JP7302676B2 - Microscope and cell culture device - Google Patents

Description

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

In the field of regenerative medicine, a multi-layered culture vessel, which is a culture vessel having a plurality of laminated observation surfaces, is sometimes used in order to culture a large amount of necessary cells. In the multi-layered culture vessel, about 10 observation surfaces are laminated, and the total thickness of all layers is 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 acquiring microscopic images of cells cultured in a multilayer culture vessel, there is no technique for efficiently observing cells by quickly focusing on all observation surfaces. Therefore, at present, the state of cells present in all layers is estimated based on the number of cells and morphological information in the bottom layer of the multilayer culture vessel.
The method of moving the objective lens over all the layers of the multilayer culture vessel and focusing on each of the layers takes time. Also, a long working microscope using the Maksutov Cassegrain Catadioptric system has been developed, but it requires movement of the reflecting mirror in the optical axis direction, and it is difficult to quickly change the focal length.

An observation device for observing an object to be observed in a multilayer culture vessel is known (Patent Document 1). The observation device described in Patent Document 1 has a storage unit that stores a movable trolley mounted with a multi-layered culture vessel containing a plurality of trays, and an optical system that outputs an image formed by the optical system. and an imaging device, and when a carriage on which the multilayer culture vessel is mounted is accommodated in the accommodation unit, the object to be observed in each tray of the multilayer culture vessel is captured by the light of the imaging device in a state where the multilayer culture vessel is mounted on the carriage. placed on the axis.

In the observation device described in Patent Document 1, the optical axes of the imaging device and the lighting device intersect the bottom surface of the tray of the multilayer culture vessel at an angle ranging from 40 degrees to 50 degrees. The device is positioned to observe all trays of the multi-layer culture vessel. Thus, when obliquely observing the bottom surface of the tray of the multilayer culture vessel, the image of the object to be observed on each tray is distorted compared to when observing from the direction perpendicular to the bottom surface of the tray.

JP 2018-139615 A

One aspect of the present invention is a microscope for observing a culture vessel in which a plurality of observation surfaces are stacked, comprising an objective lens, an imaging lens, and an optical axis between the objective lens and the imaging lens an optical member having a variable focal length disposed thereon; and changing the working distance from a first viewing plane to a second viewing plane of the plurality of viewing planes by changing the focal length of the optical member. a working distance changing section; and a lens switching section controlled by the working distance changing section ; comprises a plurality of first lenses divided into a plurality of groups having different positions in the optical axis direction, wherein the plurality of first lenses are each composed of a plurality of lens elements arranged in the optical axis direction, The optical member is configured such that the lens switching unit switches the light beam among the plurality of first lenses in a state in which the distances to the culture vessel, the objective lens, and the imaging lens on the optical axis are kept constant. In the second lens arranged on the optical axis by the lens switching unit, the second lens has a variable focal length by switching the second lens arranged on the axis, and the second lens is arranged on the image side of the objective lens. The microscope is arranged on either the object side or the image side of the focal point, and the composite principal point of the plurality of lens elements and the image side focal point of the objective lens coincide.

One aspect of the present invention is a cell culture device for observing a culture vessel in which a plurality of observation surfaces are stacked, comprising an objective lens, an imaging lens, and between the objective lens and the imaging lens an optical member having a variable focal length arranged on an optical axis; and a working distance from a first observation plane to a second observation plane among the plurality of observation planes by changing the focal length of the optical member. A working distance changing unit to be changed, a stage on which the culture vessel is placed, and a lens switching unit controlled by the working distance changing unit, wherein the optical member has a focal length in the circumferential direction of the optical axis. A plurality of first lenses different from each other are provided, the optical member includes a plurality of groups having different positions in the optical axis direction of the plurality of first lenses, and the plurality of first lenses are each arranged on the optical axis. The optical member is composed of a plurality of lens elements arranged in a direction, and the optical member is arranged to hold a constant distance on the optical axis from the culture container, the objective lens, and the imaging lens. A focal length is variable by a switching unit switching a second lens arranged on the optical axis among the plurality of first lenses, and the second lens arranged on the optical axis by the lens switching unit. wherein the second lens is disposed on either the object side or the image side of the image side focus of the objective lens, and the composite principal point of the plurality of lens elements and the image side focus of the objective lens It is a cell culture device that is consistent with

1 is a diagram showing an example of a transmission microscope according to a first embodiment; FIG. FIG. 2 is an optical path diagram showing a variable working distance configuration according to the first embodiment; FIG. 10 is an optical path diagram showing a variable working distance configuration according to a second embodiment; FIG. 10 is a configuration diagram showing an optical path of an optical system and an outline of an apparatus according to a second embodiment; FIG. 11 is an optical path diagram showing a variable working distance configuration according to a third embodiment; FIG. 11 is a configuration diagram showing an optical path of an optical system and an outline of an apparatus according to a third embodiment; FIG. 11 is a perspective view showing an example of a turret according to a third 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 in the optical system of 3rd Embodiment. It is a figure which shows the structure of the modification in the optical system of 3rd Embodiment. It is a block diagram which shows the optical path of the optical system of 4th Embodiment, and the outline|summary of an apparatus.

(First embodiment)
The first embodiment will be described in detail below with reference to the drawings. FIG. 1 is a diagram showing an example of a transmission microscope 1 according to this embodiment. A transmission microscope 1 is a microscope for observing a culture vessel having a plurality of laminated observation surfaces. The transmission microscope 1 includes an illumination optical system having a light source 3, a condenser lens 4, a field stop 5, an aperture stop 6, a condenser lens 7, and a first reflecting mirror 8, an objective lens 10, an optical It has an imaging optical system having a member 11 , a second reflecting mirror 12 and an imaging lens 13 . Then, the transmission microscope 1 can capture an image of the observation surface of the multi-layered culture device 2 placed on the stage 9 with the imaging device 14 .

The multilayer culture apparatus 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 cells cultured in each tray that constitutes the culture vessel 20 . Here, in the culture container 20, the bottom surfaces of the stacked trays serve as observation surfaces. That is, the culture vessel 20 is a multi-layered culture vessel in which a plurality of observation surfaces are laminated.

In the culture container 20, for example, 10 layers of trays, ie, observation surfaces O are stacked and have a thickness of about 20 centimeters. In the following description, each layer of the culture vessel 20 is called a first layer, a second layer, a third layer, etc. in the order of lamination. The bottom layer is the first layer and the top layer is the tenth layer. Among the layers of the culture container 20, the layers other than the top layer and the bottom layer may be called intermediate layers. Further, regarding the viewing plane O corresponding to each layer, the viewing plane corresponding to the first layer is called viewing plane O1, the viewing plane corresponding to the second layer is called viewing plane O2, and the viewing plane corresponding to the i-th layer is called viewing plane O1. It is referred to as an observation plane Oi (i=1, 2, . . . , 10). Further, the current observation plane among the plurality of observation planes O may be referred to as an observation plane Oj. That is, the observation surface Oj is the observation surface on which the focal position is adjusted among the plurality of observation surfaces O. FIG. FIG. 1 shows a state in which an observation surface O5 corresponding to the intermediate layer, which is the fifth layer, is observed as the observation surface Oj.

Further, among the observation planes O of the ten layers, as an example, the observation plane O5 corresponding to the fifth layer is used as the focusing reference plane. Here, the focus reference plane is a plane that serves as a reference for focusing of the culture container 20 . By moving the objective lens 10 relative to the stage 9 on the optical axis indicated by the dashed line in the figure, the focal position is adjusted to the focusing reference plane. Note that the observation surface of any layer other than the fifth layer of the culture container 20, which is the reference plane for focusing, may be used.

Illumination light from the light source 3 passes through the condensing lens 4 and the condenser lens 7 and becomes a substantially parallel beam. The collimated illumination light is reflected by the first reflecting mirror 8 to illuminate the cells on the observation plane Oj. The cells on the observation surface Oj are cells cultured in the tray corresponding to the observation surface Oj among the trays of each layer of the culture vessel 20 .

In the transmission microscope 1, the condenser lens 7 performs so-called Koehler illumination. Between the condenser lens 4 and the condenser lens 7, a field stop 5 is provided at a position conjugated to the observation plane Oj, and an aperture stop 6 is provided at a position conjugated to the light source 3, respectively.

The light from the observation surface Oj is condensed by the objective lens 10 and becomes a substantially parallel beam. The collimated light beam from the observation surface Oj passes through the optical member 11 , is reflected by the second reflecting mirror 12 , and enters the imaging lens 13 . Light from the viewing plane Oj is condensed onto the imaging plane 15 by the imaging lens 13 to form an enlarged image of the viewing plane Oj.
The imaging device 14 is provided at a position capable of imaging the imaging plane 15 . An enlarged image of the observation plane Oj captured by the imaging device 14 is observed on a monitor (not shown).

In order to simplify the explanation, FIG. 1 shows the light beam from the light source 3 that illuminates the culture container 20 and the imaging light beam from the observation plane Oj to the imaging plane 15 .

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

In the transmission microscope 1, the working distance WD is changed from the working distance Wmax when observing the observation surface O1 corresponding to the first layer, which is the lowest layer, to the observation surface O10 corresponding to the tenth layer, which is the uppermost layer. The working distance can be changed within the range up to the working distance Wmin of . By changing the focal length of the optical member 11, an image of an arbitrary observation plane Oi (i=1, 2, . 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. Needless to say, it is possible to construct a so-called inverted microscope in which the system and the imaging optical system are interchanged.

Here, referring to FIG. 2, taking the microscope optical system 30 as an example, the principle by which the transmission microscope 1 changes the working distance WD will be described. FIG. 2 is a diagram showing a configuration including an example of the microscope optical system 30 according to this embodiment. The microscope optical system 30 is similar to that of the transmission microscope 1 shown in FIG. It is shown.

As described above, the optical member 11 includes various lenses. A 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 this 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 the state of the reference position of the working distance WD ₀ for observing the intermediate layer among the 10 layers of the culture vessel 20. In this reference state, the optical member 11 is not arranged in the optical path. do not have. FIG. 2B shows a state in which the upper layer of the ten layers of the culture vessel 20 is observed, and a negative lens Gv1 as a conversion lens Gv is inserted on the optical axis between the objective lens 10 and the imaging lens 13. and the working distance WD ₁ is greater than the working distance WD ₀ for the intermediate layer. FIG. 2C shows a state in which the lower layer of the ten layers of the culture container 20 is observed, and the positive lens Gv2 as the conversion lens Gv is positioned on the optical axis between the objective lens 10 and the imaging lens 13. , and the working distance WD ₂ is smaller than the working distance WD ₀ in the reference state.

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

As described above, in the transmission microscope 1, the working distance WD of the objective lens 10 can be changed by changing the focal length of the optical member 11, that is, by changing the focal length _fv of the conversion lens Gv. . The working distance WD of the objective lens 10 can be changed 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. It is possible to observe and photograph an image of each layer of trays stacked in multiple layers in the culture container 20 . However, when changing the focal length (that is, the refractive power) of the conversion lens positioned on the image side of the objective lens 10, the working distance WD changes and the composite focus of the objective lens 10 and the optical member 11 (that is, the conversion lens Gv) Since the distance also changes, the imaging magnification of the microscope changes. The size of the image of each layer of the trays stacked in multiple layers in the culture container 20 is different from each other due to the change in the working distance WD. In other words, the imaging magnification changes, and therefore the field of view and the size of the image of the object to be inspected also change.

Here, a configuration that can maintain the imaging magnification β of the transmission microscope 1 constant even when the working distance WD is changed in the microscope optical system 30 will be described.
If 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 , the imaging magnification β of the transmission microscope 1 consisting of the objective lens 10 and the imaging lens 13 is given below. is given by equation (1).

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

When the distance d between the objective lens 10 and the conversion lens Gv is equal to the focal length _f1 of the objective lens 10, d= _f1 , and the combined focal length f is obtained from the above equation (2) by the equation (3 ) is derived.

That is, even if the focal length _fv of the conversion lens Gv is changed, the combined focal length f of the objective lens 10 and the conversion lens Gv remains unchanged.

Therefore, if the conversion lens Gv for making the working distance WD as a microscope objective lens variable is arranged at 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, the operation Even when the distance WD is changed, the combined focal length f of the objective lens 10 and the conversion lens Gv remains unchanged, and as a result the imaging magnification β of the microscope is kept constant.

(Second embodiment)
This state is shown in FIG. Similar to FIG. 2, FIG. 3A shows the state of the reference position at the working distance WD ₀ for observing the 10 intermediate layers of the culture container 20. FIG. FIG. 3B shows a state in which the upper layer of the ten layers of the culture container 20 is observed, and a negative lens Gv ₁ as a conversion lens Gv is placed on the optical axis between the objective lens 10 and the imaging lens 13. inserted, the working distance WD ₁ is greater than the working distance WD ₀ for the intermediate layer. FIG _. 2C shows a state in which the lower layer of the 10 layers of the culture container 20 is observed. Inserted above, the working distance WD ₂ is smaller than the working distance WD ₀ for the intermediate layer. In such a configuration, both the negative lens _Gv1 and the positive lens _Gv2 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 composite focal length of the objective lens 10 and the conversion lens Gv is kept constant, and the magnification of the microscope is kept constant.

Next, referring to 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 optical path of an optical system and an outline of an apparatus according to the second embodiment.
In this embodiment, the optical member 11 has a turret T as an example. The turret T accommodates a plurality of lenses L having different focal lengths. That is, the optical member 11 includes a plurality of lenses L with different focal lengths.

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

In the transmission microscope 1, when the focus position is adjusted to 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. FIG.

In addition to controlling the optical member 11 described above, the control unit 41 performs various controls on the microscope optical system 30 . Various controls performed by the controller 41 include, for example, adjustment of the brightness of the light source 3, movement of the stage 9, minute movement of the objective lens 10 for focusing in the direction of the optical axis, and movement of the optical axis of the imaging lens 13. Directional micro-movements and the like are included.

In the above-described second embodiment, it is possible to keep the imaging magnification constant even when the working distance is arbitrarily changed. The basic configuration for this is to arrange the conversion lens Gv for changing the working distance at the rear focal position (back focal plane F) of the objective lens 10, as shown in FIGS.
However, the focal position of the objective lens 10 is generally an important position as the pupil position of the microscope optical system, and optical elements such as special diaphragms and prisms are arranged especially in phase-contrast microscopes and differential interference microscopes. For this reason, in the second embodiment shown in FIGS. 3 and 4, since the conversion lens Gv is arranged at the rear focal position (back focal plane F) of the objective lens 10, special microscope observation such as phase contrast observation is possible. Getting it right is difficult.

(Third Embodiment)
3rd Embodiment which solves said subject is described using FIG. FIG. 5 is a schematic optical path diagram showing a variable working distance configuration according to the third embodiment. In this optical path diagram, as in FIG. 3, for ease of explanation, the microscope optical system 30 is shown as an expanded optical path diagram without including the first reflecting mirror 8 and the second reflecting mirror 12. ing. Members having the same functions as those in FIG. 3 are given the same symbols.

FIG. 5A shows the state of the reference position at the working distance WD ₀ for observing the 10 intermediate layers of the culture container 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 ten layers of the culture container 20 is observed. 13 on the optical axis. FIG. 5C shows a state in which the lower layer of the 10 layers of the culture container 20 is observed. It is inserted on the optical axis with 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 , the synthetic principal point H as the conversion lens Gv1 coincides with the rear focal plane F of the objective lens 10. FIG. Similarly, as shown in FIG. 5(C), the conversion lens Gv2 is arranged on the image side of the rear focal plane F of the objective lens 10. A composite principal point H as the lens Gv2 coincides with the rear focal plane F of the objective lens 10. FIG.
With such a configuration, the microscope can arbitrarily change the working distance while maintaining a constant imaging magnification as a microscope by means of the conversion lens. A space can be provided at the position, and advanced observation such as phase difference and differential interference is possible.

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

In the transmission microscope of this embodiment, when the focal position is adjusted to the focusing reference plane, as an example, the plurality of lenses L housed in the front stage turret T1 and the rear stage turret T2 are both arranged on the optical axis 31. not.

In addition to controlling the optical member 11 described above, the control unit 41 performs various controls on the microscope optical system 30 . 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, the front turret T1 and the rear turret T2 will be described with reference to FIG. FIG. 7 is a perspective view showing an example of the turret according to this embodiment. The front-stage turret T1 and the rear-stage turret T2 are arranged in two rows overlapping each other in the direction of the optical axis 31 .

The front turret T1 includes a plurality of storages H1. The front-stage turret T1 includes, as a plurality of storages H1, storages H1-1, storages H1-2, storages H1-3, storages H1-4, storages H1-5, and storages H1-6. . The plurality of storage units H1 are arranged in a circle.
The rear turret T2 includes a plurality of storages H2. The rear turret T2 includes, as a plurality of storages H2, a storage H2-1, a storage H2-2, a storage H2-3, a storage H2-4, a storage H2-5, and a storage H2-6. . The plurality of storage sections H2 are arranged in a circle.
The plurality of storage portions H1 and the plurality of storage portions H2 include storage portions in which the lenses L are stored and storage portions in which the lenses L are not stored. Here, the plurality of lenses L housed in the plurality of storage units have different focal lengths.

In the example of FIG. 7, among the plurality of storage units H1 provided in the front turret T1, storage unit H1-1, storage unit H1-2, storage unit H1-3, storage unit H1-4, and storage unit H1-5 contains lenses L1-1, L1-2, L1-3, L1-4, and L1-5 having different composite focal lengths. On the other hand, the lens L is not stored in the storage portion H1-6.
Lenses L2- 1, lens L2-2, lens L2-3, and lens L2-4. On the other hand, the lens L is not housed in the housing portion H2-5 and the housing portion H2-6.
Thus, in this embodiment, the number of lenses L accommodated in the front turret T1 and the rear turret T2 is nine, for example.

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

The front turret T1 and the rear turret T2 are controlled to rotate by a rotation drive section (not shown). This rotation drive section is controlled by the control section 41 . The front-stage turret T1 and the rear-stage 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 L 2 - 3 and lens L 2 - 4 are arranged on the optical axis 31 .
That is, the front-stage turret T1 and the rear-stage 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. FIG. The front turret T1 and the rear turret T2 are examples of a lens switching unit. That is, in this 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 accommodated in the turret that accommodates the lens L arranged on the optical axis 31. Let In addition, the control unit 41 moves the storage section in which the lenses of the other turret are not stored to a position on the optical axis 31, thereby withdrawing the plurality of lenses L stored in the other turret from the optical axis 31. Let

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

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

Step S10: The control unit 41 sets the first observation plane for starting imaging. The control unit 41 sets the first observation surface for starting observation based on a predetermined order. The predetermined order is, for example, the order from the tenth layer, which is the top layer of the culture container 20, to the first layer, which is the bottom layer. The control unit 41 causes the storage unit 42 to store information indicating the set viewing plane as viewing plane information.

Further, in step S<b>10 , the control unit 41 causes the storage unit 42 to store culture container information, which is information indicating the type of the culture container 20 and the like. 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 according to the type of the culture container 20 . The information indicating the lens includes, for example, the focal length of the lens.
For example, the user of the transmission microscope 1 inputs the culture vessel information to the control device 40 from an operation unit (not shown). The control unit 41 acquires the input culture container information. In addition, 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 .
In step S10, the lenses L housed in the front turret T1 and the rear turret T2 may be exchanged according to the type of culture vessel 20. FIG.

Step S20: The controller 41 changes the lens L arranged on the optical axis 31 to a desired lens corresponding to the set observation plane Oj. Based on the culture container information stored in the storage unit 42, the control unit 41 selects a lens to be arranged on the optical axis 31 from the plurality of lenses L accommodated in the front turret T1 and the rear turret T2.
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 placed 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 positions of the optical system of the objective lens 10 and the conversion lens Gv included in the optical member 11 match the position of the observation plane Oj.
When determining that the focus is achieved (step S30; YES), the control unit 41 executes the process of step S40. On the other hand, if the control unit 41 determines that it is not in focus (step S30; NO), it executes the process of step S60.

Step S40: The control section 41 causes the imaging device 14 to perform imaging. Here, imaging means generating an image of a subject (captured image) based on light detected by an imaging device on the imaging plane 15 .

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

Step S<b>60 : The control section 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 plane Oj. After that, the control unit 41 executes the process of step S40.

Step S70: The control section 41 sets the viewing plane Oj. Here, the control unit 41 sets the observation surface Oj, for example, based on the predetermined order indicated by the incubation 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 this, the control device 40 ends the imaging process.

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

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 vessel 20 in which a plurality of observation surfaces O are stacked can be easily observed. can.

Further, in the microscope according to this embodiment (in this embodiment, the transmission microscope 1), the principal point position of the optical member 11 (in this embodiment, the positions of the negative lens Gv ₁ and the positive lens Gv ₂ which are the conversion lenses Gv) ) are arranged 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 kept constant regardless of the focal length fv of the optical member 11. Therefore, the imaging magnification β of the microscope can be kept constant.

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

With this configuration, the microscope according to the present embodiment can switch the lens arranged on the optical axis 31 among the 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 (the transmission microscope 1 in the present embodiment), the lens switching section (the front turret T1 and the rear turret T2 in the present embodiment) includes a plurality of lenses (in the present embodiment, A plurality of lenses L) are divided into a plurality of sets with mutually different positions in the optical axis direction.

With this configuration, in the microscope according to the present embodiment, a plurality of lenses can be stacked and stored in the direction of the optical axis 31. Therefore, compared to the case where the plurality of lenses are not divided into a plurality of groups with mutually different positions in the optical axis direction, Therefore, a plurality of lenses can be stored compactly in the radial direction perpendicular to the optical axis 31 .

Further, in the microscope according to the present embodiment (the transmission microscope 1 in the present embodiment), the plurality of lenses (the plurality of lenses L in the present embodiment) are a plurality of lens elements arranged on the same optical axis. (in this embodiment, 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 for each of the plurality of first lenses can be increased compared to the case where the lens is a single lens. Compared to the case of a lens, it is easier to change the working distance WD according to the viewing plane Oj.

Further, in the microscope according to the present embodiment (the transmission microscope 1 in the present embodiment), one lens is arranged on the optical axis 31 by the lens switching section (the front turret T1 and the rear turret T2 in the present embodiment). In the lens, the composite principal point H of a plurality of lens elements (negative lens element and positive lens element in this embodiment) coincides with the image side focus 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 point of the objective lens 10, even if the focal length of the lens is changed, the objective lens 10 Since the combined focal length f of a plurality of lenses can be kept unchanged, when the position of the lens on the optical axis 31 deviates from the position of the image-side focal point of the objective lens 10, the working distance WD is changed. Also, the imaging magnification β of the transmission microscope 1 can be kept constant.

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

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 the focal length of the optical member 11 can be easily and quickly changed.

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

Modification 1 of the above-described third embodiment will be described below with reference to the drawings. In this modified example, the transmission microscope is provided with an oblique light stop to enable oblique illumination.
The microscope according to this modification is called a transmission microscope 1a, and the optical system of the transmission microscope 1a is called a microscope optical system 30a.

FIG. 9 is a diagram showing an example of the configuration of a 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 an oblique light stop 60a is provided. Here, the functions of the other components (light source 3, condenser lens 4, field stop 5, aperture stop 6, condenser lens 7, objective lens 10, optical member 11, and imaging lens 13) are those of the first embodiment. Same as form. A description of the same functions as those of the first embodiment will be omitted, and the description of this modification will focus on the portions that differ from those of the third embodiment.

As an example, the oblique light stop 60a is provided at the position of the aperture stop 6 and has an aperture in the peripheral portion outside the optical axis.

The oblique light diaphragm 60a has, for example, a disk-shaped opening 600a having an arcuate shape for transmitting light and a light blocking portion 601a for blocking light. For example, the opening 600a is formed in an arc shape with a central angle of 90 degrees. The width of the opening 600a is, for example, 1 millimeter. Since the aperture 600a is off the optical axis, the direction of the ray of light emitted from the light source 3 of the microscope optical system 30a can obliquely illuminate the observation surface Oj, thus performing so-called oblique illumination. This illumination method enables observation of finer patterns.

In this embodiment, the oblique light diaphragm 60a is controlled by the control unit 41a to rotate, for example, every 90 degrees around the optical axis passing through the light source and the subject. As the oblique light stop 60a rotates, the position of the aperture 600a rotates every 90 degrees around the optical axis passing through the light source and the subject. That is, the oblique light stop 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 oblique illumination light can be changed.

In the transmission microscope according to this modified example, the oblique light diaphragm 60a is rotated by the control unit 41a to change the incident angle of the illumination light by 90 degrees to capture an image of the cell, which is the subject. An image processing unit provided in the imaging device 14 synthesizes a plurality of captured images captured by changing the incident angle by 90 degrees to generate one captured image.
A captured image obtained by synthesizing a plurality of captured images captured by changing the incident angle of the illumination light has a higher contrast than when the plurality of captured images are not combined.

In the configuration of FIG. 9 above, a bandpass filter can be arranged at the position of the aperture stop 6 or in the vicinity thereof. A bandpass filter is a filter that transmits light of long wavelengths and does not transmit light of short wavelengths, where long and short wavelengths are used, as an example, by cutting wavelengths shorter than 660 nanometers to reduce 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 cell, which is the object, and to improve the image quality of the captured image.

Note that the bandpass filter in this modified example is an example and is not limited to this, and the bandpass filter may be a bandpass filter with a center of 625 nm and a bandwidth of about 20 nm. Moreover, it is also possible to use together with the light-shielding plate for oblique illumination mentioned above. Furthermore, instead of the bandpass filter, a light shielding plate may be arranged at the position of the aperture stop 6 or in the vicinity thereof.

(Modification 2 of the third embodiment)
Hereinafter, the configuration of the modified example of the above-described third embodiment will be described with reference to the drawings. In this modified example, a case will be described in which a transmission microscope is provided with a phase ring and phase contrast observation is performed.
The microscope according to this modified example is called a transmission microscope 1b, and the optical system of the transmission microscope 1b is called a microscope optical system 30b.

FIG. 10 is a diagram showing an example of the configuration of a 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, a first phase ring 70b and a second phase ring 71b are provided. There is a difference. Here, functions of other components are the same as in the third embodiment. Descriptions of functions that are the same as those of the third embodiment will be omitted, and differences will be mainly described in this modified example.

The first phase ring 70b is provided at the position of the aperture stop 6 in the illumination system and has an annular aperture.

The second phase ring 71b is arranged at a position conjugate with the position of the aperture stop 6 on the optical axis 31 in the imaging system, specifically at the rear focal position of the objective lens 10. As shown in FIG. 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 of 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 it is shifted from the pupil position. Even if the conjugate relationship between the first phase ring 70b provided at the position of the aperture stop 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, in the case of low magnification, the position Phase difference observation is possible. Also, in FIG. 10, the two phase rings 70 and 71 are shown to have substantially the same size for the sake of simplicity, but as long as they maintain a conjugate relationship with each other, the size can be appropriately changed according to the magnification. needless to say.

In the phase-contrast microscope according to this modification, phase-contrast observation becomes possible by providing the phase rings (the first phase ring 70b and the second phase ring 71b). Phase-contrast observation makes it possible to observe a clear image due to the refractive index difference even if the object to be observed is a transparent sample.

(Fourth embodiment)
A fourth embodiment of the present invention will be described below with reference to the drawings.
In the first, second and third embodiments, the focal length ( That is, the working distance WD is changed by changing the refractive power. In this embodiment, in the microscope, the optical member 11 itself as a conversion lens has a varifocal lens, and the working distance is changed according to the change in the focal length of this varifocal lens.
The microscope according to this embodiment is called a transmission microscope 1c, and the optical system of the transmission microscope 1c is called a microscope optical system 30c.

FIG. 11 is a diagram showing an example of the configuration of the microscope optical system 30c according to this 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 member 11c is different. Here, functions of other components are the same as in the first embodiment. The description of the same functions as those of the first embodiment will be omitted, and the description of this embodiment will focus on the parts that are different from those of the first embodiment.

The optical member 11c includes a varifocal lens 80c and a voltage applying section (not shown). The varifocal lens 80 c is a lens whose focal length is changed based on the voltage applied by the voltage applying section, and is installed at the image side focal plane F of the objective lens 10 on the optical axis 31 . Therefore, based on the principle described above, even if the focal length of the varifocal lens 80c is changed, the imaging magnification β of the transmission microscope according to this embodiment remains unchanged.

The varifocal lens 80c is, for example, a liquid crystal lens, 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, thereby changing the focal length. . 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. Although FIG. 11 shows the varifocal lens 80 as a biconcave negative lens, 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 with reference to FIG. 2 and the like.

A voltage application unit (not shown) applies an electric signal to the varifocal lens 80c to apply a voltage to the varifocal lens 80c, thereby switching the refractive power of the varifocal lens 80c. The voltage application section is an example of refractive power conversion means. The voltage applied by the voltage application section to the varifocal lens 80c is controlled by the control section 41c. That is, the control unit 41c changes the focal length (that is, refractive power) of the varifocal lens 80c by 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 applied voltage information 43c in the storage unit 42c of the control device 40c, for example. The applied voltage information 43 c is information indicating the applied voltage value, which is the value of the voltage applied to the varifocal lens 80 c , for each bottom surface of each layer of the culture container 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 plane O based on the read applied voltage information 43c.

The applied voltage information may be stored in a database outside the transmission microscope 1c. When the applied voltage information 43c is stored in a database outside the transmission microscope 1c, the controller 40c can communicate with this database. to control the voltage applied to the varifocal lens 80c.

In this embodiment, an example in which the focal length of the varifocal lens 80c is changed according to the viewing plane O to change the working distance WD has been described, but the present invention is not limited to this. By changing the focal length of the varifocal lens 80c and changing the relative position of the objective lens 10 to the stage 9 on the optical axis according to the observation plane O, the working distance WD may be changed. In this case, when the objective lens 10 and the varifocal lens 80 are arranged so that the distance between them is equal to the focal length of the objective lens 10, and they move integrally in the direction of the optical axis, as described above, the microscope , can be kept constant. The relative position of the objective lens 10 to the stage 9 on the optical axis is changed by the controller 41c. The controller 41c is an example of a relative position changer.

As described above, in the microscope according to the present embodiment (the transmission microscope 1c in the present embodiment), the optical member 11 includes a refractive power variable lens such as a liquid lens (the variable focus lens 80c in the present embodiment). a refractive power conversion unit (voltage applying unit in this embodiment) for switching the refractive power of the variable refractive power lens (varifocal lens 80c in this embodiment); 41) changes the refractive power of the variable refractive power lens (variable focus lens 80c in this embodiment) by the refractive power conversion means (voltage applying section in this embodiment).
Furthermore, it is possible to combine the configuration of the variable refractive power lens according to the fourth embodiment shown in FIG. 11 with the optical member 11 shown in the third embodiment shown in FIGS. In this case, when the working distance is switched by the optical member 11, by slightly changing the refractive power (that is, the focal length) of the variable refractive power lens, it is possible to quickly finely adjust the working distance. 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 mechanical driving that moves the objective lens and reflecting mirror over several tens of centimeters in the optical axis direction. Also, in the microscope according to the present embodiment, the durability of the mechanism for focusing can be improved compared to the case of mechanical driving.

Further, in the microscope according to the present embodiment (the transmission microscope 1c in the present embodiment), the stage 9 on which the culture container 20 is arranged and the relative position change for changing the relative position of the objective lens 10 on the optical axis with respect to the stage 9 It is possible to further comprise a part. For example, when changing the working distance WD, the relative position of the objective lens 10 with respect to the stage 9 on the optical axis can be changed to compensate for the insufficient amount of change in the focal length of the varifocal lens.
Incidentally, when the conversion lens is switched or the working distance WD is changed by the varifocal lens, by individually moving the objective lens 10, the focusing accuracy and the imaging performance are improved when the working distance is changed. It is possible to

In addition, the microscope in the embodiment described above may be provided in a cell culture apparatus. In other words, this cell culture apparatus is a microscope for observing a culture container in which a plurality of observation surfaces are stacked, and includes an objective lens, an imaging lens, and light between the objective lens 10 and the imaging lens 13. An optical member with a variable focal length arranged on the axis, and an operation for changing the working distance from the observation plane of the plurality of observation planes 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 vessel 20 in which a plurality of observation surfaces O are stacked can be easily observed. can be done. Moreover, it is possible to keep the imaging magnification constant even when the working distance WD is changed.

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

DESCRIPTION OF SYMBOLS 1, 1a, 1b, 1c... Transmission type microscope, 10... Objective lens, 13... Imaging lens, 31... Optical axis, 11, 11c... Optical member, 41, 41a, 41b, 41c... Control part, fv... Focal length , WD ... working distance

Claims (7)

A microscope for observing a culture vessel in which a plurality of observation surfaces are stacked,
an objective lens;
an imaging lens;
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 a first viewing plane to a second viewing plane among the plurality of viewing planes by changing the focal length of the optical member;
a lens switching unit controlled by the working distance changing unit;
with
The optical member includes a plurality of first lenses having different focal lengths in the circumferential direction of the optical axis,
wherein the optical member comprises a plurality of groups having different positions in the optical axis direction of the plurality of first lenses;
The plurality of first lenses are each composed of a plurality of lens elements arranged in the optical axis direction,
The optical member is configured such that the lens switching unit switches the light beam among the plurality of first lenses in a state in which the distances to the culture vessel, the objective lens, and the imaging lens on the optical axis are kept constant. The focal length is variable by switching the second lens arranged on the axis,
In the second lens arranged on the optical axis by the lens switching unit, the second lens is arranged on either the object side or the image side of the image side focal point of the objective lens, and A composite principal point of the plurality of lens elements and an image-side focus of the objective lens are aligned.
microscope. The lens switching unit is a turret,
A microscope according to claim 1 . a stage on which the culture vessel is placed;
3. The microscope according to claim 1 , further comprising a relative position changer for changing a relative position of said objective lens with respect to said stage on the optical axis. At the position of the aperture stop, an oblique light stop having an aperture in the peripheral part outside the optical axis is provided,
4. A microscope according to any one of claims 1-3 . A phase ring is provided at the position of the aperture stop and at the pupil position of the objective lens conjugate with the position of the aperture stop,
5. A microscope according to any one of claims 1-4 . Equipped with a bandpass filter or light shield at the position of the aperture stop,
6. A microscope according to any one of claims 1-5 . A cell culture device for observing a culture container in which a plurality of observation surfaces are stacked,
an objective lens;
an imaging lens;
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 a first viewing plane to a second viewing plane among the plurality of viewing planes by changing the focal length of the optical member;
a stage on which the culture vessel is placed;
a lens switching unit controlled by the working distance changing unit;
with
The optical member includes a plurality of first lenses having different focal lengths in the circumferential direction of the optical axis,
wherein the optical member comprises a plurality of groups having different positions in the optical axis direction of the plurality of first lenses;
The plurality of first lenses are each composed of a plurality of lens elements arranged in the optical axis direction,
The optical member is configured such that the lens switching unit switches the light beam among the plurality of first lenses in a state in which the distances to the culture vessel, the objective lens, and the imaging lens on the optical axis are kept constant. The focal length is variable by switching the second lens arranged on the axis,
In the second lens arranged on the optical axis by the lens switching unit, the second lens is arranged on either the object side or the image side of the image side focal point of the objective lens, and A composite principal point of the plurality of lens elements and an image-side focus of the objective lens are aligned.
Cell culture device.

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