JP2006003542A - Active autofocus method and system for increasing focusing accuracy on desired observation part, and microscopic device using them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve focusing at an observation position desired by an observer without false focusing in a microscopic device. <P>SOLUTION: The microscopic device is equipped with a stage 1 on which a sample being an observation object is put, observation optical systems (27, 28, 2, 3, 29 and 11) equipped with an objective 3 and obtaining the observed image of the sample S, a focusing driving means 21 for changing a distance between the stage and the objective in an optical axis direction, means 4 to 14 for detecting the position of the stage with respect to the focal position of the objective 3, and a control means 15 for determining a focusing state based on output signal from the detection means and controlling the distance to the focusing state through the focusing driving means. First focusing control is performed based on the output signal from the detection means (S11). The value of the output signal at the observation position desired by the observer near a focusing position is offset and second focusing control is performed based on a signal value obtained by adding the offset to the output signal (S12). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention generally relates to a microscope system having an autofocus function, and more particularly, to a technique for automatically focusing an observation target (also referred to as an observation object or specimen) in such a microscope system.

  2. Description of the Related Art In recent years, apparatuses that perform inspection and recording using a microscope have been automated for various functions, and an autofocus (hereinafter abbreviated as AF) function for focusing on a specimen has become an essential item for automation. . Along with this, various proposals concerning autofocus have been made.

A common problem in the autofocus function of a microscope is that there is a lack of focus (so-called “out-of-focus”) at the position where focus is desired.
For example, in the case of active AF using invisible light, one of the causes is a shift in focus position due to chromatic aberration of the objective lens. The objective lens has a characteristic that the focal position shifts depending on the wavelength of light. In other words, there is a difference between the in-focus position detected by active AF using invisible light and the in-focus position determined by the observer's eyes using visible light.

  A technique for correcting a shift in focus position due to chromatic aberration of the objective lens is disclosed as a focus adjustment device. In this focus adjustment apparatus, as shown in FIG. 14, the S-shaped focus is obtained from the normalized difference signal of the output signals of the two detection means placed at the front pin position and the rear pin position of the focus detection optical system. An error signal is obtained (see FIG. 14 (A)), and an offset V0 of the signal intensity corresponding to the focus position shift corresponding to the chromatic aberration of the objective lens is applied to the focus error signal (see FIG. 14 (B)). By performing the focusing operation, the shift of the focus position due to the chromatic aberration of the objective lens is corrected.

However, if the offset is simply applied to the focus error signal, the zero cross point becomes two points (the first point Z1 and the second point Z2 shown in FIG. 14B), and the range where AF can be applied is narrow. (Section L6). For this reason, in this focus adjustment apparatus, the AF drive region is divided into sections L3, L5, and L4 by providing threshold values T3 and T4 in the sum signal of the output signals of the two detection means (see FIG. 14 (C)). ), The focus error signal is offset for the focusing operation only in the section L5 including the first zero cross point.
JP 2001-242375 A

 However, in the above-described conventional focus adjustment device, in order to avoid the zero cross point being two points, the AF drive region is a region where the focus error signal is offset and the focusing operation is performed (section L5), It is divided into regions (sections L3 and L4) in which focusing is performed without wearing an offset. In order to set this section, threshold values T3 and T4 are provided in the sum signal of the output signals of the detection means. However, since the sum signal of the output signals of the detection means is affected by the reflectance of the sample, the threshold values T3 and T4 are not uniquely determined. In particular, the threshold value T4 is a threshold value that determines the boundary between the section L4 and the section L5. Depending on the sample, the value of the threshold T4 becomes inappropriate, and the second zero-cross point is included in the section L5, and there is a possibility that the second zero-cross point is falsely focused.

  The present invention has been made in view of the above circumstances, and is a microscope auto that can reliably focus on an arbitrary height position of a specimen without being affected by the reflectance of the specimen or individual differences of objective lenses. An object is to provide a focusing device.

  In one aspect, the present invention includes a stage on which a sample to be observed with a microscope is placed, an observation optical system that includes an objective lens to obtain an observation image of the sample, and a focusing drive that changes the distance between the stage and the objective lens in the optical axis direction. A means for detecting the position of the stage relative to the focal position of the objective lens, and a control for determining the in-focus state based on the output signal of the detecting means and controlling the distance to the in-focus state via the focusing drive means And an active autofocus method in a microscope apparatus. In the active autofocus method of the present invention, a preliminary focusing step for performing the first focusing control based on the output signal of the detection means, and the value of the output signal at the observation position desired by the observer in the vicinity of the focusing position as an offset. An offset setting step for performing the second focusing control based on a signal value obtained by adding an offset to the output signal. With such a configuration, it is possible to focus on the observation position desired by the observer without performing false focusing.

  The microscope apparatus further includes a semi-manual means that enables fine adjustment of the change by the focusing drive means by a user operation, and the offset setting step focuses on a desired observation position using the semi-manual means, The output signal value at this time is stored as an offset, and this stored offset is used in this focusing step.

  In one embodiment, the microscope apparatus includes a revolver that holds a plurality of objective lenses, and the objective lens is an objective lens selected from the plurality of objective lenses. A step of obtaining an offset corresponding to each of the plurality of objective lenses in advance and storing the offset in relation to the objective lens; and a step of selecting a desired objective lens from the plurality of objective lenses by the user prior to the focusing step; And the focusing step uses an offset stored in relation to the currently selected objective lens. Thereby, even in sample observation with a plurality of objective lenses switched, it is possible to focus on the observation position desired by the observer without false focusing.

The step of obtaining and storing the offset corresponding to each of the plurality of objective lenses in advance includes (1) performing focusing control based on the output signal of the detection means using one of the plurality of objective lenses, and (2) A focusing step to focus on the desired observation position using semi-manual means;
(3) The step of storing the value of the output signal when focusing on the desired observation position as an offset of the one objective lens, and the steps (1) to (3) are executed for all of the plurality of objective lenses. Step may be configured.

  A step of periodically determining whether or not the output signal is focused based on a signal value with an offset added, and a focus based on a signal value with an offset added to the output signal when it is determined that the output signal is not focused. And a step of performing control.

  In another aspect, the present invention provides a microscope apparatus with improved accuracy of focusing on an observation site. The microscope apparatus of the present invention includes a stage on which a sample to be observed by a microscope is placed, an observation optical system that includes an objective lens and obtains an observation image of the sample, and a focusing drive that changes the distance between the stage and the objective lens in the optical axis direction. A means for detecting the position of the stage relative to the focal position of the objective lens, and a control for determining the in-focus state based on the output signal of the detecting means and controlling the distance to the in-focus state via the focusing drive means Means for controlling the first focus based on the output signal of the detection means, and means for offsetting the value of the output signal at the observation position desired by the observer near the focus position; And means for performing second focusing control based on a signal value obtained by adding an offset to the output signal.

  In yet another aspect of the present invention, a stage on which a sample to be observed with a microscope is placed, an observation optical system for obtaining an observation image of a sample having an objective lens, and the distance between the stage and the objective lens are changed in the optical axis direction. In a microscope apparatus provided with a focusing drive means and a detection means for detecting the position of the stage with respect to the focal position of the objective lens, the in-focus state is determined based on the output signal of the detection means, and the focus driving means Provides an active autofocus system that controls the distance to focus. The active autofocus system of the present invention includes a preliminary focusing unit that performs the first focusing control based on an output signal of the detecting unit, and an offset value of the output signal at an observation position desired by an observer near the focusing position. Offset setting means for performing the second focusing control based on a signal value obtained by adding the offset to the output signal. Thereby, it becomes possible to focus on the observation position desired by the observer without false focusing.

  According to the first, sixth, and eleventh aspects of the invention, since it is possible to focus on the observation position desired by the observer without false focusing, the operability of focusing is improved, and consequently the sample observation Throughput can be improved.

According to the third and eighth aspects of the present invention, it is possible to focus on an observation position desired by an observer without false focusing even in sample observation with a plurality of objective lenses switched.
Therefore, even in sample observation with a plurality of objective lenses switched, the operability of focusing is improved, and consequently the throughput of sample observation can be improved.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention and the accompanying drawings. In addition, when showing the same element in several drawing, the same referential mark is attached | subjected.
[First Embodiment]
FIG. 1 shows an overall configuration of a microscope system according to a first embodiment of the present invention. In FIG. 1, a microscope system 100 includes an observation stage S (which includes the glass Sg, the culture solution Sc, and the cells Ss described above), a fixed stage 1 on which the observation target S is placed, an illumination light source 27, and parallel light. The generating lens 28, the mirror 29, the condensing lens 30, the plurality of objective lenses 3, the electric revolver 2 to which these objective lenses 3 can be attached and rotated, and the revolver 2 is rotated so that the arbitrary objective lens 3 can be inserted into the optical path. A revolver motor 17, a revolver position detector 18 for detecting which objective lens mounting position of the revolver 2 is currently inserted in the optical path, and a focusing motor 21 for moving the revolver 2 in the optical axis direction are provided. ing. Further, the microscope system 100 controls the entire system based on the operation unit 23 for input by the user, input information from the operation unit 23 and information on the objective lens 3 in use from the rebo hole position detection unit 18. 15, a revolver motor drive unit 16 that drives the revolver motor 17 based on a control signal from the control unit 15, and a focusing motor drive unit 20 that drives the focusing motor 21.

The observation object S is placed on the fixed stage 1 and can be observed from below with the objective lens 3.
The control unit 15 is a well-known CPU circuit, and as shown in FIG. 2, a CPU main body 30, a ROM 31 for storing a program for controlling the system, a RAM 32 for storing data necessary for control, a control signal It comprises an I / O port 33 that performs input and output, and known peripheral circuits such as an oscillator (not shown) and an address decoder that are necessary for controlling the CPU. Each peripheral device is controlled from the I / O port 33 and the data bus 34. Further, the control unit 15 may include a nonvolatile memory (not shown) so that various setting data generated after the system introduction can be stored.

  The operation unit 23 includes various operation switches so that the observer can input operations necessary for AF such as AF start / stop and objective lens switching and glass thickness. Furthermore, it is preferable that the operation unit 23 is provided with a JOG dial 24 so that fine adjustment of the vertical movement of the revolver 2 can be easily performed semi-manually in a known manner.

  The microscope system 100 includes a laser driving unit 19, a reference light source 4, a collimating lens 5, a light projecting side stopper 6, a polarizing beam splitter, a polarizing beam splitter (PBS) 7, a condensing lens group 8, and a chromatic aberration correcting lens group. 9, λ / 4 plate 10, dichroic mirror 11, condensing lens group 12 that collects the return light that has passed through PBS 7, light receiving sensor 13 that receives light from condensing lens group 12, and outputs of light receiving sensor 13 A signal processing unit 14 that is processed as described later and passed to the control unit 15, a chromatic aberration correction lens driving motor 26 that moves the chromatic aberration correction lens group 9 to the optical axis method, and a chromatic aberration correction lens driving unit 25 are provided.

  As the reference light source 4 used for autofocus, a light source in the visible light wavelength region such as an infrared laser is used. The laser driving unit 19 turns on the light source 4 while controlling the strength of the reference light source 4. The laser light from the reference light source 4 passes through a collimating lens 5 for maintaining parallel light, and half of the light beam diameter is cut by the light projecting side stopper 6. Thereafter, only the P-polarized light component is reflected by the PBS 7 and guided to the sample side.

  The light beam once condensed by the condenser lens group 8 passes through the chromatic aberration correction lens group 9. The chromatic aberration correction lens group 9 is moved in the optical axis direction by the chromatic aberration correction lens group driving motor 26, thereby making it possible to correct the chromatic aberration of the observation light and the infrared laser. It becomes.

  The light that has passed through the chromatic aberration correction lens group 9 is polarized by 45 ° when passing through the λ / 4 plate 10 and enters the dichroic mirror 11. Since the dichroic mirror 11 reflects only the infrared region, the laser beam is reflected.

  The reflected light beam forms a spot-shaped image on the observation object S by the objective lens 3. Then, the light beam reflected by the observation object S passes through the objective lens 3 and the dichroic mirror 11 and is further polarized by 45 ° when passing through the λ / 4 plate 10 again to switch to the S polarization component. Further, the light enters the PBS 7 through the chromatic aberration correction lens group 9 and the condenser lens group 8. At this time, since the light flux is an S-polarized component, it passes through the PBS 7 as it is and forms an image on the light receiving sensor 13 after passing through the condenser lens group 12.

  The light receiving sensor 13 is composed of a two-divided photodiode installed around the optical axis. When the observation object S is at the focus position of the objective lens 3, the spot imaged on the light receiving sensor 13 has a narrow range and high signal intensity as shown in FIG. 3B, and is above the focus position (rear focus position). 3A, the intensity distribution is biased toward the range B as shown in FIG. 3A, and when it is at the lower side (front pin position), the intensity distribution is biased toward the range A as shown in FIG. 3C. It is converted into the sensor signal shown in (a). The converted detection signal is divided into ranges A and B by the signal processing unit 14, and the sum of the intensity in each range is calculated. Therefore, as shown in FIG. 4A, when the horizontal axis is the relative position of the observation object S with respect to the focus position of the objective lens 3, and the vertical axis is the light intensity incident on each light receiving sensor, the image is symmetrical with respect to the focus position. Two curves A and B can be detected.

This signal is input to the control unit 15. The control unit 15 calculates A + B as shown in FIG. 4B from the input A and B signals (1).
And K. (AB) / (A + B) as shown in (c) (2)
Is calculated. However, K is a constant such that the value of Expression (2) falls within an appropriate range. In particular, the signal of FIG. 4C shows a characteristic like an S curve, and the value is referred to as an evaluation function value (hereinafter abbreviated as Ef value). FIGS. 4B and 4C are graphs (b) and (c) showing how the values of the expressions (1) and (2) change depending on the relative position of the observation target S with respect to the focal point of the objective lens 3. . The control unit 15 determines the in-focus position direction based on the sign of the Ef value. For example, when the AF operation is started from the position P1 in FIG. 4, since the sign of the Ef value is positive, the objective lens 3 is controlled to be lowered, and when the AF operation is started from the position P2. Since the sign of the Ef value is negative, control for raising the objective lens 3 is performed, focus control is performed so that the Ef value finally becomes 0, and the observation object S is led to focus.

As described above, the active AF optical system is realized by controlling the lighting of the laser light and detecting the reflected light of the laser beam projected onto the specimen.
The illumination light for observation passes from the illumination light source 27 through the lens 28, is reflected by the mirror 29, and is collected by the lens 30, and then irradiates the observation object S from above. The light transmitted through the sample passes through the objective lens 3 and passes through the dichroic mirror 11 to become observation light.

  Based on the apparatus configuration as described above, in the present embodiment, for example, when focusing on an arbitrary height position of a biological specimen such as a cell as shown in FIG. This will be described with reference to the flowchart of FIG.

FIG. 6 is a flowchart showing an outline of the AF operation of the present embodiment.
When the observer gives an instruction to start AF to the control unit 15 via the operation unit 23, first, in step S11, a normal focus error signal F1 = (A−B). / (A + B) (in the example of FIG. 5, a solid line focus error signal), AF is applied to focus on the glass surface of the observation sample S.

  Next, in step S12, an offset O (see FIG. 5) corresponding to the focus error signal intensity corresponding to the distance from the glass surface to the in-focus position desired by the observer is (A−B) / (A + B). AF is applied based on this signal (in the example of FIG. 5, dotted focus error signal F2 = (A−B) / (A + B) −O) to focus on the in-focus position desired by the observer. Let

The offset O is a parameter set in advance before performing the focusing operation. Here, the offset O setting operation will be described with reference to the flowchart of FIG. 9A.
First, in step S101, AF is applied based on a normal focus error signal F1 to focus on the glass surface of the observation sample S. This operation is the same as that in step S11, and details thereof will be described later.

Next, in step S102, the observer operates the JOG dial 24 to move the objective lens 3 so that the desired position is in focus.
However, in step S102, when the observer moves the objective lens so as to focus on the desired position, the movement of the objective lens is controlled so that the focus error signal F does not exceed the offset upper limit value Omax. This offset upper limit value Omax is the maximum value of the offset O that does not interfere with the focus determination when performing AF based on the focus error signal F2 in step S12, and is measured in advance according to the objective lens to be used. For example, as shown in FIG. 9B, the data is stored in the ROM 31, RAM 32 of the control unit 15, or the above-described non-illustrated nonvolatile memory. FIG. 9B shows a table for storing the ID of each objective lens and the offset upper limit value Omax in association with each other.

In step S103, the focus error signal intensity at the in-focus position desired by the observer is stored as an offset O in the RAM 32 or the nonvolatile memory.
In this way, the offset O is set.

Here, returning to the description of the AF operation of the present embodiment, the details of the operations of step S11 and step S12 will be described.
FIG. 7 is a flowchart showing details of the operation in step S11 of FIG. In FIG. 7, first, in step S110, the AF operation is started based on the focus error signal F1, and in step S111, it is determined whether or not the focus is achieved. For example, as shown in FIG. 10, thresholds J and −J are set in advance for the + side and the − side of the focus error signal, and the focus error signal intensity is set to the threshold −J. ˜J (in-focus determination range) and the sign of the focus error signal intensity is reversed, that is, in the area d in the figure, it is determined that the in-focus state is obtained. If it is not the area d in the figure, it is determined that the subject is not in focus. As the threshold value J, an optimal value is measured in advance according to the objective lens to be used, and is stored in the ROM 31, RAM 32 or nonvolatile memory of the control unit 15.

  When it is determined that the in-focus state is not in focus in step S111, the focusing motor 21 is driven via the focusing motor drive unit 20 in step S112, and the revolver 2 and the objective lens 3 are moved in the optical axis direction. Move to. When the focus error signal is negative, the drive direction is driven in the direction in which the objective lens 3 approaches the observation sample Ss (upward), and when the focus error signal is positive, the objective lens 3 is moved away from the observation sample Ss (downward). Direction).

Then, in-focus determination is performed again in step S111.
When the operation of Step S111 and Step S112 is repeated and the glass surface of the observation material Ss comes to the focal position of the objective lens, it is determined that the in-focus state is determined in Step S111, and the focusing motor drive unit is determined in Step S113. The focusing motor 21 that has been driven through 20 is stopped.

As described above, AF is applied based on the normal focus error signal F to focus on the glass surface of the observation material Ss.
FIG. 8 is a flowchart showing details of the operation in step S12 of FIG. In FIG. 8, first, in step S120, this time, an AF operation is started based on the focus error signal F2 with the offset added, and in step S111, it is determined whether or not the focus is achieved. The focus determination method is the same as the method described in the focus determination in step S111, and thus description thereof is omitted.

  When it is determined that the in-focus state is not in focus in step S121, the focusing motor 21 is driven via the focusing motor drive unit 20 in step S122, and the revolver 2 and the objective lens 3 are moved in the optical axis direction. Move to. The method for determining the drive direction is the same as the method for determining the drive direction described in step S112. In the present embodiment, since the focus position desired by the observer is located above the glass surface, the focus error signal F2 is negative. Therefore, the objective lens 3 is driven in a direction (upward) approaching the observation sample Ss.

Then, in-focus determination is performed again in step S121.
The operations in steps S121 and S122 are repeated, and when the in-focus position desired by the observer of the observation material Ss comes to the focal position of the objective lens, it is determined that the in-focus determination is made in step S121, and the process proceeds to step S123. Then, the focusing motor 21 that has been driven via the focusing motor drive unit 20 is stopped.

As described above, AF is applied on the basis of the focus error signal F2 including the offset O corresponding to the focus error signal intensity corresponding to the distance from the glass surface to the in-focus position desired by the observer. Focus on the focal position.
[Action]
In the focus adjustment device of the prior art, in order to avoid false focusing due to a plurality of zero-cross points of the focus error signal including the offset, a threshold value is provided to the sum signal of the detector, and AF is applied based on the normal focus error signal The AF drive area is divided into a section and a section where AF is performed based on a focus error signal with an offset. However, since the sum signal of the detector is affected by the reflectance of the observation sample, if the threshold value is inappropriate, the AF drive area is not properly divided, and there is a possibility of false focusing.

However, according to the microscope autofocus device of the present embodiment, the glass surface is once focused based on the normal focus error signal F1 in which the zero cross point is not two. After that, based on the focus error signal F2 including an offset O corresponding to the focus error signal intensity corresponding to an arbitrary height position desired by the observer from the glass surface, the observer focuses on an arbitrary height position desired by the observer. Let That is, it is not necessary to provide a threshold for the sum signal of the detector that is affected by the reflectance of the observation sample. Therefore, it is possible to reliably focus at an arbitrary height position desired by the observer without being affected by the reflectance of the sample.
[effect]
According to the microscope autofocus device of the first embodiment, it is possible to reliably focus on an arbitrary height position desired by the observer without being affected by the reflectance of the specimen. There is no need to re-apply AF due to the focus, the operability of focusing is improved, and the efficiency of sample observation can be improved.
[Second Embodiment]
In the second embodiment of the present invention, after focusing on an arbitrary height position of a biological specimen such as a cell as shown in the first embodiment, the focus position is continuously maintained. .

Since the configuration of the microscope autofocus device of the present embodiment is the same as the device configuration shown in the first embodiment, the description thereof is omitted.
The AF operation of this embodiment will be described with reference to the flowcharts of FIGS.

  FIG. 11 is a flowchart showing an outline of the AF operation of the present embodiment. In FIG. 11, when the observer gives an instruction to start AF to the control unit 15 via the operation unit 23, first, in the operation of step S21, the glass surface of the observation sample Ss is temporarily aligned based on the focus error signal F1. After focusing, in step S22, based on the focus error signal F2, the viewer focuses on the desired focus position.

Since the operations of Step S21 and Step S22 are the same as the operations of Step S11 and Step S12 shown in the first embodiment, detailed description thereof is omitted.
Next, in step S23, the focus position of the objective lens 3 is set to the in-focus position desired by the observer by performing control to continue the AF based on the focus error signal F2 until the observer stops the AF operation. maintain.

Details of this operation will be described with reference to the flowchart of FIG.
First, in step S231, it is confirmed whether an AF stop instruction has been issued.
If an AF stop instruction has been issued, the focusing motor is stopped in step S235, and the AF operation ends.

  If the AF stop instruction has not been issued, in-focus determination based on the focus error signal F2 is performed in step S232. Since the focus determination method is the same as the focus determination method shown in the first embodiment, the description thereof is omitted.

  If it is determined in step S232 that the subject is in focus, the focusing motor is stopped in step S233, and the operations in steps S231, S232, and S233 are repeated until an AF stop instruction is issued.

  However, if the distance between the observation sample Ss and the objective lens 3 changes beyond the in-focus determination range due to mechanical drift or the like, it is determined in step S232 that the in-focus state is not achieved, and in step S234. The focusing motor is driven to move the objective lens 3 in the optical axis direction. Since the method for determining the drive direction is the same as the method shown in the first embodiment, the description thereof is omitted.

Then, in-focus determination is performed again in step S232, and the operations in steps S232 and S234 are repeated until the in-focus position desired by the observer is in focus.
In this way, the focus position of the objective lens 3 is maintained at the focus position desired by the observer until the observer stops the AF operation.
[Action]
According to the microscope autofocus device of the present embodiment described above, as in the microscope autofocus device shown in the first embodiment, any desired height desired by the observer can be obtained without being affected by the reflectance of the specimen. It is possible to reliably focus on the vertical position. In addition, the focus can be maintained at an arbitrary height position desired by the observer until the observer stops the AF operation.
[effect]
According to the microscope autofocus device of the second embodiment, in addition to the effects shown in the first embodiment, the AF operation also follows a change in the distance between the observation sample and the objective lens due to a temperature change or the like. Thus, it is possible to observe the sample stably without observing the focus of the observed image even when observed for a long time.
[Third Embodiment]
In the third embodiment of the present invention, a plurality of objective lenses are switched, and the sample is observed by focusing on an arbitrary height position of a biological specimen such as a cell as shown in the first embodiment. is there.

The configuration of the microscope autofocus device according to the present embodiment is the same as the device configuration shown in the examples described so far, and a description thereof will be omitted.
As for the AF operation, as in the embodiments described so far, the AF operation can be focused on an arbitrary height position desired by the observer, either once or continuously.

  In the microscope autofocus device according to the present embodiment, the focus error signal offset is set for all objective lenses attached to the revolver body 2 before the AF operation is started. It is a different point.

The offset setting operation of this embodiment will be described with reference to the flowchart of FIG.
First, in step S300, the revolver body 2 is rotated, and one of the plurality of objective lenses 3 (assumed to be the objective lens 3a) is inserted into the optical path. At this time, in order to avoid a collision between the objective lens and the observation sample Ss or the stage 1, the revolver body 2 may be rotated after the objective lens is once moved below the optical axis.

  Next, in step S301, AF is applied based on the normal focus error signal F1 to focus on the glass surface of the observation sample Ss. This operation is the same as that in step S101 shown in the first embodiment, and detailed description thereof is omitted.

  Next, in step S302, the observer operates the JOG dial 24 to move the objective lens 3 so that the desired position is in focus. This operation is the same operation as step S102 shown in the first embodiment.

  Next, in step S303, the focus error signal intensity at the in-focus position desired by the observer is stored in the RAM 32 as an offset when the objective lens 3a is used. The RAM 32 has a storage area for storing offsets for all the objective lenses 3 attached to the revolver body 2, and can store the offset for each objective lens to be used.

  In step S304, it is confirmed whether offsets have been set for all objective lenses attached to the revolver body 2. If not all are set, the objective lenses are switched again in step S300 ( For example, by switching to the objective lens 3b), the offset at the time of using the objective lens 3b is stored in the RAM 32 by the operations in steps S301 to S303.

By performing the above operation on all the objective lenses attached to the revolver body 2, the offset when using each objective lens attached to the revolver body is set.
[Action]
According to the microscope autofocus device of this embodiment, since the offset of the focus error signal is held for all objective lenses attached to the revolver, even if the objective lens is switched by rotating the revolver. It is possible to focus on an arbitrary height position desired by the observer in a single shot or continuously. That is, an optimum focusing operation can be performed for each objective lens.
[effect]
According to the microscope autofocus device of the third embodiment, the effects shown in the first embodiment and the second embodiment can be obtained even in sample observation with switching of the objective lens.

  The above is merely an embodiment for explaining the present invention. Therefore, it is easy for those skilled in the art to make various changes, modifications, or additions to the above-described embodiments in accordance with the technical idea or principle of the present invention.

  For example, in the above description, the example in which the collation operation is performed by moving the objective lens or the revolver in the optical axis direction is shown, but the present invention is also applied to a microscope system that performs collation by moving the stage in the optical axis direction. Of course, the same applies.

1 is a diagram illustrating an overall configuration of a microscope autofocus device according to a first embodiment of the present invention. The figure which shows the circuit structure of a control part. The figure which shows the laser spot on the light reception sensor in the case of a back pin position. The figure which shows the laser spot on the light reception sensor in the case of a focusing position. The figure which shows the laser spot on the light reception sensor in the case of a front pin position. The figure which shows the sum signal and focus error signal which are calculated from the output signal of a light receiving sensor. The figure which shows the relationship between the focus error signal which considered the normal focus error signal and offset, and the focus position of an objective lens. 3 is a flowchart showing an outline of an AF operation of the microscope autofocus device according to the first embodiment of the present invention. The flowchart which shows the detail of operation | movement of step S11 in the flowchart of FIG. The flowchart which shows the detail of operation | movement of step S12 in the flowchart of FIG. The flowchart which shows offset setting operation | movement. The table which correlates and stores ID of each objective lens and the offset upper limit value Omax is shown. The figure which shows the focus determination range in a focus error signal. 7 is a flowchart showing an outline of an AF operation of a microscope autofocus device according to a second embodiment of the present invention. The flowchart which shows the detail of operation | movement of step S23 in the flowchart of FIG. The flowchart which shows the offset setting operation | movement of the microscope autofocus apparatus which concerns on 3rd Embodiment of this invention. The figure explaining the characteristic of the focus adjustment apparatus of a prior art.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Stage 2 Revolver 3, 3a, 3b Objective lens 4 Reference light source 5 Collimate lens 6 Light emission side stopper 7 Polarization beam splitter (PBS)
8 Condensing lens group 9 Chromatic aberration correction lens group 10 λ / 4 plate 11 Dichroic mirror 12 Condensing lens group 13 Light receiving sensor 14 Signal processing unit 15 Control unit 16 Revolver motor drive unit 17 Revolver motor 18 Revo hole position detection unit 19 Laser drive unit 20 Focusing motor drive unit 21 Focusing motor 23 Operation unit 24 JOG dial 25 Chromatic aberration correction lens drive unit 26 Chromatic aberration correction lens group drive motor 27 Illumination light source 28, 40 Lens 29 Mirror 30 CPU body 31 ROM
32 RAM
33 I / O port 34 Data bus S Observation sample

Claims (11)

A stage on which a sample to be observed with a microscope is placed; an observation optical system that includes an objective lens to obtain an observation image of the sample; a focusing drive means that changes a distance between the stage and the objective lens in an optical axis direction; Detection means for detecting the position of the stage with respect to the focal position of the objective lens, and control for determining the in-focus state based on the output signal of the detection means and controlling the distance to the in-focus state via the focusing drive means And an active autofocus method in a microscope apparatus comprising means,
A preliminary focusing step for performing a first focusing control based on an output signal of the detection means;
An offset setting step for offsetting the value of the output signal at an observation position desired by an observer near the in-focus position;
An active autofocus method with improved focusing accuracy for an observation site, comprising a main focusing step for performing second focusing control based on a signal value obtained by adding the offset to the output signal. The microscope apparatus further includes semi-manual means that enables fine adjustment of the change by the focusing drive means by a user operation,
The offset setting step includes the step of focusing on the desired observation position using the semi-manual means, and storing the value of the output signal at this time as the offset,
2. The active autofocus method according to claim 1, wherein the main focusing step uses the stored offset. The microscope apparatus includes a revolver that holds a plurality of objective lenses, and the objective lens is an objective lens selected from the plurality of objective lenses;
Obtaining an offset corresponding to each of the plurality of objective lenses in advance and storing the offset in relation to the objective lens;
Prior to the focusing step, a user selects a desired objective lens from the plurality of objective lenses, and the focusing step is related to the currently selected objective lens. 2. The active autofocus method according to claim 1, wherein a stored offset is used. Preliminarily obtaining and storing an offset corresponding to each of the plurality of objective lenses,
(1) performing focusing control based on an output signal of the detection means using one of the plurality of objective lenses;
(2) a focusing step of focusing on the desired observation position using the semi-manual means;
(3) storing the value of the output signal at the time of focusing on the desired observation position as an offset of the one objective lens;
4. The active autofocus method with improved accuracy of focusing on an observation site according to claim 3, wherein the steps (1) to (3) are performed on all of the plurality of objective lenses. Periodically determining whether or not the output signal is in focus based on a signal value obtained by adding the offset;
4. The method according to claim 1, further comprising a step of performing focusing control based on a signal value obtained by adding the offset to the output signal when it is determined that focusing is not performed. Active autofocus method with improved focusing accuracy. A stage on which a sample to be observed under a microscope is placed;
An observation optical system that includes an objective lens and obtains an observation image of the sample;
Focusing drive means for changing the distance between the stage and the objective lens in the optical axis direction;
Detecting means for detecting the position of the stage relative to the focal position of the objective lens;
Control means for determining the in-focus state based on the output signal of the detection means, and controlling the distance to the in-focus state via the focusing drive means;
The control means is
Preliminary focusing means for performing first focusing control based on an output signal of the detection means;
An offset setting means for offsetting the value of the output signal at an observation position desired by an observer near the in-focus position;
A microscope apparatus with improved accuracy of focusing on an observation site, comprising: a focusing unit that performs second focusing control based on a signal value obtained by adding the offset to the output signal. Semi-manual means for enabling fine adjustment of the change by the focusing drive means by user operation,
The offset setting means comprises means for focusing on the desired observation position using the semi-manual means, and storing the value of the output signal at this time as the offset,
The microscope apparatus according to claim 6, wherein the focusing means uses the stored offset. The microscope apparatus includes a revolver that holds a plurality of objective lenses, and the objective lens is an objective lens selected from the plurality of objective lenses;
The control means is
Means for preliminarily determining an offset corresponding to each of the plurality of objective lenses and storing the offset in relation to the objective lens, and the focusing step is related to the currently selected objective lens. The microscope apparatus according to claim 6, wherein a stored offset is used. Means for preliminarily obtaining and storing an offset corresponding to each of the plurality of objective lenses,
(1) means for performing focusing control based on an output signal of the detection means using one of the plurality of objective lenses;
(2) focusing means for focusing on the desired observation position using the semi-manual means;
(3) means for storing the value of the output signal when focusing on the desired observation position as an offset of the one objective lens;
9. The microscope apparatus according to claim 8, wherein all of the plurality of objective lenses comprise means for executing the steps (1) to (3). Means for periodically determining whether or not the output signal is in focus based on a signal value taking the offset into account;
9. The method according to claim 6, further comprising means for performing focusing control based on a signal value obtained by adding the offset to the output signal when it is determined that focusing is not performed. Microscope device with improved focusing accuracy. A stage on which a sample to be observed with a microscope is placed; an observation optical system for obtaining an observation image of the sample having an objective lens; a focusing drive means for changing a distance between the stage and the objective lens in an optical axis direction; In a microscope apparatus comprising a detection means for detecting the position of the stage with respect to the focal position of the objective lens, a focusing state is determined based on an output signal of the detection means, and the distance is adjusted via the focusing drive means. An active autofocus system that controls the focus state,
Preliminary focusing means for performing first focusing control based on an output signal of the detection means;
An offset setting means for offsetting the value of the output signal at an observation position desired by an observer near the in-focus position;
A microscope active autofocus system, comprising: a focusing unit that performs a second focusing control based on a signal value obtained by adding the offset to the output signal.

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