JP3431300B2 - Automatic focus detection system for microscope - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an optical device for optically acquiring an image of an object, and more particularly to an automatic focus detecting device in a microscope.

[0002]

2. Description of the Related Art Conventionally, there is an automatic focus detection device which detects a focus state of an object image and drives a stage or a lens to a focus position. Among such automatic focus detection devices, when the contrast of the object image is low and focus detection is impossible, a method for searching for a lens position or stage position where focus detection is possible while moving the lens or stage (low-conscan method)
There is one.

For example, Japanese Patent Laid-Open No. 1-187520 discloses that
An apparatus is disclosed which determines whether or not a lens has been set immediately after, and determines the direction and method of low contrast scanning based on the determination result. This operation control suppresses an increase in focusing time due to low contrast scanning. In addition to this, various methods have been developed for the low contrast scan method.

[0004]

However, it is difficult to apply the above-mentioned conventional focus detection technique to a microscope. That is, in a microscope, the objective lens is set (mounted) and the objective lens is set (exchanged) when the magnification is changed.
Therefore, the focusing direction of the object image is not always constant.
Therefore, the direction of low contrast scan cannot be determined only by determining whether or not the lens has just been set.

Further, since the microscope has various thicknesses of the sample to be inspected, it is difficult to determine the direction of the low contrast scan by judging whether or not the sample has just been set. Furthermore, the depth of focus of the objective lens of the microscope (the range in which the subject image can be observed) is in the range of several to several tens [μm] in most cases. However, if the low-conscan is performed bidirectionally as in the past, the time required for focus detection becomes long and the focusing time becomes long.

Therefore, an object of the present invention is to detect the focusing direction of an object image in a short time without adding complicated hardware in low-con scan control of a microscope, and focus detection in the entire range in a short time. An object of the present invention is to provide an inexpensive microscope automatic focus detection device that can be used for various purposes.

[0007]

The present invention has taken the following means in order to achieve the above object.

The object to be observed can be illuminated with an illumination optical system.
The object image emitted from the objective lens by
To project on the light receiving part consisting of multiple light receiving elements,
Output signal corresponding to the light intensity distribution of the object image from the optical element
And output these output signals according to a predetermined evaluation function.
The focus degree evaluation value is calculated by arithmetic processing and based on the focus degree evaluation value.
Then, the focus state of the imaging optical system with respect to the object is detected.
An automatic focus detection device for a microscope for output, the present invention corresponding to claim 1 comprises a field stop the disposed object image position conjugate in an optical path of the illumination optical system, a stop diameter of the field stop A field diaphragm drive unit that changes based on an opening / closing control command, a low contrast determination unit that determines whether a contrast value of an object image is insufficient for focus detection from an output signal output from the light receiving unit, and the low contrast determination unit. If it is determined by the means that the contrast is insufficient, an opening / closing control command is given to the driving means to reduce the diaphragm diameter until a field diaphragm image is projected on the light receiving element array, and the diaphragm diameter control. The first field stop image acquired by the means while the field stop image is projected onto the light receiving element array.
Contrast value and a second contrast value obtained after changing the relative distance between the object and the objective lens while maintaining the aperture diameter set by the aperture diameter control means, and the focus direction is determined. A focusing direction detecting means for detecting is provided.

[0009]

According to a second aspect of the present invention, a field diaphragm arranged at a position conjugate with the object image on the optical path of the illumination optical system and a diaphragm diameter of the field diaphragm are changed based on an opening / closing control command. A field diaphragm drive means for controlling the aperture stop, an aperture stop provided on the optical path of the illumination optical system for changing the numerical aperture of the illumination optical system, and an aperture stop drive for changing the aperture diameter of the aperture diaphragm based on an opening / closing control command. Means, first low contrast determining means for determining whether or not the contrast value of the object image is insufficient for focus detection from the output signal output from the light receiving section, and the first low contrast determining means determines insufficient contrast. Then, the aperture stop diameter control means for narrowing the aperture stop by giving an opening / closing control command to the aperture stop drive means and the object image again in the state where the aperture diameter is stopped by the aperture stop diameter control means. A second low contrast determining means for determining whether or not the contrast value is insufficient for focus detection, and if the second low contrast determining means determines that the contrast is insufficient, an opening / closing control command is given to the field stop driving means. A field stop diameter control means for narrowing the field stop diameter until the field stop image is projected onto the light receiving portion, and a field stop obtained by the stop diameter control means while the field stop image is projected onto the light receiving element array. Comparing the first contrast value of the image with the second contrast value obtained after changing the relative distance between the object and the objective lens while maintaining the diaphragm diameter set by the diaphragm diameter control means. A focusing direction detecting means for detecting the focusing direction is provided. In the present invention corresponding to claim 3 , the illumination optical system is an epi-illumination optical system,
A field diaphragm arranged at a position conjugate with the object image on the optical path of the epi-illumination optical system, a field diaphragm driving means for changing a diaphragm diameter of the field diaphragm based on an opening / closing control command, and a switching means for opening / closing the driving means. A stop diameter control means for narrowing the stop diameter until a field stop image is projected on the light receiving element array by giving a control command, and a state in which a field stop image is projected on the light receiving element array by the stop diameter controlling means. The first contrast value of the field stop image acquired in step 1) and the second contrast value acquired after changing the relative distance between the object and the objective lens while maintaining the stop diameter set by the stop diameter control means. A focusing direction detecting means for detecting the focusing direction by comparing with the value is provided. According to a fourth aspect of the present invention, the focus state is detected by obtaining the focus degree evaluation value by using the low frequency image or the field stop image instead of the object image.

[0011]

The present invention exerts the following actions by taking the above means . Oite to the present invention Rokonsu
When performing the can, the low-frequency image generated by the low-frequency image generating means at a conjugate position with the object image is projected onto the light receiving portion by the low-frequency image projecting means.

Here, since the spatial frequency of the low-frequency image is set to a value sufficiently smaller than that of the object image, even if the defocus amount is large enough to be in the low contrast state, it corresponds to the change of the defocus amount. Then there is a sufficiently detectable change in the power spectrum.

Therefore, the first contrast value of the low-frequency image acquired in a state where the low-frequency image is projected onto the light-receiving portion by the low-frequency image projecting means, and the low-frequency image is projected onto the light-receiving portion as an object. By comparing the second contrast value obtained after changing the relative distance with the objective lens, it is possible to detect the in-focus position by a so-called hill climbing method or an optical path difference method.

As a result, even in the low contrast state, the bidirectional low contrast scan is not performed, and only the projection of the low frequency image onto the light receiving portion, the detection of the second contrast value, and the contrast comparison are performed to achieve accurate matching. Since the focus direction can be detected,
The time required to detect the in-focus position during low contrast can be shortened.

In the present invention, low frequency image projection is preferred.
A field stop is used as the shadow means. In this case, given the switching control command from the throttle size control means to the drive means low frequency
The diaphragm diameter is narrowed until a field diaphragm image as an image is projected on the light receiving element array.

Then, an output signal is fetched from the light-receiving element in a state where the field-diameter image is projected on the light-receiving element array by the aperture diameter control means, and the contrast value of the field-iris image is obtained. Further, after the relative distance between the object and the objective lens is changed while maintaining the diaphragm diameter set by the diaphragm diameter control means, the output signal of the light receiving element is captured again,
The contrast value of the field stop image is obtained. In the focusing direction detection means, the former is used as the first contrast value and the latter is used as the second contrast value, and both are compared,
The focusing direction is detected based on the comparison result.

To detect the low contrast state, an object image is projected.
The contrast value of the object image is
This is used to determine whether the focus detection level is reached.
A computer determination means is used. Also, a case where the observation target object of low contrast, be recognized that the contrast of the object image is lower by low contrast recognition means that given the contrast information of the object image from the outside
Good .

[0018]

Furthermore, if it is determined that the insufficient contrast in low contrast determination unit, may be opening and closing control command from the aperture stop size control means to the aperture stop driving means are provided aperture stop is strangled. By this control, imaging optics
The depth of focus of the system becomes deep . In such a state, whether the contrast value of the object image is insufficient for focus detection again.
It is determined depending on whether b bacon determining means. And if it is determined that the controller <br/> last missing field stop diameter down to field stop image given field switching control command from the throttle size control means to the field stop driving means is projected onto the light receiving portion To be

It should be noted that the illumination optical system is preferably a drop optical system.
Illumination optics are used. Furthermore, instead of the object image
Obtain the in-focus evaluation value using the low-frequency image or field stop image.
Focus on the low frequency image or field stop image itself
You may ask.

[0021]

EXAMPLES Examples of the present invention will be described below. FIG. 1 is a diagram showing an embodiment in which an automatic focus detection device according to the present invention is applied to an epi-illumination observation microscope. In the epi-illumination observation microscope shown in the figure, a stage holding member 2 is attached to a body portion 1 of a microscope main body so as to be vertically slidable, and a stage 3 is attached to the stage holding member 2.
A revolver 4 is rotatably attached to the tip of the body 1 located above the stage 3, and a predetermined objective lens 5 is arranged on the optical axis. An eyepiece tube 8 is provided on the optical axis of the objective lens 5 arranged on the optical axis, and the optical axis is branched into the eyepiece 7 side and the photographing unit 8 side. An end portion of an epi-illumination fluorescent light projection tube 9 is inserted between the revolver 4 and the eyepiece tube 8. A light source box 10 is attached to the other end of the light projecting tube 9. A field stop (FS) 11 and an aperture stop (AS) 12 are arranged on the optical path formed in the light projecting tube 9.

The stage holding member 2 is a stage driving device 2 via a transmission mechanism incorporated in the body portion 1 of the microscope body.
Is bound to 1. The driving force generated by the stage driving device 21 is converted into a force for vertically moving the stage holding member 2 by the transmission mechanism.

The field stop 11 and the aperture stop 12 are FS / A.
The diaphragm diameter is controlled by the S drive circuit 22. The photographing unit 8 includes an image sensor 13
An image forming lens 14 for forming an image of the sample S on the image sensor 13 and an illuminance monitor 15 for monitoring the illuminance of the sample image. Image sensor 13
The charge storage time and read timing of the image sensor are controlled by the image sensor drive circuit 23.

At the output stage of the image sensor 13, an analog signal processing circuit 24 and an evaluation function calculator 25 are connected in series. The evaluation calculation result by the evaluation function calculator 25 is C
Inputting to PU26. Then, the CPU 26 sends a control signal to the stage driving device 21 based on the result of the evaluation calculation,
By moving the stage 3 in the vertical direction, the relative distance between the sample S and the objective lens 35 is adjusted to obtain automatic focusing.

An external controller 27 is connected to the CPU 26. The external controller 27 is a device having a switch and a jog dial used by a spectroscope to control an electric part of the microscope. When the microscoper performs an operation such as starting or interrupting the automatic focusing operation using the external controller 27, the information is sent to the CPU 26, and in response thereto, the CPU 26 performs an operation corresponding to the operation.

Next, the operation of this embodiment configured as described above will be described. The illumination light emitted from the light source box 10 passes through the epi-illumination fluorescent light projection tube 9 and the objective lens 5 and is applied to the sample S. The fluorescence image emitted from the sample S passes through the objective lens 5, and a part of the fluorescence image is incident on the eyepiece lens 7 and is observed by a spectroscope. A part of the image is projected on the image sensor 13 for focus adjustment.

The image sensor drive circuit 23 includes the CPU 2
By the control from 6, the driving pulse is always output to the image sensor 13 at proper timing. Image sensor 1
Reference numeral 3 converts the received image into an electric signal at a timing according to the input drive pulse. The image signal converted into an electric signal by the image sensor 13 is processed by the analog signal processing circuit 24, and is further processed by the evaluation function calculator 25 by the CPU.
The evaluation calculation is performed according to the calculation formula designated by 26. The CPU 26 sends a control signal to the stage drive device 21 based on the result of the evaluation calculation to adjust the relative distance between the sample S and the objective lens 5 to obtain automatic focusing.

Further, the CPU 26 outputs an open / close control signal to the FS / AS drive circuit 22 to open / close the field stop 11 and the aperture stop 12 as needed. The focus detection operation of this embodiment configured as described above will be described in detail with reference to FIG.

When the automatic focusing operation (hereinafter referred to as "AF") is started (S1), the output of the image sensor 13 is read (S2), and the output of the image sensor 13 is suitable for the range of the analog processing circuit 24. Check if there is (S
3).

If the range is not compatible, the CPU 26 gives the image sensor drive circuit 23 a command to set the storage time so that the range is compatible. This operation is performed until the output of the image sensor 13 matches the range of the analog processing circuit 24.

When the range is suitable, it is determined whether the contrast value of the optical image of the sample S output from the image sensor 13 is equal to or higher than the focus detectable contrast value (so-called low contrast) (S5). If it is determined in step S5 that the contrast is low and focus detection cannot be performed, the low contrast processing of FIG. 3 described later is performed (S).
6).

On the other hand, if it is determined in step S5 that focus detection is possible, the stage 3 is driven based on the defocus amount output from the evaluation function calculator 25 (S7). Such a series of AF is repeated until the sample S reaches the in-focus position, and ends when the defocus amount of the evaluation function calculator 25 reaches the in-focus state (S8, S).
9).

The low contrast processing (step S6) will be described in detail with reference to FIG. When it is determined in step S5 that it is a low contrast, the CPU 26 controls the FS / AS drive circuit 22 to reduce the diameter of the field stop 11 by a predetermined amount (S10). When the diameter of the field diaphragm 11 is completely closed (step S11), the diameter of the field diaphragm 11 is returned to the diameter at the time when it is determined to be low contrast, and then the bidirectional scanning is started (step S12).

On the other hand, when the diameter of the field stop 11 is not fully closed, it is detected whether the FS image is projected on the image sensor 13 (S13). This series of control operations of the aperture diameter is repeated until the FS image is projected on the image sensor 13. Then, the contrast value of the FS image at the time when the FS image is projected on the image sensor 13 is stored in memory (because it is a low contrast, the contrast value obtained here is F
Then, the stage drive device 21 is controlled to lower the stage 3 by a predetermined amount (S14).

Next, the contrast value of the FS image after the stage 3 is lowered is detected and compared with the contrast value of the previously stored FS image (S15). As a result of this comparison,
If the contrast value is increased, it is determined that the focus position of the sample S is below the current stage position because the sample S and the FS image have a conjugate relationship (S16). If the contrast value has decreased, it can be determined that the focus position of the sample S is above the current stage position (S16 ').

When the focusing direction of the sample S is confirmed, the diameter of the field stop 11 is returned to the value at the time of low contrast determination (S1
7) Then, the search for the sample S is started (S18). here,
The reason why the FS image can be searched will be described. Generally, the imaging optical system has a limit in the spatial frequency that can be identified by a sensor or a light receiving system such as visual inspection. This frequency is called the cutoff frequency. Furthermore, the spatial frequency and its defocus characteristics are determined by the imaging optical system, and this is
I call it a characteristic. In the MD characteristics, the power spectrum of most spatial frequencies decreases as the defocus amount increases. However, the relationship between the increase of the defocus amount at the spatial frequency and the decrease amount of the power spectrum is that the decrease amount of the power spectrum corresponding to the increase of the defocus amount becomes large as the cutoff frequency is approached, and it is difficult to identify in the light receiving system. Become. That is, if the spatial frequency is low, the amount of decrease in the power spectrum is small even if the defocus amount is large, and it is easy to identify by the light receiving system.

FIG. 4 shows this model. The horizontal axis of FIG. 4 represents the defocus amount, and the vertical axis represents the power spectrum. In the figure (a), the spatial frequency is 400 lines /
The MD characteristic of mm is shown, and the figure (b) shows the MD characteristic of 50 lines / mm. Since the spatial frequency of the FS image is very low, it is possible to detect even if the defocus amount is considerably large. Therefore, it is possible to search by the FS image even in the low contrast state outside the depth of focus.

As described above, according to the present invention, even if the sample S is outside the depth of focus range and in the low contrast state, the contrast of the FS image having a very small spatial frequency, that is, the edge of the FS image is detected to detect the sample. Since the focus direction of S is detected, the low contrast scan can be suppressed to the minimum and high-speed AF can be performed.

In the present embodiment, the focusing direction at the time of low contrast is detected by a so-called hill climbing search using the FS image. However, if the focusing direction is detected by using the optical path difference optical system, the same result is obtained. The effect can be obtained.

Further, in the present embodiment, the FS image is used only for detecting the focusing direction at the time of low contrast, but it may be focused on the FS image itself. In that case, after focusing on the FS once, F
If S is opened and the sample image is focused, the accuracy is higher.
F can be realized.

Furthermore, in the present embodiment, the AF control is performed by driving the stage, but the same applies when driving the objective lens. Next, a second embodiment of the present invention will be described.

The optical system and the hardware of this embodiment are the same as those of the first embodiment, and only the focus detection operation by the CPU 26 is different. Here, the operation of this embodiment will be described with reference to FIG.

In the present embodiment, when the sample is a low-contrast material such as liquid crystal, the speculum operator instructs the CPU 26 in advance via the external controller 27 to the CPU 26.

When AF is started (S20), the CPU2
6 detects whether a low contrast mode instruction is input from the external controller 27 (S21). When the low contrast mode is not designated, the CPU 26 performs the normal AF control as in the first embodiment (S22).

On the other hand, when the low contrast mode is designated, the CPU 26 narrows the field stop 11 by a predetermined amount by the FS / AS drive circuit 22 (S23). When the narrowed field stop 11 is closed, the field stop diameter is set to a predetermined value related to the objective lens magnification and bidirectional scanning is started until the FS image can be recognized (S24,
S25, S26). Before detection of the FS image is completed by performing bidirectional scanning before the field stop diameter is fully closed (S2
7) The stage is driven based on the defocus amount of the FS image so as to focus on the FS image (S28). This focusing operation is repeated until the FS image is focused (S2).
9). The AF operation ends when the FS image is focused (S30).

According to the present embodiment as described above, it is possible to perform high-precision and high-speed AF even for a subject that cannot be focused by the normal contrast method. Further, in the present embodiment, the AF operation is performed using the FS image. However, if the control of narrowing the aperture diaphragm than usual is combined so that the depth of focus is increased and the subject image or the FS image is easily searched for, the synergistic effect is obtained. This enables even faster AF.

In the embodiment of the present invention, the epi-illumination microscope system has been described, but the present invention can be implemented by replacing it with a transmission microscope system. Further, in the present invention, the focusing control is performed by the FS image, but when there is another space, the same effect can be obtained by projecting an extremely low frequency optical image at a position conjugate with the subject image. You can The present invention is not limited to the above embodiments, and various modifications can be made without departing from the gist of the present invention.

[0048]

As described in detail above, according to the present invention, even when the sample is out of the depth of focus range and the low-constant state, the low-constant scan is suppressed to the minimum, and high-speed AF is possible. According to the present invention, even if the sample has a low contrast that cannot be obtained by the usual contrast method, high-accuracy and high-speed AF is possible.
Is possible.

According to the present invention, higher speed AF is possible by performing AF using the field stop image only when the object cannot be searched even if the depth of focus is increased by the aperture stop.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an automatic focus detection device for a microscope according to a first embodiment of the present invention.

FIG. 2 is a flowchart showing a focus detection operation of the microscope automatic focus detection apparatus according to the first embodiment.

FIG. 3 is a flowchart of a low contrast process in the focus detection operation.

FIG. 4 is a diagram showing MD characteristics at different spatial frequencies.

FIG. 5 is a flowchart showing focus detection operation of the microscope automatic focus detection apparatus according to the second embodiment.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Body part of microscope main body, 3 ... Stage, 5 ... Objective lens, 7 ... Eyepiece lens, 8 ... Imaging unit, 10 ... Light source box, 11 ... Field stop, 12 ... Aperture stop, 13 ... Image sensor, 21 ... Stage Drive device, 22 ... FS / A
S drive circuit, 23 ... Image sensor drive circuit, 24 ... Analog signal processing circuit, 25 ... Evaluation function calculator, 26 ... C
PU.

Claims (4)

(57) [Claims] 1. An object image emitted from an objective lens by illuminating an object to be observed with an illumination optical system is projected by an image forming optical system onto a light receiving section composed of a plurality of light receiving elements, and each of the light receiving elements outputs the image. Obtaining output signals corresponding to the light intensity distribution of the object image, performing arithmetic processing on these output signals according to a predetermined evaluation function to obtain a focus degree evaluation value, and based on the focus degree evaluation value, the above-mentioned result for the object is obtained. In an automatic focus detection device for a microscope that detects a focus state of an image optical system, a field stop disposed at a position conjugate with the object image on the optical path of the illumination optical system, and an opening / closing control command for a stop diameter of the field stop. A field stop driving means for changing the contrast value of the object image, a low contrast judging means for judging whether or not the contrast value of the object image is insufficient for focus detection from the output signal output from the light receiving section, If the contrast determination means determines that the contrast is insufficient, an opening / closing control command is given to the driving means to reduce the aperture diameter until a field stop image is projected on the light receiving element array, and the aperture diameter control means. The first contrast value of the field stop image acquired by the diameter control means in a state where the field stop image is projected on the light receiving element array, and the object while maintaining the stop diameter set by the stop diameter control means. An automatic focus detection device for a microscope, comprising: a focusing direction detection unit that detects a focusing direction by comparing a second contrast value obtained after changing a relative distance with the objective lens. 2. An object image emitted from an objective lens by illuminating an object to be observed with an illumination optical system is projected by an imaging optical system onto a light receiving section composed of a plurality of light receiving elements, and each of the light receiving elements outputs the image. Obtaining output signals corresponding to the light intensity distribution of the object image, performing arithmetic processing on these output signals according to a predetermined evaluation function to obtain a focus degree evaluation value, and based on the focus degree evaluation value, the above-mentioned result for the object is obtained. In an automatic focus detection device for a microscope that detects a focus state of an image optical system, a field stop disposed at a position conjugate with the object image on the optical path of the illumination optical system, and an opening / closing control command for a stop diameter of the field stop. Based on the opening / closing control command, the aperture stop driving means for changing the aperture diameter of the aperture stop provided on the optical path of the illumination optical system and changing the numerical aperture of the illumination optical system. An aperture stop driving means for changing, the contrast value of the object image from an output signal output from the light receiving unit determines whether or not the missing detection focus 1
And a first low contrast determining means, if the first low contrast determining means determines that the contrast is insufficient, an aperture stop diameter control means for giving an opening / closing control command to the aperture stop driving means to reduce the aperture stop, and the aperture stop diameter control Second contrast control means for determining whether or not the contrast value of the object image is insufficient for focus detection again when the aperture diameter is narrowed by the means, and if the second contrast control means determines that the contrast is insufficient. A field stop diameter control means for narrowing the field stop diameter until a field stop image is projected onto the light receiving part by giving an opening / closing control command to the field stop drive means; The object and the objective lens while maintaining the first contrast value of the field diaphragm image acquired in a state of being projected on the row and the diaphragm diameter set by the diaphragm diameter control means. And an in-focus direction detecting means for detecting an in-focus direction by comparing with a second contrast value acquired after changing the relative distance of the microscope automatic focus detection apparatus. 3. An object image emitted from an objective lens by illuminating an object to be observed with an illumination optical system is projected by an imaging optical system onto a light receiving section composed of a plurality of light receiving elements, and each of the light receiving elements outputs the image. Obtaining output signals corresponding to the light intensity distribution of the object image, performing arithmetic processing on these output signals according to a predetermined evaluation function to obtain a focus degree evaluation value, and based on the focus degree evaluation value, the above-mentioned result for the object is obtained. In a microscope automatic focus detection device for detecting a focus state of an image optical system, the illumination optical system is an epi-illumination optical system, a field of view disposed at a position conjugate with the object image on the optical path of the epi-illumination optical system. A diaphragm, a field diaphragm driving unit that changes the diaphragm diameter of the field diaphragm based on an opening / closing control command, and an opening / closing control command to the driving unit until a field diaphragm image is projected on the light receiving element array. A diaphragm diameter control means for narrowing the diaphragm diameter, a first contrast value of the field diaphragm image obtained by the diaphragm diameter control means in a state where the field diaphragm image is projected on the light receiving element array, and the diaphragm diameter control means. Focusing direction detecting means for detecting a focusing direction by comparing a second contrast value obtained after changing the relative distance between the object and the objective lens while maintaining the aperture diameter set in 1. Microscope automatic focus detection device characterized by 4. The focus state is detected by obtaining the focus degree evaluation value by using the low-frequency image or the field stop image instead of the object image. The automatic microscope focus detection device according to any one of 3 to 3 .