JP5108627B2 - Observation apparatus and observation method - Google Patents

Description

  The present invention relates to an observation apparatus such as a microscope having an automatic focus control function and an observation method. For example, an image of a sample held on a thin transparent member such as a multiwell plate, a petri dish, or a microscope specimen, particularly a multiwell. The present invention relates to an observation apparatus and an observation method suitable for acquiring an image of a cell sample cultured on a plate.

  Conventionally, in an image acquisition system of an observation apparatus that acquires images of cells or the like cultured on a multiwell plate, various automatic focus detection methods have been adopted to automatically focus on cells in the multiwell plate. Yes.

  The automatic focus detection method includes an active type and a passive type. In the active type automatic focus detection method, a light spot or mark is projected onto an object, and a focusing signal is formed depending on the position or shape of the light spot. On the other hand, in the passive automatic focus detection method, a focusing signal is formed from an image signal of an object received by an image sensor.

  However, the passive automatic focus detection method requires more time for focus detection than the active type, and it is essential to illuminate to obtain an image of the target cell. There is concern about cell damage caused by illumination light. For this reason, in the above-described image acquisition system, it is desirable to adopt an active auto focus detection method that is advantageous from the viewpoint of detection time.

As an image acquisition system that employs an active automatic focus detection method, for example, there is one described in Patent Document 1 below.
JP-T-2002-541430

  In the system described in Patent Document 1, an optical reference point in the vicinity of the bottom surface of the bottom of the microplate well is detected by active autofocus detection, and the detected optical reference point is fixed to a target layer in the well. The lower surface of the microplate well is moved in the longitudinal direction by the distance, thereby optically focusing the target layer of the microplate well.

However, the active automatic focus detection method employed in the system described in Patent Document 1 performs focus detection on an optical reference point in the vicinity of the lower surface of the bottom of the microplate well depending on the objective lens used for observation. It may not be possible.
For example, when the magnification of the objective lens is lowered, the depth of focus increases. However, the bottom of a transparent member such as a multi-plate well or petri dish that holds a sample is thin. For this reason, when a low-magnification objective lens is used, the focus detection accuracy is deteriorated and the focus detection is likely to be hindered. On the other hand, when the magnification of the objective lens is increased, the depth of focus becomes shallow, but the exit pupil diameter becomes small and the field of view becomes dark. As a result, when a high-magnification objective lens is used, the amount of light for forming a focusing signal used for automatic focus detection is insufficient, and this tends to hinder focus detection. For this reason, for an objective lens having a high degree of low magnification or high magnification, focus detection cannot be performed with the active automatic focus detection method described in Patent Document 1, and focus detection takes time. A passive autofocus detection method must be adopted.

  The present invention has been made in view of the above problems, and an observation apparatus and an observation device capable of aligning a focus position with an observation position in a short time even when an objective lens that is not applicable to active automatic focus detection is used. It aims to provide a method.

In order to achieve the above object, an observation apparatus according to the present invention is an observation apparatus that automatically focuses an objective lens on a specimen placed in a container and observes the specimen, and has a predetermined reference at the bottom of the container. A reference surface focusing objective lens for focusing on a surface, a sample observation objective lens for observing a predetermined observation position on the sample spaced a predetermined distance from the reference surface, and the reference surface focusing objective An objective lens switching means capable of switching between a lens and the specimen observation objective lens and inserting it into the observation optical path; and prior to inserting the specimen observation objective lens into the observation optical path, the objective lens switching means inserted into the observation optical path and line cormorant active autofocusing means auto focus detection of an active type to match the focus position of the reference plane focusing objective lens to the reference plane, an objective for focusing the reference plane if inserted in the observation light path When lens is focused on the reference surface, a position information detection means for detecting the relative position information in the optical axis direction between the reference plane and the reference plane focusing objective lens, the position information detecting means Relative position information in the optical axis direction between the reference plane and the reference plane focusing objective lens detected via the optical axis, a focusing position of the reference plane focusing objective lens, and the specimen observation objective lens And the relative position information in the optical axis direction with respect to the in-focus position of the specimen so that the in-focus position of the objective lens for specimen observation inserted in the observation optical path matches the observation position. It has a specimen observation objective lens automatic focusing means for adjusting the relative distance between the observation objective lens and the container.

Further, in the observation apparatus of the present invention, the specimen observation objective lens automatic focusing means includes optical reference directions of the reference plane and the reference plane focusing objective lens detected via the position information detection means. relative position information, the average thickness of the bottom portion of the container, the average thickness of the specimen, and if the focus position and the specimen observation objective lens of the reference plane focusing objective lens in Based on the relative position information in the optical axis direction with respect to the focal position, the specimen observation objective is adjusted so that the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position. It is preferable to adjust the relative distance between the lens and the container.

In the observation apparatus of the present invention, the specimen observation objective lens Autofocus Asete stage, the optical axis of the position information detected via the detecting means is said reference plane and the reference plane focusing objective lens Relative position information in the direction, average thickness of the bottom of the container, average thickness of the sample, and the focus position of the reference lens focusing objective lens and the specimen observation objective lens Based on the relative position information in the optical axis direction with respect to the in-focus position, the in-focus position of the objective lens for sample observation inserted in the observation optical path matches the observation position. The relative distance between the objective lens and the container is adjusted, and further, passive automatic focus detection is performed via the specimen observation objective lens, and the in-focus position of the specimen observation objective lens is set to the observation position. as match, the target present observation objective lens and the container Preferably, adjusting the relative distance.

In the observation apparatus of the present invention, the specimen observation objective lens Autofocus Asete stage, the optical axis of the position information detected via the detecting means is said reference plane and the reference plane focusing objective lens relative position information in the direction, and based on the relative position information in the optical axis direction between the focus position of the focus position and the specimen observation objective lens of the reference plane focusing objective lens, the The relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the reference plane. The relative distance between the specimen observation objective lens and the container is set so that the in-focus position of the specimen observation objective lens coincides with the observation position. Is preferably adjusted.

In order to achieve the above object, an observation method according to the present invention is an observation method for automatically focusing an objective lens on a specimen placed in a container and observing the specimen, through an objective lens switching means. Then, a reference plane focusing objective lens for focusing on a predetermined reference plane at the bottom of the container is inserted into the observation optical path, and then the reference plane focusing objective is connected via an active automatic focusing means. Active type automatic detection is performed through a lens, and the in-focus position of the reference surface focusing objective lens inserted in the observation optical path is made coincident with the reference surface, and then the position information detecting means is used to reference the reference plane focusing objective lens to detect the position information of the relative optical axis in the optical axis direction between, the reference plane and the reference plane focusing objective lens when focusing on the reference plane An objective lens focusing step for surface focusing, and the objective lens Via the switching means, the reference plane focusing objective lens is switched to the specimen observation objective lens for observing a predetermined observation position in the specimen separated from the reference plane by a predetermined distance, and then inserted into the observation optical path. The relative position information in the optical axis direction of the reference surface and the reference surface focusing objective lens detected via the position information detection means via the specimen observation objective lens automatic focusing means; Using the relative position information in the optical axis direction between the in-focus position of the reference plane focusing objective lens and the in-focus position of the specimen observation objective lens, the specimen observation inserted into the observation optical path A specimen observation objective lens focusing step for adjusting a relative distance between the specimen observation objective lens and the container so that a focus position of the objective lens matches the observation position. Yes.

Further, in the observation method according to the present invention, the specimen observation objective focusing step includes the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Is switched to a specimen observation objective lens for observing a predetermined observation position and inserted into the observation optical path, and then detected via the position information detection means via a specimen observation objective lens automatic focusing means. Relative positional information of the reference surface and the reference surface focusing objective lens in the optical axis direction, the average thickness of the bottom of the container, the average thickness of the sample, and the reference surface focusing Focusing of the specimen observation objective lens inserted in the observation optical path on the basis of relative position information in the optical axis direction between the in-focus position of the objective objective lens and the in- focus position of the specimen observation objective lens The position is consistent with the observation position To manner, it is preferable to adjust the relative distance between the target present observation objective lens and the container.

Further, in the observation method according to the present invention, the specimen observation objective focusing step includes the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Is switched to a specimen observation objective lens for observing a predetermined observation position and inserted into the observation optical path, and then detected via the position information detection means via a specimen observation objective lens automatic focusing means. Relative positional information of the reference surface and the reference surface focusing objective lens in the optical axis direction, the average thickness of the bottom of the container, the average thickness of the sample, and the reference surface focusing Focusing of the specimen observation objective lens inserted in the observation optical path on the basis of relative position information in the optical axis direction between the in-focus position of the objective objective lens and the in- focus position of the specimen observation objective lens The position is consistent with the observation position As to to adjust the relative distance between the target present observation objective lens and container, and then performs a passive auto-focus detection via target present observation objective lens, of the target present observation objective lens It is preferable to adjust the relative distance between the specimen observation objective lens and the container so that the in-focus position matches the observation position.

Further, in the observation method according to the present invention, the specimen observation objective focusing step includes the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Is switched to a specimen observation objective lens for observing a predetermined observation position and inserted into the observation optical path, and then detected via the position information detection means via a specimen observation objective lens automatic focusing means. Relative positional information in the optical axis direction between the reference surface and the reference surface focusing objective lens, and the in-focus position of the reference surface focusing objective lens and the in-focus position of the specimen observation objective lens Based on the relative position information in the optical axis direction, the relative distance between the specimen observation objective lens and the container is set so that the in-focus position of the specimen observation objective lens coincides with the reference plane. Adjust, then It performs passive auto focus detection via target present observation objective lens, as the focal position of the target present observation objective lens coincides with the observation position, relative to the target the observation objective lens and the container It is preferable to adjust the general distance.

  ADVANTAGE OF THE INVENTION According to this invention, even if it uses the objective lens which cannot be applied to active type automatic focus detection, the observation apparatus and the observation method which can align a focus position to an observation position in a short time are obtained.

Prior to the description of the embodiments, the configuration and operational effects of the present invention will be described.
The observation apparatus according to the present invention provides a specimen observation for observing a predetermined observation position on a specimen separated from a reference plane by a reference plane focusing objective lens for focusing on a predetermined reference plane at the bottom of the container. The objective lens for specimen observation and the objective lens for reference surface focusing can be switched and inserted into the observation optical path via the objective lens switching means. In addition, it is configured so that the in-focus position of the reference surface focusing objective lens inserted in the observation optical path coincides with the reference surface by performing active automatic focus detection via the active automatic focusing means. ing.

  As described above, when a reference surface focusing objective lens for focusing on a predetermined reference surface at the bottom of the container is provided separately from the specimen observation objective lens, the reference surface focusing objective lens has an active type. By using a lens applicable to automatic focus detection, the in-focus position of the reference surface focusing objective lens can be matched with the reference surface without taking time.

  In addition, the observation apparatus of the present invention provides a reference plane focusing objective lens on the reference plane when the reference plane focusing objective lens inserted in the observation optical path is focused on the reference plane via the position information detection means. And a reference surface focusing objective lens on the reference plane detected via the position information detection means, through the specimen observation objective lens automatic focusing means, Relative position information in the optical axis direction and relative position information in the optical axis direction with respect to the in-focus position of the specimen observation objective lens at the in-focus position of the reference surface focusing objective lens. Thus, the relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position.

For this reason, according to the observation device of the present invention, the objective lens for sample observation cannot perform active automatic focus detection (as a result, the conventional observation system employing the active focus detection device cannot focus on the observation position. ) Even the specimen observation objective lens can be focused on the observation position.
In addition, as described above, active auto focus detection can be performed through the reference plane focusing objective lens up to the position of the reference plane, so that passive auto focus detection is performed at all stages. Compared to performing focusing and performing focusing, the focusing time can be greatly reduced.
In particular, when a plurality of specimen observation objective lenses having different focal lengths (different magnifications) are exchanged at any time for observation, based on the position information of the reference plane focused by the reference plane focusing objective lens. If it is possible to focus, the time required for focusing can be shortened.

  Here, in the observation apparatus according to the first aspect of the invention, the specimen observation objective lens automatic focusing means inserts the specimen observation objective lens into the observation optical path and sets the focal position of the specimen observation objective lens as the observation position. In order to match, relative position information in the optical axis direction with respect to the reference surface focusing objective lens on the reference surface detected via the position information detecting means, at the in-focus position of the reference surface focusing objective lens In addition to the relative position information in the optical axis direction with respect to the in-focus position of the objective lens for specimen observation, the position information of the observation position is obtained based on the average thickness of the bottom of the container and the average thickness of the specimen. It is preferable to do so.

Relative position information in the optical axis direction with respect to the in-focus position of the specimen observation objective lens at the in-focus position of the reference surface focusing objective lens is known. In addition, the average thickness of the bottom of the container used to inspect the specimen and the average thickness of the specimen are also the average of the normalized thickness of the bottom of the container and the average of the general thickness of the specimen. It is a value and can be obtained as known information before inspection.
Therefore, the relative position information in the optical axis direction with respect to the reference surface focusing objective lens on the reference surface obtained by performing active type automatic focus detection via the reference surface focusing objective lens is stored in the container. By adding the average thickness of the bottom of the lens and the average thickness of the sample, position information of the observation position relative to the reference plane focusing objective lens in the optical axis direction can be obtained. Here, the position with the barrel in the observation optical path of the reference surface focusing objective lens and the specimen observation objective lens switched via the objective lens switching means is located at the same height position in the optical axis direction.
Therefore, the distance from the position of the reference lens focusing objective lens to the observation position, which is the position information of the obtained observation position, and the focus of the specimen observation objective lens from the position of the specimen observation objective lens By moving the specimen observation objective lens inserted in the observation optical path in the optical axis direction by the difference from the position, the in-focus position of the specimen observation objective lens can be matched with the observation position.

  Therefore, according to the observation apparatus of the first aspect of the present invention, after the in-focus position of the reference surface focusing objective lens is made to coincide with the reference surface, the active and passive types of the specimen observation objective lens are used. Without performing any automatic focus detection, the in-focus position can be made to coincide with the observation position calculated using a known value, so that the in-focus operation can be made very quick.

  By the way, in the observation apparatus of the first invention, the average thickness of the bottom of the container, which is added to the position information of the reference surface when the in-focus position of the reference lens focusing objective lens is matched with the reference surface, The average thickness of the specimen is not the actual measured value of the individual container or specimen. However, when there is a large error in the thickness of the bottom of the container, or when there is a large individual difference in the thickness of the sample placed inside the container, the average thickness of the bottom of the container is Even if the average thickness of the sample is added, there is a possibility that the focus position of the objective lens for sample observation is greatly deviated from the observation position.

  Therefore, in order to increase the focusing accuracy of the objective lens for specimen observation to the observation position, in the observation apparatus of the second invention, the objective lens automatic focusing means for specimen observation is detected via the position information detecting means. Relative position information in the optical axis direction with respect to the reference surface focusing objective lens in the measured reference surface, the average thickness of the bottom of the container, the average thickness of the sample, and the reference surface for focusing The focus position of the sample observation objective lens inserted in the observation optical path is observed based on the relative position information in the optical axis direction with the focus position of the sample observation objective lens at the focus position of the objective lens. After adjusting the relative distance between the specimen observation objective lens and the container so as to match the position, passive automatic focus detection is further performed via the specimen observation objective lens. Focus position is the same as observation position As to, it is preferable to adjust the relative distance between the vessel in the objective lens specimen observation.

  In this way, the time spent for passive autofocus detection via the specimen observation objective lens is very short, so it is much shorter than focusing using only passive autofocus detection. In time, it is possible to match the in-focus position to the desired observation position, and even if there is a large error in the shape of the container, or even if there is a large individual difference in the thickness of the specimen placed inside the container, sample observation The objective lens can be focused on the observation position with high accuracy.

  In addition, when observing a specimen, the thickness of the bottom of the container and the thickness of the specimen may vary greatly. In such a case, relative position information in the optical axis direction with respect to the reference surface focusing objective lens on the reference surface detected through the position information detecting means as in the second invention, and the container Relative to the focal position of the objective lens for specimen observation at the focal position of the objective lens for focusing on the reference plane in the optical axis direction Based on the correct position information, adjust the relative distance of the specimen observation objective lens to the container so that the in-focus position of the specimen observation objective lens matches the observation position. Even if you are in a rush, it may take a long time.

The present in the observation apparatus of the third invention, the optical axis direction between the specimen observation objective lens Autofocus Asete stage, the reference plane focusing objective lens in the detected reference surface via the position information detecting means And the relative position information in the optical axis direction with respect to the in-focus position of the specimen observation objective lens at the in-focus position of the reference surface focusing objective lens. The relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens inserted into the reference plane coincides with the reference plane. It is preferable to perform automatic focus detection of the mold and adjust the relative distance between the specimen observation objective lens and the container so that the focus position of the specimen observation objective lens coincides with the observation position.
That is, in the observation apparatus according to the third aspect of the invention, after the in-focus position of the specimen observation objective lens is made to coincide with the reference plane, the in-focus position is made to coincide with the observation position further through passive automatic focus detection. I have to.
In this way, even if the thickness of the bottom of the container or the thickness of the specimen varies greatly, it is possible to focus with high accuracy without taking too much time.

In addition, as the reference plane in the observation apparatus of the present invention, any one of the surface on the sample side at the bottom of the container and the surface on the side opposite to the sample can be applied. However, according to the first aspect of the invention, in the observation apparatus of the second invention, when the surface of the specimen-side at the bottom of the container and the reference plane, the objective lens Autofocus Asete stage specimen observation, the container bottom The relative position information in the optical axis direction between the reference surface detected by the position information detecting means and the reference surface focusing objective lens without taking into account the average thickness of the sample, the average of the sample A sample inserted into the observation optical path based on the thickness and relative positional information in the optical axis direction with the focus position of the sample observation objective lens at the focus position of the reference surface focusing objective lens What is necessary is just to adjust the relative distance with the container in the objective lens for sample observation so that the focus position of the objective lens for observation may correspond to an observation position. In the observation apparatus according to the third aspect of the invention, the configuration in which the surface on the specimen side at the bottom of the container is used as the reference surface has no individual difference in the thickness of the bottom of the container, and the individual difference in the thickness of the specimen. Effective when is large.

First Embodiment FIG. 1 is an explanatory diagram schematically showing the overall configuration of an observation apparatus according to a first embodiment of the present invention, FIG. 2 is a flowchart showing a procedure of a focusing operation in the observation apparatus of FIG. 1, and FIG. FIG. 2 is a state explanatory diagram showing a procedure of focusing operation in the observation apparatus of FIG. 1, (a) is an initial state in which a reference plane focusing objective lens is inserted into an observation optical path, and (b) is a reference by an active focus detection method. (C) is a state in which the objective lens for focusing on the surface is focused on the reference plane, (c) is a state in which the objective lens to be inserted into the observation optical path is switched from the objective lens for focusing on the reference plane to the objective lens for specimen observation, Position information relative to the reference surface when the surface focusing objective lens is focused on the reference surface, the average thickness of the bottom of the container, the average thickness of the sample, and for reference surface focusing Relative in the optical axis direction to the focus position of the objective lens for specimen observation at the focus position of the objective lens Based on the Do positional information shows a state of being focus the objective lens specimen observed in the observation position.

The observation apparatus according to the first embodiment includes a reference plane focusing objective lens 1, a specimen observation objective lens 2, an objective lens switching unit 3, an active automatic focusing unit 4, and a position information detection unit ( not shown). And a specimen observation objective lens automatic focusing means 6.

  The reference surface focusing objective lens 1 is an objective lens used for focusing on a predetermined reference surface in the bottom portion 11a of the microwell plate 11 which is a container for accommodating the specimen 10. Examples of the predetermined reference surface include a surface 11a1 on the sample 10 side in the bottom portion 11a or a surface 11a2 on the opposite side to the sample 10. In the observation apparatus of the first embodiment, the opposite surface 11a2 is used as a reference surface. In addition, the reference surface focusing objective lens 1 has a focal depth and NA that can be focused on a predetermined reference surface 11a2 in the bottom 11a of the microplate well 11 by performing active autofocus detection described later. I have.

  The specimen observation objective lens 2 is an objective lens used for observing a predetermined observation position 10 a in the specimen 10. The predetermined observation position 10a is, for example, an interface between the sample 10 and the culture solution that is separated from the reference surface 11a2 of the sample 10 by a predetermined distance.

  The objective lens switching means can be switched between the reference surface focusing objective lens 1 and the specimen observation objective lens 2 and inserted into the observation optical path, for example, a switching member (not shown) such as a revolver or a slider, and a switching member. The switching member driving unit 3a to be driven and a switching member driving unit 3a drive control circuit (not shown) provided inside the control unit 9 are configured.

The active autofocus means is means for performing active autofocus detection and matching the focus position of the reference surface focusing objective lens 1 inserted in the observation optical path with the reference surface 11a2. In the observation apparatus of FIG. 1, a laser diode 4a, a half mirror 4b, a collimator lens 4c, a half mirror 4d, a pinhole 4j, a photodiode 4e, a laser diode driver 4f, and a received light signal processor 4g The Z stage 4h, the Z stage driving unit 4i, the laser diode driving unit 4f drive control circuit (not shown) and the Z stage driving unit 4i drive control circuit (not shown) provided inside the control unit 9 are provided. It is configured.
Then, the active type automatic focusing means emits laser light of a predetermined wavelength from the laser diode 4a via the laser diode driving unit 4f, and the half mirror 4b, the collimating lens 4c, the half mirror 4d, and the reference surface focusing objective lens. 1 is directed toward the container 11 as convergent light, and the reflected light from the container 11 is passed through the reference surface focusing objective lens 1, half mirror 4d, collimator lens 4c, half mirror 4b, and pinhole 4j. Then, light is received by the photodiode 4e. Then, a drive control circuit (not shown) for the Z stage drive unit 4i provided inside the control unit 9 passes through the Z stage drive unit 4i so that the light receiving area at the photodiode 4e is minimized. 4h is moved in the Z direction so that the in-focus position of the reference surface focusing objective lens 1 coincides with the reference surface 11a2.
In the example of FIG. 1, the Z stage 4h is configured to move the objective lens in the Z direction, but the container 11 may be configured to be movable in the Z direction.

  For example, the position information detecting means detects the amount of driving in the Z direction by the Z stage driving unit 4i provided in the control unit 9, and also detects the focusing position from the position where the reference lens focusing objective lens 1 is mounted. And a circuit (not shown) for calculating the position of the reference surface 11a2 in the Z direction on the basis of the focal distance L1. Then, the relative position in the optical axis direction of the reference surface focusing objective lens 1 on the reference surface 11a2 when the reference surface focusing objective lens 1 inserted in the observation optical path is focused on the reference surface 11a2. It has a function to detect information.

Objective lens autofocus unit for specimen observation, provided in the control unit 9, when focusing on the reference surface 11a2 in the reference plane 11a2 detected via the position information detection means, the reference plane focusing objective lens Relative position information Z1 + L1 in the direction of the optical axis based on the in-focus position L1 from the position of the body 1 to the in-focus position , the average thickness T1 of the bottom 11a of the container 11, and the average thickness of the specimen 10 Based on T2 and the focal distance L2 from the barrel position of the specimen observation objective lens 2 to the focusing position, the focusing position of the specimen observation objective lens 2 inserted in the observation optical path matches the observation position 10a. As described above, the rotation direction and amount of rotation of the Z stage drive unit 4i are controlled to adjust the relative distance between the sample observation objective lens 2 and the container 11 (not shown).

  In addition, in FIG. 1, 5a is an imaging lens, 5b is a mirror, 5c is an image sensor, 5d is a signal processing unit for processing a signal imaged by the image sensor, and 6a is in the XY direction with the container 11 placed thereon. An XY stage 6b to be moved is an XY stage driving unit. The signal processing unit and the XY stage driving unit 6 b are respectively connected to the control unit 9 and controlled in operation through a control circuit provided inside the control unit 9.

The observation apparatus of the first embodiment is configured to focus the observation position 10a of the sample observation objective lens 2 through the control unit 9 according to the procedure shown in FIGS. It is assumed that a desired well is disposed on the observation optical path.
First, the reference lens focusing objective lens 1 is inserted into the observation optical path via the objective lens switching means (step S11). At this stage, the focus position of the reference surface focusing objective lens 1 is away from the reference surface 11a2 by Δ1 as shown in FIG.

  Next, active automatic focus detection is performed via the active automatic focusing means, and as shown in FIG. 3B, the in-focus position of the reference surface focusing objective lens 1 inserted in the observation optical path. Is matched with the reference plane 11a2 (step S12).

Next, when the reference plane focusing objective lens 1 is focused on the reference plane 11a2 via the position information detection means, the reference plane 11a2 is relative to the reference plane focusing objective lens 1 in the optical axis direction. Position information is detected (step S13). Specifically, the position Z1 in the Z direction of the reference plane focusing objective lens 1 is obtained by detecting the driving amount Δ1 in the Z direction by the Z stage driving unit 4i. Then, the relative position in the Z direction of the reference surface 11a2 is obtained by adding the focal distance L1 of the reference surface focusing objective lens 1 to the position Z1 in the Z direction of the reference surface focusing objective lens 1. Z1 + L1 is obtained.

Next, the reference lens focusing objective lens 1 is switched to the specimen observation objective lens 2 via the objective lens switching means and inserted into the observation optical path (step S14). At this stage, the focus position of the reference surface focusing objective lens 1 is Δ2 away from the reference surface 11a2, as shown in FIG.

Next, through the objective lens Autofocus Asete stage specimen observation, the relative position in the optical axis direction of the objective lens 1 for focusing the reference plane if the reference surface 11a2, which is detected via the position information detecting means Information Z1 + L1, the average thickness of the bottom 11a of the container 11 (here, T1), the average thickness of the sample 10 (here, T2), and the focal distance L2 of the sample observation objective lens 2 3 (d), the relative distance between the specimen observation objective lens 2 and the container 11 is set so that the in-focus position of the specimen observation objective lens 2 coincides with the observation position 10a. Adjust (step S15).

According to the observation apparatus of the first embodiment, the reference surface 11a2 is focused using the reference surface focusing objective lens 1 capable of active automatic focus detection, and the reference surface focusing on the reference surface 11a2 at that time is performed. The position information Z1 + L1 + T1 + T2 of the observation position 10a is calculated based on the position information Z1 + L1 relative to the objective lens 1, and the alignment information based on the position information Z1 + L1 + T1 + T2 of the observation position 10a and the focal distance L2 of the sample observation objective lens 2 is calculated. Since the difference Δ2 + T1 + T2 with respect to the focal position Z1 + L2 is moved in the Z direction, even if an objective lens that is not applicable to active autofocus detection is used as the objective lens 2 for sample observation, the focal position can be adjusted to the observation position in a short time. An observation apparatus and an observation method that can be aligned are obtained.

Second Embodiment FIG. 4 is a flowchart showing the procedure of the focusing operation in the observation apparatus according to the second embodiment of the present invention, and FIG. 5 is a state explanatory diagram showing the procedure of the focusing operation in the observation apparatus of the second embodiment. (A) is an initial state in which the reference surface focusing objective lens is inserted into the observation optical path, (b) is a state in which the reference surface focusing objective lens is focused on the reference surface by an active focus detection method, c) The objective lens inserted into the observation optical path is switched from the reference lens focusing objective lens to the specimen observation objective lens, and (d) is the reference when the reference surface focusing objective lens is focused on the reference plane. Position relative to the surface, the average thickness of the bottom of the container, the average thickness of the sample, and the focusing position of the objective lens for specimen observation at the focal position of the objective lens for focusing on the reference plane Objective observation lens based on relative position information in the optical axis direction A state of being focused on the observation position, and shows a state, in which is focused through the objective lens specimen observed in the observation position by (e) the passive focus detection method from the position of (d).

In observation device of the second embodiment, the objective lens Autofocus Asete stage specimen observation, in the optical axis direction between the objective lens 1 for focusing the reference plane if the position information detecting means is detected via the reference surface 11a2 Based on the relative position information Z1 + L1, the average thickness T1 of the bottom portion 11a of the container 11, the average thickness T2 of the specimen 10, and the focal distance L2 of the objective lens 2 for specimen observation, it is inserted into the observation optical path. The relative distance of the specimen observation objective lens 2 from the container 11 is adjusted so that the in-focus position of the specimen observation objective lens 2 coincides with the observation position 10a. 2 is used to adjust the relative distance between the specimen observation objective lens 2 and the container 11 so that the focus position of the specimen observation objective lens 2 coincides with the observation position 10a. I will do it It is configured.
Control for passive automatic focus detection is performed by a control unit 9 that detects a driving amount in the Z direction by the Z stage driving unit 4i based on a signal obtained via the signal processing unit 5 shown in FIG. It is configured to be performed by a control circuit (not shown) provided in the interior of the device.
Other configurations are substantially the same as those of the observation apparatus of the first embodiment.

And the observation apparatus of 2nd embodiment is comprised so that the focusing to the observation position 10a of the objective lens 2 for sample observation may be performed in the procedure shown in FIG.4 and FIG.5 via the control part 9. FIG.
First, the reference lens focusing objective lens 1 is inserted into the observation optical path via the objective lens switching means (step S21). At this stage, the focus position of the reference surface focusing objective lens 1 is separated from the reference surface 11a2 by Δ1 as shown in FIG.

  Next, active automatic focus detection is performed via the active automatic focusing means, and as shown in FIG. 5B, the in-focus position of the reference surface focusing objective lens 1 inserted in the observation optical path. Is matched with the reference plane 11a2 (step S22).

Next, when the reference plane focusing objective lens 1 is focused on the reference plane 11a2 via the position information detection means, the reference plane 11a2 is relative to the reference plane focusing objective lens 1 in the optical axis direction. Position information is detected (step S23). Specifically, the position Z1 of the reference lens focusing objective lens 1 in the Z direction is obtained by detecting the driving amount Δ1 in the Z direction by the Z stage driving unit 4i shown in FIG. Then, the relative position in the Z direction of the reference surface 11a2 is obtained by adding the focal distance L1 of the reference surface focusing objective lens 1 to the position Z1 in the Z direction of the reference surface focusing objective lens 1. Z1 + L1 is obtained.

Next, the reference lens focusing objective lens 1 is switched to the specimen observation objective lens 2 via the objective lens switching means and inserted into the observation optical path (step S24). At this stage, the focus position of the reference surface focusing objective lens 1 is Δ2 away from the reference surface 11a2, as shown in FIG.

Next, through the objective lens Autofocus Asete stage specimen observation, the relative position in the optical axis direction of the objective lens 1 for focusing the reference plane if the reference surface 11a2, which is detected via the position information detecting means Information Z1 + L1, the average thickness of the bottom 11a of the container 11 (here, T1), the average thickness of the sample 10 (here, T2), and the focal distance L2 of the sample observation objective lens 2 5 (d), the relative distance between the sample observation objective lens 2 and the container 11 is set so that the in-focus position of the sample observation objective lens 2 coincides with the observation position 10a. Adjust (step S25).
The processing procedure so far is the same as that of the observation apparatus of the first embodiment.

Here, the observation device of the second embodiment, the objective lens Autofocus Asete stage specimen observation, further performs passive auto focus detection through a specimen observation objective lens 2, an objective lens for specimen observation 2 The relative distance between the sample observation objective lens 2 and the container 11 is adjusted so that the in-focus position coincides with the observation position 10a (step S26).

  Used in the adjustment of the relative distance from the container 11 in the sample observation objective lens (steps S15 and S25) performed through the specimen observation objective lens automatic focusing means of the first and second embodiments. The thickness T1 of the bottom 11a of the container 11 and the thickness T2 of the specimen 10 are the average value of the standardized thickness of the bottom 11a of the container 11 and the average value of the general thickness of the specimen 10, It is not a value obtained by measuring the individual containers 11 and the specimen 10. However, if there is a large error in the thickness of the bottom 11a of the container 11 or a solid difference in the thickness of the specimen 10 placed inside the container 11, the observation position 10a is determined based on the position information of the predetermined reference surface 11a2. Even if the position information Z1 + L1 is calculated and the position information Z1 + L1 + T1 + T2 of the observation position 10a is obtained by adding the thickness T1 of the bottom 11a of the container 11 and the thickness T2 of the specimen 10, as shown in FIG. 5 (d). There is a possibility that the position obtained by the calculation process is greatly deviated from the actual observation position 10a, and the in-focus position of the sample observation objective lens 2 is largely deviated from the observation position 10a.

  However, according to the observation apparatus of the second embodiment, after performing the same position adjustment as that of the observation apparatus of the first embodiment, further, by performing passive automatic focus detection via the objective lens 2 for sample observation. Since the specimen observation objective lens 2 is focused on the observation position 10a, the time required for the passive automatic focus detection via the specimen observation objective lens is small, and the passive automatic focus detection is sufficient. The focusing position can be made to coincide with the desired observation position 10a in a much shorter time than focusing using only the sample, and when the shape error of the container 11 is large, or the specimen 10 placed inside the container 11 Even when the individual difference in thickness is large, the specimen observation objective lens can be focused on the observation position 10a with high accuracy.

Third Embodiment FIG. 6 is a flowchart showing the procedure of the focusing operation in the observation apparatus according to the third embodiment of the present invention, and FIG. 7 is a state explanatory diagram showing the procedure of the focusing operation in the observation apparatus of the third embodiment. (A) is an initial state in which the reference surface focusing objective lens is inserted into the observation optical path, (b) is a state in which the reference surface focusing objective lens is focused on the reference surface by an active focus detection method, c) The objective lens inserted into the observation optical path is switched from the reference lens focusing objective lens to the specimen observation objective lens, and (d) is the reference when the reference surface focusing objective lens is focused on the reference plane. Based on positional information relative to the surface and relative positional information in the optical axis direction relative to the focal position of the objective lens for specimen observation at the focal position of the objective lens for focusing on the reference plane, for specimen observation When the objective lens is focused on the reference plane, (e) Shows a state, the obtained by focusing the objective lens specimen observed in the observation position by blanking type focus detection system.

In the observation apparatus of the third embodiment, the surface 11a1 on the specimen 10 side in the bottom portion 11a is used as a reference surface.
Also, the observation device of the third embodiment, the optical axis of the objective lens Autofocus Asete stage specimen observation, an objective lens 1 for focusing the reference plane if the reference surface 11a1, which is detected via the position information detecting means Based on the relative position information Z1 ′ + L1 and the focal distance L2 of the specimen observation objective lens 2, the focal position of the specimen observation objective lens 2 inserted in the observation optical path matches the reference plane 11a1. As described above, the relative distance of the specimen observation objective lens 2 from the container 11 is adjusted, and further, passive automatic focus detection is performed via the specimen observation objective lens 2, and the specimen observation objective lens 2 is aligned. The relative distance between the specimen observation objective lens 2 and the container 11 is adjusted so that the focal position coincides with the observation position 10a.
That is, in the observation apparatus of the third embodiment, after the in-focus position of the specimen observation objective lens 2 is made to coincide with the reference plane 11a1, the in-focus position is made to coincide with the observation position 10a via the passive automatic focus detection. I try to let them.

And the observation apparatus of 3rd embodiment is comprised so that the focusing to the observation position 10a of the objective lens 2 for sample observation may be performed through the control part 9 in the procedure shown in FIG.6 and FIG.7.
First, the reference lens focusing objective lens 1 is inserted into the observation optical path via the objective lens switching means (step S31). At this stage, the focus position of the reference surface focusing objective lens 1 is separated from the reference surface 11a1 by Δ1 ′, as shown in FIG.

  Next, active automatic focus detection is performed via the active automatic focusing means, and as shown in FIG. 7B, the in-focus position of the reference surface focusing objective lens 1 inserted in the observation optical path. Is matched with the reference plane 11a1 (step S32).

Next, when the reference plane focusing objective lens 1 is focused on the reference plane 11a1 via the position information detection means, the reference plane 11a1 is relative to the reference plane focusing objective lens 1 in the optical axis direction. Position information is detected (step S33). Specifically, the position Z1 ′ of the reference plane focusing objective lens 1 in the Z direction is obtained by detecting the driving amount Δ1 ′ in the Z direction by the Z stage driving unit 4i shown in FIG. Then, by adding the in-focus distance L1 of the reference surface focusing objective lens 1 to the position Z1 ′ of the reference surface focusing objective lens 1 in the Z direction, the reference surface 11a1 in the Z direction can be relatively compared. A position Z1 ′ + L1 is obtained.

Next, the reference lens focusing objective lens 1 is switched to the specimen observation objective lens 2 via the objective lens switching means and inserted into the observation optical path (step S34). At this stage, the focus position of the reference surface focusing objective lens 1 is Δ2 ′ away from the reference surface 11a1 as shown in FIG. 7C.

Next, through the objective lens Autofocus Asete stage specimen observation, the relative position in the optical axis direction of the objective lens 1 for focusing the reference plane if the reference surface 11a1, which is detected via the position information detecting means Based on the information Z1 ′ + L1 and the focal distance L2 of the specimen observation objective lens 2, as shown in FIG. 7 (d), the in-focus position of the specimen observation objective lens 2 matches the reference position 11a1. Then, the relative distance between the sample observation objective lens 2 and the container 11 is adjusted (step S35).

Here, the observation apparatus of the third embodiment, the objective lens Autofocus Asete stage specimen observation, further performs passive auto focus detection through a specimen observation objective lens 2, an objective lens for specimen observation 2 The relative distance between the sample observation objective lens 2 and the container 11 is adjusted so that the in-focus position coincides with the observation position 10a (step S36).

  When observing a specimen, the thickness of the bottom of the container and the thickness of the specimen may vary greatly. In such a case, relative position information in the optical axis direction with the reference surface focusing objective lens on the reference surface detected via the position information detection unit as in the observation apparatus of the second embodiment, The relative thickness in the direction of the optical axis of the average thickness of the bottom of the container, the average thickness of the specimen, and the focal position of the objective lens for reference observation at the focal position of the objective lens for focusing on the reference plane Based on the correct position information, adjust the relative distance of the specimen observation objective lens to the container so that the in-focus position of the specimen observation objective lens matches the observation position. Even if you are in a rush, it may take a long time.

  However, according to the observation apparatus of the third embodiment, the specimen surface side at the bottom of the container is adopted as the reference surface, and after the in-focus position of the objective lens for specimen observation is made coincident with the reference surface, the passive type is further used. Since the in-focus position is matched to the observation position via automatic focus detection, even if the thickness of the bottom of the container or the thickness of the specimen differs greatly, it can be adjusted with high accuracy without taking time. Can be burnt.

  INDUSTRIAL APPLICABILITY The present invention is useful in the biological and medical fields in which cell observation and analysis are performed using an image of a sample held on a thin transparent member, particularly an image of a cell sample cultured on a multiwell plate.

It is explanatory drawing which shows roughly the whole structure of the observation apparatus concerning 1st embodiment of this invention. It is a flowchart which shows the procedure of the focusing operation | movement in the observation apparatus of FIG. FIG. 2 is a state explanatory diagram showing a procedure of focusing operation in the observation apparatus of FIG. 1, (a) is an initial state in which a reference plane focusing objective lens is inserted into an observation optical path, and (b) is a reference by an active focus detection method. (C) is a state in which the objective lens for focusing on the surface is focused on the reference plane, (c) is a state in which the objective lens to be inserted into the observation optical path is switched from the objective lens for focusing on the reference plane to the objective lens for specimen observation, and (d) is the reference. Position information relative to the reference surface when the surface focusing objective lens is focused on the reference surface, the average thickness of the bottom of the container, the average thickness of the sample, and for reference surface focusing This shows a state in which the specimen observation objective lens is focused on the observation position based on relative position information in the optical axis direction with respect to the focal position of the specimen observation objective lens at the focal position of the objective lens. . It is a flowchart which shows the procedure of the focusing operation | movement in the observation apparatus concerning 2nd embodiment of this invention. It is a state explanatory diagram showing the procedure of the focusing operation in the observation apparatus of the second embodiment, (a) is an initial state in which the reference surface focusing objective lens is inserted in the observation optical path, (b) is an active focus detection method (C) is a state in which the reference lens focusing objective lens is focused on the reference plane, and (c) is a state in which the objective lens inserted into the observation optical path is switched from the reference surface focusing objective lens to the specimen observation objective lens. Is the relative position information with respect to the reference surface when the reference lens focusing objective lens is focused on the reference surface, the average thickness of the bottom of the container, the average thickness of the sample, and the reference surface alignment. Based on the relative position information in the optical axis direction with the focus position of the specimen observation objective lens at the focus position of the focus objective lens, the specimen observation objective lens is focused on the observation position, ( e) shows the objective lens for specimen observation from the position of (d) by the passive focus detection method. A state of being focused, shows. It is a flowchart which shows the procedure of the focusing operation | movement in the observation apparatus concerning 3rd embodiment of this invention. It is a state explanatory diagram showing the procedure of the focusing operation in the observation apparatus of the third embodiment, (a) is an initial state in which the reference surface focusing objective lens is inserted into the observation optical path, (b) is an active focus detection method (C) is a state in which the reference lens focusing objective lens is focused on the reference plane, and (c) is a state in which the objective lens inserted into the observation optical path is switched from the reference surface focusing objective lens to the specimen observation objective lens. Is the relative position information with respect to the reference surface when the reference surface focusing objective lens is focused on the reference surface, and the in-focus position of the specimen observation objective lens at the in-focus position of the reference surface focusing objective lens Based on the relative position information in the optical axis direction, the specimen observation objective lens is focused on the reference plane. (E) shows specimen observation using the passive focus detection method from the position (d). 2 shows a state where the objective lens is focused on the observation position.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reference surface focusing objective lens 2 Sample observation objective lens 3a Switching member drive part 4a Laser diode 4b Half mirror 4c Collimate lens 4d Half mirror 4e Photo diode 4f Laser diode drive part 4g Light reception signal processing part 4h Z stage 4i Z stage Drive unit 4j Pinhole 5a Imaging lens 5b Mirror 5c Image sensor 5d Signal processing unit 6a XY stage 6b XY stage drive unit 9 Control unit 10 Sample 10a Observation position 11 Microwell plate 11a Bottom portion 11a1 Surface 11a2 on the bottom portion 11a 11a2 Surface on the opposite side of the specimen 10 at the bottom 11a L1 A focal distance from the position where the reference lens focusing objective lens 1 is mounted to the focusing position L2 From the position where the specimen observation objective lens 2 is mounted to the focusing position Confocal distance

Claims (8)

An observation apparatus for automatically focusing an objective lens on a specimen placed in a container and observing the specimen,
A reference surface focusing objective lens for focusing on a predetermined reference surface at the bottom of the container;
A specimen observation objective lens for observing a predetermined observation position in the specimen separated from the reference plane by a predetermined distance;
An objective lens switching means capable of switching between the reference lens focusing objective lens and the specimen observation objective lens and inserting it into the observation optical path;
Prior to inserting the specimen observation objective lens into the observation optical path, active automatic focus detection is performed so that the focus position of the reference plane focusing objective lens inserted into the observation optical path is coincident with the reference plane. and line cormorant active-type automatic focusing means,
Relative position information in the optical axis direction between the when observed inserted the reference plane focusing objective lens in the optical path is focused on the reference surface, the reference plane and the reference plane focusing objective lens Position information detecting means for detecting
Relative position information in the optical axis direction of the reference plane and the reference plane focusing objective lens detected via the position information detection means, the in-focus position of the reference plane focusing objective lens, and the Using the relative position information in the optical axis direction with respect to the focal position of the specimen observation objective lens, the focal position of the specimen observation objective lens inserted in the observation optical path matches the observation position. As described above, the specimen observation objective lens automatic focusing means for adjusting the relative distance between the specimen observation objective lens and the container,
An observation apparatus comprising: The specimen observation objective lens automatic focusing means includes relative position information in the optical axis direction of the reference plane and the reference plane focusing objective lens detected via the position information detection means, the container bottom average thickness of the average thickness of the specimen, and relative to the optical axis direction of the focus position of the focus position and the specimen observation objective lens of the reference plane focusing objective lens The relative distance between the specimen observation objective lens and the container so that the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position based on the general position information. The observation apparatus according to claim 1, wherein the observation device is adjusted. The specimen observation objective lens Autofocus Asete stage, the relative position information in the optical axis direction between the position information detected via the detecting means is said reference plane and the reference plane focusing objective lens, the average thickness of the bottom of the container, the average thickness of the specimen, and in the optical axis direction between the focus position of the focus position and the specimen observation objective lens of the reference plane focusing objective lens Based on the relative position information, the relative position between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position. The distance is adjusted, and further, passive automatic focus detection is performed via the specimen observation objective lens, and the specimen observation objective is adjusted so that the in-focus position of the specimen observation objective lens coincides with the observation position. characterized by adjusting the relative distance between the lens and the container Observation apparatus according to claim 1 or 2. The specimen observation objective lens Autofocus Asete stage, the relative position information in the optical axis direction between the position information detected via the detecting means is said reference plane and the reference plane focusing objective lens, and Based on the relative position information in the optical axis direction between the in-focus position of the reference lens focusing objective lens and the in-focus position of the specimen observation objective lens, the specimen observation inserted into the observation optical path The relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the objective lens coincides with the reference plane, and further, a passive-type automatic through the specimen observation objective lens is adjusted. The focus detection is performed, and the relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens coincides with the observation position. The observation apparatus according to 1. An observation method for automatically focusing an objective lens on a sample placed in a container and observing the sample,
Through the objective lens switching means, a reference surface focusing objective lens for focusing on a predetermined reference surface at the bottom of the container is inserted into the observation optical path, and then through the active automatic focusing means, Active type automatic detection is performed through the reference surface focusing objective lens, the in-focus position of the reference surface focusing objective lens inserted in the observation optical path is made coincident with the reference plane, and then position information detection is performed. When the reference plane focusing objective lens is focused on the reference plane via the means, the relative optical axis direction of the reference plane and the reference plane focusing objective lens in the optical axis direction is increased. An objective lens focusing step for focusing on a reference surface for detecting position information;
Via the objective lens switching means, the reference lens focusing objective lens is switched to a specimen observation objective lens for observing a predetermined observation position in the specimen separated from the reference plane by a predetermined distance and inserted into the observation optical path. and, then, through the objective lens automatic focusing device for specimen observation, relative to the optical axis direction of the detected said reference surface and said reference surface focusing objective lens through the position information detecting means Using the positional information and relative positional information in the optical axis direction between the in-focus position of the reference lens focusing objective lens and the in-focus position of the specimen observation objective lens, the optical axis is inserted into the observation optical path. A specimen observation objective lens focusing step for adjusting a relative distance between the specimen observation objective lens and the container so that a focal position of the specimen observation objective lens coincides with the observation position;
An observation method characterized by comprising: The specimen observation focusing step for observing a predetermined observation position on the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Switch to use the objective lens inserted in the observation light path, then the objective for through the objective lens automatic focusing device for specimen observation, detected the reference plane and the reference plane focusing through the position information detecting means Position information relative to the lens in the optical axis direction, average thickness of the bottom of the container, average thickness of the sample, and in-focus position of the reference lens focusing objective lens and the sample Based on relative position information in the optical axis direction with respect to the in-focus position of the observation objective lens, the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position. , a target present observation objective lens The method of observation according to claim 5, characterized in that for adjusting the relative distance between the vessel. The specimen observation focusing step for observing a predetermined observation position on the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Switch to use the objective lens inserted in the observation light path, then the objective for through the objective lens automatic focusing device for specimen observation, detected the reference plane and the reference plane focusing through the position information detecting means Position information relative to the lens in the optical axis direction, average thickness of the bottom of the container, average thickness of the sample, and in-focus position of the reference lens focusing objective lens and the sample Based on relative position information in the optical axis direction with respect to the in-focus position of the observation objective lens, the in-focus position of the specimen observation objective lens inserted in the observation optical path matches the observation position. , a target present observation objective lens The relative distance from the container is adjusted, and then passive automatic focus detection is performed via the specimen observation objective lens so that the in-focus position of the specimen observation objective lens coincides with the observation position. The observation method according to claim 5, wherein a relative distance between the specimen observation objective lens and the container is adjusted. The specimen observation focusing step for observing a predetermined observation position on the specimen separated from the reference plane by a predetermined distance from the reference plane focusing objective lens via the objective lens switching means. Switch to use the objective lens inserted in the observation light path, then the objective for through the objective lens automatic focusing device for specimen observation, detected the reference plane and the reference plane focusing through the position information detecting means Relative positional information in the optical axis direction with respect to the lens, and relative positional information in the optical axis direction between the in-focus position of the reference lens focusing objective lens and the in-focus position of the specimen observation objective lens The relative distance between the specimen observation objective lens and the container is adjusted so that the in-focus position of the specimen observation objective lens coincides with the reference plane, and then the specimen observation objective is adjusted. Pass through lens It performs automatic focus detection type, and characterized in that the focus position of the target present observation objective lens so as to coincide with the observation position, adjusting the relative distance between the target present observation objective lens and the container The observation method according to claim 5.

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