JPH07333511A - Microscope - Google Patents

Abstract

PURPOSE:To obtain the focal plane moving mechanism of a microscope which is produced at low cost and does not vibrate a specimen moving a supporting means, thereby changing a distance between an observation means and an objective lens and changing the focal plane observed by the observation means. CONSTITUTION:An imaging device 13 is fixed and held by an imaging device holding member 14 and engaged with a guide member 15. The holding member 14 slides in the guide member 15 and is moved by a rack pinion mechanism in an optical axis direction. By moving the holding member 14 supporting the imaging device 13 so as to move with respect to the objective lens 4, the distance between the imaging device 13 and the objective lens 4 is changed and the focal plane observed by the imaging device 13 is changed. Since the focal plane is moved by the movement of the imaging device 13 without depending on the movement of the objective lens 4 or a specimen stage, the specimen such as an isolation cell is prevented from being moved by the vibration caused in the case of moving the objective lens 4 or the specimen stage, and micromanipulation is facilitated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a focus moving mechanism for a microscope.

[0002]

2. Description of the Related Art In conventional microscopes, when observing different parts in the depth direction of a thick sample or a sample with uneven surface, the focal plane moves in the depth direction according to the part to be observed. I was doing The focal plane is moved by changing the distance between the stage on which the sample is placed and the objective lens. When the objective lens is fixed, the movement is performed by a mechanism that moves the stage up and down with respect to the microscope body. The same was true when the objective lens was moved.

An example of a conventional focal plane moving mechanism will be described with reference to FIG. An arm 2 is provided in the microscope body 1, and an objective lens 4 is attached to a revolver 3 attached to the arm 2. A rack and pinion 8 mechanism (not shown) is provided on the column 5 of the microscope main body 1, and the coarse / fine adjustment handle 9 is provided.
The rotation of moves the stage 7 up and down, and the distance between the sample 10 placed on the stage 7 and the objective lens 4 can be relatively changed. An eyepiece lens 12 is attached to the arm 2 to observe the image of the sample 10 with the naked eye, or photoelectric conversion is performed by an image sensor 13 arranged on the image plane of the sample 10 by the objective lens 4 to output an electric signal. It has become.

Such a method of moving the focal plane is a general focusing method of a conventional microscope, and when focusing is performed immediately after the specimen is placed on the stage, it is necessary to use the same thickness as cells. When observing a thick sample at different depths, or when observing concaves and convexes of a sample having irregularities such as the resist pattern of a silicon wafer, the stage or objective lens should be changed each time. The focal plane was moved up and down by a coarse and fine adjustment mechanism.

In recent years, the use of confocal microscopes has been increasing. However, while moving the focal planes using the confocal microscope, images at each focal plane are acquired, and these images are processed by a computer. Even when reconstructing a three-dimensional image or creating a multi-focus image, the focal plane is moved by moving the stage or the objective lens up and down using a uniaxial coarse / fine movement mechanism or a fine movement Z stage using a piezoelectric element.

[0006]

However, in the conventional method of moving the stage to change the focal plane, micromanipulation is performed while observing a thick sample such as a cell while changing the depth. In this case, if the stage is moved to change the focal plane, even a slight vibration due to the stage movement is transmitted to the sample, and isolated cells easily move, which makes micromanipulation extremely difficult. .

Even when the objective lens is moved instead of the stage to change the focal plane, when an immersion type objective lens mediated by water or oil is used to increase the resolution, the objective lens is also moved when the objective lens is moved. A slight vibration was transmitted to the sample, which caused the same problem as the movement of the stage.

Further, in the case where a plurality of sectioning images are acquired using a confocal microscope to reconstruct a three-dimensional image, even if the cells are fixed to the sample, the feeding accuracy and running of the stage and the objective lens Since the accuracy becomes the reproduction accuracy after reconstructing the three-dimensional image as it is, it is necessary that the upper and lower feeding accuracy and running accuracy of the stage and the objective lens are high accuracy which is about half or less of the resolution of the objective lens. For example, when the numerical aperture of the objective lens is NA = 1, the resolution is about 0.3 μm, so the required feed accuracy and running accuracy are about 0.15 μm or less. There was also the problem that it would be very expensive.

In view of the above problems, it is an object of the present invention to provide a focal plane moving mechanism of a microscope which can be manufactured at low cost and which does not give vibration to a sample.

[0010]

According to a first aspect of the present invention, there is provided a microscope including an objective lens and an observation means for observing an image of a sample formed by the objective lens, wherein the observation means is the objective lens. And a supporting means for movably supporting the object, and by moving the supporting means, the distance between the observing means and the objective lens is changed to change the focal plane observed by the observing means. It is a thing.

It is preferable that the observing means is an image pickup device which forms an image of a sample by the objective lens and outputs an electric signal.

It is also preferable that the observing means is an eyepiece lens that magnifies and observes the image of the sample formed by the objective lens.

According to a fourth aspect of the present invention, an objective lens for converting the light emitted from the sample into a parallel light beam, a second objective lens for converging the parallel light beam to form an image of the sample, and the second objective lens A microscope comprising an observation means for observing an image of the sample formed by an objective lens, comprising a support means for movably supporting the second objective lens with respect to the objective lens, and moving the support means. By changing the distance between the second objective lens and the observation means,
The focal plane observed by the observing means is changed.

According to a fifth aspect of the present invention, an objective lens for converting the light emitted from the sample into a parallel light beam, a relay optical system provided in the optical path of the parallel light beam, and the parallel light beam is converged on a pinhole. In the confocal microscope, the relay optical system includes the objective lens, and a condensing lens that forms an image of the sample by using the condensing lens and an observation unit that observes the image of the sample formed by the condensing lens. A supporting means for movably supporting the moving means, and changing the distance between the relay optical system and the objective lens by moving the supporting means to change the focal plane observed by the observing means. Is.

[0015]

According to the invention described in claim 1, the observation means of the microscope is supported by the support means, and when the support means moves with respect to the objective lens, the distance between the observation means and the objective lens is changed, and the observation means. The focal plane to be observed at is changed.

In a fourth aspect of the invention, the second objective lens is supported by the supporting means, and when the supporting means moves with respect to the objective lens, the distance between the second objective lens and the observing means changes, The focal plane observed by the observation means is changed.

According to the invention described in claim 5, the relay optical system is supported by the supporting means, and when the supporting means moves with respect to the objective lens, the distance between the relay optical system and the objective lens is changed, and the observing means. The focal plane to be observed at is changed.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a sectional view of a microscope equipped with this embodiment, and FIG. 2 is a conceptual diagram showing an optical system of this embodiment. An arm 2 is provided in the microscope body 1, and an objective lens 4 is attached to a revolver 3 attached to the arm 2. A stage 7 is movably provided on a column 5 of the microscope body 1, and a sample 10 is mounted on the stay. An eyepiece lens 12 is attached to the arm 2 to observe the image of the sample 10 with the naked eye, or photoelectric conversion is performed by an image sensor 13 arranged on the image plane of the sample 10 by the objective lens 4 to output an electric signal. It has become.

The image pickup device 13 is fixedly held by the image pickup device holding member 14 and engaged with the guide member 15. The image sensor holding member 14 is slidable in the guide member 15 and is movable in the optical axis direction by a known mechanism such as a rack and pinion mechanism.

Next, the operation will be described. When observing one part 16a of the cell 16 at a position as shown in FIG. 2A, the object plane 17 of the part 16a has a conjugate relationship with the image plane 18 formed by the objective lens 4, and the image of the part 16a is An image is formed on the image pickup surface 13a of the image pickup device 13 on the image pickup surface 18. Next, in order to observe the other part 16b of the cell 16, FIG.
As shown in (b), the image sensor 13 is moved with respect to the objective lens 4. The object plane 19 and the imaging plane 20 have a conjugate relationship, and the image of the portion 16b of the cell 16 is imaged on the imaging plane 13b.

In this way, the objective lens 4 and the image pickup device 13 are
When the lens barrel length is changed by changing the distance between the object planes 17, the magnification of the objective lens 4 is set between the distance d1 between the object planes 17 and 19 and the distance d2 between the image planes 18 and 20. M
Then, the following relationship is established. d1 = d2 / M ² (1) For example, when using a 40 × objective lens, the image sensor 1
When 3 is moved along the optical axis of 16 mm, the focal plane of the portion moved by 10 μm inside the cell 16 can be observed. A 40 × objective lens is used, and the image sensor 13 is
When it moves by mm, the focal plane moves by about 40 μm on the object surface, and if the cell has an ordinary size, it can focus on almost all the depth. 20 mm to 30 m on the image plane
Even if it is moved by m, the performance of the formed image is practically not deteriorated.

In the present invention, the focal plane is moved by the movement of the image pickup device and not by the movement of the objective lens or the sample stage. Therefore, the vibration generated when the objective lens or the sample stage is moved causes the isolated cells and the like to move. There is no problem that the sample moves, and micromanipulation etc. can be easily performed.

Further, since the running accuracy of the image pickup device is proportional to the lateral magnification of the microscope, the running accuracy required when the resolution of the objective lens (NA = 1, 40 times) is 0.3 μm is 6 μm and the running mechanism of the image pickup device. Is easy and cheap to manufacture.

Next, a second embodiment will be described with reference to FIG. In the present embodiment, instead of the image pickup device in the above-described embodiment, the eyepiece lens is moved to change the lens barrel length to move the focal plane, and the detailed description of the same or similar points will be omitted. An observer visually observes the image of the sample 10 formed by the objective lens 4 through the eyepiece lens 12. The eyepiece lens 12 is supported by the guide member 15 and is movable up and down with respect to the objective lens 4. When the eyepiece lens 12 is moved upward with respect to the objective lens 4, the focal plane moves to the shallow portion of the sample 10.

Next, a third embodiment will be described with reference to FIG. In this embodiment, the focal plane is moved without changing the lens barrel length, and the detailed description of the same or similar points as the above-mentioned embodiments will be omitted. The light from the sample 10 becomes a parallel light flux by the objective lens 4, and the image formed through the second objective lens 21 is observed by the image sensor 13. The second objective lens 21 is supported by the guide member 15 and is movable up and down with respect to the objective lens 4. The second objective lens 21 is replaced with the objective lens 4
When moved upward with respect to, the focal plane moves to a portion of the sample 10 at a shallow position. 20 mm to 30 m on the image plane
Even if it is moved by m, the performance of the formed image is practically not deteriorated.

Since the running accuracy of the image sensor is proportional to the lateral magnification of the microscope, the running accuracy required when the resolution of the objective lens (NA = 1, 40 times) is 0.3 μm is 6 μm, and the running mechanism of the image sensor is Easy to manufacture at low cost.

According to the present embodiment, the focal plane to be observed is moved by moving the second objective lens without moving the objective lens or the sample stage, and the sample does not move due to vibration.

Next, a fourth embodiment will be described with reference to FIG. In this embodiment, as in the previous embodiment, the focal plane is moved without changing the lens barrel length. Detailed description of the same or similar points as those of the above-described embodiments will be omitted. The light from the sample 10 becomes a parallel light flux by the objective lens 4, and forms an image on the image sensor 13 via the focusing lens 22. The focusing lens 22 is formed of a plurality of unit lenses 22a and 22b, and changes the interval between them to move the focal plane in the depth direction of the sample 10.

Next, a fifth embodiment will be described with reference to FIG. This embodiment relates to a laser scanning confocal microscope. Detailed description of the same or similar points as those of the above-described embodiments will be omitted. The laser light emitted from the laser 25 is reflected by the beam splitter 26, and the scanning unit 2
7 is scanned, relayed by the second relay lens 28 and the first relay lens 29, and then converged by the objective lens 4 to irradiate the sample 10. The fluorescence emitted from the sample 10 travels backward, passes through the objective lens 4, the first relay lens 29, the second relay lens 28, and the scanning unit 27, passes through the beam splitter 26, and the condenser lens 30.
Are collected by the pinhole detector 31 and detected by the pinhole detector 31.

At least one of the relay lenses 28 and 29 is formed of a plurality of unit lenses, and the distance between the plurality of unit lenses can be changed. The interval between the plurality of unit lenses is changed to move the focal plane in the depth direction of the sample 10. At least one of the relay lenses 28 and 29 may be movable with respect to the objective lens 4. Even if the image plane is moved by 20 mm to 30 mm, the performance of the image formed is practically not deteriorated.

According to this embodiment, the focal plane to be observed is moved by moving the relay lens without moving the objective lens or the sample stage, and the sample does not move due to vibration. Micromanipulation such as electrophysiology and patch clamp can be performed easily. Further, a highly accurate three-dimensional image can be obtained.

Since the feed accuracy of the relay lens required to faithfully reproduce a three-dimensional image is proportional to the square of the vertical magnification of the microscope, that is, the horizontal magnification, it is often used in such applications.
With a double objective lens, the vertical magnification becomes 1600 times, and the required accuracy is very low, about 0.25 mm, and the relay lens feed mechanism can be manufactured at low cost.

[0033]

According to the invention described in claim 1, when the observation means of the microscope is supported by the support means and the support means moves with respect to the objective lens, the distance between the observation means and the objective lens changes, Since the focal plane observed by the observation means is changed, the focal plane of the microscope is moved by the inexpensive moving mechanism without vibrating the sample.

In the invention according to claim 4, the second objective lens is supported by the supporting means, and when the supporting means moves with respect to the objective lens, the distance between the second objective lens and the observing means changes, Since the focal plane observed by the observation means is changed, the focal plane of the microscope is moved by the inexpensive moving mechanism without vibrating the sample.

According to a fifth aspect of the present invention, the relay optical system is supported by the supporting means, and when the supporting means moves with respect to the objective lens, the distance between the relay optical system and the objective lens changes, and the observing means. Since the focal plane to be observed at is changed,
An inexpensive moving mechanism moves the focal plane of the microscope without applying vibration to the specimen.

[Brief description of drawings]

FIG. 1 is a conceptual side view of an embodiment of the present invention.

FIG. 2 is a diagram showing an optical path of an embodiment of the present invention.

FIG. 3 is a diagram showing an optical path of a second embodiment of the present invention.

FIG. 4 is a conceptual side view of the third embodiment of the present invention.

FIG. 5 is a diagram showing an optical path according to a fourth embodiment of the present invention.

FIG. 6 is a diagram showing an optical path according to a fifth embodiment of the present invention.

FIG. 7 is a conceptual side view of a conventional example.

[Explanation of symbols]

 1 ... Microscope body 4 ... Objective lens 10 ... Specimen 12 ... Eyepiece 13 ... Image pickup element 14 ... Image pickup element holding member 15 ... Guide member 16 ... Cell 17 ... 19 Object plane 18 20 ... Image plane 21 ... Second objective lens 22 ... Focusing lens 25 ... Laser 26 ... ... Beam splitter 27 ... Scanning unit 28, 29 ... Relay lens 30 ... Condensing lens 31 ... Pinhole detector

Claims (5)

[Claims] 1. A microscope comprising an objective lens and an observation means for observing an image of a sample formed by the objective lens, comprising a supporting means for movably supporting the observation means with respect to the objective lens. Then, the microscope is characterized in that by moving the supporting means, the distance between the observing means and the objective lens is changed to change the focal plane observed by the observing means. 2. The microscope according to claim 1, wherein the observing means is an image pickup device which forms an image of a sample by the objective lens and outputs an electric signal. 3. The microscope according to claim 1, wherein the observing means is an eyepiece lens which magnifies and observes an image of the sample formed by the objective lens. 4. An objective lens for converting light emitted from a sample into a parallel light beam, a second objective lens for converging the parallel light beam to form an image of the sample, and the above-mentioned image formed by the second objective lens. In a microscope including an observation means for observing an image of a sample, there is provided a support means for movably supporting the second objective lens with respect to the objective lens, and the second means is provided by moving the support means. A microscope characterized in that a focal plane observed by the observation means is changed by changing a distance between an objective lens and the observation means. 5. An objective lens for converting light emitted from a sample into a parallel light beam, a relay optical system provided in an optical path of the parallel light beam, and the parallel light beam being converged on a pinhole to form an image of the sample. In a confocal type microscope including a condensing lens for imaging and an observing means for observing the image of the sample formed by the condensing lens, the relay optical system is movably supported with respect to the objective lens. A microscope having a supporting means, wherein the distance between the relay optical system and the objective lens is changed by moving the supporting means to change a focal plane observed by the observing means.