the electrostrictive element 6 including the transient period,
In order to prevent inappropriate screens from being displayed on the monitor TV, it is preferable to open and close the gate switch 20 provided between the TV camera 3 and the monitor TVIO based on a time control signal. In addition, the gate switch 20
It is further preferable that an image immediately before the gate switch 20 is closed be displayed on the monitor TVIO when the gate switch 20 is closed. With the configuration of this embodiment, the laser beam can be projected near the center of the living cell while accurately grasping the position of the living cell. It is considered to be an extremely preferable method for material transfer into the interior of the body. Note that, as in this embodiment, since the projection of the laser beam is controlled by blocking the laser beam itself, either pulsed or continuous laser beams can be used.

FIG. 7 is a schematic diagram of a third embodiment of the present invention, and is an example of a laser drilling device similar to each of the embodiments described above. In this embodiment as well, the laser beam is projected at the position indicated by the light pen, as in the second embodiment, but the focal depth position of the laser beam 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 has a light pen main body 15a.
It has a photosensitive section 15c that is movably provided in the front and back of the cavity 15b at the tip of the monitor, and light from the monitor TVIO is guided to the photodetector 15d through this light guiding section 15c. A pressure detection element 15e is provided behind the light guide section 15c. This pressure detection element 15e is a light guide portion 15e.
and a spring 15d, and generates a voltage value between opposing surfaces in accordance with the pressure applied between the light pen main body 15a and the light guide portion 15c. That is, monitor TV with light pen 15.
(7) A signal with a value corresponding to the pressing force is emitted to the monitor TVIO when IO is instructed, together with a detection signal of fluorescence emission during electron beam scanning.

A voltage signal emitted from the light pen 15 corresponding to the pressing force is sent to the focal depth position 'a 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 a focal depth position with respect to the sample 2 corresponding to the pressing force of the light pen 15 on the monitor TVIO.

In this embodiment, by adding control of the "depth of the laser focus" corresponding to the "depth of the scalpel", three-dimensional control becomes possible, and processing accuracy can be dramatically improved. High-precision cell surgery becomes possible, such as laser irradiation aimed at a specific organ inside a cell, such as the cell nucleus, at a specific depth, or drilling aimed at the inner surface of a biological membrane.

FIG. 9 is a schematic diagram of a fourth embodiment of the present invention.

It is well known that even if completely identical optical systems are used, different wavelengths will be focused at different depth positions.

If the present invention is used in such a case, light can be focused on the same spot on the sample even if the wavelengths are different. Although laser beams 34, 35, and 36 having different wavelengths are emitted from the laser oscillators 31, 32, and 33, only one type of laser beam is projected onto the sample 2 by shutters 37, 38, and 39. Shutter 37.38.3
The opening and closing of 9 is controlled by a changeover switch 40. This changeover switch simultaneously changes the driving voltage generated from the electrostrictive element driving device 9 according to the wavelength of the projected laser beam, so that the focal depth position with respect to the sample 2 does not vary depending on the laser beam wavelength.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of the first embodiment of the present invention, FIG. 2 is a signal timing diagram in the first embodiment, FIG. 3 is a schematic diagram of the second embodiment of the present invention, and FIG. 4 is a schematic diagram of the second embodiment of the present invention. Figure 5 is a diagram showing the relationship between the voltage applied to the electrostrictive element and the output of the photoelectric converter; Figure 6 is a diagram showing the relationship between the output of the phase sensitive detector and the output of the electrostrictive element. 7 is a schematic diagram of the third embodiment of the present invention; FIG. 8 is a sectional view of an embodiment of the light pen used in the third embodiment; FIG. 9 The figure is a schematic diagram of a fourth embodiment of the present invention. ■... nHk mirror body, 1a... Objective lens lb... Lens barrel, 2... Sample, 3.
...TV camera 4.31, 32.33...
・Laser oscillator 5.34, 35.36... Laser light 6... Electrostrictive element, 7... Master signal generator 8... Delay trigger circuit 9... Electrostrictive element drive device, 10... Monitor TV 11... Shutter 12... Shutter drive circuit 13... Movement stage 14. 37.38.39... 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 beam 26 ... Photoelectric converter, 27... Oscillator 29... Phase sensitive detector 30... Focal depth position control signal generator 40...
...Choice switch. Fig. 1 Fig. 5 Gai bronze Fig. 6

Claims (1)

[Claims] A time-division optical adjustment method, characterized in that a distance between an objective optical system shared by a first optical system and a second optical system and a sample is changed in a time-division manner.