JP7156190B2 - Cell observation device - Google Patents

Description

The present invention relates to a cell observation device.

A digital holography device capable of observing the state of cells in the process of culturing pluripotent stem cells such as ES cells and iPS cells is known. The digital holography device includes a laser light source that emits coherent light, a half mirror that splits the coherent light into object light and reference light, an image sensor that obtains a hologram image from the object light and the reference light, a half mirror and an image sensor. An object light optical member (mirror and lens) placed on the optical path of the object light between the sensor and a reference light optical member (mirror and lens) placed on the optical path of the reference light between the half mirror and the image sensor There is a digital holography device including a mirror and a beam expander (see, for example, Patent Document 1). In the digital holography device described in Patent Document 1, it is necessary to equalize the path length of the object light and the reference light in order to obtain a clear hologram image.

JP-A-10-268740

However, in the digital holography device described in Patent Document 1, simply assembling the laser light source, the half mirror, the image sensor, the optical member for the object beam, and the optical member for the reference beam does not make the path length of the object beam and the path length of the reference beam are not equal. Therefore, the positions of these members constituting the digital holography device are finely adjusted to equalize the path length of the object light and the path length of the reference light. In this way, when assembling the digital holography device, fine adjustments are made, and there is a problem that the assembling work becomes complicated accordingly.

An object of the present invention is to provide a cell observation device that can be assembled easily and accurately (to the extent that clear interference fringes can be detected).

An aspect of the present invention is a cell observation device for observing cells,
a light source that emits coherent light;
Between a first core portion disposed on the coherent light emitting side with respect to the light source and through which object light caused by the coherent light and directed toward the cell passes, and the object light caused by the coherent light an optical waveguide having a second core portion through which the reference light that generates interference fringes passes;
A detection unit that detects the interference fringes,
the first core portion has a first incident surface on which the object light is incident and a first exit surface from which the object light is emitted;
the second core portion has a second incident surface on which the reference light is incident and a second exit surface from which the reference light is emitted;
Cell observation, wherein the path length of the object light from the first incident surface to the first exit surface and the path length of the reference light from the second incident surface to the second exit surface are the same length. Regarding the device.

According to the present invention, an optical waveguide in which the first core portion and the second core portion have the same total length can be used as a part of the components constituting the cell observation device. By simply incorporating this optical waveguide, each path length becomes equal, so that the cell observation device can be assembled easily and accurately (to the extent that clear interference fringes can be detected).

FIG. 1 is a side view showing the first embodiment of the cell observation device of the present invention. FIG. 2 is an enlarged view of area [A] and area [B] surrounded by two-dot chain lines in FIG. 3 is a vertical cross-sectional view of a position regulating portion provided in the cell observation device shown in FIG. 1. FIG. 4 is a view (plan view) seen from the direction of arrow C in FIG. 3. FIG. FIG. 5 is a side view showing a second embodiment of the cell observation device of the invention.

The cell observation device of the present invention will now be described in detail based on preferred embodiments shown in the accompanying drawings.
<First Embodiment>
FIG. 1 is a side view showing the first embodiment of the cell observation device of the present invention. FIG. 2 is an enlarged view of area [A] and area [B] surrounded by two-dot chain lines in FIG. 3 is a vertical cross-sectional view of a position regulating portion provided in the cell observation device shown in FIG. 1. FIG. 4 is a view (plan view) seen from the direction of arrow C in FIG. 3. FIG. In the following, for convenience of explanation, the upper side in FIGS. 1 to 3 is called "upper (or upper)" and the lower side is called "lower (or lower)" (the same applies to FIG. 5).

The cell observation device 1 shown in FIG. 1 is an off-axis type device that captures a holographic image obtained from interference fringes and observes the state of a cell Q. As shown in FIG. In recent years, in the field of regenerative medicine, for example, in the process of culturing pluripotent stem cells such as ES cells and iPS cells, the state of cell Q has been noninvasively measured and analyzed. The cell observation device 1 is suitable for use in this field of regenerative medicine.

The cell observation device 1 includes a light source 2 , a light dividing section 3 , an optical waveguide 4 , a light superimposing section 5 , a detecting section 6 , a position restricting section 7 and a control section 8 . The cell observation device 1 also includes a support section 11 that supports the culture plate 10 . The culture plate 10 contains cells Q to be observed. The configuration of each part will be described below.

The light source 2 has a laser diode 21 that emits coherent light (laser light) L<b>0 and a drive current supply section 22 that supplies drive current to the laser diode 21 .
The wavelength of the coherent light L0 emitted from the laser diode 21 is preferably 600 nm to 800 nm, for example.
Further, the drive current supply unit 22 can adjust the intensity of the coherent light L0 emitted from the laser diode 21, the coherence length, and the like.

A light splitting section 3 is arranged on the side from which the coherent light L0 is emitted with respect to the light source 2 . The light splitting unit 3 is not particularly limited, and a dichroic mirror is used as an example in this embodiment. When the light splitting unit 3 is composed of a dichroic mirror, the coherent light L0 incident on the light splitting unit 3 has a specific wavelength component reflected therefrom and becomes object light (measurement light) L1 directed toward the cell Q. Light of the remaining wavelength components is transmitted and becomes reference light L2. Thereby, the coherent light L0 can be split into the object light L1 and the reference light L2. Therefore, both the object light L1 and the reference light L2 are light caused by the coherent light L0.

An optical waveguide 4 is arranged adjacent to the light splitting section 3 . The optical waveguide 4 has a first core portion (first path) 41 through which the object light L1 passes, a second core portion (second path) 42 through which the reference light L2 passes, and a clad portion 43 .
As shown in FIG. 2, the first core portion 41 has an elongated shape and is provided on one side thereof with a first entrance surface 411 on which the object light L1 is incident, and on the other side thereof. and a first exit surface 412 for exiting.
The second core portion 42 has an elongated shape, and is provided on one side thereof with a second incident surface 421 on which the reference light L2 is incident, and is provided on the other side with a second emission surface 422 from which the reference light L2 is emitted. and

The first core portion 41 and the second core portion 42 are arranged inside the clad portion 43 . The refractive indices of the first core portion 41 and the second core portion 42 are higher than the refractive index of the clad portion 43 . As a result, the object light L1 incident from the first incident surface 411 is totally reflected at the boundary between the first core portion 41 and the clad portion 43, and can be emitted from the first exit surface 412 as it is. Similarly, the reference light L2 incident from the second incident surface 421 can be totally reflected at the boundary between the second core portion 42 and the clad portion 43 and can be emitted from the second exit surface 422 as it is.

The materials for forming the first core portion 41, the second core portion 42, and the clad portion 43 are not particularly limited. Silicon dioxide (SiO ₂ ) can be used as a constituent material of the cladding portion 43 . In this case, to manufacture the optical waveguide 4, first, a plate member made of silicon is prepared. Then, masking (resist) is applied to the portions of the plate member where the first core portion 41 and the second core portion 42 are to be formed by photolithography, and heat treatment is applied to the exposed portions. As a result, the exposed portion is oxidized to silicon dioxide and functions as the clad portion 43 . On the other hand, the portions other than the cladding portion 43 remain made of silicon and thus function as the first core portion 41 and the second core portion 42 .

The optical superimposing section 5 is arranged apart from the optical waveguide 4 . The light superimposing unit 5 superimposes the object light L1 emitted from the first emission surface 412 and the reference light L2 emitted from the second emission surface 422 . The light superimposing unit 5 is not particularly limited, and a dichroic mirror is used as an example in this embodiment. When the light superimposing unit 5 is composed of a dichroic mirror, the object light L1 incident on the light superimposing unit 5 is reflected toward the detecting unit 6, and the reference light L2 incident on the light superimposing unit 5 is transmitted and detected as it is. Head to part 6. As a result, the object light L1 and the reference light L2 overlap and interfere with each other, and are received by the light receiving surface 61 of the detection unit 6 as they are. Interference fringes (interference images) are generated on the light receiving surface 61 by interference between the object light L1 and the reference light L2.

A support portion 11 is arranged between the first emission surface 412 of the optical waveguide 4 and the light superimposing portion 5 . The support portion 11 can horizontally support the culture plate 10 . Further, the object light L1 can pass through the culture plate 10 together with the cells Q on the way from the first emission surface 412 to the light superimposing section 5 . This passage changes the phase of the object light L1. Thereby, interference fringes can be generated with the reference light 15 whose phase does not change.

The detection unit 6 has a light receiving surface 61 that receives the object light L1 and the reference light L2, and can detect interference fringes. The detector 6 is not particularly limited, and for example, a CCD image sensor can be used.
The control section 8 is electrically connected to the light source 2 and the detection section 6 and controls their operations.

The interference fringes detected by the detector 6 , that is, the two-dimensional light intensity distribution data on the light receiving surface 61 are transmitted to the controller 8 . Then, the control unit 8 executes predetermined arithmetic processing on this data to calculate phase information, intensity information, etc. of the entire observation target region (cell Q). Further, the control unit 8 creates a phase image and an intensity image of the entire observation target region based on the calculated phase information, intensity information, and the like. Note that a known algorithm disclosed in, for example, International Publication No. 2016/084420 can be used when calculating phase information or creating a phase image.
Moreover, unnecessary interference fringes caused by the culture plate 10 may also be included on the light receiving surface 61 . In this case, for example, the unnecessary interference fringes can be reduced by performing known frequency adjustment disclosed in International Publication No. 2017/204013.

As described above, the detector 6 detects interference fringes. The interference fringes are preferably sharp. Therefore, in the cell observation apparatus 1, the first path length PL1 of the object light L1 from the first incident surface 411 to the light receiving surface 61 and the second path length PL2 of the reference light L2 from the second incident surface 421 to the light receiving surface 61 and must have the same length (equals). The cell observation device 1 is configured such that the first path length PL1 and the second path length PL2 are the same length. This configuration will be described below. In addition, "the same length" means that the total length of the two first core portions 41 and the total length of the second core portion 42 are within an error equal to or less than the coherence length (coherent length) of the light source (laser diode 21). Say something (hereafter, common).

The object light L<b>1 emitted from the light splitting section 3 passes through the first core section 41 . Since the first core portion 41 is a part of the optical waveguide 4, the total length of the first core portion 41, that is, the path length PL1-1 of the object light L1 from the first entrance surface 411 to the first exit surface 412 is maintained constant.
Similarly, the reference light L2 emitted from the light splitting section 3 passes through the second core section 42 . Since the second core portion 42 is a part of the optical waveguide 4, the total length of the second core portion 42, that is, the path length PL2-1 of the reference light L2 from the second entrance surface 421 to the second exit surface 422 is maintained constant.

In the cell observation device 1 (optical waveguide 4), the path length PL1-1 and the path length PL2-1 are the same length. The method for making both lengths the same is not particularly limited, and a normal micromachine manufacturing technique can be used. As a result, since micron-level processing is possible in both wet and dry processes, the optical waveguide 4 can be manufactured with an error equal to or less than the coherence length.

As described above, the cell observation device 1 has the position regulating section 7 . The position regulating portion 7 is a member that regulates the positional relationship of the light source 2 , the light splitting portion 3 , the optical waveguide 4 , the light superimposing portion 5 and the detecting portion 6 . As shown in FIGS. 3 and 4, the position regulating portion 7 includes a light source mounting portion 71, a light splitting portion mounting portion 72, an optical waveguide mounting portion 73, a light superimposing portion mounting portion 74, and a detection portion mounting portion 75. and

The light source mounting portion 71 is configured by a concave portion, and the light source 2 can be detachably mounted by inserting and removing the light source 2 into and from the concave portion. Then, in a state where the light source 2 is attached to the light source attachment portion 71, the light source 2 is positioned and fixed.
The light splitting section mounting portion 72 is configured by a recess communicating with the light source mounting section 71, and the light splitting section 3 can be detachably mounted by inserting and removing the light splitting section 3 into and out of the recess. In the state where the light splitting section 3 is attached to the light splitting section attachment section 72, positioning and fixing with respect to the light splitting section 3 are performed.

The optical waveguide mounting portion 73 is composed of a concave portion communicating with the optical splitting portion mounting portion 72, and the optical waveguide 4 can be detachably mounted by inserting and removing the optical waveguide 4 into and from the concave portion. Then, in a state where the optical waveguide 4 is attached to the optical waveguide attachment portion 73, positioning and fixing with respect to the optical waveguide 4 are performed.
The light superimposing section mounting section 74 is formed of a recess, and the light superimposing section 5 can be detachably mounted by inserting and removing the light superimposing section 5 into and out of the recess. Then, in a state in which the light superimposing section 5 is mounted on the light superimposing section mounting section 74, positioning and fixing with respect to the light superimposing section 5 are performed.

The detection section mounting section 75 is formed of a recess communicating with the light superimposing section mounting section 74, and the detection section 6 can be detachably mounted by inserting and removing the detection section 6 into and from the recess. Then, in a state in which the detection section 6 is attached to the detection section attachment section 75, the detection section 6 is positioned and fixed.
Further, the position regulating portion 7 has a communicating portion 76 that communicates the optical waveguide mounting portion 73 and the light superimposing portion mounting portion 74 . The communication portion 76 is also formed of a concave portion, and the reference light L2 from the second emission surface 422 of the optical waveguide 4 to the light superimposing portion 5 can pass through.

With such a position regulating portion 7, the path length PL1-2 of the object light L1 from the first output surface 412 to the light receiving surface 61 and the path length PL2- of the reference light L2 from the second output surface 422 to the light receiving surface 61 are determined. 2 have the same length, the positional relationship between the optical waveguide 4, the light superimposing portion 5, and the detecting portion 6 can be regulated.
As shown in FIGS. 3 and 4, when the X-axis, Y-axis, and Z-axis are set to be orthogonal to each other, the X-axis direction, Y-axis direction, and Z-axis direction of the light source mounting portion 71 to the detection portion mounting portion 75 are set. is preferably within ±5 μm in any direction, for example.

As described above, in the cell observation device 1, the path length PL1-1 and the path length PL2-1 are the same length, and the path length PL1-2 and the path length PL2-2 are the same length. . Therefore, the first path length PL1, which is the sum of the path lengths PL1-1 and PL1-2, and the second path length PL2, which is the sum of the path lengths PL2-1 and PL2-2, are also the same length. It will be As a result, the interference fringes detected by the detector 6 can be kept clear.

Further, when assembling the cell observation device 1 , the optical waveguide 4 in which the first core portion 41 and the second core portion 42 have the same total length is used as a part of the components constituting the cell observation device 1 . Further, the light source 2 is inserted into the light source mounting portion 71, the light splitting portion 3 is inserted into the light splitting portion mounting portion 72, the optical waveguide 4 is inserted into the optical waveguide mounting portion 73, and the light superimposing portion is mounted into the light superimposing portion mounting portion 74. Only by a simple assembly of inserting the part 5 and inserting the detection part 6 into the detection part mounting part 75, fine adjustment of the positions of the light source 2 to the detection part 6 is omitted, and the light source 2 to the detection part 6 can be accurately mounted. Can be easily placed in position. Accordingly, the cell observation device 1 can be assembled easily and accurately, that is, to the extent that clear interference fringes can be detected, regardless of the assembly ability of the assembly operator.

As shown in FIG. 2, the first entrance surface 411 and the first exit surface 412 are parallel, and the second entrance surface 421 and the second exit surface 422 are parallel (hereafter referred to as "parallel state"). ”). Further, the angle θ1 formed between the first incident surface 411 and the second incident surface 421 is a right angle, and the angle θ2 formed between the first exit surface 412 and the second exit surface 422 is a right angle (hereinafter this state will be referred to as “Right angle”). As a result, when designing the cell observation device 1, for example, if the positional relationship of the light splitting section 3, the optical waveguide 4, the light superimposing section 5, and the detecting section 6 on the position regulating section 7 is determined in advance, the light guiding section The object light L1 and the reference light L2 can be designed to be directed to the detection unit 6 by setting the wave path 4 in a parallel state and a perpendicular state.

<Second embodiment>
FIG. 5 is a side view showing a second embodiment of the cell observation device of the invention.
Hereinafter, the second embodiment of the cell observation device of the present invention will be described with reference to this figure, but the description will focus on the differences from the above-described embodiment, and the description of the same items will be omitted.
This embodiment is the same as the first embodiment, except that it has a condensing section.

As shown in FIG. 5, in the present embodiment, the cell observation device 1 includes a condensing section 9 that is arranged between the first exit surface 412 and the light superimposing section and condenses the object light L1. The condensing section 9 has a biconvex lens 91 and a fixing section 92 that fixes the biconvex lens 91 on the light superimposing section 5 .
For example, when the culture plate 10 is relatively large or the light superimposing portion 5 is relatively large, the optical distance (the path length of the object light L1) from the cell Q of the culture plate 10 to the detecting portion 6 increases. , the resolution of the detection unit 6 may be lowered. In this case, the focal length (work distance) of the object light L1 can be adjusted by the condensing section 9 to prevent the resolution of the detecting section 6 from being lowered.

Although the illustrated embodiment of the cell observation device of the present invention has been described above, the present invention is not limited to this, and each part constituting the cell observation device may have any configuration capable of exhibiting similar functions. can be replaced with Moreover, arbitrary components may be added.
Also, the cell observation device of the present invention may be a combination of any two or more configurations (features) of the above embodiments.

[Aspect]
It will be appreciated by those skilled in the art that the multiple exemplary embodiments described above are specific examples of the following aspects.

(Section 1) A cell observation device according to one aspect includes:
A cell observation device for observing cells,
a light source that emits coherent light;
Between a first core portion disposed on the coherent light output side with respect to the light source and through which object light caused by the coherent light and directed toward the cell passes, and the object light caused by the coherent light an optical waveguide having a second core portion through which the reference light that generates interference fringes passes;
A detection unit that detects the interference fringes,
the first core portion has a first incident surface on which the object light is incident and a first exit surface from which the object light is emitted;
the second core portion has a second incident surface on which the reference light is incident and a second exit surface from which the reference light is emitted;
The path length of the object light from the first incident surface to the first exit surface and the path length of the reference light from the second incident surface to the second exit surface are the same.

According to the cell observation device described in Item 1, the optical waveguide in which the first core portion and the second core portion have the same total length can be used as part of the components constituting the cell observation device. By simply incorporating this optical waveguide, each path length becomes equal, so that the cell observation device can be assembled easily and accurately.

(Section 2) In the cell observation device according to Section 1,
The detection unit has a light receiving surface that receives the object light and the reference light,
The path length of the object light from the first exit surface to the light receiving surface and the path length of the reference light from the second exit surface to the light receiving surface are the same.

According to the cell observation device described in item 2, the path length of the object light from the first incident surface to the light receiving surface and the path length of the reference light from the second incident surface to the light receiving surface are the same length. can be done. As a result, the interference fringes detected by the detector can be kept clear.

(Section 3) In the cell observation device according to Section 1 or 2,
A light splitting unit is provided between the light source and the optical waveguide and splits the coherent light into the object light and the reference light.

According to the cell observation device described in Item 3, it is possible to generate object light and reference light resulting from coherent light.

(Item 4) In the cell observation device according to any one of items 1 to 3,
the first incident surface and the first exit surface are parallel,
The second entrance surface and the second exit surface are parallel.

According to the cell observation device described in Item 4, when designing the cell observation device, the design can be such that the object light and the reference light are directed toward the detection unit.

(Item 5) In the cell observation device according to any one of items 1 to 4,
an angle formed by the first incident surface and the second incident surface is a right angle;
The angle formed by the first exit surface and the second exit surface is a right angle.

According to the cell observation device described in item 5, when designing the cell observation device, the object light and the reference light can be designed to be directed toward the detection unit.

(Section 6) In the cell observation device according to any one of Sections 1 to 5,
A position regulating portion that regulates the mutual positional relationship between the light source, the optical waveguide, and the detection portion is provided.

According to the cell observation device according to item 6, when assembling the cell observation device, positioning with respect to the light source, the optical waveguide, and the detection section can be easily performed, so that fine adjustment of the light source, the optical waveguide, and the detection section can be performed. can be omitted.

(Section 7) In the cell observation device according to Section 6,
The position regulating section includes a light source mounting section to which the light source is detachably mounted, an optical waveguide mounting section to which the optical waveguide is detachably mounted, and a detection section mounting section to which the detection section is detachably mounted. and have

According to the cell observation device described in item 7, the path length of the object light from the first emission surface to the light receiving surface is the same as the path length of the reference light from the second emission surface to the light receiving surface. can be done.

(Item 8) In the cell observation device according to any one of items 1 to 7,
A light superimposing unit 5 is arranged between the optical waveguide and the detecting unit and superimposes the object light and the reference light.

According to the cell observation device described in Item 8, the object light and the reference light can be superimposed and received by the detection unit as they are.

(Section 9) In the cell observation device according to Section 8,
A condensing section is provided between the first exit surface and the light superimposing section and condenses the object light.

According to the cell observation device according to the ninth aspect, it is possible to adjust the focal length of the object light and prevent deterioration of resolution in the detection unit.

REFERENCE SIGNS LIST 1 cell observation device 2 light source 21 laser diode 22 drive current supply section 3 light splitting section 4 optical waveguide 41 first core section 411 first incidence surface 412 first emission surface 42 second core section 421 second incidence surface 422 second emission Surface 43 Cladding Part 5 Light Superimposing Part 6 Detecting Part 61 Light Receiving Surface 7 Position Regulating Part 71 Light Source Mounting Part 72 Light Splitting Part Mounting Part 73 Optical Waveguide Mounting Part 74 Light Superimposing Part Mounting Part 75 Detector Mounting Part 8 Control Part 9 Condensing Part 91 Biconvex lens 92 Fixing part 10 Culture plate 11 Supporting part L0 Coherent light (laser light)
L1 Object light (measurement light)
L2 Reference beam PL1 First path length PL1-1 Path length PL1-2 Path length PL2 Second path length PL2-1 Path length PL2-2 Path length Q Cell θ1 angle θ2 angle

Claims (9)

A cell observation device for observing cells,
a light source that emits coherent light;
Between a first core portion disposed on the coherent light emitting side with respect to the light source and through which object light caused by the coherent light and directed toward the cell passes, and the object light caused by the coherent light an optical waveguide having a second core portion through which the reference light that generates interference fringes passes;
A detection unit that detects the interference fringes,
the first core portion has a first incident surface on which the object light is incident and a first exit surface from which the object light is emitted;
the second core portion has a second incident surface on which the reference light is incident and a second exit surface from which the reference light is emitted;
Cell observation, wherein the path length of the object light from the first incident surface to the first exit surface and the path length of the reference light from the second incident surface to the second exit surface are the same length. Device. The detection unit has a light receiving surface that receives the object light and the reference light,
The path length of the object light from the first emission surface to the light receiving surface and the path length of the reference light from the second emission surface to the light receiving surface are the same length. Cell observation device. 3. The cell observation device according to claim 1, further comprising a light splitting unit arranged between the light source and the optical waveguide and splitting the coherent light into the object light and the reference light. the first incident surface and the first exit surface are parallel,
The cell observation device according to any one of claims 1 to 3, wherein the second entrance surface and the second exit surface are parallel. an angle formed by the first incident surface and the second incident surface is a right angle;
The cell observation device according to any one of claims 1 to 4, wherein an angle formed by said first exit surface and said second exit surface is a right angle. The cell observation device according to any one of claims 1 to 5, further comprising a position regulating section that regulates the mutual positional relationship between the light source, the optical waveguide, and the detection section. The position regulating section includes a light source mounting section to which the light source is detachably mounted, an optical waveguide mounting section to which the optical waveguide is detachably mounted, and a detection section mounting section to which the detection section is detachably mounted. and, the cell observation device according to claim 6. The cell observation device according to any one of claims 1 to 7, further comprising a light superimposing section arranged between the optical waveguide and the detecting section for superimposing the object light and the reference light. 9. The cell observation device according to claim 8, further comprising a condensing section arranged between the first exit surface and the light superimposing section for condensing the object light.

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