JP2003005088A - Focusing device for microscope and microscope having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a focusing device for a microscope which is capable of easily focusing a position where the contrast of the image of a specimen is highest regardless of the brightness of the specimen. SOLUTION: This device has regulating means 11 and 38 which regulate the positional relation of the specimen and an objective lens 12, means 16, 17, 32 to 36 which capture the image of the specimen formed by means of the objective lens as an image and detect the positional relation when the contrast of the image is highest, means 37 which stores the reference of the positional relation, means 36 which has the positional relation of the highest contrast detected and sets the positional relation obtained by this detection as the reference, means 16, 21 to 24 which irradiate the specimen with detecting light through the objective lens, means 16, 25 to 30 which detect the reflected light from the specimen irradiated with the detecting light through the objective lens, means 31 to 36 which detect the deviation of the positional relation with respect to the reference and control means 36 which controls the regulating means in accordance with the detected deviation.

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 active system has been known as a focusing device for a microscope.
The active method irradiates the sample with the detection light and receives the reflected light from the sample irradiated with the detection light,
This is a method of focusing based on the shift of the light receiving position of the reflected light. According to the active method, focusing can be performed at high speed. Further, since the detection light is emitted by itself, focusing can be performed regardless of the brightness of the sample.

By the way, in order to perform focusing by using the active method, it is necessary to detect the shift of the light receiving position of the reflected light from the sample, and there is some standard for detecting the shift of the light receiving position. is necessary. Therefore, in the active method, if the reference is not optimally set, the shift of the light receiving position cannot be accurately detected, and the image of the sample cannot be observed with good contrast even if focusing is performed. That is,
In the active method, whether or not the image of the sample can be observed with good contrast is determined depending on the setting of the reference. Conventionally, the standard has been set by the operator's visual observation.

[0004]

However, in the prior art, in setting the reference of the active method, the result of visual observation by the operator was used, which is troublesome and has a problem of poor reproducibility. . An object of the present invention is to provide a focusing device for a microscope that can easily focus on a position where the contrast of the image of the sample is maximum regardless of the brightness of the sample, and a microscope including the same. It is in.

[0005]

A focusing device for a microscope according to the present invention includes adjusting means for adjusting the positional relationship between a sample and an objective lens, and an image of the sample formed through the objective lens, which is taken in as an image. The position detecting means for detecting the positional relationship when the contrast of the image becomes maximum, the storage means for storing the reference of the positional relationship, and the position detecting means are controlled,
A setting unit that detects the positional relationship that maximizes the contrast and sets the positional relationship obtained by the detection as a reference of the storage unit, an irradiation unit that irradiates the sample with the detection light through the objective lens, and the detection light Based on the light receiving means for receiving the reflected light from the irradiated sample through the objective lens and the light receiving signal output from the light receiving means and the reference stored in the storage means, the deviation of the positional relationship with respect to the reference is detected. It is provided with a positional deviation detecting means and a control means for controlling the adjusting means based on the deviation detected by the positional deviation detecting means.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the drawings. The embodiment of the present invention is claim 1.
~ Corresponding to claim 4. 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. The focusing device (21 to 38) incorporated in the afocal system between the eyepiece lens 12 and the eyepiece lens 13.

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.
Taking the microscope 10 of FIG. 1 as an example, the transmission type illumination device is arranged below the stage 11, and the epi-illumination type illumination device is arranged above the stage 11.

In the microscope 10, a dichroic mirror 16 and a half mirror 17 are arranged between the objective lens 12 and the eyepiece lens 13 (afocal system). The dichroic mirror 16 reflects the infrared light La and Lb and transmits the visible light Lc. The half mirror 17
Part of the visible light Ld is reflected and the other part Le is transmitted.
The dichroic mirror 16 and the half mirror 17 are optical elements arranged to incorporate the focusing devices (21 to 38).

Now, the focusing device (21
38) will be specifically described. Focusing device (2
1 to 38) 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, and a memory 37.

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.
A motor driver 42 for rotating the DC motor 41 based on a control signal from the rotary encoder 43, a rotary encoder 43 for detecting the rotation angle of the DC motor 41, and an up-and-down counter for counting the vertical movement of the stage 11 based on the detection result of the rotary encoder 43. A down counter 44 is provided. The count result of the up / down counter 44 is output to the CPU 36 as a vertical movement signal.

Further, the drive unit 38 includes a jog dial 45 attached to the stage 11 for manual fine adjustment,
A rotary encoder 46 that detects the rotation angle of the jog dial 45, and an up / down counter that counts the vertical movement of the stage 11 based on the detection result of the rotary encoder 46.
A down counter 47 is provided. The count result of the up / down counter 47 is used as a fine adjustment signal by the CPU.
36 is output.

The stage 11 is the D of the drive unit 38 described above.
When the C motor 41 or the jog dial 45 rotates,
It moves up and down according to the rotation angle. And stage 11
The sample placed on the top also moves up and down together with the cover glass 14 and the slide glass 15, and the positional relationship between the sample and the objective lens 12 is adjusted. The stage 11 and the drive unit 38 are
It corresponds to "adjusting means" in the claims.

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 shielded by the first pupil limiting mask 24, then passes through the half mirror 25, and is emitted toward the dichroic mirror 16. The light Lf emitted from the infrared emission units (21 to 24) is
It is slit-like 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). Infrared emission part (21 ~
24) and the dichroic mirror 16 correspond to the "irradiating means" in the claims.

Since the sample on the stage 11 is covered with the cover glass 14, the infrared emitting section (21 to 21).
24) to the dichroic mirror 16 and the objective lens 12
The slit-shaped infrared light Lh guided via and is reflected on the surface of the cover glass 14. Therefore, the description of “irradiating the sample with the detection light” in the present invention means that “in the case where the cover glass 14 is present on the sample, the detection light is actually irradiated onto the surface of the cover glass 14”. I mean.

The reflected light Lk is transmitted to 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.

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.
An area sensor arranged two-dimensionally may be used.

In the infrared light receiving section (25 to 30), the infrared light Lg from the dichroic mirror 16 is reflected by the half mirror 25 and then passed through the second objective lens 26, the relay lens system 27, and the cylindrical lens 29. Pass, CC
An image is formed on the imaging surface of the D sensor 30. At this time, CCD
A slit image is formed on the imaging surface of the sensor 30. C
The position where the slit image is formed on the imaging surface of the CD sensor 30 moves in the longitudinal direction of the line sensor 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 charge is accumulated in each light receiving portion according to the slit image formed on the image pickup surface. Then, the charges accumulated in each light receiving portion of the CCD sensor 30 are transferred through the CCD and output to the signal processing portion 31 as an image pickup signal. A start pulse is input to the CCD sensor 30 from the CPU 36 when the output of the image pickup signal to the signal processing unit 31 is started. Dichroic mirror 16 and infrared receiver (25-30)
Corresponds to "light receiving means" in the claims.

The image pickup signal output from the CCD sensor 30 to the signal processor 31 will be described with reference to FIG. The horizontal axis of FIG. 3 represents the light receiving position (time at the time of output) at the CCD sensor 30, and the vertical axis represents the voltage value of the image pickup signal.

The image pickup signal output from the CCD sensor 30 to the signal processing unit 31 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 position on the image pickup surface of the CCD sensor 30 where the slit image is formed. 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 slit image on the imaging surface moves, the peak position (time tp) in the imaging signal also shifts.

For example, when the stage 11 moves toward the rear pin side, the peak position (time tp) of the image pickup signal shifts toward the time ts side (solid line (a) → dotted line (b)).
Conversely, when the stage 11 moves toward the front pin side, the position of the peak of the image pickup signal (time tp) shifts toward the time te side (solid line (a) → dotted line (c)). Next, the signal processing unit 31 will be described.

The signal processing section 31, as shown in FIG.
C-cut circuit 51 and programmable gain amplifier
(PGA) 52, integrating circuits 53 and 54, and 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 the time ts to the predetermined time tk as shown in FIGS. 4 (a) and 4 (b). The integration range of the integration circuit 54 is a range from a predetermined time tk to a time te. The predetermined time tk is an integration boundary line and is arbitrarily set according to a control signal from the CPU 36. The setting of the predetermined time tk will be described later.

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 peak position (time tp) in the image pickup signal shifts (FIG. 3).
Therefore, the integrated value signal output from the integration circuit 53
The magnitude of (A) and the magnitude of the integrated value signal (B) output from the integrating circuit 54 change. When the peak of the imaging signal appears before the predetermined time tk (FIG. 4 (a), tp
<Tk), the integrated value signal (A) becomes larger than the integrated value signal (B) (A> B). The vertical movement position (z position) of the stage 11 at this time is defined as z1.

On the contrary, when the peak of the image pickup signal appears after the predetermined time tk (FIG. 4 (b), tp> tk), the integrated value signal
(A) is smaller than the integrated value signal (B) (A <B). The z position of the stage 11 at this time is defined as z2. And
When the peak of the imaging signal appears almost at the same time as the predetermined time tk (FIG. 5, tp = tk), the integrated value signal (A) and the integrated value signal
(B) is equal to each other (A = B). The z position of the stage 11 at this time is set to z0.

The difference circuit 55 connected to the subsequent stage of the above-mentioned integrating circuits 53 and 54 is an integrated value signal from the integrating 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
When> B (the stage 11 is z1, the peak of the imaging signal is t
p <tk), the difference data has a positive value. Further, as shown in FIG. 5, when the integrated value signal is A = B (z0, tp = tk),
The difference data becomes zero. When the integrated value signal is A <B (z2, 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 so that the value of the difference data becomes zero on the basis of the value of the difference data (FIG. 6) described above, and the value of the difference data. When becomes zero, the stage 11 is stopped. The value of the difference data becomes zero when the peak of the image pickup signal output from the CCD sensor 30 appears at substantially the same time as the predetermined time tk (FIG. 5, tp = tk).

That is, the control based on the value of the above-mentioned difference data (FIG. 6) is performed by the stage 11 when the peak of the image pickup signal appears at substantially the same time as the predetermined time tk (FIG. 5, tp = tk).
Is performed with reference to the z position (z0) of. As described above, the predetermined time tk which is the integration boundary line can be said to be a parameter indicating the reference of the z position of the stage 11 (the positional relationship between the sample and the objective lens 12). The value of the difference data (FIG. 6) described above indicates the deviation of the z position of the stage 11 from the reference. Hereinafter, the predetermined time tk is referred to as “reference time tk” and the difference data is referred to as “positional shift data”.

Since the reference time tk can be arbitrarily set according to the control signal from the CPU 36, the reference time tk
The stage 11 can be stopped at an arbitrary z position by changing the set value of. The set value of the reference time tk is stored in the memory 37. The signal processing unit 31 and the CPU 36 correspond to "positional deviation detecting means" in the claims, and the CPU 36 corresponds to "controlling means", "setting means", and "monitoring means". The memory 37 corresponds to "storage means".

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 a sensor in which a plurality of light receiving parts are 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 is imaged on the image pickup surface of the CCD sensor 34. To be done. The visible light Le is a part of visible light generated from a sample illuminated by an illumination device (not shown), passing through the objective lens 12 and the dichroic mirror 16, and reaching the half mirror 17. For this reason,
A sample image is formed on the imaging surface of the CCD sensor 34.

The other part Ld of the visible light that has reached the half mirror 17 is reflected by the half mirror 17 and imaged by the eyepiece lens 13. Therefore, the contrast of the sample image formed on the imaging surface of the CCD sensor 34 is
This is the same as the contrast of the sample image observed through the eyepiece lens 13. 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 charges accumulated in each light receiving portion of the CCD sensor 34 are transferred through the CCD and output to the signal processing portion 35 as an image signal. Next, the 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 indicates 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 the level of the contrast of the sample image, it changes when the position of the sample changes due to the vertical movement of the stage 11, and becomes the maximum at a certain z position (zc). At this time, a sample image with high contrast can be satisfactorily observed on the eyepiece 13 side.

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. The control based on the value of the contrast data (FIG. 7) is performed to set the reference time tk described above.

The dichroic mirror 16, the half mirror 17, the visible light receiving parts (32 to 34), the signal processing part 35 and the CP.
U36 corresponds to the "position detecting means" in the claims. Next, the focusing operation in the microscope 10 configured as described above will be described using the flowchart in FIG. When the microscope 10 is powered on and the focusing mode is set to the autofocus mode, the CPU 36 executes the focusing operation according to the procedure of the flowchart shown in FIG. In this flowchart, when the focusing operation is started, a certain reference time t has already been reached.
It is assumed that k (FIGS. 4 and 5) is stored in the memory 37. The reference time tk already stored in the memory 37 at this point is set, for example, at the time of the previous observation.

The CPU 36 first determines whether or not the objective lens 12 has been switched (step S1), and if the determination result is "N", determines whether or not the stage 11 is largely defocused. The determination (step S2) is performed. Then, if the determination result of step S2 is also “N”, the processes of steps S3 to S5 are executed. Also, CP
When the determination result of either step S1 or step S2 is “Y”, the U36 executes the processes of steps S6 to S10 and then executes the processes of steps S4 and S5.

By the way, the determination as to whether the objective lens 12 has been switched (step S1) is made by operating the objective lens switching button provided on the microscope 10 or detecting the rotation of the revolver. Also, it is determined whether the stage 11 is largely defocused (step S
2) is the positional deviation data output from the signal processing unit 31
This is performed depending on whether the absolute value of (FIG. 6) is larger or smaller than the predetermined value.

First, the processing of steps S3 to S5 will be described. In step S3, after irradiating the sample with infrared light (Lh), the CPU 36 takes in the positional deviation data (FIG. 6) output from the signal processing unit 31 and sets the stage so that the value of the positional deviation data becomes zero. 11 drive unit 38 is controlled. Here, when loading the positional deviation data from the signal processing unit 31, the setting unit in the CPU 36 instructs the integration circuits 53 and 54 of the signal processing unit 31 to store the reference time tk (see FIG. The control signal for setting (FIG. 5) is output.

More specifically, the CPU 36 controls the CC
A start pulse is output to the D sensor 30, and the CCD
The output of the imaging signal from the sensor 30 to the signal processing unit 31 is started (time ts) to start time measurement, and at the same time, the integration circuit 5
Instruct 3 to start integration. Then, at the reference time tk stored in the memory 37, the CPU 36 instructs the integration circuit 53 to end the integration and instructs the integration circuit 54 to start the integration. Further, at time te when the output of the image pickup signal from the CCD sensor 30 ends, the CPU 36 instructs the integration circuit 54 to end the integration.

As a result, the signal processing unit 31 to the CPU 36
The positional shift data (FIG. 6) indicating the shift of the z position of the stage 11 with respect to the reference is output to. In addition, stage 2
The reference of the z position of 1 is when the image pickup signal peaks at almost the same time as the reference time tk stored in the memory 37 (FIG. 5).
Is the z position (z0) of the stage 11. In this way, when the positional deviation data (FIG. 6) based on the reference time tk stored in the memory 38 is obtained, the CPU 36 causes the driving unit 3 of the stage 11 to set the positional deviation data to zero.
Control eight.

For example, when the positional deviation data has a positive value (FIG. 4 (a)), the CPU 36 moves the stage 11 toward the front pin side and outputs the positional deviation data output from the signal processing unit 31. Are sequentially taken in and the stage 11 is stopped when the positional deviation data becomes zero. On the contrary, when the positional deviation data has a negative value (FIG. 4B), the CPU 36
While moving the stage 11 toward the rear pin side, the positional shift data output from the signal processing unit 31 is successively taken in, and when the positional shift data becomes zero, the stage 11 is stopped.

As described above, since the value of the positional deviation data (FIG. 6) includes information on the direction in which the stage 11 should be moved, the search for actually moving the stage 11 up and down to search for a position where the focus is actually achieved. No action required. Therefore, the focusing operation (control based on the value of the positional deviation data) in step S3 is performed at high speed. As a result of step S3, the stage 11 is quickly positioned at the reference (z0) of the z position corresponding to the reference time tk stored in the memory 37. Since the positioning in step S3 is performed by irradiating infrared light by itself, the same result can be obtained regardless of whether the sample is bright or dark.

In the next step S4, the CPU 36
Judges whether or not the observer has operated the jog dial 45. If the judgment result is "N", the process returns to step S1. If the judgment result is "Y", the process proceeds to the next step S5.

When the observer operates the jog dial 45, the CPU 36 reads the fine adjustment signal output from the up / down counter 47 of the drive unit 38 in step S5, and reads the reference time stored in the memory 37. Finely adjust tk (correction unit). That is, the reference (z0) of the z position of the stage 11 is finely adjusted according to the manual fine adjustment amount by the observer.

Therefore, when the process of step S3 is executed next, the positional deviation data is fetched from the signal processing unit 31 based on the reference time tk in which the amount of manual fine adjustment by the observer is reflected, and this position is read. The drive unit 38 of the stage 11 is controlled so that the value of the deviation data becomes zero. Next, the processing of steps S6 to S10 will be described. The processing of steps S6 to S10 is executed only when the objective lens 12 is switched or the stage 11 is largely defocused. The process of step S6 is the same as the process of step S3 described above.

In step S7, the CPU 36 takes in the contrast data from the signal processing unit 35 and determines whether the sample is bright or dark. Then, when the sample is bright enough to extract the contrast data (Y in step S7), the processes of steps S8 to S10 are executed, and the step of changing the reference time tk is executed. In step S8, the CPU 36 controls the drive unit 38 of the stage 11 with reference to the contrast data (FIG. 7) loaded from the signal processing unit 35. In other words, the stage 11 is driven up / down within a certain vertical movement range (search operation),
The z position (zc) that maximizes the contrast data is detected, and the vertical movement of the stage 11 is stopped at the z position (step S9).

At this time, on the eyepiece lens 13 side, a sample image with high contrast can be satisfactorily observed. Next, in step S10, the CPU 36 fetches the positional deviation data (FIG. 6) from the signal processing unit 31 in the state where the stage 11 is stopped at the z position (zc) where the contrast data is maximum, and the positional deviation data is acquired. The set value of the reference time tk is changed so that the value of is zero.

For example, when the positional deviation data has a positive value (FIG. 4 (a)), the CPU 36 sets the reference time tk to the time ts.
While shifting toward the side, the positional deviation data output from the signal processing unit 31 is sequentially taken in, and the reference time tk when the positional deviation data becomes zero is stored in the memory 37. On the contrary, when the positional deviation data has a negative value (FIG. 4 (b)), the CPU 36 moves the reference time tk toward the time te side and outputs the positional deviation data output from the signal processing unit 31. The reference time tk when the positional deviation data becomes zero is stored in the memory 37 one after another.

By setting and updating the reference time tk in this way, the z position (zc) at which the contrast data corresponding to the new sample state becomes maximum is stored in the memory 37 as the z position (sample and sample) of the stage 11. It is stored as a reference of (positional relationship with the objective lens 12). Therefore,
When executing the process of step S3 at the next observation, the positional deviation data is fetched from the signal processing unit 31 based on the reference time tk newly set in step S10, and the value of the positional deviation data is zero. The drive unit 38 of the stage 11 is controlled so that

As a result, the stage 11 carries out step S8.
Z position of the maximum contrast data detected in
It is positioned at (zc). If it is determined in step S7 that the sample is too dark to extract the contrast data, the process proceeds to step S4 without executing the processes of steps S8 to S10 for updating the contrast data. Then, the set value of the reference time tk is updated according to the operation of the jog dial 45 by the observer (steps S4 and S5). That is, a parameter (tk) newly indicating the z position where the contrast data becomes maximum.
The setting of is not changed and the already stored reference time tk is used.

As described above, according to the microscope 10 of this embodiment, the reference time tk set in the memory 37 is set.
In order to control the drive unit 38 of the stage 11 so that the positional shift data is fetched from the signal processing unit 31 based on the (parameter indicating the z position where the contrast data is maximum) and the value of the positional shift data becomes zero. Step S
3) Regardless of the brightness of the sample, it is possible to quickly focus on the z position where the contrast of the sample image is maximum.

Further, when the objective lens 12 is switched or the stage 11 is largely defocused,
To change the setting of the reference time tk (parameter indicating the z position where the contrast data becomes maximum) in the memory 37
(Step S10) The sample image with the maximum contrast can always be observed. In addition, when manual fine adjustment is performed by the observer, the memory 3 is adjusted based on the fine adjustment amount.
Since the set value of the reference time tk within 7 is finely adjusted (step S5), even if the depth of focus is shallow with respect to the thickness of the sample, it is possible to satisfactorily observe an arbitrary target object of the sample with the best focus.

In the above-described embodiment, when the sample is dark, the set value of the reference time tk is updated according to the operation of the jog dial 45 by the observer. It is also possible to switch to the interference observation state) and execute the processing of steps S8 to S10 to update the set value of the reference time tk. further,
In the above-described embodiment, an example in which only the reference time tk (integral boundary line) is used as a parameter indicating the reference of the z position of the stage 11 (positional relationship between the sample and the objective lens 12) has been described. Not limited to

For example, the reference time tk (integral boundary line) is fixed at an arbitrary time between the times ts and te, and the stage 1
When setting and changing the reference of the z position of 1 (positional relationship between the sample and the objective lens 12), the integrated value output from the integrating circuits 53 and 54 according to the fixed reference time tk (integral boundary line). It is conceivable to add a predetermined offset signal to the signals (A) and (B) and the difference signal output from the difference signal 55. Further, predetermined offset data may be added to the difference data output from the A / D converter 56.

Further, in the above-described embodiment, an example of the microscope 10 for observing a biological specimen with fluorescence by epi-illumination or transmissive illumination is described, but the present invention is not limited to the biological specimen.
For example, it can be applied to a microscope for observing a semiconductor wafer by epi-illumination. 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.

The position detecting means for detecting the positions of the sample and the objective lens when the contrast data becomes maximum may be incorporated in the alignment device.
It may also be attached to the outside of the device.

[0064]

As described above, according to the present invention,
The specimen can be easily focused.

[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 a reference time.

FIG. 5 is a diagram showing a state in which a peak of an image pickup signal and a reference time almost match each other.

FIG. 6 is a diagram showing how difference data (positional deviation 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 a flowchart showing a focusing operation in the microscope of this 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 29 Cylindrical lens 30,34 CCD sensor 31, 35 Signal processing unit 33 Intermediate variable power lens 36 CPU 37 memory 38 Drive

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) G02B 7/04 F

Claims (4)

[Claims] 1. An adjusting device for adjusting a positional relationship between a sample and an objective lens, and the position when the image of the sample formed through the objective lens is captured as an image and the contrast of the image is maximized. A position detecting unit that detects a relationship, a storage unit that stores the reference of the positional relationship, and a position detecting unit that controls the position detecting unit to detect the positional relationship that maximizes the contrast. Setting means for setting a positional relationship as the reference of the storage means, irradiation means for irradiating the specimen with detection light through the objective lens, and reflection light from the specimen irradiated with the detection light for the objective lens Based on a light receiving means for receiving light via the light receiving means, and a light receiving signal output from the light receiving means and the reference stored in the storage means, the positional relationship with respect to the reference is determined. A focusing device for a microscope, comprising: a positional deviation detecting means for detecting a deviation; and a control means for controlling the adjusting means on the basis of the deviation detected by the positional deviation detecting means. 2. The focusing device for a microscope according to claim 1, wherein at least whether the deviation detected by the position deviation detecting means is larger than a predetermined value and whether the objective lens is switched. A monitoring means for monitoring one of the settings, wherein the setting means, based on a result of monitoring by the monitoring means, when the deviation is larger than the predetermined value, or
A focusing device for a microscope, wherein when the objective lens is switched, the position detecting means is controlled and the positional relationship detected by the position detecting means is set as the reference of the storage means. 3. The focusing device for a microscope according to claim 1, wherein the adjusting unit includes a fine adjusting unit that finely adjusts a positional relationship between the sample and the objective lens, and the setting unit. A microscope focusing device, comprising: a correction unit that corrects the reference stored in the storage unit based on the amount of fine adjustment by the fine adjustment unit. 4. An image forming optical system having an objective lens and an eyepiece lens for forming an image of a sample, and the 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.