WO2021132395A1 - Microscope and cell culture device - Google Patents

Abstract

A microscope for observing a culture vessel that has a plurality of layered observation surfaces. The microscope comprises an objective lens, an image formation lens, an optical member that is arranged on the optical axis between the objective lens and the image formation lens and has a variable focal length, and a working distance modification part that, by changing the focal length of the optical member, changes the working distance from an observation surface from among the plurality of observation surfaces to the objective lens.

Description

Microscope and cell culture device

The present invention relates to a microscope and a cell culture device.
The present application claims priority based on Japanese Patent Application No. 2019-233715 filed in Japan on December 25, 2019, the contents of which are incorporated herein by reference.

In the field of regenerative medicine, in order to cultivate a large amount of necessary cells, a multi-layer culture vessel, which is a culture vessel in which a plurality of observation surfaces are laminated, may be used. In the multi-layer culture vessel, about 10 observation surfaces are laminated, and all layers have a total thickness of about 20 cm. In order to understand the quality and quantity of cells in culture, it is necessary to acquire microscopic images of cells. In the acquisition of microscopic images of cells cultured in a multi-layer culture vessel, there is no technique for efficiently observing cells by quickly focusing on all observation surfaces. Therefore, the current situation is that the state of cells existing in all layers is estimated based on the number of cells and morphological information in the lowermost layer of the multi-layer culture vessel.
The method of moving the objective lens across all layers of the multi-layer culture vessel and focusing on each layer is time consuming. Further, a long-acting microscope using the Maksutov Cassegrain Catadioptric method has been developed, but it is necessary to move the reflecting mirror in the optical axis direction, and it is difficult to change the focal length quickly.

An observation device for observing an object to be observed in a multi-layer culture vessel is known (Patent Document 1). The observation device described in Patent Document 1 has an accommodating portion for accommodating a movable trolley on which a multi-layer culture container containing a plurality of trays is mounted, and an optical system, and outputs an image formed by the optical system. When a trolley equipped with an imaging device and a multi-layer culture container is mounted in a storage portion, the observed object on each tray of the multi-layer culture container is the light of the imaging device with the multi-layer culture container mounted on the trolley. Placed on the axis.

In the observation device described in Patent Document 1, the image pickup device and the illumination device are provided so that the optical axes of the image pickup device and the illumination device intersect the bottom surface of the tray of the multilayer culture vessel at an angle in the range of 40 to 50 degrees. The device is placed to observe all trays of the multilayer culture vessel. In this way, when observing from an angle with respect to the bottom surface of the tray of the multilayer culture container, the image of the object to be observed in each tray is distorted as compared with the case of observing from the direction perpendicular to the bottom surface of the tray.

JP-A-2018-139615

One aspect of the present invention is a microscope for observing a culture vessel in which a plurality of observation surfaces are stacked, and is an optical axis between an objective lens, an imaging lens, and the objective lens and the imaging lens. An optical member having a variable focal length arranged in the optical member and a working distance change for changing the working distance from the observation surface to the objective lens among the plurality of observation surfaces by changing the focal length of the optical member. It is a microscope equipped with a part.

One aspect of the present invention is a microscope for observing a culture vessel in which a plurality of observation surfaces are stacked, and is an optical axis between an objective lens, an imaging lens, and the objective lens and the imaging lens. An optical member having a variable focal length arranged in the optical member and a working distance change for changing the working distance from the observation surface to the objective lens among the plurality of observation surfaces by changing the focal length of the optical member. It is a cell culture apparatus including a microscope including a unit and a stage on which the culture container is arranged.

It is a figure which shows an example of the transmission type microscope which concerns on 1st Embodiment. It is an optical path diagram which shows the structure of the variable working distance which concerns on 1st Embodiment. It is an optical path diagram which shows the structure of the variable working distance which concerns on 2nd Embodiment. It is a block diagram which shows the outline of the optical path and the apparatus of the optical system which concerns on 2nd Embodiment. It is an optical path diagram which shows the structure of the variable working distance which concerns on 3rd Embodiment. It is a block diagram which shows the outline of the optical path and the apparatus of the optical system which concerns on 3rd Embodiment. It is a perspective view which shows an example of the turret which concerns on 3rd Embodiment. It is a figure which shows an example of the imaging process which concerns on 3rd Embodiment. It is a figure which shows the structure of the modification of the optical system of 3rd Embodiment. It is a figure which shows the structure of the modification of the optical system of 3rd Embodiment. It is a block diagram which shows the outline of the optical path and the apparatus of the optical system of 4th Embodiment.

(First Embodiment)
Hereinafter, the first embodiment will be described in detail with reference to the drawings. FIG. 1 is a diagram showing an example of a transmission microscope 1 according to the present embodiment. The transmission microscope 1 is a microscope for observing a culture vessel in which a plurality of observation surfaces are stacked. The transmission type microscope 1 includes an illumination optical system having a light source 3, a condenser lens 4, a field aperture 5, an aperture aperture 6, a condenser lens 7, and a first reflecting mirror 8, an objective lens 10, and optics. It has a member 11, a second reflecting mirror 12, and an imaging optical system including an imaging lens 13. Then, the transmission microscope 1 can take an image of the observation surface of the multilayer culture apparatus 2 placed on the stage 9 by the imaging apparatus 14.

The multi-layer culture device 2 includes a culture container 20, and the culture container 20 is configured by stacking a plurality of trays. The transmission microscope 1 observes and images the cells to be cultured in each tray constituting the culture vessel 20. Here, in the culture vessel 20, the bottom surfaces of the stacked trays serve as observation surfaces. That is, the culture container 20 is a multi-layer culture container in which a plurality of observation surfaces are laminated.

As an example, the culture container 20 has 10 layers of trays, that is, observation surfaces O stacked on top of each other, and has a thickness of about 20 cm. In the following description, each layer of the culture vessel 20 will be referred to as a first layer, a second layer, a third layer, and the like in the order in which they are laminated. The bottom layer is the first layer, and the top layer is the tenth layer. Of the layers of the culture vessel 20, layers other than the uppermost layer and the lowermost layer may be referred to as an intermediate layer. Further, regarding the observation surface O corresponding to each layer, the observation surface corresponding to the first layer is referred to as the observation surface O1, the observation surface corresponding to the second layer is referred to as the observation surface O2, and the observation surface corresponding to the i-th layer is referred to as the observation surface O2. It is called the observation surface Oi (i = 1, 2, ..., 10). Further, among the plurality of observation surfaces O, the current observation surface may be referred to as an observation surface Oj. That is, the observation surface Oj is an observation surface in which the focal positions are aligned among the plurality of observation surfaces O. FIG. 1 shows a state in which the observation surface O5 corresponding to the intermediate layer, which is the fifth layer, is observed as the observation surface Oj.

Further, as an example of the observation surface O of the 10 layers, the observation surface O5 corresponding to the 5th layer is used as the focusing reference surface. Here, the focusing reference plane is a plane that serves as a focusing reference for the culture vessel 20. The objective lens 10 moves relative to the stage 9 on the optical axis indicated by the alternate long and short dash line in the figure, so that the focal position is aligned with the focusing reference plane. The observation surface of any layer other than the fifth layer of the culture vessel 20 on the focusing reference surface may be used.

The illumination light from the light source 3 passes through the condenser lens 4 and the condenser lens 7 and becomes a substantially parallel luminous flux. The illumination light that has become a parallel luminous flux is reflected by the first reflecting mirror 8 and illuminates the cells on the observation surface Oj. The cells on the observation surface Oj are cells to be cultured on the tray corresponding to the observation surface Oj among the trays of each layer of the culture vessel 20.

In the transmission microscope 1, so-called Koehler illumination is performed by the condenser lens 7. Between the condenser lens 4 and the condenser lens 7, a field diaphragm 5 is provided at a position conjugate with the observation surface Oj, and an aperture diaphragm 6 is provided at a position conjugate with the light source 3.

The light from the observation surface Oj is focused by the objective lens 10 to become a substantially parallel luminous flux. The light from the observation surface Oj, which has become a parallel luminous flux, passes through the optical member 11 and is then reflected by the second reflecting mirror 12 and incident on the imaging lens 13. The light from the observation surface Oj is focused on the image formation surface 15 by the imaging lens 13, and a magnified image of the observation surface Oj is formed.
The imaging device 14 is provided at a position where the imaging surface 15 can be imaged. The enlarged image of the observation surface Oj imaged by the image pickup apparatus 14 is observed by a monitor (not shown).

Note that, in FIG. 1, for the sake of simplicity, the light rays illuminating the culture vessel 20 from the light source 3 and the image forming rays from the observation surface Oj to the imaging surface 15 are shown.

The optical member 11 is arranged on the optical axis between the objective lens 10 and the imaging lens 13. As will be described later, the optical member 11 includes various lenses and has a variable focal length. That is, the optical member 11 is arranged on the optical axis between the objective lens 10 and the imaging lens 13, and the focal length is variable. Then, the working distance WD is changed by changing the focal length of the optical member 11.

In the transmission microscope 1, when observing the observation surface O10 corresponding to the tenth layer, which is the uppermost layer, from the working distance Wmax when observing the observation surface O1 corresponding to the first layer, which is the lowest layer, in the working distance WD. The working distance can be changed in the range up to Wmin. Then, by changing the focal length of the optical member 11, an image of an arbitrary observation surface Oi (i = 1, 2, ..., 10) among the observation surfaces corresponding to the 10 layers is formed on the image plane 15. It is possible to form.
In the microscope shown in FIG. 1, the illumination optical system illuminates from below the stage 9, and the imaging optical system including the objective lens 10 is provided above the stage 9, but the illumination optics with respect to the stage. Needless to say, it can be configured as a so-called inverted microscope in which the system and the imaging optical system are exchanged.

Here, with reference to FIG. 2, the principle of the transmission microscope 1 changing the working distance WD will be described by taking the microscope optical system 30 as an example. FIG. 2 is a diagram showing a configuration including an example of the microscope optical system 30 according to the present embodiment. The microscope optical system 30 is the same as the transmission type microscope 1 shown in FIG. 1, but for the sake of simplicity, the first reflecting mirror 8 and the second reflecting mirror 12 are not included as a developed optical path diagram. It is shown.

As described above, the optical member 11 includes various lenses. The lens included in the optical member 11 is called a conversion lens Gv. The focal length of the optical member 11 is the focal length of the conversion lens Gv. The optical member 11 is arranged between the objective lens 10 and the imaging lens 13 on the optical axis 31.

_{FIG. 2A shows a state of a reference position of the working distance WD 0} for observing the intermediate layer among the 10 layers of the culture vessel 20, and in this reference state, the optical member 11 is arranged in the optical path. Absent. FIG. 2B shows a state in which the upper layer of the 10 layers of the culture vessel 20 is observed, and the negative lens Gv1 is inserted as the conversion lens Gv on the optical axis between the objective lens 10 and the imaging lens 13. The working distance WD ₁ is larger than _{the working distance WD 0 in} the case of the intermediate layer. FIG. 2C shows a state in which the lower layer of the 10 layers of the culture vessel 20 is observed, and the positive lens Gv2 is on the optical axis between the objective lens 10 and the imaging lens 13 as the conversion lens Gv. The working distance WD ₂ is smaller than the working distance WD _{0 in the reference state.}

In the configuration of this embodiment, the focal length of the conversion lens is switched between negative and positive for the observation of the upper layer and the lower layer with the reference state as the intermediate layer, but the reference position is set as the lower layer and the conversion lens Gv is used. It is possible to observe the upper layer portion by sequentially shortening the negative focal length of a certain negative lens Gv1, that is, gradually increasing the negative refractive power. It is also possible to observe the lower layer portion by gradually shortening the positive focal length of the positive lens Gv2, which is the conversion lens Gv, that is, gradually increasing the positive refractive power with the reference position as the upper layer.

In transmission microscope 1, as described above, by changing the focal length of the optical member 11, i.e., by changing the focal length f _v of the conversion lens Gv, it is possible to change the working distance WD of the objective lens 10 .. By changing the focal length of such an optical member 11, specifically the conversion lens Gv, in other words, the refractive power of the conversion lens, the working distance WD of the objective lens 10 can be changed, and as a result, it is shown in FIG. It is possible to observe and photograph an image of each layer of trays stacked in multiple layers in such a culture container 20. However, when the focal length (that is, the refractive force) of the conversion lens located on the image side of the objective lens 10 is changed, the working distance WD changes and the combined focal length of the objective lens 10 and the optical member 11 (that is, the conversion lens Gv) is changed. Since the distance also changes, the imaging magnification of the microscope changes. Then, the size of the image of each layer of the trays stacked in multiple layers in the culture vessel 20 will be different from each other due to the change of the working distance WD. That is, the imaging magnification changes, and therefore the observation field of view and the size of the image of the test object also change, which may be very inconvenient and cause a big problem for comparative examination of each layer.

Here, a configuration will be described in which the imaging magnification β of the transmission microscope 1 can be maintained constant even if the working distance WD is changed in the microscope optical system 30.
The imaging magnification β of the transmission type microscope 1 composed of the objective lens 10 and the imaging lens 13 is as follows, assuming that the focal length of the objective lens 10 is the focal length f ₁ and the focal length of the imaging lens 13 is the focal length f _2. It is given by the equation (1) of.

Further, in general, the combined focal length f of the two lenses when a second lens (here, the conversion lens Gv of the focal length fv) is added as the objective lens is the distance d between the objective lens 10 and the conversion lens Gv. Then, it is expressed by the following equation (2).

Distance d between the objective lens 10 and the conversion lens Gv is the equal to the focal length _{f 1} of the objective lens 10, d = _{f 1,} and the combined focal length f, the above equation (2), the formula (3 ) Is derived.

_{That is, even when the focal length f v} of the conversion lens Gv is changed, the combined focal length f between the objective lens 10 and the conversion lens Gv does not change.

Therefore, if the conversion lens Gv for making the working distance WD as a microscope objective lens variable is arranged on the image side focal position of the objective lens 10, that is, on the optical axis of the rear focal plane F of the objective lens 10, it operates. Even when the distance WD is changed, the combined focal distance f between the objective lens 10 and the conversion lens Gv does not change, and as a result, the imaging magnification β as a microscope is kept constant.

(Second embodiment)
This situation is shown in FIG. Similar to FIG. 2, FIG. 3 (A) shows a state of a reference position at a _{working distance of WD 0 for observing the intermediate layer of 10 layers of the culture vessel 20.} FIG. 3B shows a state in which the upper layer of the 10 layers of the culture vessel 20 is observed, and the negative lens Gv ₁ as the conversion lens Gv is on the optical axis between the objective lens 10 and the imaging lens 13. Inserted, the working distance WD ₁ is larger than _{the working distance WD 0 in} the case of the intermediate layer. FIG. 2C shows a state in which the lower layer portion of the 10 layers of the culture vessel 20 is observed, and the positive lens Gv ₂ is the optical axis between the objective lens 10 and the imaging lens 13 as the conversion lens Gv. Inserted above, the working distance WD ₂ is smaller than _{the working distance WD 0 in} the case of the intermediate layer. In such a configuration, both the negative lens Gv ₁ and the positive lens Gv ₂ as the conversion lens Gv are arranged on the rear focal point of the objective lens 10, that is, on the optical axis of the rear focal plane F of the objective lens. As described above, the combined focal distance between the objective lens 10 and the conversion lens Gv is maintained constant, and the magnification as a microscope is maintained constant.

Next, in FIG. 4, the configuration of the optical member 11 and the control of changing the focal length of the optical member 11 will be described. FIG. 4 is a configuration diagram showing an outline of an optical path and an apparatus of an optical system according to a second embodiment.
In this embodiment, as an example, the optical member 11 includes a turret T. A plurality of lenses L having different focal lengths are housed in the turret T. That is, the optical member 11 includes a plurality of lenses L having different focal lengths.

The control device 40 is, for example, a computer and includes a control unit 41. The control unit 41, on the optical axis 31 of the plurality of lenses L, by switching the lens disposed at the focal position of the objective lens 10 changes the focal length f _v of the optical member 11. The control unit 41 retracts a lens other than the lens arranged on the optical axis 31 among the plurality of lenses L from the optical axis 31. In FIG. 4, as an example, a positive lens among the plurality of lenses L is arranged on the optical axis 31. The control unit 41 is an example of a working distance changing unit. That is, the control unit 41 changes the working distance WD of by changing the focal length f _v of the optical member 11 from the viewing plane Oj of the plurality of the observation plane O to the objective lens 10.

In the transmission microscope 1, when the focal position is aligned with the focusing reference plane, as an example, none of the plurality of lenses L housed in the turret T is arranged on the optical axis 31.

The control unit 41 performs various controls on the microscope optical system 30 in addition to the control of the optical member 11 described above. Various controls performed by the control unit 41 include, for example, adjusting the brightness of the light source 3, moving the stage 9, minute movement of the objective lens 10 for focusing in the optical axis direction, and the optical axis of the imaging lens 13. Includes minute movements in the direction.

In the second embodiment described above, the imaging magnification can be kept constant even when the working distance is arbitrarily changed. As shown in FIGS. 3 and 4, the basic configuration for this is to arrange the conversion lens Gv for changing the working distance at the rear focal position (rear focal plane F) of the objective lens 10.
However, the focal position of the objective lens 10 is generally an important position as the pupil position of the microscope optical system, and in particular, in a phase-contrast microscope or a differential interference microscope, an optical element such as a special aperture or a prism is arranged. Therefore, in the second embodiment shown in FIGS. 3 and 4, the conversion lens Gv is arranged at the rear focal position (rear focal plane F) of the objective lens 10, so that special microscopic observation such as phase difference can be performed. It's difficult to do it right.

(Third Embodiment)
A third embodiment for solving the above problems will be described with reference to FIG. FIG. 5 is a schematic optical path diagram showing a configuration in which the working distance is variable according to the third embodiment. Also in this optical path diagram, as in FIG. 3, for simplification of explanation, the first reflecting mirror 8 and the second reflecting mirror 12 are not included in the microscope optical system 30 and are shown as a developed optical path diagram. ing. Members having the same functions as those in FIG. 3 are designated by the same symbols.

FIG. 5A shows a state of a reference position at a _{working distance of WD 0 in} order to observe the intermediate layer of 10 layers of the culture vessel 20. In this reference state, the optical member 11 is not arranged in the optical path. FIG. 5B shows a state in which the upper layer of the 10 layers of the culture vessel 20 is observed, and the conversion lens Gv1 having the negative lens element and the positive lens element in order from the object side is the objective lens 10 and the imaging lens. It is inserted on the optical axis between 13 and 13. Then, FIG. 5C shows a state in which the lower layer portion of the 10 layers of the culture vessel 20 is observed, and the conversion lens Gv2 having the positive lens element and the negative lens element is connected to the objective lens 10 in order from the object side. It is inserted on the optical axis between the image lens 13 and the image lens 13.

What is important here is that, as shown in FIG. 5B, the conversion lens Gv1 is arranged on the object side of the rear focal plane F of the objective lens 10, but the combination of the negative lens element and the positive lens element is important. Therefore, the composite main point H as the conversion lens Gv1 coincides with the rear focal plane F of the objective lens 10. Similarly, as shown in FIG. 5C, the conversion lens Gv2 is arranged on the image side of the rear focal plane F of the objective lens 10, but is converted by the combination of the positive lens element and the negative lens element. The combined principal point H as the lens Gv2 coincides with the rear focal plane F of the objective lens 10.
With such a configuration, the rear focal position of the objective lens 10, that is, the image side pupil of the microscope, can be arbitrarily changed while maintaining the imaging magnification as a microscope constant by the conversion lens. A space can be provided at the position, and advanced observation such as phase difference and differential interference contrast is possible.

The entire apparatus of the third embodiment will be described below. FIG. 6 is a configuration diagram showing an outline of an optical path and an apparatus of an optical system according to a third embodiment. Members having the same functions as those in FIG. 4 described above are designated by the same reference numerals, and detailed description thereof will be omitted as they are duplicated.
The control device 40 is, for example, a computer and includes a control unit 41. Control unit 41, by switching the lens disposed on the optical axis 31 of the plurality of lenses L, it changes the focal length f _v of the optical member 11. The control unit 41 retracts a lens other than the lens to be arranged on the optical axis 31 among the plurality of lenses L from the optical axis 31. In FIG. 6, as an example, the lens L1-1 of the plurality of lenses L is arranged on the optical axis 31. The control unit 41 is an example of a working distance changing unit. That is, the control unit 41 changes the working distance WD of by changing the focal length f _v of the optical member 11 from the viewing plane Oj of the plurality of the observation plane O to the objective lens 10.

In the transmission microscope of the present embodiment, when the focal position is aligned with the focusing reference plane, as an example, the plurality of lenses L housed in the front turret T1 and the rear turret T2 are all arranged on the optical axis 31. Not done.

The control unit 41 performs various controls on the microscope optical system 30 in addition to the control of the optical member 11 described above. The various controls performed by the control unit 41 include, for example, adjustment of the brightness of the light source 3, movement of the stage 9, movement of the objective lens 10 in the optical axis direction, movement of the imaging lens 13 in the optical axis direction, and the like. ..

Here, with reference to FIG. 7, the front turret T1 and the rear turret T2 will be described. FIG. 7 is a perspective view showing an example of the turret according to the present embodiment. The front turret T1 and the rear turret T2 are arranged in two rows so as to overlap in the direction of the optical axis 31.

The front turret T1 includes a plurality of storage portions H1. The front turret T1 includes a storage unit H1-1, a storage unit H1-2, a storage unit H1-3, a storage unit H1-4, a storage unit H1-5, and a storage unit H1-6 as a plurality of storage units H1. .. The plurality of storage portions H1 are arranged and provided in a circular shape.
The rear turret T2 includes a plurality of storage portions H2. The rear turret T2 includes a storage unit H2-1, a storage unit H2-2, a storage unit H2-3, a storage unit H2-4, a storage unit H2-5, and a storage unit H2-6 as a plurality of storage units H2. .. The plurality of storage portions H2 are arranged and provided in a circular shape.
The plurality of storage portions H1 and the plurality of storage portions H2 include a storage portion in which the lens L is stored and a storage portion in which the lens L is not stored. Here, the plurality of lenses L housed in the plurality of storage portions have different focal lengths from each other.

In the example of FIG. 7, among the plurality of storage portions H1 provided in the front turret T1, the storage unit H1-1, the storage unit H1-2, the storage unit H1-3, the storage unit H1-4, and the storage unit H1-5 A lens L1-1, a lens L1-2, a lens L1-3, a lens L1-4, and a lens L1-5 having different synthetic focal lengths are housed in each. On the other hand, the lens L is not stored in the storage unit H1-6.
Of the plurality of storage units H2 provided in the rear turret T2, the storage unit H2-1, the storage unit H2-2, the storage unit H2-3, and the storage unit H2-4 have lenses L2- with different synthetic focal lengths. 1. Lens L2-2, lens L2-3, and lens L2-4 are stored respectively. On the other hand, the lens L is not stored in the storage unit H2-5 and the storage unit H2-6.
As described above, in the present embodiment, the number of the plurality of lenses L housed in the front-stage turret T1 and the rear-stage turret T2 is nine as an example.

As described above, the front turret T1 and the rear turret T2 overlap in the direction of the optical axis 31 and are arranged in two rows. That is, the positions of the front turret T1 and the rear turret T2 in the optical axis direction are different from each other. The plurality of lenses L are divided into two sets depending on whether they are housed in either the front turret T1 or the rear turret T2 whose positions in the optical axis direction are different from each other. Therefore, the plurality of lenses L are provided in a plurality of sets having different positions in the optical axis direction.

The front turret T1 and the rear turret T2 rotate under the control of a rotation drive unit (not shown). This rotation drive unit is controlled by the control unit 41. The front turret T1 and the rear turret T2 rotate to rotate the lens L1-1, the lens L1-2, the lens L1-3, the lens L1-4, the lens L1-5, the lens L2-1, and the lens L2-2. , Lens L2-3, and lens L2-4 are arranged on the optical axis 31.
That is, the front turret T1 and the rear turret T2 are controlled by the control unit 41 to switch the lens arranged on the optical axis 31 among the plurality of lenses L. The front turret T1 and the rear turret T2 are examples of the lens switching unit. That is, in the present embodiment, the lens switching unit is a turret.

Here, the control unit 41 retracts the lenses other than the lens L arranged on the optical axis 31 from the optical axis 31 among the plurality of lenses L housed in the turret in which the lens L arranged on the optical axis 31 is stored. Let me. Further, the control unit 41 moves the storage unit in which the lens of the other turret is not stored to a position on the optical axis 31, so that the plurality of lenses L stored in the other turret are retracted from the optical axis 31. Let me.

In the example of FIG. 7, the lens L1-1 housed in the storage part H1-1 of the front turret T1 is arranged on the optical axis 31, and the storage part H2-6 in which the lens of the rear turret T2 is not stored is the optical axis. It is arranged on 31. In this case, the light beam enters the lens L1-1 at the position of the front turret T1 and passes through the hole of the accommodating portion H2-6 at the position of the rear turret T2.

Next, with reference to FIG. 8, an imaging process, which is a process of observing the observation surface O corresponding to each layer of the culture vessel 20 by the control device 40, will be described. FIG. 8 is a diagram showing an example of an imaging process according to the present embodiment. Before the start of the imaging process shown in FIG. 8, the observation surface Oj is set as the focusing reference surface by the control unit 41. Here, the focusing reference plane is the observation plane O6 corresponding to the sixth layer of the culture vessel 20 as described above. Before the start of the imaging process shown in FIG. 8, none of the plurality of lenses L housed in the front turret T1 and the rear turret T2 is arranged on the optical axis 31.

Step S10: The control unit 41 sets the first observation surface to start imaging. The control unit 41 sets the first observation surface to start observation based on a predetermined order. The predetermined order is, for example, the order from the 10th layer, which is the uppermost layer of the culture vessel 20, to the 1st layer, which is the lowest layer. The control unit 41 stores the information indicating the set observation surface in the storage unit 42 as the observation surface information.

Further, in step S10, the control unit 41 stores the culture container information, which is information indicating the type of the culture container 20, in the storage unit 42. This culture container information includes information indicating a lens for focusing on the observation surface O corresponding to each layer of the culture container 20, depending on the type of the culture container 20. The information indicating the lens includes, for example, the focal length of the lens.
The culture container information is input to the control device 40 from the operation unit (not shown), for example, by the user of the transmission microscope 1. The control unit 41 acquires the input culture container information. The culture container information includes information indicating a predetermined order for observing the observation surface O corresponding to each layer of the culture container 20.
Depending on the type of the culture vessel 20, the lenses L housed in the front turret T1 and the rear turret T2 may be replaced in step S10.

Step S20: The control unit 41 changes the lens L arranged on the optical axis 31 to a desired lens according to the set observation surface Oj. The control unit 41 selects a lens to be arranged on the optical axis 31 among a plurality of lenses L housed in the front turret T1 and the rear turret T2 based on the culture container information stored in the storage unit 42.
Here, the control unit 41 rotates the front turret T1 via the rotation drive unit and rotates the rear turret T2 via the rotation drive unit according to the lens L to be arranged, and arranges the lens L on the optical axis 31. change.

Step S30: The control unit 41 determines whether or not the observation surface Oj is in focus. Here, focusing means that the focal position of the optical system of the objective lens 10 and the conversion lens Gv included in the optical member 11 coincides with the position of the observation surface Oj.
When determining that the control unit 41 is in focus (step S30; YES), the control unit 41 executes the process of step S40. On the other hand, when it is determined that the control unit 41 is out of focus (step S30; NO), the control unit 41 executes the process of step S60.

Step S40: The control unit 41 causes the imaging device 14 to perform imaging. Here, the imaging is to generate an image (captured image) of the subject based on the light detected by the image pickup device on the imaging surface 15.

Step S50: The control unit 41 determines whether or not all the observation surfaces from the observation surface O1 to the observation surface O10 have been photographed. Here, the control unit 41 makes a determination based on, for example, a predetermined order indicated by the culture container information stored in the storage unit 42 and the observation surface information.
When the control unit 41 determines that the imaging of all the observation surfaces is completed (step S50; YES), the control device 40 ends the imaging process. On the other hand, when it is determined that the imaging of all the observation surfaces is not completed (step S50; NO), the control unit 41 executes the process of step S70.

Step S60: The control unit 41 adjusts the objective lens 10. Here, the control unit 41 finely adjusts the objective lens 10 to bring the transmission microscope 1 into focus on the observation surface Oj. After that, the control unit 41 executes the process of step S40.

Step S70: The control unit 41 sets the observation surface Oj. Here, the control unit 41 sets the observation surface Oj based on, for example, a predetermined order indicated by the culture container information stored in the storage unit 42 and the observation surface information. After that, the control unit 41 executes the process of step S20 again.
With the above, the control device 40 ends the imaging process.

As described above, the microscope according to the present embodiment (in the present embodiment, the transmission type microscope 1) is a microscope for observing the culture vessel 20 in which a plurality of observation surfaces O are stacked, and is an objective lens. 10, an imaging lens 13, an optical member 11, and a working distance changing unit (control unit 41 in the present embodiment) are provided.

With this configuration, in the microscope according to the present embodiment, the working distance WD can be easily changed according to the observation surface O, so that the culture container 20 in which a plurality of observation surfaces O are stacked can be easily observed. it can.

Further, in the microscope according to the present embodiment (transmission microscope 1 in the present embodiment), the position of the principal point of the optical member 11 (in the present embodiment, the position of the negative lens Gv ₁ and the positive lens Gv _{2 which are conversion lenses Gv).} ) Is arranged so as to coincide with the image-side focal position 32 of the objective lens 10.

With this configuration, in the microscope according to the present embodiment, when the focal length fv of the optical member 11 is changed, the combined focal length of the objective lens 10 and the optical member 11 is maintained constant regardless of the focal length fv of the optical member 11. Therefore, the imaging magnification β as a microscope can be maintained constant.

Further, in the microscope according to the present embodiment (transmission microscope 1 in the present embodiment), the optical member 11 includes a plurality of first lenses (a plurality of lenses L in the present embodiment) having different focal lengths. The microscope (transmission type microscope 1 in this embodiment) includes a lens switching unit (front turret T1 and rear turret T2 in this embodiment).
The lens switching unit (in the present embodiment, the front turret T1 and the rear turret T2) is controlled by the working distance changing unit (control unit 41 in the present embodiment) and has a plurality of lenses (in the present embodiment, a plurality of lenses). Of L), the second lens arranged on the optical axis 31 is switched.

With this configuration, in the microscope according to the present embodiment, it is possible to switch the lens arranged on the optical axis 31 among a plurality of lenses having different focal lengths, so that the focal length of the optical member 11 can be changed quickly.

Further, in the microscope according to the present embodiment (transmission microscope 1 in the present embodiment), the lens switching unit (the front turret T1 and the rear turret T2 in the present embodiment) has a plurality of lenses (in the present embodiment, the transmission type microscope 1). A plurality of lenses L) are provided in a plurality of sets having different positions in the optical axis direction.

With this configuration, in the microscope according to the present embodiment, since a plurality of lenses can be stacked and stored in the direction of the optical axis 31, the plurality of lenses are not divided into a plurality of sets having different positions in the optical axis direction. Therefore, a plurality of lenses can be compactly stored in the radial direction perpendicular to the optical axis 31.

Further, in the microscope according to the present embodiment (transmission microscope 1 in the present embodiment), the plurality of lenses (plurality of lenses L in the present embodiment) are a plurality of lens elements arranged on the same optical axis. (In this embodiment, it is composed of a negative lens element and a positive lens element).

With this configuration, in the microscope according to the present embodiment, the types of focal length values of the plurality of first lenses can be increased as compared with the case where the lens is a single lens, so that the number of lenses is one. Compared with the case of a lens, it is easy to change the working distance WD according to the observation surface Oj.

Further, in the microscope according to the present embodiment (transmission type microscope 1 in the present embodiment), one lens switching portion (in the present embodiment, the front turret T1 and the rear turret T2) is arranged on the optical axis 31. In the lens, the composite principal point H of the plurality of lens elements (in the present embodiment, the negative lens element and the positive lens element) coincides with the image side focal point of the objective lens 10.

With this configuration, in the microscope according to the present embodiment, when the position of the lens on the optical axis 31 deviates from the position of the image-side focal length of the objective lens 10, even if the focal length of the lens is changed, the objective lens 10 and the objective lens 10 are used. Since the combined focal length f with a plurality of lenses can be made invariant, the working distance WD is changed when the position of the lens on the optical axis 31 deviates from the position of the image side focal length of the objective lens 10. However, the imaging magnification β of the transmission type microscope 1 can be kept constant.

Further, in the microscope according to the present embodiment (transmission microscope 1 in the present embodiment), the lens switching unit is a turret (in the present embodiment, the front turret T1 and the rear turret T2).

With this configuration, in the microscope according to the present embodiment, the second lens arranged on the optical axis 31 can be switched by rotating the turret, so that the focal length of the optical member 11 can be changed easily and quickly.

(Modification 1 of the third embodiment)
Hereinafter, the modification 1 of the third embodiment described above will be described in detail with reference to the drawings. In this modification, a case where the transmission microscope is provided with a bandpass filter to improve the image quality of the captured image will be described.
The microscope according to this modification is referred to as a transmission microscope 1a, and the optical system of the transmission microscope 1a is referred to as a microscope optical system 30a.

Hereinafter, a modification 1 of the third embodiment described above will be described with reference to the drawings. In this modification, the transmission microscope is equipped with an oblique light diaphragm, and oblique illumination is possible.
The microscope according to this modification is referred to as a transmission microscope 1a, and the optical system of the transmission microscope 1a is referred to as a microscope optical system 30a.

FIG. 9 is a diagram showing an example of the configuration of the microscope optical system 30a according to this modified example. Comparing the microscope optical system 30a (FIG. 9) according to the present modification with the microscope optical system 30 (FIG. 6) according to the third embodiment, the difference is that the oblique light diaphragm 60a is provided. Here, the functions of the other components (light source 3, condenser lens 4, field diaphragm 5, aperture diaphragm 6, condenser lens 7, objective lens 10, optical member 11, and imaging lens 13) are the first implementation. It is the same as the form. The description of the same function as that of the first embodiment is omitted, and in this modified example, a part different from that of the third embodiment will be mainly described.

The oblique light diaphragm 60a is provided at the position of the aperture diaphragm 6 as an example, and has an aperture at a peripheral portion outside the optical axis.

The oblique light diaphragm 60a has, for example, a disk-shaped opening 600a on an arc that transmits light and a light-shielding portion 601a that blocks light. As an example, the opening 600a is formed in an arc shape having a central angle of 90 degrees. The width of the opening 600a is, for example, 1 millimeter. As for the direction of the light beam of the irradiation light from the light source 3 of the microscope optical system 30a, since the opening 600a is off the optical axis, the observation surface Oj can be illuminated obliquely, and so-called oblique illumination is performed. This illumination method enables observation of finer patterns.

In the present embodiment, the oblique light diaphragm 60a is controlled by the control unit 41a and, as an example, rotates every 90 degrees around the optical axis passing through the light source and the subject. As the oblique light diaphragm 60a rotates, the position of the opening 600a rotates every 90 degrees around the optical axis passing through the light source and the subject. That is, the oblique light diaphragm 60a can rotate the position of the opening 600a around the optical axis passing through the light source and the subject. Here, the oblique light diaphragm 60a can rotate the position of the opening 600a by a predetermined angle around the optical axis passing through the light source and the subject. By rotating the position of the opening 600a, the incident direction of the oblique illumination light can be changed.

In the transmission electron microscope according to this modification, the oblique light diaphragm 60a is rotated by the control unit 41a to change the incident angle of the illumination light by 90 degrees, and an image of a cell as a subject is imaged. The image processing unit provided in the image pickup apparatus 14 synthesizes a plurality of captured images captured by changing the incident angle by 90 degrees to generate one captured image.
In the captured image obtained by synthesizing a plurality of captured images captured by changing the incident angle of the illumination light, the contrast is higher than in the case where the plurality of captured images are not combined.

In the configuration of FIG. 9 above, the bandpass filter can be arranged at or near the position of the aperture diaphragm 6. A bandpass filter is a filter that transmits long-wavelength light and does not transmit short-wavelength light, where long-wavelength and short-wavelength are, for example, in cells by cutting wavelengths shorter than 660 nanometers. It is possible to remove noise light that is likely to occur. Specifically, it is possible to reduce the diffraction and scattering of the irradiation light applied to the cells that are the subject, and it is possible to improve the image quality of the captured image.

The bandpass filter in this modification is an example, and the bandpass filter is not limited to this, and the bandpass filter may be a bandpass filter having a center of 625 nm and a bandwidth of about 20 nm. It can also be used in combination with the above-mentioned light-shielding plate for oblique lighting. Further, instead of the bandpass filter, a light-shielding plate may be arranged at or near the position of the aperture diaphragm 6.

(Modification 2 of the third embodiment)
Hereinafter, the configuration of the modified example of the third embodiment described above will be described with reference to the drawings. In this modification, a case where the transmission microscope is provided with a phase ring to perform phase difference observation will be described.
The microscope according to this modification is referred to as a transmission microscope 1b, and the optical system of the transmission microscope 1b is referred to as a microscope optical system 30b.

FIG. 10 is a diagram showing an example of the configuration of the microscope optical system 30b according to this modified example. Comparing the microscope optical system 30b (FIG. 10) according to the present modification with the microscope optical system 30 (FIG. 6) according to the first embodiment, the first phase ring 70b and the second phase ring 71b are provided. The difference is that they are. Here, the functions of the other components are the same as those of the third embodiment. The description of the same function as that of the third embodiment is omitted, and in this modification, different parts will be mainly described.

The first phase ring 70b is provided at the position of the opening diaphragm 6 in the lighting system and has a ring-shaped opening.

The second phase ring 71b is arranged on the optical axis 31 in the imaging system at a position conjugate with the position of the aperture diaphragm 6, specifically, at the position of the rear focal point of the objective lens 10. The image-side focal position 32 of the objective lens 10 is also the pupil position of the objective lens 10.

The second phase ring 71b has an opening having the same shape as the opening of the first phase ring 70b. The second phase ring 71b provides a predetermined phase difference and transmittance characteristics.
Here, the vicinity of the pupil position includes a range in which the second phase ring 71b gives a predetermined phase difference and transmittance characteristics even when the second phase ring 71b deviates from the pupil position. Even if the conjugate relationship between the first phase ring 70b provided at the position of the aperture diaphragm 6 in the illumination system and the second phase ring 71b provided at the pupil position of the objective lens 10 deviates to some extent, the position is high in the case of low magnification. Phase difference observation is possible. Further, in FIG. 10, for the sake of simplicity, the two phase rings 70 and the phase ring 71 are shown as having substantially the same size, but as long as the conjugate relationship with each other is maintained, the sizes may change as appropriate according to the magnification. Needless to say, is necessary.

In the phase-contrast microscope according to this modification, phase-contrast observation is possible by providing a phase ring (first phase ring 70b and second phase ring 71b). By phase difference observation, even if the observation target is a transparent sample, it is possible to observe a clear image due to the difference in refractive index.

(Fourth Embodiment)
Hereinafter, a fourth embodiment of the present invention will be described with reference to the drawings.
In the first, second and third embodiments described above, in the transmission microscope, the focal length of the optical member 11 is determined by exchanging the lens arranged on the optical axis among the plurality of lenses housed in the turret. That is, the working distance WD was changed by changing the refractive power). In the present embodiment, in the microscope, the optical member 11 itself as a conversion lens has a variable focal length lens, and the working distance is changed according to a change in the focal length of the variable focal length lens.
The microscope according to this embodiment is referred to as a transmission microscope 1c, and the optical system of the transmission microscope 1c is referred to as a microscope optical system 30c.

FIG. 11 is a diagram showing an example of the configuration of the microscope optical system 30c according to the present embodiment. Comparing the microscope optical system 30c (FIG. 11) according to the present embodiment with the microscope optical system 30 (FIG. 2) according to the first embodiment, the optical members 11c are different. Here, the functions of the other components are the same as those of the first embodiment. The description of the same function as that of the first embodiment is omitted, and in this embodiment, a part different from that of the first embodiment will be mainly described.

The optical member 11c includes a varifocal lens 80c and a voltage application unit (not shown). The varifocal lens 80c is a lens whose focal length is changed based on the voltage applied by the voltage application unit, and is installed at the position of the image-side focal plane F of the objective lens 10 on the optical axis 31. Therefore, based on the above-mentioned principle, even if the focal length of the varifocal lens 80c is changed, the imaging magnification β of the transmission microscope according to the present embodiment does not change.

The varifocal lens 80c is a liquid crystal lens as an example, and changes the refractive index by changing the angle of the crystal of the liquid crystal according to the magnitude of the voltage applied to the liquid crystal, and as a result, the focal length is changed. .. The variable focus lens 80c is an example of a variable refractive power lens, and can be a liquid lens such as a liquid crystal lens. In FIG. 11, the varifocal lens 80 is displayed as a biconcave negative lens, but it may be a negative lens or a positive lens. If the focal length is variable, the working distance WD can be changed within the variable range, as described above in FIG. 2 and the like.

The voltage application unit (not shown) applies a voltage to the varifocal lens 80c by applying an electric signal to the varifocal lens 80c to switch the refractive power of the varifocal lens 80c. The voltage application unit is an example of the refractive power conversion means. The voltage applied by the voltage application unit to the varifocal lens 80c is controlled by the control unit 41c. That is, the control unit 41c changes the focal length (that is, the refractive power) of the varifocal lens 80c depending on the voltage application unit. The control unit 41c is an example of a working distance changing unit.

The value of the voltage applied to the varifocal lens 80c is stored as the applied voltage information 43c in the storage unit 42c of the control device 40c as an example. The applied voltage information 43c is information indicating the applied voltage value, which is the value of the voltage applied to the varifocal lens 80c, for each bottom surface of each layer of the culture vessel 20. The control unit 41c reads the applied voltage information 43c from the storage unit 42c, and controls the voltage applied to the varifocal lens 80c according to the observation surface O based on the read applied voltage information 43c.

The applied voltage information may be stored in an external database of the transmission microscope 1c. When the applied voltage information 43c is stored in an external database of the transmissive microscope 1c, the control device 40c and this database can communicate with each other, and the control unit 41c reads the applied voltage information from this database and reads the applied voltage information. The voltage applied to the varifocal lens 80c is controlled based on the above.

In the present embodiment, an example in which the focal length of the varifocal lens 80c is changed according to the observation surface O and the working distance WD is changed has been described, but the present invention is not limited to this. The working distance WD may be changed by changing the focal length of the varifocal lens 80c according to the observation surface O and changing the relative position of the objective lens 10 on the optical axis with respect to the stage 9. In this case, if the distance between the objective lens 10 and the varifocal lens 80 is arranged equal to the focal length of the objective lens 10 and the lens 10 is integrally moved in the optical axis direction, the microscope is as described above. The image magnification can be kept constant. The relative position of the objective lens 10 on the optical axis with respect to the stage 9 is changed by the control unit 41c. The control unit 41c is an example of a relative position change unit.

As described above, in the microscope according to the present embodiment (transmission type microscope 1c in the present embodiment), the optical member 11 is a variable optical power lens such as a liquid lens (varifocal lens 80c in the present embodiment). It is provided with a refractive power conversion means (voltage application unit in the present embodiment) for switching the refractive power of the variable refractive power lens (varifocal lens 80c in the present embodiment), and a working distance changing unit (control unit in the present embodiment). 41) changes the refractive power of the variable refractive power lens (variable focus lens 80c in the present embodiment) by the refractive power conversion means (voltage application unit in the present embodiment).
Further, the configuration of the variable refractive power lens according to the fourth embodiment shown in FIG. 11 can be combined with the optical member 11 shown in the third embodiment shown in FIGS. 5 and 6. In this case, when the working distance is switched by the optical member 11, the working distance can be finely adjusted quickly by slightly changing the refractive power (that is, the focal length) of the variable refractive power lens. High-precision observation can be performed at high speed.

With this configuration, in the microscope according to the present embodiment, the working distance WD can be changed more quickly than in the case of mechanically driving the objective lens or the reflection mirror over several tens of centimeters in the optical axis direction. Further, in the microscope according to the present embodiment, the durability of the mechanism for focusing can be improved as compared with the case where mechanical driving is performed.

Further, in the microscope according to the present embodiment (transmission microscope 1c in the present embodiment), the relative position of the stage 9 in which the culture vessel 20 is arranged and the relative position of the objective lens 10 on the optical axis with respect to the stage 9 are changed. It is possible to further provide a unit. For example, when the working distance WD is changed, the relative position of the objective lens 10 on the optical axis with respect to the stage 9 can be changed to compensate for the insufficient amount of change in the focal length of the varifocal lens.
By adding individual movements of the objective lens 10 when switching the conversion lens or changing the working distance WD by the variable focus lens described above, the focusing accuracy and imaging performance when changing the working distance are improved. It is possible to do.

The microscope in the above-described embodiment may be provided in the cell culture apparatus. That is, this cell culture apparatus is a microscope for observing a culture vessel in which a plurality of observation surfaces are stacked, and the light between the objective lens, the imaging lens, the objective lens 10 and the imaging lens 13. An optical member having a variable focal length arranged on the axis and an operation of changing the working distance from the observation surface of the plurality of observation surfaces O to the objective lens 10 by changing the focal length fv of the optical member 11. A microscope including a distance changing unit and a stage on which the culture vessel is arranged is provided.
In the cell culture apparatus provided with the microscope in the above-described embodiment, the working distance WD can be easily changed according to the observation surface O, so that the culture container 20 in which a plurality of observation surfaces O are stacked can be easily observed. Can be done. Moreover, the imaging magnification can be kept constant even when the working distance WD is changed.

Although one embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to the above, and various design changes and the like are made without departing from the gist of the present invention. It is possible to do.

1, 1a, 1b, 1c ... Transmission microscope, 10 ... Objective lens, 13 ... Imaging lens, 31 ... Optical axis, 11, 11c ... Optical member, 41, 41a, 41b, 41c ... Control unit, fv ... Focal length , WD ... Working distance

Claims (15)

A microscope for observing a culture vessel in which multiple observation surfaces are stacked.
With the objective lens
Imaging lens and
An optical member having a variable focal length arranged on the optical axis between the objective lens and the imaging lens,
A working distance changing unit that changes the working distance from the observation surface to the objective lens among the plurality of observation surfaces by changing the focal length of the optical member.
A microscope equipped with. The principal point position of the optical member is arranged so as to coincide with the image-side focal position of the objective lens.
The microscope according to claim 1. The optical member includes a plurality of lenses having different focal lengths.
A lens switching unit for switching a lens arranged on the optical axis among the plurality of lenses controlled by the working distance changing unit is provided.
The microscope according to claim 1 or 2. The microscope according to claim 3, wherein the lens switching unit includes the plurality of lenses in a plurality of sets having different positions in the optical axis direction. The plurality of lenses are composed of a plurality of lens elements arranged on the same optical axis.
The microscope according to claim 3 or 4. In one lens arranged on the optical axis by the lens switching unit, the composite principal point of the plurality of lens elements and the image-side focal point of the objective lens coincide with each other.
The microscope according to claim 5. The lens switching unit is a turret.
The microscope according to any one of claims 3 to 6. The stage on which the culture container is placed and
The microscope according to any one of claims 1 to 7, further comprising a relative position changing portion for changing the relative position of the objective lens on the optical axis with respect to the stage. An oblique light diaphragm having an aperture at a peripheral portion outside the optical axis is provided at the position of the aperture diaphragm.
The microscope according to any one of claims 1 to 8. A phase ring is provided at the position of the aperture diaphragm and the pupil position of the objective lens conjugate with the position of the aperture diaphragm.
The microscope according to any one of claims 1 to 9. A bandpass filter or shading plate is provided at the position of the aperture stop.
The microscope according to any one of claims 1 to 10. The optical member includes a refractive power variable lens and a refractive power conversion means for switching the refractive power of the refractive power variable lens.
The working distance changing unit changes the refractive power of the variable refractive power lens by the refractive power conversion means.
The microscope according to claim 1 or 2. The microscope according to claim 12, wherein the variable refractive power lens is a liquid lens, and the refractive power conversion means switches the refractive power of the variable refractive power lens by applying an electric signal to the liquid lens. The stage on which the culture vessel is placed and
A relative position changing portion for changing the relative position of the objective lens on the optical axis with respect to the stage is further provided.
The microscope according to claim 12 or 13. A microscope for observing a culture vessel in which multiple observation surfaces are stacked.
With the objective lens
Imaging lens and
An optical member having a variable focal length arranged on the optical axis between the objective lens and the imaging lens,
A working distance changing unit that changes the working distance from the observation surface to the objective lens among the plurality of observation surfaces by changing the focal length of the optical member.
The stage on which the culture container is placed and
A cell culture device equipped with a microscope.

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