JPH09189849A - Focus detection device for microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To drastically enlarge the restriction of the wavelength area of an optical image that the focusing accuracy of a focus detection means is secured with respect to a peripheral system. SOLUTION: By projecting one part of the optical image of a sample S illuminated by a light source for transmission 3 or a light source for vertical illumination 4 on an image sensor 103 from an objective lens 5 by an image forming optical system having a coupling lens 102, an output signal corresponding to the light intensity distribution of the image of the sample is obtained. The output signal is arithmetically operated by an analog signal processing circuit 11 and an evaluation function computing unit 12 according to a prescribed evaluation function. Then, this device is provided with a photographic system 9 being as the peripheral system having the image forming optical system being different from a focus detection unit 10 with respect to the focus detection unit 10 constituted so that the focusing state of the image forming optical system with respect to the sample S is detected based on an obtained focusing evaluation value. Then, the length of an optical path in the detection unit 10 is changed with respect to the photographic system 9 by moving the parallel flat prism 20 of a focusing unit 101.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a focus detecting device used in a microscope.

[0002]

2. Description of the Related Art Conventionally, there are microscopes that can photograph an observation image of a sample or can observe it on a television (TV) screen. Those that enable such photographing and TV observation. It is said that focusing is difficult when a low-magnification objective lens is used as the objective lens of the microscope body.

The reason for this is that when a low-magnification objective lens is used, the NA (numerical aperture) on the sample side of the objective lens is small, so the depth of field becomes deep, and focusing is performed while viewing the observation image through the eyepiece. , With the addition of a focus correction function by the eye, it may appear that the image is in focus even if it is out of focus. On the other hand, on the image side of the objective lens, the depth of focus becomes shallow and the defocus becomes noticeable. Points.

Therefore, in a microscope employing such a low-magnification objective lens, a focus detection device for a microscope for detecting a focus is considered to be one of important applications.

Japanese Unexamined Patent Publication No. 61-143710 discloses an example of such a focus detecting device for a microscope, which enables quick focusing even on a sample surface having a height difference. ing.

On the other hand, in the case of observing a living tissue or a living cell using a microscope, the living tissue or the living cell is fluorescently stained, and the fluorescence observation is carried out in this state. In this case, an optical image of infrared light is generated from these living tissues and living cells. However, when taking a picture or observing a television image of such infrared light,
It is impossible to focus with the naked eye, which also requires a focus detection device for a microscope.
As described above, the focus detection device used in the microscope needs to be able to detect the focus from visible light to non-visible light, including general applications.

[0007]

However, as a practical problem, in the case of enabling photography and TV observation, color correction of image forming optical systems of the photography device, the TV camera device, and the focus detection device is insufficient. As a result, chromatic aberration occurs, which makes it difficult to secure focus accuracy.

FIG. 7 is a model showing the change of the focus position with respect to the wavelength of the light image in a certain photographic system. The vertical axis shows the focus position of the imaging optical system and the horizontal axis shows the wavelength of the light image. In this case, as is clear from the figure, it is understood that the focus position with respect to the reference position is changed due to the chromatic aberration of the imaging optical system.

FIG. 8 shows the model a shown in FIG.
The model b showing the change of the focus position with respect to the wavelength of the AF (autofocus) system is added by a broken line. In this case, since the AF system and the photographic system have different imaging optical systems, as shown in FIG. The change curves of the focus position with respect to the wavelength of the optical image of the AF system and the photographic system do not match.

In such a case, the focusing accuracy guaranteed range c of the photographic system and the AF system is 460 to 460, assuming that the same focal shift is allowed up to ± 1.0 [mm] in consideration of the depth of focus. Focusing accuracy of AF is guaranteed in the wavelength range of 600 [nm], but at 460 [nm] or less or 600 [nm] or more, the distance between the two curves is ±.
Since it exceeds 1.0 [mm], the focusing accuracy of AF cannot be guaranteed. That is, a photograph taken by AF becomes so-called out-of-focus in a range of 460 [nm] or less or 600 [nm] or more.

In the case where the AF system and the photographic system are combined in this way, since the imaging optical system of the system is different, there is always a wavelength limitation as a guarantee condition of the focusing accuracy for the above reason. Therefore, there is a problem that the original objective performance of AF cannot be exhibited in the wavelength range where the focus shift due to chromatic aberration occurs.

The present invention has been made in view of the above circumstances, and a focus for a microscope capable of greatly enlarging the limitation of the wavelength range of an optical image that guarantees the focusing accuracy of the focus detection means for a peripheral system such as a photographic system. An object is to provide a detection device.

[0013]

According to the first aspect of the present invention,
Focus detecting means for detecting the focus state of the image forming optical system with respect to the sample based on an in-focus evaluation value for the output of the light receiving means, the image forming optical system forming an observation light image of the sample on the light receiving means. And a peripheral system having a coupling optical system different from the image forming optical system of the focus detecting means, and means for determining an optical path length or a chromatic aberration correction amount of the focus detecting means with respect to the peripheral system.

According to a second aspect of the present invention, in the first aspect, the optical path length or the chromatic aberration correction amount of the focus detecting means with respect to the peripheral system is determined based on the wavelength of the sample image.

According to a third aspect of the present invention, there is provided an image forming optical system for forming an observation light image of the sample on the light receiving means, and the image forming on the sample is performed based on a focus degree evaluation value for the output of the light receiving means. Focus detecting means for detecting the focus state of the optical system, a peripheral system having a coupling optical system different from the image forming optical system of the focus detecting means, and the image forming optical system or the image forming optical system according to the degree of focus of the focus detecting means. Servo means for driving at least one of the sample side to perform a focused focus search, means for determining an offset amount according to the wavelength of the sample image, and focus focus search by the servo means to determine that the focus detection means is in focus And a means for driving at least one of the imaging optical system and the sample by the determined offset amount with respect to the position.

As a result, according to the first aspect of the present invention,
For peripheral systems with different focus detection means and imaging optical system, it becomes possible to correct the focus deviation between the focus detection means and the peripheral system related to the wavelength of the optical image caused by insufficient chromatic aberration correction. It is possible to ensure the focusing accuracy of the focus detection means for the system.

Further, according to the second aspect of the present invention, for a peripheral system having a different focus detection means and imaging optical system, a focus deviation between the focus detection means and the peripheral system with respect to the wavelength of the optical image caused by insufficient chromatic aberration correction. , It becomes possible to automatically correct based on the wavelength of the sample image.

Further, according to the third aspect of the invention, for a peripheral system in which the focus detection means and the imaging optical system are different from each other, a focus shift between the focus detection means and the peripheral system with respect to the wavelength of the optical image caused by insufficient chromatic aberration correction. Further, it becomes possible to make correction by further driving the focus detection means by a fixed offset amount from the position determined to be in focus.

[0019]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows an example in which the microscope focus detection apparatus of the present invention is applied to an epi-illumination / transmission observation photographic microscope.

In the figure, reference numeral 1 is a microscope body, and a stage 2 is provided on the microscope body 1. The stage 2 mounts a sample S, which is an observation sample, and is vertically movable in the optical axis direction.

A transmission light source 3 for a transmission microscope is disposed below the stage 2, and an epi-illumination light source 4 for an epi-fluorescence microscope is disposed above the stage 2 for transmission. In the case of the speculum, the light flux from the transmissive light source 3 is transmitted through the sample S to obtain an observation light image through the objective lens 5, and in the case of the epifluorescence spectroscope, the light flux from the episcopic light source 4 is reflected by the sample S. An observation light image is obtained by reflecting the light through the objective lens 5.

The light flux passing through the objective lens 5 is given to the cube lens 7 of the light projecting tube 6, and a part of the light flux passing through the cube lens 7 is given to the eyepiece lens 8 as well as the photographic system 9 and the focus detector. I am giving it to 10.

Here, the cube lens 7 is such that the wavelength of the excitation light can be selected when the epi-illumination fluorescence microscope is used. Further, the photographic system 9 has an image forming lens 91, and an observation light image from the objective lens 5 is formed on the film surface via the image forming lens 91. Also, the focus detector 1
0 is the parfocal adjustment unit 101 and the imaging lens 10.
2. The image sensor 1 having the image sensor 103 and the observation light image from the objective lens 5 through the image forming lens 102.
It is made to image on 03.

The parfocal adjustment unit 101 includes a focus detector 1
The focus detector 8 adjusts the stage position determined to be in focus so that the photograph S of the sample S of the photographic system 9 is in focus at the stage position determined to be 0. The details will be described later. Further, the image sensor 103 outputs an analog signal corresponding to a voltage corresponding to the incident light amount of the projected light image and the accumulation time.

An analog signal processing circuit 11 is connected to the image sensor 103, and the analog signal processing circuit 11 is connected.
The evaluation function calculator 12 is connected to the CPU 13 and the CPU 13
Are connected.

The analog signal processing circuit 11 amplifies the analog signal from the image sensor 103 and executes analog processing such as filter processing. Further, the evaluation function calculator 12 takes in the analog signal processed by the analog signal processing circuit 11, calculates the defocus amount indicating the focus degree of the sample S based on a predetermined evaluation function, and outputs the defocus signal. Is transmitted to the CPU 13.

The CPU 13 controls the analog signal output from the image sensor 103 to match the range of the analog signal processing circuit 11, and focuses the sample S on the basis of the defocus signal from the evaluation function calculator 12. A signal indicating the movement amount and the movement direction of the stage 2 as described above is transmitted to the stage driving device 14.
The stage drive device 14 moves the stage 2 in the vertical direction on the basis of a movement amount signal and a movement direction signal from the CPU 13 to perform focus adjustment.

The CPU 13 is the external controller 1
5, Cube drive / detector 16, Camera controller 1
7 is connected, and by the control start switch from the external controller 15, in addition to the focus detection control described above, cube drive control by the excitation light cube drive / detector 16, and
The camera controller 17 controls the camera exposure of the photo system 9.

FIG. 2 shows the above-mentioned parfocal adjustment unit 101.
2 shows the detailed configuration of the. In this case, the parfocal adjustment unit 101 includes the wedge-shaped prisms 21 and 22 made of different materials.
The parallel plate prism 20 is formed by vertically stacking and integrating the above. The parallel plate prism 20 is supported in the support frame 23, and the support frame 23 can be moved in parallel in the direction of the arrow in the figure. ing.

The support frame 23 has a side wall 231.
The operation shaft 25 is provided through the side wall 231 through the threaded portion 24, and the knob 251 of the operation shaft 25 is rotated to change the amount of protrusion of the shaft tip in the support frame 21.
The amount of movement of the parallel plate prism 20 in the direction indicated by the arrow in the support frame 23 can be adjusted.

A hole 232 through which an optical image from the objective lens 5 passes is formed on the bottom surface of the support frame 23, and a holding plate for pressing the parallel plate prism 20 from above is formed on the side wall 231. 26 is provided.

Therefore, the knob 251 is turned to set the optical axis of the incident light to the parallel plate prism 20 to, for example, a or b.
If the position is set to, the ratio of the optical path length passing through the parallel plate prism 20 is changed from the ratio of the thicknesses of the two wedge-shaped prisms 21 and 22 made of different materials, so that the optical path length of the photographic system 9 is changed. Since the optical path length of the focus detector 10 with respect to can be changed, this makes it possible to adjust the stage position which the focus detector 8 determines to be in focus so that the photograph S of the sample S in the photographic system 9 is brought into focus.

A scale (not shown) indicating the amount of rotation of the knob 251 is provided at or near the knob 251 for rotating the operating shaft 25, and the amount of movement of the parallel plate prism 20 can be confirmed by this scale. It is like this.

In this case, in order to manually adjust the parfocal adjustment unit 101, the adjustment amount is obtained by relying on the knob 251 based on the wavelength of the optical image of the sample S, that is, the sample image. The wavelength of the sample image is almost determined by the type of the cube lens 7, and the wavelength is adjusted based on this.
When the wavelength is unknown, the spectral characteristics of the image are examined by a well-known method, and adjustment is performed based on the result.

If the wavelength of the light image of the sample S and the amount of rotation of the knob 251 are checked in advance according to the photographic system 9 to be used and the wavelength of the light image is described as the scale, the operation is further performed. It is easy to do.

On the other hand, in order to automatically adjust the parfocal adjustment unit 101, a driving source such as a motor of the knob 251 is connected and driven. In this case, the external controller 15 inputs information about the cube lens 7 to be used, wavelength information about the sample image, and the like to the CPU 13, and based on this information, the CPU 13 commands the drive source to control the control amount.

The information of the cube lens 7 to be used may be the one sent from the cube drive detector 16 to the CPU 13. Next, the first configured as described above
The operation of this embodiment will be described with reference to the flowchart shown in FIG.

First, in step 301, the speculum operator sets the parfocal adjustment unit 101 from a known sample wavelength. The setting method of the parfocal adjustment unit 101 in this case is specifically performed as shown in FIG. In this case, FIGS. 7A and 7B are models in which the optical path length of the AF by the parfocal adjustment unit 101 with respect to the camera is changed. Similar to FIG. The axis shows the wavelength of the sample. The solid line in these figures shows the focus position with respect to the incident wavelength of the photographic system a and the broken line shows the AF system b. Next, FIG. 6B shows the focus position curve of the AF system b shown in FIG. 3A moved by changing the optical path length in the y-axis direction. That is, when the focus position allowance is set to 1.0 [mm] by changing the optical path length of the AF system, the focusing accuracy guarantee ranges c ′ and c ″ are obtained, which are compared with FIG. During ~
About 425 to 455 [nm] shown in (i) and (ii) in the figure
It is possible to guarantee the focusing accuracy in the range of about 600 to 650 [nm] indicated by. That is, the microscope operator or (i) and (i
When performing photography or observation using the AF of i), the parfocal adjustment unit 101 is set to a predetermined value.

In this way, the microscope person can move the parfocal unit 10
When AF is started in step 302 by a signal from the external controller 15 after setting 1 to a predetermined value, the analog image signal from the image sensor 103 is read in step 303, and the analog signal of the image sensor 103 is read in step 304. Check whether the signal is within the range of the analog signal processing circuit 11.

If the range does not match, the storage time of the image sensor 103 is controlled until the range matches. On the other hand, when the range is suitable, in step 305, the defocus amount indicating the focus degree is calculated from the signal of the image sensor 103 according to a predetermined evaluation function. Then, in step 306, focus determination is performed based on the calculated defocus amount, and when it is determined that focus is not achieved, in step 307, the stage is driven according to the defocus amount, and the process returns to step 303. Then, a series of operations including driving of the stage are repeated until it is determined that the focus is achieved. At step 306, the focus is determined, and the process proceeds to step 308 to end the control.

Therefore, with this arrangement, the transmissive light source 3
Alternatively, at least a part of the light image of the sample S illuminated by the epi-illumination light source 4 is projected from the objective lens 5 onto the image sensor 103 by the imaging optical system having the coupling lens 102, and the output corresponding to the light intensity distribution of the sample image is output. Along with obtaining the signals, these output signals are arithmetically processed by the analog signal processing circuit 11 and the evaluation function calculator 12 in accordance with a predetermined evaluation function, and based on the focus degree evaluation value obtained here, the imaging optical system of the sample S For the focus detector 10 for detecting the focus state, a photographic system 9 is provided as a peripheral system having an imaging optical system different from the focus detector 10, and the focus detector 10 for the photographic system 9 is provided. Since the optical path length is changed by moving the parallel plate prism 20 of the parfocal adjustment unit 101, chromatic aberration correction is insufficient. Therefore, it becomes possible to correct the focus shift between the focus detector 10 and the photographic system 9 relating to the wavelength of the optical image, and ensure the focusing accuracy of the focus detector 10 for the photographic system 9 regardless of the wavelength of the optical image. You can

In the above-described embodiment, the focusing accuracy guarantee range is set by the focusing adjustment unit 101 by changing the optical path length. However, this can be done by moving the image sensor 103 itself, The same effect can be obtained by using a chromatic aberration correction lens for each wavelength.

In the above-described embodiment, the adjustment amount is obtained by rotating the knob 251 of the parallel plate prism 20 of the parfocal adjustment unit 101, but the same parts as those in FIG. As shown in FIG. 5, the pick 25
1 may be configured as a push-in / pull-out type having a click mechanism. In this case, the operating shaft 25 having the knob 251
A plurality of click grooves 252 are provided on the
A ball plunger 27 that engages with 52 is provided on the support frame 21 side. In this way, if the click grooves 252 are provided at the respective positions according to the wavelength of the image that is frequently observed, the adjustment can be further simplified. (Second Embodiment) Next, a second embodiment of the present invention will be described. In this case, the schematic configuration of the microscope focus detection apparatus according to the second embodiment is similar to that described in the first embodiment with reference to FIG. Is omitted.

Then, the method of guaranteeing the focusing accuracy by the wavelength of the object, which is the feature of the second embodiment having such a configuration, will be described with reference to the flowchart shown in FIG. First, in step 601, AF is started by a signal from the external controller 15. Then, step 602
Then, the analog image signal from the image sensor 103 is read, and in step 603, it is checked whether the analog signal of the image sensor 103 matches the range of the analog signal processing circuit 11.

If the range does not match, the storage time of the image sensor 103 is controlled until the range matches. On the other hand, when the range is suitable, in step 604, the defocus amount indicating the degree of focus is calculated from the signal of the image sensor 103 according to a predetermined evaluation function. Then, in step 605, the in-focus determination is performed based on the calculated defocus amount, and when it is determined that the out-of-focus state is not achieved, the stage is driven in accordance with the defocus amount in step 606, and the process returns to step 602. Then, a series of operations including the stage driving are repeated until it is determined that focus is achieved.

On the other hand, if it is determined that the object is in focus, step 607
Then, the wavelength of the object is detected directly from the excitation cube information, the focus position shift amount due to the wavelength of the object is detected in step 608, and the stage is driven by the calculated offset amount in step 609. Control proceeds to 610 and control ends.

Accordingly, in this case, the focus detection is performed by the servo system on the stage 2 on which the subject S is placed, based on the focus signal indicating the focus degree by the focus detector 10, and the focus is determined. Since the stage 2 is forcibly driven by the offset amount determined by the wavelength of the sample image with respect to the vertical position of the stage 2, the focus detector 10 and the photographic system 9 relating to the wavelength of the optical image caused by insufficient chromatic aberration correction. Can be corrected by further driving a fixed offset amount from the position determined to be in focus, and the focusing accuracy of the focus detector 10 with respect to the photographic system 9 can be secured regardless of the wavelength of the optical image. It is also possible to eliminate the need for a special unit such as a parfocal adjustment unit, and it is possible to realize further cost reduction.

In the above embodiment, the stage forced drive of the focus shift amount due to the wavelength is performed after the focusing control. However, if this is an element of the evaluation function, the objective drive operation can be omitted even if the forced drive operation is omitted. Can be achieved. Further, in the above-described embodiment, the focus control is performed by driving the stage, but the same effect can be obtained by controlling this by driving the lens.

The above description is based on the embodiments, but the present invention also includes the following inventions. (1) In the focus detecting device for a microscope according to claim 1,
The optical path length or the chromatic aberration correction amount of the focus detection means with respect to the peripheral system is determined by the information of the excitation light cube or the filter as the information of the illumination optical system that illuminates the sample with the illumination light.

By doing so, it is possible to automatically correct the focus shift between the focus detection means and the peripheral system relating to the wavelength of the optical image caused by the insufficient correction of chromatic aberration, from the information of the illumination optical system.

[0051]

As described above, according to the present invention, the limitation of the wavelength range of the optical image that guarantees the focusing accuracy in the focus detection means can be greatly expanded and is suitable for peripheral systems such as a photographic system. Focus detection with high focus accuracy can be realized.

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of a parfocal adjustment unit used in the first embodiment.

FIG. 3 is a flowchart for explaining the operation of the first embodiment.

FIG. 4 is a diagram for explaining a setting method of the parfocal adjustment unit according to the first embodiment.

FIG. 5 is a diagram showing a schematic configuration of another example of the parfocal adjustment unit used in the first embodiment.

FIG. 6 is a flowchart for explaining the operation of the second embodiment of the present invention.

FIG. 7 is a model diagram showing a change in focus position with respect to a wavelength of an optical image of a conventional photographic system.

8 is a diagram in which a model showing changes in the focus position with respect to the wavelength of the AF system is added to the model shown in FIG.

[Explanation of Codes] 1 ... Microscope main body, 2 ... Stage, 3 ... Transmission light source, 4 ... Epi-illumination light source, 5 ... Objective lens, 6 ... Projector tube, 7 ... Cube lens, 8 ... Eyepiece lens, 9 ... Photo System, 91 ... Coupled lens, 10 ... Focus detector, 101 ... Parfocal adjustment unit, 102 ... Imaging lens, 103 ... Image sensor, 11 ... Analog signal processing circuit, 12 ... Evaluation function calculator, 13 ... CPU, 14 ... Stage drive device, 15 ... External controller, 16 ... Cube drive / detector, 17 ... Camera controller, 20 ... Parallel plate prism, 21, 22 ... Wedge type prism, 23 ... Support frame, 231 ... Side wall, 232 ... Hole part , 24 ... screw part, 25 ... operating shaft, 251 ... knob, 252 ... click groove, 26 ... pressing plate, 27 ... ball plunger.

Claims (3)

[Claims] 1. An image forming optical system for forming an observation light image of a sample on a light receiving unit, and a focus state of the image forming optical system for the sample based on a focus degree evaluation value for an output of the light receiving unit. A focus detecting means for detecting, a peripheral system having a coupling optical system different from the image forming optical system of the focus detecting means, and means for determining an optical path length or a chromatic aberration correction amount of the focus detecting means with respect to the peripheral system are provided. A focus detection device for a microscope, which is characterized in that 2. The focus detecting apparatus for a microscope according to claim 1, wherein the optical path length or the chromatic aberration correction amount of the focus detecting means with respect to the peripheral system is determined based on the wavelength of the sample image. 3. An image forming optical system for forming an observation light image of a sample on a light receiving means, and a focus state of the image forming optical system for the sample is determined based on a focus degree evaluation value for an output of the light receiving means. Focus detecting means for detecting, a peripheral system having a coupling optical system different from the image forming optical system of the focus detecting means, and at least one of the image forming optical system or the sample side depending on the degree of focus of the focus detecting means. A servo means for driving a focusing point by driving the driving means, a means for determining an offset amount according to the wavelength of the sample image, and a position for determining a focus point by the focus detecting means by the servo means. A focus detecting apparatus for a microscope, comprising: a means for driving at least one of the image forming optical system and the sample by the determined offset amount.