JP2003005021A - Focusing device for microscope and microscope equipped with the same - Google Patents

Abstract

(57) [Problem] To provide a focusing device capable of focusing on a position where an operator can observe a high-contrast image favorably, and a microscope provided with the same. SOLUTION: A first automatic adjusting means (11, 11) for automatically adjusting a relative positional relationship between a sample and an objective lens based on contrast of an image of the sample formed through the objective lens.
16, 17, 32 to 36, 38) and an irradiation unit that irradiates the sample with detection light via the objective lens, and automatically detects the positional relationship based on the reflected light from the sample irradiated with the detection light. Adjust to the second
Automatic adjustment means (11, 16, 21 to 31, 36, 38), and offset storage means (36, 37, 71) for storing a predetermined offset amount,
A third automatic adjusting means (11, 36, 38) for automatically adjusting the positional relationship based on the offset amount after the adjustment by the second automatic adjusting means and before the adjustment by the first automatic adjusting means.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a focus detection device for a microscope used for observing a specimen, and a microscope equipped with the same.

[0002]

2. Description of the Related Art Conventionally, a structure adopting an image contrast method is known as a focusing device for a microscope. The image contrast method is a method of capturing an image of a sample as an image and performing focusing so that the contrast of the image is maximized.

[0003]

However, quick focus adjustment is difficult only with the image contrast method.

In addition, when the operator observes the image of the specimen after focusing using the above-mentioned conventional apparatus,
It was not always possible to satisfactorily observe a high-contrast image. This is because the optical system related to the formation of the image of the sample is configured separately for the focusing device and the operator's observation (eyepiece etc.), and generally the respective image forming positions are displaced (mechanical error ). The deviation of the image formation position may occur not only when the device is assembled but also due to aging.

An object of the present invention is to provide a microscope focusing device capable of focusing on a position where an operator can satisfactorily observe a high-contrast image, and a microscope equipped with the same.

[0006]

The microscope focusing device of the present invention automatically determines the relative positional relationship between the sample and the objective lens based on the contrast of the image of the sample formed through the objective lens. A first automatic adjustment means for adjusting to, and an irradiation unit that irradiates the sample with the detection light through the objective lens, and based on the reflected light from the sample irradiated with the detection light,
Second automatic adjustment means for automatically adjusting the positional relationship, offset storage means for storing a predetermined offset amount,
A third automatic adjustment means for automatically adjusting the positional relationship based on the offset amount stored in the offset storage means after the adjustment by the second automatic adjustment means and before the adjustment by the first automatic adjustment means. It is a thing.

Further, the microscope focusing device of the present invention automatically adjusts the relative positional relationship between the sample and the objective lens based on the contrast of the image of the sample formed through the objective lens. 1 adjustment means, second adjustment means for finely adjusting the positional relationship based on manual operation from the outside, deviation between the positional relationship after adjustment by the first adjustment means and the positional relationship after adjustment by the second adjustment means Detecting means, first storage means for storing the deviation detected by the first detecting means, and first storage after the adjustment by the first adjusting means and before the adjustment by the second adjusting means. It is provided with a third adjusting means for determining whether or not the deviation is stored in the means, and if the deviation is stored, automatically adjusting the positional relationship based on the deviation.

The microscope according to the present invention has an objective lens and an eyepiece lens, and is arranged between the objective lens and the eyepiece lens, and an imaging optical system for forming an image of a specimen. The microscope focusing device according to any one of claims 1 to 7.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the drawings.

Embodiments of the present invention include claims 1 to 5.
Corresponding to. As shown in FIG. 1, the microscope 10 of the present embodiment includes a stage 11 on which a sample (not shown) to be observed is mounted, an objective lens 12 and an eyepiece lens 13 that form a magnified image of the sample, and an objective lens. Focusing device incorporated in the afocal system between the eyepiece 12 and the eyepiece 13
(21-39).

Here, the sample (not shown) is the cover glass 1.
It is a biological specimen (for example, a cell) sandwiched between 4 and a slide glass 15. Although not shown, the microscope 10
An illuminating device that illuminates the sample placed on the stage 11 is also provided. The lighting device is a transmissive type or an epi-illumination type.
The transmissive illumination device is arranged below the stage 11, and the epi-illumination device is arranged above the stage 11.

Although only one objective lens 12 is shown in FIG. 1, the microscope 10 of this embodiment can be observed by a plurality of objective lenses 12 having different magnifications. The plurality of objective lenses 12 are mounted on an electric revolver (not shown). The electric revolver (not shown) is connected to the drive unit 18 that rotationally drives the electric revolver. Furthermore, microscope 1
At 0, a dichroic mirror 16 and a half mirror 17 are arranged between the objective lens 12 and the eyepiece lens 13 (afocal system). Dichroic mirror 1
Reference numeral 6 reflects infrared light La and Lb and transmits visible light Lc. The half mirror 17 reflects a part Ld of visible light and transmits another part Le. The dichroic mirror 16 and the half mirror 17 are optical elements arranged to incorporate the focusing devices (21 to 39).

Now, the focusing device (21
39) will be specifically described. Focusing device (2
1 to 39) is a drive unit 38 that drives the stage 11,
An infrared emitting section (21 to 24) that emits slit-shaped infrared light Lf (detection light) toward the dichroic mirror 16 and an infrared receiving section (25 to 25) that receives the infrared light Lg from the dichroic mirror 16. 30), a signal processing unit 31 that processes output signals from the infrared light receiving units (25 to 30), and a visible light receiving unit (32 to 3) that receives the visible light Le from the half mirror 17.
4), a signal processing unit 35 that processes output signals from the visible light receiving units (32 to 34), a CPU 36, a memory 37,
It is composed of an input unit 39.

First, the drive section 38 of the stage 11 will be described. As shown in FIG. 2, the drive unit 38 includes a DC motor 41 attached to the stage 11 and a CPU 36.
The motor driver 42 that rotates the DC motor 41 based on the vertical movement control signal from the motor, the rotary encoder 43 that detects the rotation angle of the DC motor 41, and the vertical movement of the stage 11 based on the detection result of the rotary encoder 43. An up / down counter 44 is provided. The count result of the up / down counter 44 is output to the CPU 36 as a vertical movement position signal.

The stage 11 is the DC motor 41 described above.
When is rotated, it moves up and down according to the rotation angle. The specimen placed on the stage 11 is also covered by the cover glass 1.
4. It moves up and down together with the slide glass 15, and the positional relationship between the sample and the objective lens 12 is adjusted. In addition, the drive unit 38
Is provided with a limit sensor 45. The limit sensor 45 is a sensor that detects a vertical movement limit point of the stage 11, and is provided to avoid contact between the objective lens 12 and the cover glass 14.

Next, the infrared emitting sections (21-24) will be described. As shown in FIG. 1, the infrared emitting units (21 to 24) are composed of an LED light source 21, a slit member 22, a collector lens 23, and a first pupil limiting mask 24.
The LED light source 21 emits infrared light. Slit member 2
2 has a rectangular elongated slit opening 22a. The longitudinal direction of the slit opening 22a is perpendicular to the paper surface. The collector lens 23 collects the diffused light and converts it into parallel light.

The first pupil limiting mask 24 shields half of the pupil from light. The area shielded by the first pupil limiting mask 24 is a surface including the optical axis 23a of the collector lens 23 and parallel to the longitudinal direction of the slit aperture 22a (a surface perpendicular to the paper surface).
It is one (upper side in the figure) of the two areas divided by. In the infrared emitting section (21 to 24), the infrared light from the LED light source 21 passes through the slit opening 22a,
The light is converted into parallel light by the collector lens 23, half of which is blocked by the first pupil limiting mask 24, and then the light is emitted toward the dichroic mirror 16. The light Lf emitted from the infrared emission units (21 to 24) is slit-shaped infrared light.

The light Lf from the infrared emission section (21-24) is
When it reaches the dichroic mirror 16, it is reflected there.
(Light La) enters the objective lens 12 through one (a left side in the figure) of a region divided by a surface including the optical axis 12a of the objective lens 12, and enters the stage 1 through the objective lens 12.
The sample above 1 is irradiated (light Lh). In addition, stage 11
Since the upper sample is covered with the cover glass 14, the slit-shaped infrared light Lh guided from the infrared emission section (21 to 24) through the dichroic mirror 16 and the objective lens 12 is covered by the cover glass 14. Is projected onto the surface of and reflected there.

The reflected light Lk is reflected by the objective lens 1
2 becomes parallel light and passes through the optical axis 12 of the objective lens 12.
The light is incident on the dichroic mirror 16 through the other (right side in the figure) of the area divided by the plane including a (light L
b), where it is reflected. The light reflected by the dichroic mirror 16 travels toward the infrared light receiving section (25 to 30) (light Lg).

Next, the infrared light receiving section (25 to 30) will be described. The infrared light receiving part (25 to 30) is a half mirror 25.
A second objective lens 26, a relay lens system 27, a second pupil limiting mask 28, a cylindrical lens 29,
It is composed of a CCD sensor 30. Half mirror 25
Reflects a part of the infrared light Lg and transmits a part of the infrared light Lf. The second objective lens 26 collects the parallel light, converts the parallel light into an imaging light, and forms a slit image. The relay lens system 27 relays the slit image formed by the second objective lens 26, and re-images it on the imaging surface of the CCD sensor 30 via the cylindrical lens 29.

The second pupil limiting mask 28 shields half of the pupil from light. The area shielded by the second pupil limiting mask 28 corresponds to the area shielded by the first pupil limiting mask 24 described above. The cylindrical lens 29 has a refracting action only in a predetermined direction. The CCD sensor 30 is a line sensor in which a plurality of light receiving parts are arranged one-dimensionally.
The CCD sensor 30 may be an area sensor arranged two-dimensionally.

In the infrared receiving section (25 to 30), the infrared light Lg from the dichroic mirror 16 is reflected by the half mirror 25, and then, through the second objective lens 26 and the relay lens system 27, a cylindrical lens. The light enters the image sensor 29, and the cylindrical lens 29 compresses the slit image only in a predetermined direction (the longitudinal direction of the slit image is compressed) and forms an image on the imaging surface of the CCD sensor 30.

At this time, a compressed slit image is formed on the image pickup surface of the CCD sensor 30. The position where the compressed slit image is formed on the imaging surface of the CCD sensor 30 moves in the longitudinal direction of the CCD sensor 30 when the position of the sample or the cover glass 14 changes due to the vertical movement of the stage 11. In the CCD sensor 30, electric charges are accumulated in each light receiving portion according to the compressed slit image formed on the imaging surface. Then, the charges accumulated in each light receiving portion of the CCD sensor 30 are transferred through the CCD and output to the first signal processing unit 31 (slit projection type AF control circuit) as an image pickup signal.

It should be noted that the CCD sensor 30 starts to output the image pickup signal to the first signal processing section 31 when the CPU 36.
A start pulse is output to. Here, the image pickup signal output from the CCD sensor 30 to the first signal processing unit 31 will be described with reference to FIG. The horizontal axis of FIG. 3 is
The light receiving position of the CCD sensor 30 is shown, and the peak position (light receiving position) of the image pickup signal at each time t is shown. The vertical axis of FIG. 3 represents the voltage value of the image pickup signal.

From CCD sensor 30 to first signal processor 31
The image pickup signal output to (1) has one peak (time tp) as shown by the solid line (a) in FIG. 3, for example. The time tp at which a peak appears in the image pickup signal corresponds to the light receiving position where the compressed slit image is formed on the image pickup surface of the CCD sensor 30. Therefore, when the positions of the sample and the cover glass 14 change due to the vertical movement of the stage 11 and the formation position of the compression slit image on the imaging surface moves, the light receiving position (time tp) showing the peak in the imaging signal also shifts. That is, the focus of the objective lens 12 is adjusted by moving the stage 11 up and down.

For example, when the stage 11 moves toward the rear pin side, the light receiving position (at the time t
p) shifts toward the time ts side (solid line (a) → dotted line (b)). On the contrary, when the stage 11 moves toward the front pin side, the light receiving position (time tp) showing the peak of the image pickup signal
Shifts toward the time te side (solid line (a) → dotted line
(c)).

Next, the first signal processing section 31 (slit projection type AF control circuit) will be described. First signal processing unit 3
As shown in FIG. 2, 1 comprises a DC cut circuit 51, a programmable gain amplifier (PGA) 52, integrating circuits 53 and 54, a difference circuit 55, and an A / D converter 56. . The DC cut circuit 51 cuts the DC component of the image pickup signal output from the CCD sensor 30. The PGA 52 normalizes the peak voltage of the image pickup signal from which the DC component is cut so as to be constant.

The integrating circuits 53 and 54 integrate the voltage value of the standardized image pickup signal. However, the integration range of the integration circuit 53 is the range from time ts to the integration boundary line (time tk) as shown in FIGS. 4 (a) and 4 (b). The integration range of the integration circuit 54 is from the integration boundary line (time tk) to the time te.
The range is up to. The integration boundary line (time tk) is the CPU 3
It is set in accordance with the control signal from 6.

As described above, when the position of the sample or the cover glass 14 changes due to the vertical movement of the stage 11, the light receiving position (time tp) showing the peak in the image pickup signal shifts (see FIG. 3), so that the integrating circuit 53. The magnitude of the integrated value signal (A) output from the integrated circuit and the magnitude of the integrated value signal (B) output from the integration circuit 54 change. When the peak of the imaging signal appears before the integration boundary line (time tk)
(FIG. 4A, tp <tk), the integrated value signal (A) is larger than the integrated value signal (B) (A> B). The vertical movement position (Z position) of the stage 11 at this time is Za.

On the contrary, when the peak of the image pickup signal appears after the integration boundary line (time tk) (FIG. 4 (b), tp> tk), the integration value signal (A) is better than the integration value signal (B). Become smaller (A
<B). The Z position of the stage 11 at this time is Zb. Then, the peak of the imaging signal is the integration boundary line (time tk).
And (t = tk in FIG. 5), the integrated value signal (A) and the integrated value signal (B) are equal to each other (A =
B). The Z position of the stage 11 at this time is Z ₁ .

The difference circuit 55 connected to the subsequent stage of the integration circuits 53 and 54 is an integrated value signal from the integration circuit 53.
(A-) and the integrated value signal (B) from the integration circuit 54 (A-
B) is calculated and the difference signal is output. A / D converter 5
6 converts the difference signal from the difference circuit 55 into digital data and outputs it to the CPU 36 as difference data. here,
The difference data output from the A / D converter 56 to the CPU 36 will be described with reference to FIG. The horizontal axis of FIG. 6 represents the Z position of the stage 11, and the vertical axis represents the value of the difference data.

For example, as shown in FIG. 4A, the integrated value signal is A
> B (stage 11 is Za, image signal peak is t
p <tk), the difference data has a positive value. Further, when the integrated value signal is A = B as shown in FIG. 5 (Z ₁ , tp = tk), the difference data becomes zero. When the integrated value signal is A <B (Zb, tp> tk) as shown in FIG. 4B, the difference data has a negative value.

Although the details will be described later, the CPU 36 controls the drive unit 38 of the stage 11 based on the value of the above-mentioned difference data (FIG. 6) so that the value of the difference data becomes zero, and the value of the difference data is changed. When becomes zero, the stage 11 is stopped (Z position = Z ₁ ). At this time, the microscope 10 of the present embodiment
Then, the surface of the cover glass 14 coincides with the focal plane of the objective lens 12 (see FIG. 8A).

Here, the position control of the stage 11 based on the value of the above-mentioned difference data (FIG. 6) is referred to as "slit projection type AF.
Control. Further, the stop position (Z ₁ ) of the stage 11 by the slit projection type AF control is referred to as “focus position Z ₁ ”.
In addition, the stage 11, the drive unit 38, the infrared emission unit (21 to 2
4), dichroic mirror 16, infrared receiving section (25-3
0), the signal processor 31, and the CPU 36 correspond to the "fourth adjusting means" and the "second automatic adjusting means" in the claims. The infrared emitting parts (21 to 24) and the dichroic mirror 16 correspond to the "irradiating part" in the claims.

Next, the visible light receiving portions (32 to 34) will be described. As shown in FIG. 1, the visible light receiving portions (32 to 34) include a second objective lens 32, an intermediate variable magnification lens 33, and a C lens.
It is composed of a CD sensor 34. Second objective lens 32
Collimates the parallel light (visible light Le) from the half mirror 17 and converts it into imaging light. The intermediate variable magnification lens 33 switches the image forming magnification. The CCD sensor 34 is an area sensor in which a plurality of light receiving parts are two-dimensionally arranged.

In the visible light receiving portions (32 to 34), the visible light Le transmitted through the half mirror 17 passes through the second objective lens 32 and the intermediate variable magnification lens 33 and forms an image on the image pickup surface of the CCD sensor 34. To be done. In addition, the visible light Le is generated from the sample and is generated by the objective lens 12 and the dichroic mirror 1.
It is a part of visible light which has passed through 6 and 6 and has reached the half mirror 17. Therefore, a sample image is formed on the image pickup surface of the CCD sensor 34. As is well known, the contrast of the sample image changes when the position of the sample changes due to the vertical movement of the stage 11.

In the CCD sensor 34, electric charge is accumulated in each light receiving portion according to the contrast of the sample image formed on the image pickup surface. Then, the electric charge accumulated in each light receiving portion of the CCD sensor 34 is transferred through the CCD and output to the second signal processing portion 35 as an image signal. Next, the second signal processing unit 35 will be described.

The signal processing unit 35, as shown in FIG.
A C-cut circuit 61, a PGA 62, a bandpass filter (BPF) 63, a differentiating circuit 64, an integrating circuit 65,
It is composed of an A / D converter 66. The DC cut circuit 61 cuts the DC component of the image signal output from the CCD sensor 34. The PGA 62 normalizes the peak voltage of the image signal from which the DC component is cut so as to be constant.

The BPF 63 removes unnecessary frequency components (low frequency components) from the standardized image signal. Differentiator circuit 6
4 and the integration circuit 65 integrate the voltage value of the image signal (high frequency component) output from the BPF 63. The A / D converter 66 converts the image signal from the integration circuit 65 into digital data and outputs it to the CPU 36 as contrast data.

From the A / D converter 66 to the CPU
The contrast data output to 36 will be described with reference to FIG. 7. The horizontal axis of FIG. 7 is the Z of the stage 11.
The position is shown, and the vertical axis shows the value of the contrast data. Since the value of the contrast data indicates whether the contrast of the sample image is high or low, it changes when the position of the sample changes due to the vertical movement of the stage 11, and becomes maximum at a certain Z position (Z ₂ ).

As will be described later in detail, the CPU 36 controls the drive unit 38 of the stage 11 so that the value of the contrast data becomes maximum based on the value of the above-mentioned contrast data (FIG. 7), and the contrast data becomes maximum. When the value is reached, the stage 11 is stopped (Z position = Z ₂ ). At this time, in the microscope 10 of the present embodiment, the arbitrary surface 10a in the sample matches the focal plane of the objective lens 12 (FIG. 8).
(See (b)).

Here, the position control of the stage 11 based on the value of the above-mentioned contrast data (FIG. 7) is called "image contrast type AF control". In addition, the stop position (Z ₂ ) of the stage 11 by the image contrast AF control is referred to as “focus position Z ₂ ”. The stage 11, the drive unit 38,
The dichroic mirror 16, the half mirror 17, the visible light receiving portion (32 to 34), the second signal processing portion 35, and the CPU 36 correspond to the "first adjusting means" and the "first automatic adjusting means" in the claims.

Next, the drive section 18 of the electric revolver (not shown in FIG. 1) to which the objective lens 12 is attached will be described with reference to FIG. As shown in FIG. 2, the drive unit 18 is provided with a DC motor 46 attached to the electric revolver 12 a and a motor driver 47 that rotates the DC motor 46 based on a rotation control signal from the CPU 36.

The electric revolver 12a rotates according to the rotation of the DC motor 46 described above. Then, the plurality of objective lenses 12 mounted on the electric revolver 12 a also rotate together, and any one objective lens 12 is positioned on the observation optical path of the microscope 10. Among the revolver holes (for example, 6) of the electric revolver 12a, the drive unit 18 has the numbers (1 to 6) of the revolver holes positioned on the observation optical path of the microscope 10.
A sensor (not shown) for detecting is also provided.

At the end of the description of the structure of the microscope 10, the input unit 3
9 will be described. As shown in FIG. 2, the input unit 39 includes a keyboard 71 and an objective lens changeover switch 72.
An up / down fine adjustment switch 73, a focus correction memory switch 74, a focus correction permission switch 75, and a focus control start switch 76 are provided. In the microscope 10 of this embodiment, the keyboard 71 is used when inputting the thickness G (FIG. 8A) of the cover glass 14. Data regarding the thickness G of the cover glass 14 input from the keyboard 71 is stored in the memory 37. The area of the memory 37 that stores the thickness data of the cover glass 14 (“offset amount” in claims) is referred to as “memory _MG ”.

The objective lens changeover switch 72 is the microscope 1
It is used when switching the objective lens 12 positioned on the 0 observation optical path to another objective lens 12. CPU
Reference numeral 36 controls the drive unit 18 of the electric revolver 12a based on the switching signal input from the objective lens switching switch 72, and positions the revolver hole designated by the switching signal on the observation optical path of the microscope 10.

The up / down fine adjustment switch 73 is used when finely adjusting the vertical movement of the stage 11 by manual operation. The CPU 36 controls the drive unit 38 of the stage 11 based on the fine adjustment signal input from the up / down fine adjustment switch 73, and positions the stage 11 as designated by the fine adjustment signal.

The up / down fine adjustment switch 73
The operation is performed while the operator observes the image of the sample through the eyepiece lens 13 of the microscope 10. Then, when an image with high contrast can be satisfactorily observed by the operator, the operation of the up / down fine adjustment switch 73 ends, and the stage 11 stops (Z position = Z ₃ ). At this time, in the microscope 10 of the present embodiment, the arbitrary surface 10b in the sample matches the focal plane of the objective lens 12 (FIG. 8C).
reference).

In general, the contrast of the sample image observed through the eyepiece lens 13 is determined by the above-mentioned CCD sensor 34.
The contrast of the sample image formed on the image pickup surface is different (when the stage 11 is stopped). This is because the optical system related to the formation of the sample image has a different structure. The sample image observed through the eyepiece lens 13 is also the CCD sensor 3
Also in the sample image formed on the imaging surface of No. 4, visible light (Lc) generated from the sample and passing through the objective lens 12 and the dichroic mirror 16 is divided into two by the half mirror 17 (Ld,
Le), one light Ld becomes a sample image through the eyepiece lens 13, and the other light Led is the second objective lens 3
2. Since the sample image is formed through the intermediate variable power lens 33, the contrasts thereof do not match.

Therefore, as a result of the manual operation by the up / down fine adjustment switch 73, the stopped position (Z ₃ ) of the positioned stage 11 is controlled by the image contrast AF control based on the value of the contrast data (FIG. 7) described above. The stage 11 is displaced from the stop position (focus position Z ₂ ). The stop position (Z ₃ ) of the stage 11 that is manually operated is referred to as “focus position Z ₃ ”.

The microscope 1 of this embodiment will be described in detail later.
At 0, the focus position Z by the image contrast AF control
₂ and the focus position Z ₃ due to manual operation N (= Z ₃ −
The vertical movement of the stage 11 can be automatically corrected based on Z ₂ ). In addition, the stage 11, the drive unit 38, the CP
The U36 and the up / down fine adjustment switch 73 correspond to the "second adjusting means" in the claims.

The focus correction memory switch 74 detects the shift N (= Z ₃ −Z ₂ ) between the above-mentioned focus positions Z ₂ and Z ₃ , and then the data relating to this shift N (hereinafter referred to as “correction data N”). ) Is stored in the memory 37. Here, the area of the memory 37 that stores the correction data N is for each objective lens 12 when the shift N (= Z ₃ −Z ₂ ) between the focus positions Z ₂ and Z ₃ is detected, that is, the objective lens. It is individually secured for each number (1 to 6) of the revolver holes to which 12 is attached.

The CPU 36 uses the focus correction memory switch 74.
Is operated, the number a (= 1 to 6) of the revolver hole positioned in the observation optical path of the microscope 10 is confirmed, and the number a
Area of the memory 37 corresponding to (hereinafter "memory M _N [a]")
Say. The correction data N is stored in (see FIG. 9). The focus correction memory switch 74, the CPU 36, the memory M
_N [a] corresponds to the "first storage means" in the claims.

The focus correction permission switch 75 is based on the correction data N stored in the memory M _N [a].
This is used when setting whether or not the vertical movement of the stage 11 is automatically corrected. When the focus correction permission switch 75 is operated, the CPU 36 "permits" when performing automatic correction.
Of the memory 36, or the data of “prohibition” when automatic correction is not performed, is stored in a predetermined area of the memory 36 (hereinafter “memory _Mo ”
I said).

The focus control start switch 76 is used for the microscope 10
It is used when instructing the start of focusing control in.
When the focusing control start switch 76 is operated, the CPU 36 controls the slit projection type AF control and the image contrast type AF.
Control, and further, automatic correction control based on the correction data N (FIG. 9) described above are sequentially executed. In this way, CP
U36 is input from the control program and various data stored in the memory 37, the difference data from the first signal processing unit 31 (FIG. 6), the contrast data from the second signal processing unit 35 (FIG. 7), and the input unit 39. The drive unit 38 of the stage 11 and the drive unit 18 of the electric revolver 12a described above are controlled by appropriately referring to the various signals thus generated.

The memory 37 is the memory M described above.
_G , memory M _N [a], memory M _o , slit projection type A
A memory M ₁ for storing the data of the focus position Z ₁ (FIG. 8A) obtained by the F control, and an image contrast AF
A memory M ₂ for storing data of a focusing position Z ₂ (FIG. 8B) obtained by control, and a memory M for storing data of a focusing position Z ₃ (FIG. 8C) obtained by manual operation. ₃
And the deviation M between the back surface of the cover glass 14 and the in-focus position Z _2.
A memory M _M for storing data (hereinafter referred to as “difference data M”) regarding (= Z ₂ −Z ₁ −G) is included. CP
The U36 and the memory M _M correspond to the "second storage means" in the claims.

Further, the memory 37 also stores data (FIG. 10A) for limiting the search range in the image contrast AF control. The data of the search range (lower limit, upper limit) is represented by the count value of the up / down counter 44 that constitutes the drive unit 38 of the stage 11. Incidentally, one count of the up / down counter 44 of this embodiment corresponds to 0.05 μm.

The area of the memory 37 for storing the data of the search range (FIG. 10A) is provided for each objective lens 12.
That is, it is individually secured for each number (1 to 6) of the revolver hole in which the objective lens 12 is mounted. Further, in the memory 37, regarding the seven objective lenses 12 that may be used in the microscope 10, as shown in FIG. 10B, a focusable range (μm) set according to each magnification. And the data of the search range converted into the count value of the up / down counter 44 are stored in advance.

The data in the search range shown in FIG.
It is data of the objective lens 12 actually attached to the electric revolver 12a, and is associated with the numbers (1 to 6) of the revolver holes in which the respective objective lenses 12 are attached, and FIG.
It is copied from the data regarding the seven objective lenses 12 shown in (b). The CPU 36 refers to the data of the search range (lower limit, upper limit) shown in FIG. 10A during the image contrast AF control (described later).

The search range data shown in FIG. 10A may be stored in an electronic element (IC or the like) built in the objective lens itself for each objective lens 12, and the data is a signal. It is read into the microscope body by communication.
The above-mentioned up / down counter 44, CPU 36
Corresponds to "first detecting means" and "second detecting means" in the claims. In addition, the stage 11, the drive unit 38, the CPU 36
Corresponds to "third adjusting means", "fifth adjusting means", and "third automatic adjusting means". The CPU 36 corresponds to the "blocking means" in the claims. Keyboard 71, CPU36,
Memory M _G corresponds to "offset storage unit".

Next, the microscope 10 configured as described above is used.
The focusing operation in the above will be described with reference to the flowcharts of FIGS. The procedure shown in FIGS. 12 and 13 (steps S21 to S40) is the same as the procedure shown in FIG.
The process of step S11 in (steps S1 to S17) is shown in detail. When the microscope 10 is powered on, the CPU 36 first initializes each unit (the memory 37 and the like) of the microscope 10 (step S1 in FIG. 11). By this initialization, the memories M _G , M _N [1] to M _N [6],
“0” is input to M ₁ , M ₂ , M ₃ , and M _M , and data indicating “permitted” is stored in the memory M _o .

After the initialization, the CPU 36 makes settings based on the input signal from the input section 39 (steps S2 to S9 in FIG. 11), and then the focus control start switch.
If the (SW) 76 is input (Y in step S10), focus control is executed (step S11) (steps S21 to S40 in FIGS. 12 and 13). First, there is no input from the objective lens changeover switch 72 (N in step S2),
There is input from the keyboard 71 (Y in step S5),
A case in which the focus control start switch 76 is input (step S10 is Y) and the focus control (step S11) is executed when there is no input from the focus correction permission switch 75 (step S8 is N) will be described. .

In this case, prior to the start of focusing control (step S11), the thickness G of the cover glass 14 is input from the keyboard 71 (step S6), and data relating to this thickness _G is stored in the memory MG. (Step S7). As a result, the memory M _G ≠ 0. Other settings are the same as those at the time of initialization (step S1), and the memory M _N [1] to
“0” is input to M _N [6], M ₁ , M ₂ , M ₃ , and M _M , and data indicating “permitted” is stored in the memory M _o .

Now, when the focusing control (step S11) is started, the CPU 36 stores the data of the focusing positions Z ₁ and Z ₂ (FIGS. 8A and 8B) in step S21 of FIG. Initialization of the memories M ₁ and M ₂ is performed again. Next, CP
U36 controls the drive unit 38 of the stage 11 by the slit projection AF control so that the value of the difference data (FIG. 6) becomes zero, and performs the focus position Z ₁ detection process (step S22, S23). When the focus position Z ₁ is detected (Y in step S23), the process by the slit projection AF control is ended, the count value of the up / down counter 44 at this point is read, and the data of the focus position Z ₁ is read. Is stored in the memory M ₁ (step S24).

Slit projection type AF control (step S22,
As a result of the processing in S23), the surface of the cover glass 14 is positioned on the focal plane of the objective lens 12 (FIG. 8).
(a)). Considering that the focal plane of the objective lens 12 moves up and down relative to the stage 11 that is stopped, the slit projection AF control (steps S22 and S23)
The processing by means will proceed as shown by the arrow in FIG.

Next, the CPU 36 moves the stage 11 up and down to the z position at which the above-mentioned image contrast AF control is started. Therefore, it is judged whether or not the difference data M is stored in the memory M _M (step and S25), performs the determination data regarding thickness G of the cover glass 14 is whether it is stored in the memory M _G (step S26).

At this time, since the memory M _M = 0 (N in step S25) and the memory M _G ≠ 0 (Y in step S26), the CPU 36 moves the stage 11 up and down by the thickness G of the cover glass 14. It is moved (step S27). This process will be described with reference to FIG. 14. The vertical movement of the cover glass 14 by the thickness G proceeds as indicated by the arrow. As a result, the back surface of the cover glass 14 is positioned on the focal plane of the objective lens 12.

Then, the CPU 36 starts the image contrast AF control from this position. That is, the drive unit 38 of the stage 11 is controlled so that the value of the contrast data (FIG. 7) is maximized, and the focus position Z ₂ is detected (steps S29 to S33). This search operation is
It progresses from the back surface of the cover glass 14 as shown by the arrow in FIG.

While the search operation is in progress, the CPU 36
Is monitoring the output signal from the limit sensor 45
(Step S30), when the limit signal indicating the vertical movement limit point (limit position) of the stage 11 is output (S3)
If 0 is Y), focusing is impossible and error processing is performed (step S40), and focusing control ends. In addition, the CPU 36
Determines whether or not the difference data M is stored in the memory M _M (step S31). If it is stored (S31 is Y), the process proceeds to step S32, and the search operation currently in progress. Is determined to exceed the search range shown in FIG. 10 (a). However, since the memory M _M = 0 (N in step S31) at the present moment, step S
The process of 32 will be described later.

Then, when the CPU 36 detects the in-focus position Z ₂ by the above search operation (Y in step S33), the processing by the image contrast AF control is ended. Since the stage 11 has passed the focus position Z ₂ at this point, the CPU 36 moves the stage 11 up and down by the amount passed (step S34). This process proceeds as shown by the arrow in FIG. as a result,
The focal plane of the objective lens 12 is the arbitrary plane 10 in the specimen.
a is positioned (FIG. 8 (b)).

Next, the CPU 36 updates the CPU at this point.
/ The count value of the down counter 44 is read and stored in the memory M ₂ as data of the focus position Z ₂ (step S35). Further, CPU 36 is the basis of the thickness G of the data of the cover glass 14 in the memory M _G, and focus position Z ₁ of the data in the memory M _1, in the focus position Z ₂ data in the memory M ₂ Te, the difference data M seeking _{(= Z 2 -Z 1 -G)} , and stores in the memory M _M (step S36).

In the next step S37, the CPU 36
Confirms the contents of the memory M _o set according to the operation of the focus correction permission switch 75 (step S89 in FIG. 11), and if data indicating “permitted” is stored in the memory M _o (S37). If Y), the process proceeds to step S38, and if the data indicating "prohibition" is stored (S37 is N), the focusing control is ended.

[0073] In the present time, in the memory M _o "permission"
Is stored (Y in S37), the CP
In step S38, the U36 determines whether or not the correction data N is stored in the memory M _N [a], and if it is stored (S38 is Y), the step S3 is performed.
Proceed to 9. However, at this moment, the memory M _N [a] =
Since it is 0 (S38 is N), the CPU 36 executes the step S
The focus control ends without executing the process of 39. The process of step S39 will be described later.

After the focusing control is completed in this way, C
The PU 36 stores the memory M in step S12 of FIG.
Check the contents of ₂ . Then, focusing on the memory M ₂ position Z ₂
If the data is stored (Y in S12), the process proceeds to step S13, and if it is not stored (N in S12), the process returns to step S2. Focused on the memory M ₂ position Z ₂
The case where the data is stored (Y in S12) means that the focusing control in step S11 is normally completed. Further, when the data of the focus position Z ₂ is not stored in the memory M ₂ (N in S12), it means that the focus control of step S11 is not normally completed, that is, error processing (see FIG. 1).
This is the case when the process is finished by step S40) of 3.

Next, the processing (steps S13 to S17) in the case where the focusing control of the above-mentioned step S11 is normally completed and the data of the focusing position Z ₂ is stored in the memory M ₂ will be described. At this time, as described above, the arbitrary surface 10a in the sample is positioned on the focal plane of the objective lens 12 (FIG. 8B, focusing position Z ₂ ), and the CCD sensor 34
The contrast of the sample image formed on the image pickup surface (FIG. 1) is maximum.

However, the contrast of the sample image observed through the eyepiece lens 13 is not always the maximum. Therefore, in step S13, the CPU 36 determines whether or not there is an input from the up / down fine adjustment switch 73, and when there is no input (S13 is N), the process returns to step S2. On the other hand, when the up / down fine adjustment switch 73 is input (S13 is Y), the CPU 36
Controls the drive unit 38 of the stage 11 based on the fine adjustment signal from the up / down fine adjustment switch 73 to move the stage 11 up and down as specified by the fine adjustment signal (step S14). The operator operates the up / down fine adjustment switch 73 by operating the eyepiece lens 13 of the microscope 10.
It is performed while observing the image of the specimen through. The vertical movement of the stage 11 by this manual operation proceeds as shown by the arrow in FIG. The operation of the up / down fine adjustment switch 73 ends when the operator can observe a high-contrast image well. At this time, the focal plane of the objective lens 12 has an arbitrary surface 10b in the sample.
Are positioned (FIG. 8 (c)).

Next, the CPU 36 determines whether or not the focus correction memory switch 74 is input (step S15),
If there is no input (N in S15), the process returns to step S2. Then, if the focus correction memory switch 74 is input (Y in S15), the CPU 36 operates at this time.
/ The count value of the down counter 44 is read and stored in the memory M ₃ as the data of the focus position Z ₃ (step S16).

[0078] Further, CPU 36 has a data position Z ₃ focusing the memory M _3, based on the focus position Z ₂ data in the memory M _2, correction data N (= Z ₃ -Z ₂₎ Is calculated and stored in the memory M _N [a] corresponding to the number a of the revolver hole of the objective lens 12 (step S17). Then, the process returns to step 2. As described above, the memory _MG
≠ 0, memory M _N [a], M _M = 0, memory M _o = “enable”.
_The difference data M (= Z ₂ −Z ₁ −G) is stored in M.
(Step S36 of FIG. 13). As a result, the memory M _M ≠ 0
Becomes Further, if the processing at the step S17 in FIG. 11, the memory M _N [a], the correction data N (= Z ₃ -
Z ₂ ) is stored. As a result, the memory M _M ≠ 0.

Next, focusing control under the settings of memories M _G , M _N [a], M _M ≠ 0 and memory M _o = “permitted” (step S11 in FIG. 11) (see FIGS. 12 and 13). Steps S21 to S4
0) will be described. In the focusing control this time, the processing proceeds according to the arrows 1 through 3 shown in FIG.
The CPU 36 executes the processing of steps S21 to S24 similarly to the previous time, detects the focusing position Z ₁ by the slit projection AF control (arrow in FIG. 15), and stores the data of the focusing position Z _{1 in} the memory M ₁ To memorize. At this time, the objective lens 1
The surface of the cover glass 14 is positioned on the focal plane of 2 (FIG. 8A).

Next, the CPU 36 executes the process of step S25 as in the previous time, and this time, the memory M _M ≠ 0 (S25
Is Y), the process proceeds to step S28, and the stage 11 is moved up and down by the sum of the thickness G of the cover glass 14 and the difference data M (= Z ₂ −Z ₁ −G) (arrow in FIG. 15). ). As a result, on the focal plane of the objective lens 12, the focus position Z detected by the previous image contrast AF control is detected.
₂ The surface 10a of the sample in FIG. 8 (b) is positioned. Then, from this position, the image contrast formula A of this time
F control is started. That is, the search operation of the image contrast AF control this time is performed from the surface 10a of the sample as shown in FIG.
Will proceed as indicated by the arrow.

While the search operation is in progress (steps S29 to S33), the CPU 36 executes the processing of step S30 (monitoring the output signal from the limit sensor 45) as in the previous time. Further, this time, since the difference data M is stored in the memory M _M (Y in S31), the process proceeds to step S32, and it is determined whether or not the search operation currently in progress exceeds the search range shown in FIG. 10 (a). Is determined.

The CPU 36 reads the data corresponding to the number a (= 1 to 6) of the revolver hole positioned in the observation optical path of the microscope 10 from the data of the search range shown in FIG. The search operation is performed while referring to the data in the search range. Then, if the search range is exceeded (S32 is Y), focusing is impossible, error processing is performed (step S40), and focusing control ends.

Focus position Z ₂ without exceeding the search range
When the CPU 36 has finished detecting (Y in step S33), the CPU 36
Ends the processing by the image contrast AF control and executes the processing of steps S34 to S36 as in the previous time. That is, the stage 11 is moved up and down by the amount of passing the focus position Z ₂ (arrow in FIG. 15, S34), and the data of the current focus position Z ₂ is stored in the memory M _2.
(S35), and stores the current difference data M a (= Z ₂ -Z ₁ -G) in the memory M _M (S36).

Next, the CPU 36 causes the memory M_OTo
Confirm that the data that indicates `` OK '' is stored.
Memory S with step S37 Y)_NCorrection data in [a]
N (= Z ₃-Z₂) Is stored (step
If S38 is Y), the process proceeds to step S39. And
Memory M_NCorrection data N (= Z stored in [a]₃−
Z₂), The stage 11 is moved up and down (see FIG. 15).
Arrow). Then, the focus control process ends.

At this time, on the focal plane of the objective lens 12,
Focus position Z ₃ detected by the previous manual operation (Fig. 8
The vicinity of the surface 10b of the sample in (c)) is positioned.
Then, after the focusing control is completed, the CUP 36 executes the processing of steps S12 to 17 of FIG.
The data in the memory M _N [a] is stored as appropriate. As described above, the memories M _G , M _N [a], M _M ≠ 0, and the memory M _o =
When the focusing operation under the setting of “permitted” is completed, the difference data M (= Z ₂ −Z ₁ − stored in the memory M _M is stored.
G) is updated according to the current data. In addition, FIG.
When the process of step S17 is executed, the memory M
_N [a] the stored correction data N (= Z ₃ -Z ₂₎ is also updated in accordance with the current data.

Therefore, the objective lens changeover switch 72
If there is no input from step S2 of FIG. 11 (N in step S2 of FIG. 11) and no input from the focus correction permission switch 75 (step S8 is N), then the above-mentioned memories _MG , _MN [a], M _M ≠ 0,
The focusing operation under the setting of memory M _o = “permitted” is repeatedly executed. Then, in response to the operation of the focus correction permission switch 75 (Y in step S8 of FIG. 11), the content of the memory _Mo is changed to "prohibition" (step S8).
Then, the CPU 36, during the repetition of the focusing operation,
Processing of steps S38 and S39 of FIG. 13 (correction data N
Without performing the automatic correction processing of the stage 11 based on
Focusing control (step S11 in FIG. 11) is ended.

If the objective lens changeover switch 72 is input (Y in step S2 of FIG. 11), the CPU 36
The objective lens 12 positioned on the observation optical path of the microscope 10 is controlled to be switched to another objective lens 12.
(Step S3), then, the memory M _M for storing the correction data M is initialized (step S4). As a result, M _M =
It becomes 0. In the focusing control in this case, when the other settings are the memories M _G and M _N [a] ≠ 0 and the memory M _o = “permitted”, the process proceeds according to the arrows from FIG. .

As described above, according to the microscope 10 of this embodiment, the image contrast type AF control (see FIG. 1) is performed under the setting of the memory M _o = “permitted” and the memory M _N [a] ≠ 0.
After steps S29 to S34 of 3), the correction data N in the memory M _N [a] (the focus position Z ₃ determined by manual operation up to the previous time and the focus position Z _{2 determined} by the image contrast AF control).
The vertical movement of the stage 11 is automatically corrected on the basis of the difference (step S39) (step S39).

Further, according to the microscope 10 of the present embodiment,
By setting changes the stored contents of the memory M _o from "permission" to "prohibition", since it not to perform automatic correction of the stage 11 based on the correction data N, rather than the eyepiece 13 an image of the specimen For example, it is possible to deal with the case of displaying on a TV monitor for observation. Further, according to the microscope 10 of the present embodiment, the slit projection type AF control (steps S22 and S23 in FIG. 12) is executed before the image contrast type AF control, and then the stage 11 is moved up and down by a predetermined amount. Automatic correction (step S27 in FIG. 12)
Alternatively, since S28), the range of the search operation by the image contrast AF control can be narrowed. As a result, the time required for focusing control (step S11 in FIG. 11) can be shortened, and a microscope image can be acquired in a short time.

Particularly, after the slit projection type AF control is executed, the thickness G of the cover glass 14 and the difference data M (= Z ₂ −
Z ₁ -G) to the case of automatically correcting the vertical movement of only the stage 11 amount that the sum of (the arrow) in FIG. 15, the range of the search operation by the image contrast type AF control can be reliably narrowed, if The focus control time and the microscope image acquisition time can be surely shortened.

In the above-described embodiment, an example of the microscope 10 for performing fluorescence observation of a biological specimen by epi-illumination or transmissive illumination has been described, but the present invention is not limited to biological specimen observation, for example, a semiconductor wafer is observed by epi-illumination. It can also be applied to a microscope. Further, in the above-described embodiment, the example in which the stage 11 is moved up and down in order to adjust the positional relationship between the sample and the objective lens 12 has been described, but the objective lens 12 may be moved up and down, or the objective lens 12 and the objective lens 12. Both the sample and the sample may be moved up and down.

[0092]

As described above, according to the present invention,
It enables quick focus adjustment and improves operability.

[Brief description of drawings]

FIG. 1 is a diagram showing an overall configuration of a microscope of this embodiment.

FIG. 2 is a block diagram showing an electrical configuration of the microscope of this embodiment.

FIG. 3 is a diagram showing how a peak of an image pickup signal shifts when a positional relationship between a sample and an objective lens changes.

FIG. 4 is a diagram illustrating a positional relationship between a peak of an image pickup signal and an integration boundary line.

FIG. 5 is a diagram showing a state in which a peak of an image pickup signal and an integration boundary line substantially coincide with each other.

FIG. 6 is a diagram showing how the difference data changes according to the position of the stage.

FIG. 7 is a diagram showing how the contrast data changes according to the position of the stage.

FIG. 8 is an explanatory diagram showing three types of in-focus positions in the microscope of this embodiment.

9 is an explanatory diagram of a memory that stores correction data N. FIG.

FIG. 10 is an explanatory diagram of a memory that defines a search range in image contrast AF control.

FIG. 11 is a flowchart showing a focusing operation in the microscope of this embodiment.

FIG. 12 is a flowchart showing a focusing operation in the microscope of this embodiment.

FIG. 13 is a flowchart showing a focusing operation in the microscope of this embodiment.

FIG. 14 is an explanatory diagram showing a stage of a focusing operation in the microscope of the present embodiment.

FIG. 15 is an explanatory diagram showing a stage of a focusing operation in the microscope of the present embodiment.

[Explanation of symbols]

10 microscope 11 stages 12 Objective lens 13 eyepiece 14 cover glass 15 Slide glass 16 dichroic mirror 17,25 Half mirror 21 LED light source 22 Slit member 23 Condenser lens 24 First pupil restriction mask 26, 32 Second objective lens 27 Relay lens system 28 Second pupil restriction mask 19 Cylindrical lens 30,34 CCD sensor 31 First Signal Processing Unit 35 Second Signal Processing Unit 33 Intermediate variable power lens 36 CPU 37 memory 18,38 Drive unit 39 Input section

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G02B 7/11 DF term (reference) 2F065 AA06 DD09 FF66 GG07 GG21 JJ02 JJ25 LL08 LL28 LL46 MM03 PP24 QQ03 QQ14 QQ51 2H051 AA11 BA44 BA45 BA70 CB20 CB22 DA02 DA31 2H052 AD09 AD19 AF03 AF06 AF14

Claims (5)

[Claims] 1. A first automatic adjustment means for automatically adjusting a relative positional relationship between the sample and the objective lens based on a contrast of an image of the sample formed through the objective lens, and the objective. A second unit that has an irradiation unit that irradiates the sample with detection light via a lens, and that automatically adjusts the positional relationship based on reflected light from the sample irradiated with the detection light
Automatic adjusting means, offset storing means for storing a predetermined offset amount, and stored in the offset storing means after the adjustment by the second automatic adjusting means and before the adjustment by the first automatic adjusting means. A focusing device for a microscope, comprising: a third automatic adjusting means for automatically adjusting the positional relationship based on the offset amount. 2. A first adjusting means for automatically adjusting a relative positional relationship between the sample and the objective lens based on a contrast of an image of the sample formed through the objective lens, and an external adjusting means. Second adjustment means for finely adjusting the positional relationship based on a manual operation, and a deviation between the positional relationship after adjustment by the first adjusting means and the positional relationship after adjustment by the second adjusting means is detected. A first detection unit; a first storage unit that stores the deviation detected by the first detection unit; and a first storage unit after the adjustment by the first adjustment unit and before the adjustment by the second adjustment unit. A third adjusting means for determining whether or not the shift is stored in the storage means, and if the shift is stored, automatically adjusting the positional relationship based on the shift. For microscopes featuring Point alignment device. 3. The microscope focus position detecting apparatus according to claim 2, further comprising a blocking unit that blocks the adjustment of the positional relationship by the third adjusting unit. 4. The focusing device for a microscope according to claim 2, further comprising an irradiation unit that irradiates the specimen with detection light via the objective lens, and the detection light irradiates the specimen. Fourthly, the positional relationship is automatically adjusted based on the reflected light from the sample.
Adjusting means, second detecting means for detecting a deviation between the positional relationship after the adjustment by the fourth adjusting means and the positional relationship after the adjustment by the first adjusting means, and the second detecting means. It is determined whether or not the deviation is stored in the second storage means after the adjustment by the fourth adjusting means and before the adjustment by the first adjusting means. A fifth focusing means for automatically adjusting the positional relationship based on the deviation when the deviation is stored, and a focusing device for a microscope. 5. An image forming optical system having an objective lens and an eyepiece lens for forming an image of a specimen; and an image pickup optical system arranged between the objective lens and the eyepiece lens. A microscope including the focusing device for a microscope according to item 1.