JP6853728B2 - Sample container and imaging method using it - Google Patents

Description

The present invention relates to a technique of detecting an interference light component between the reflected light from an imaging object and a reference light to perform imaging, and particularly to a sample container suitable for the imaging technique.

In the technical fields of medicine and biochemistry, observation of cells and microorganisms cultured in a container is performed. As a method of observing cells or the like to be observed without affecting them, a technique of imaging cells or the like using a microscope or the like has been proposed. As one of such techniques, there is one using optical coherence tomography (OCT) technique. In this technique, low coherence light emitted from a light source is incident on an imaged object as illumination light, and interference light between reflected light (signal light) from the imaged object and reference light having a known optical path length is detected. Therefore, the intensity distribution of the reflected light from the imaged object in the depth direction is obtained and a tomographic image is formed.

When optically observing or imaging a biochemical sample such as a cell, the biochemical sample is generally subjected to imaging in a state of being contained in a sample container together with a liquid. Imaging of such a biochemical sample can be performed, for example, through the wall surface of the sample container. However, many transparent materials suitable for imaging through such a wall surface have a relatively high refractive index. Therefore, the reflection of light on the wall surface of the container may affect the imaging result.

In order to deal with such a problem, for example, in the technique described in Patent Document 1, an antireflection coating is applied to the transparent bottom surface of the container that holds the sample. The antireflection coat in this technique is provided on the lower surface of the bottom surface of the container, that is, a portion corresponding to the interface between the bottom surface of the container and the outside air. This is because the difference in the refractive index between the container material and the air is larger than the difference in the refractive index between the container material and the liquid, so the reflection at the interface between the container and the outside air is more remarkable. Is.

Japanese Unexamined Patent Publication No. 2011-232056 (for example, FIG. 3)

On the other hand, in OCT imaging, the reflected light from the wall surface of the container acts as pseudo reference light and interferes with the signal light to generate ghost-like noise called autocorrelation noise, which causes image deterioration. When the object to be imaged is a biochemical sample such as a cell, the object to be imaged is a translucent body and its reflectance is low, so that the reflected light on the wall surface of the container has a large influence. In particular, when the image-imaging object is located close to the container wall surface, the reflected light from the image-imaging object may be masked by the strong reflected light from the container wall surface, making it undetectable.

In this case, the problem is the reflection from the wall surface of the container located close to the object to be imaged, that is, the surface of the container surface on the side in contact with the liquid. However, this point is overlooked in the above-mentioned conventional technique, and as a result, the above-mentioned conventional technique cannot achieve the purpose of reducing autocorrelation noise in OCT imaging.

The present invention has been made in view of the above problems, and in a technique for imaging an imaging object in a container by utilizing the interference between the reflected light from the imaging object and the reference light, the reflected light from the wall surface of the container is used. It is an object of the present invention to provide a technique capable of reducing the resulting autoreflective noise.

One aspect of the present invention is a sample container having a storage space capable of storing a liquid containing a biochemical sample to be imaged, and in order to achieve the above object, the bottom surface portion forming the bottom surface of the storage space is A light-transmitting base material and a reduced reflection that is provided on the inner surface of the base material facing the storage space and has an intermediate refractive index between the refractive index of the base material and the refractive index of the liquid. The sample container has a layer, and the sheet member having the base material and the antireflection layer is in close contact with one end surface of a plate-shaped member in which a plurality of through holes are arranged, and one end of the through holes is formed. by closing, in which the formation of the storage space.

In the invention configured as described above, the antireflection layer is provided on the inner surface of the bottom surface of the sample container located closer to the biochemical sample in the storage space. Therefore, light reflection at the interface between the bottom surface of the sample container and the liquid in the container is suppressed, and autocorrelation noise caused by the reflected light at the interface and masking against the reflected light from the imaging object are reduced, which is good. It is possible to perform imaging with image quality.

Further, in another aspect of the present invention, an illumination light is incident on a biochemical sample to be imaged, which is stored together with a liquid in the storage space of the sample container according to the present invention, through the bottom surface portion, and the raw light is incident. The step of receiving the interference light generated by the interference between the reflected light reflected by the chemical sample and emitted through the bottom surface portion and the reference light, and the tomographic image of the biochemical sample based on the received light amount of the interference light. It is an imaging method including a step of creating.

In the invention configured in this way, the reflected light at the interface between the container and the liquid is obtained by imaging the biochemical sample through the bottom surface provided with the antireflection layer on the surface of the sample container on the side in contact with the liquid. It is possible to perform imaging with good image quality by reducing masking for autocorrelation noise and reflected light from the imaged object due to the above.

As described above, according to the present invention, the antireflection layer is provided on the bottom surface of the sample container that comes into contact with the liquid in the container. Therefore, it is possible to reduce the light reflection at the interface between the container and the liquid, and it is possible to suppress the autocorrelation noise caused by the reflected light and the masking of the reflected light from the imaged object to perform imaging with good image quality. Can be done.

It is a figure which shows one configuration example of the image pickup apparatus suitable for carrying out the image pickup method which concerns on this invention. It is a figure explaining the image pickup principle in this image pickup apparatus. It is a figure which shows the specific configuration example of the OCT apparatus. It is a figure which shows typically the influence of the reflection at the bottom part of a well. It is a figure which shows the other structural example of the antireflection layer. It is a figure which shows the whole structure of a well plate. It is a flowchart which shows the content of the imaging process suitable for cell observation.

FIG. 1 is a diagram showing a configuration example of an imaging device suitable for carrying out the imaging method according to the present invention. This imaging device 1 uses cells cultured in medium M, spheroids (cell agglomerates) composed of a large number of cells, tissue-like structures, etc. (hereinafter, collectively referred to as “cells, etc.”) as imaging objects. An image is taken, and the obtained tomographic image is image-processed to create a stereoscopic image of the object to be imaged. Although an example in which a spheroid in a medium is used as an imaging object will be described here, the imaging object is not limited to this. In order to show the directions in each of the following figures in a unified manner, the XYZ orthogonal coordinate axes are set as shown in FIG. Here, the XY plane represents the horizontal plane. Further, the Z axis represents the vertical axis, and more specifically, the (−Z) direction represents the vertical downward direction.

The image pickup apparatus 1 includes a holding unit 10. The holding portion 10 holds the well plate 11 provided with a plurality of recesses called wells W capable of holding the liquid in the internal storage space in a substantially horizontal posture with its opening surface facing upward. A predetermined amount of an appropriate medium M is stored in the well W of the well plate 11, and the spheroid Sp is cultured in the medium near the bottom Wa of the well W. FIG. 1 shows a state in which the medium M and the spheroid Sp are supported only on a part of the wells W, but the spheroid Sp is cultured in each of the plurality of wells W provided on one well plate 11.

The imaging unit 20 is arranged below the well plate 11 held by the holding unit 10. The imaging unit 20 uses an optical coherence tomography (OCT) device capable of capturing a tomographic image of an object to be imaged in a non-contact, non-destructive (non-invasive) manner. As will be described in detail later, the imaging unit 20 which is an OCT device includes a light source 21 that generates illumination light for an imaging object, a beam splitter 22, an object optical system 23, a reference mirror 24, a spectroscope 25, and the like. It includes a photodetector 26.

Further, the image pickup device 1 further includes a control unit 30 that controls the operation of the device, and a drive control unit 40 that controls the movable mechanism of the image pickup unit 20. The control unit 30 includes a CPU (Central Processing Unit) 31, an A / D converter 32, a signal processing unit 33, a 3D restoration unit 34, an interface (IF) unit 35, an image memory 36, and a memory 37.

The CPU 31 controls the operation of the entire device by executing a predetermined control program, and the control program executed by the CPU 31 and the data generated during the processing are stored in the memory 37. The A / D converter 32 converts a signal output from the photodetector 26 of the image pickup unit 20 according to the amount of received light into digital data. The signal processing unit 33 performs signal processing described later based on the digital data output from the A / D converter 32 to create a tomographic image of the imaged object. The 3D restoration unit 34 has a function of creating a stereoscopic image (3D image) of an imaged object to be imaged based on the image data of a plurality of captured tomographic images. The image data of the tomographic image created by the signal processing unit 33 and the image data of the stereoscopic image created by the 3D restoration unit 34 are appropriately stored and saved in the image memory 36.

The interface unit 35 is responsible for communication between the image pickup device 1 and the outside. Specifically, the interface unit 35 has a communication function for communicating with an external device and a user interface function for receiving operation input from the user and notifying the user of various information. For this purpose, the interface unit 35 has an input device 351 such as a keyboard, a mouse, and a touch panel capable of accepting operation inputs related to device function selection and operating condition setting, and a fault created by the signal processing unit 33. A display unit 352 made of, for example, a liquid crystal display, which displays various processing results such as an image and a stereoscopic image created by the 3D restoration unit 34, is connected.

Further, the CPU 31 gives a control command to the drive control unit 40, and the drive control unit 40 causes the movable mechanism of the image pickup unit 20 to perform a predetermined operation in response to the control command. As will be described next, by combining the scanning movement of the image pickup unit 20 executed by the drive control unit 40 and the detection of the amount of received light by the photodetector 26, the spheroid (cell clump) Sp, which is the object to be imaged, is A tomographic image is acquired.

FIG. 2 is a diagram illustrating an imaging principle in this imaging device. More specifically, FIG. 2A is a diagram showing an optical path in the imaging unit 20, and FIG. 2B is a diagram schematically showing a state of tomographic imaging of spheroids. As described above, the imaging unit 20 functions as an optical coherence tomography (OCT) device.

In the image pickup unit 20, a low coherence light beam L1 containing a wide-band wavelength component is emitted from a light source 21 having a light emitting element such as a light emitting diode or a superluminescent diode (SLD). The light beam L1 is incident on the beam splitter 22 and branches, and a part of the light L2 is directed to one well W as shown by the broken line arrow, and a part of the light L3 is directed to the reference mirror 24 as shown by the alternate long and short dash arrow. Head.

The light L2 directed toward the well W enters the well W via the object optical system 23. More specifically, the light L2 emitted from the beam splitter 22 is incident on the well bottom surface Wa via the object optical system 23. The object optical system 23 has a function of converging the light L2 directed from the beam splitter 22 toward the well W to an imaging object (in this case, a spheroid Sp) in the well W, and condensing the reflected light emitted from the imaging object. It has a function of directing the beam splitter 22. Although the object optical system 23 is typically represented by a single objective lens in the figure, it may be a combination of a plurality of optical elements.

The object optical system 23 is movably supported in the Z direction by a focus adjusting mechanism 41 provided in the drive control unit 40. As a result, the focal position of the object optical system 23 with respect to the object to be imaged can be changed in the Z direction. Hereinafter, the focal position of the object optical system 23 in the depth direction (Z direction) may be referred to as "focus depth". The optical axis of the object optical system 23 is parallel to the vertical direction and therefore perpendicular to the flat bottom surface Wa of the well. Further, the arrangement of the object optical system 23 is determined so that the incident direction of the illumination light on the object optical system 23 is parallel to the optical axis and the optical center thereof coincides with the optical axis.

When the user gives the setting information of the focal depth via the input device 351, the focal adjustment mechanism 41 changes the focal position of the object optical system 23 according to the setting information. The focus adjustment mechanism 41 can change the focal position by, for example, moving the objective lens included in the object optical system 23 in the optical axis direction. The amount of change in the focal position per step may be adjusted in predetermined step units.

Unless the spheroid Sp is transparent to the light L2, the light L2 incident through the well bottom surface Wa is reflected on the surface of the spheroid Sp. On the other hand, when the spheroid Sp has a certain degree of transparency with respect to the light L2, the light L2 enters the inside of the spheroid Sp and is reflected by the structure inside the spheroid Sp. By using, for example, near infrared rays as the light L2, it is possible to allow the incident light to reach the inside of the spheroid Sp. The reflected light from the spheroid Sp is radiated in various directions as scattered light. Among them, the light L4 radiated within the focusing range of the object optical system 23 is focused by the object optical system 23 and sent to the beam splitter 22.

The reference mirror 24 uses a mirror drive mechanism 42 provided in the drive control unit 40 to make its reflecting surface perpendicular to the incident direction of the light L3, and in a direction along the incident direction (Y direction in the figure). It is supported so that it can be moved. The light L3 incident on the reference mirror 24 is reflected by the reflecting surface and heads toward the beam splitter 22 as light L5 traveling in the opposite direction along the incident optical path. This light L5 serves as a reference light. By changing the position of the reference mirror 24 by the mirror drive mechanism 42, the optical path length of the reference light changes. The position of the reference mirror 24 is automatically set according to the purpose of imaging, and is appropriately changed according to the setting input from the user via the input device 351.

The reflected light L4 reflected by the surface or the internal reflecting surface of the spheroid Sp and the reference light L5 reflected by the reference mirror 24 are incident on the photodetector 26 via the beam splitter 22. At this time, interference due to the phase difference occurs between the reflected light L4 and the reference light L5, but the spectral spectrum of the interference light differs depending on the depth of the reflecting surface. That is, the spectral spectrum of the interference light has information in the depth direction of the imaged object. Therefore, the reflected light intensity distribution in the depth direction of the imaged object can be obtained by detecting the amount of light by splitting the interference light for each wavelength and Fourier transforming the detected interference signal. The OCT imaging technique based on such a principle is called a Fourier domain OCT (FD-OCT).

In the imaging unit 20, a spectroscope 25 is provided on the optical path of the interference light from the beam splitter 22 to the photodetector 26. As the spectroscope 25, for example, one using a prism, one using a diffraction grating, or the like can be used. The interference light is separated by the spectroscope 25 for each wavelength component and received by the photodetector 26.

By Fourier transforming the interference signal output from the photodetector 26 according to the interference light detected by the photodetector 26, the reflection of the spheroid Sp in the depth direction at the incident position of the light beam L2, that is, in the Z direction. The light intensity distribution is required. By scanning the light beam L2 incident on the well W in the X direction, the reflected light intensity distribution in a plane parallel to the XZ plane can be obtained, and from the result, a tomographic image of a spheroid Sp having the plane as a cross section can be created. Can be done. Hereinafter, in the present specification, a series of operations for acquiring one tomographic image It in a cross section parallel to the XZ plane by beam scanning in the X direction will be referred to as one imaging.

Further, by taking a tomographic image each time while changing the beam incident position in the Y direction in multiple stages, as shown in FIG. 2B, the spheroid Sp was tomographically imaged in a cross section parallel to the XZ plane. A large number of tomographic images It can be obtained. If the scanning pitch in the Y direction is reduced, image data having a resolution sufficient to grasp the three-dimensional structure of the spheroid Sp can be obtained. Beam scanning in the X and Y directions is a method of changing the beam incident position in the XY direction using an optical component that changes the optical path, such as a galvanometer mirror (not shown), and a well provided with a well W carrying a spheroid Sp. This can be realized by a method of moving either the plate 11 or the imaging unit 20 in the XY directions to change their relative positions.

In the above explanation of the principle, the beam is a beam that splits the light from the light source 21 into the illumination light and the reference light in the imaging unit 20 and the function of combining the signal light and the reference light to generate interference light. It is realized by the splitter 22. On the other hand, in recent years, an optical fiber coupler as illustrated below may be used as an OCT apparatus capable of performing such demultiplexing / combining functions.

FIG. 3 is a diagram showing a specific configuration example of the OCT apparatus. In addition, in the following description, in order to facilitate understanding, the same reference numerals are given to the same or corresponding configurations as those in the above-mentioned principle diagram. Unless otherwise specified, its structure and function are basically the same as those in the above principle diagram, and detailed description thereof will be omitted. Further, since the OCT imaging principle for detecting the interference light by the optical fiber coupler is basically the same as the above, detailed description thereof will be omitted.

In the configuration example shown in FIG. 3A, the imaging unit 20a includes an optical fiber coupler 220 as a demultiplexer / combiner instead of the beam splitter 22. One of the optical fibers 221 constituting the optical fiber coupler 220 is connected to the light source 21, and the low coherence light emitted from the light source 21 is branched into light to the two optical fibers 222 and 223 by the optical fiber coupler 220. Will be done. The optical fiber 222 constitutes an object-based optical path. More specifically, the light emitted from the end of the optical fiber 222 enters the object optical system 23 via the collimator lens 223. The reflected light (signal light) from the object to be imaged is incident on the optical fiber 222 via the object optical system 23 and the collimator lens 223.

The other optical fiber 224 constitutes a reference system optical path. More specifically, the light emitted from the end of the optical fiber 224 enters the reference mirror 24 via the collimator lens 225. The reflected light (reference light) from the reference mirror 24 is incident on the optical fiber 224 via the collimator lens 225. The signal light propagating in the optical fiber 222 and the reference light propagating in the optical fiber 224 interfere with each other in the optical fiber coupler 220, and the interfering light is incident on the photodetector 26 via the optical fiber 226 and the spectroscope 25. According to the above principle, the intensity distribution of the reflected light in the image pickup object can be obtained from the interference light received by the photodetector 26.

Also in the example shown in FIG. 3B, the optical fiber coupler 220 is provided in the image pickup unit 20b. However, the optical fiber 224 is not used, and the collimator lens 223 and the beam splitter 227 are provided for the optical path of the light emitted from the optical fiber 222. Then, according to the above-mentioned principle, the object optical system 23 and the reference mirror 24 are arranged in the two optical paths branched by the beam splitter 227, respectively. In such a configuration, the signal light and the reference light are combined by the beam splitter 227, and the interference light generated thereby is guided to the photodetector 26 through the optical fibers 222 and 226.

In these examples, in FIG. 1, a part of the optical path of each light traveling in space is replaced with an optical fiber, but the operating principle is the same. Also in these examples, the focus adjustment mechanism 41 moves the object optical system 23 in the approaching / separating direction with respect to the well plate 11 to adjust the focal depth of the object optical system 23 with respect to the image-imaging object. is there. Further, the mirror drive mechanism 42 moves the reference mirror 24 along the incident direction of the light, so that the optical path length of the reference light can be changed.

Hereinafter, problems in OCT imaging when the spheroid Sp, which is an imaging target, is present near the bottom surface of the well, and a solution thereof will be described. Up to now, the bottom surface of the well W has been simply referred to as "well bottom surface Wa", but in the following, it is defined more strictly as follows. That is, among the main surfaces forming the flat bottom surface of the well W, the upper main surface facing the storage space in the well W is referred to as "upper bottom surface Wa". Further, the main surface on the opposite side, that is, the lower main surface which forms the lower surface of the well plate 11 and faces the imaging unit 20, is referred to as "lower bottom surface Wb".

FIG. 4 is a diagram schematically showing the effect of reflection on the bottom surface of the well. More specifically, FIG. 4A is a diagram illustrating a mode of reflection of illumination light incident from below. Further, FIGS. 4 (b) and 4 (c) are diagrams schematically showing an example of a tomographic image obtained by imaging. Further, FIG. 4D is a diagram showing a structure of a well bottom surface portion for reducing reflection.

As shown in FIG. 4A, consider the case where the spheroid Sp is in the vicinity of the upper bottom surface Wa in the medium M stored in the well W. At this time, when the illumination light is incident from below the well W, the reflected light Lb reflected by the lower bottom surface Wb of the well W as shown by the broken line arrow in the figure and the upper bottom surface Wa of the well W as shown by the solid line arrow. The reflected reflected light La and the reflected light Ls reflected on the surface or the internal reflecting surface of the spheroid Sp can be generated.

Here, the refractive index of air is about 1, and the refractive index of resin, glass, or the like used as a transparent material constituting the bottom surface of the well W is about 1.5. Further, the medium M generally contains water as a main component, and although a slight change in the refractive index occurs due to the addition of some chemical substance, the medium M has a refractive index substantially equal to that of water. Here, typically, the value of the refractive index of water, 1.33, is regarded as the refractive index of the medium M. On the other hand, the spheroid Sp, which is the object to be imaged, can be regarded as a translucent body in a liquid, and its refractive index is close to that of water or a medium, and its light reflectance is low. Therefore, when comparing the intensities of the reflected light from each position in the depth direction, as shown in the right figure of FIG. 4A, the reflection at the lower bottom surface Wb, which is the interface between the air and the bottom surface of the well, is the strongest. This is followed by reflection at the upper bottom Wa, which is the interface between the medium M and the bottom of the well. Then, the reflected light from the spheroid Sp is generally weaker than these.

The reflected light from the lower bottom surface Wb can be detected separately from the reflected light from the spheroid Sp if the thickness of the bottom surface of the well W is sufficiently larger than the size of the spheroid Sp. While the diameter of the spheroid Sp is about 100 μm at the maximum, the thickness of the bottom surface of the well W is generally larger than 100 μm. Therefore, even if the reflected light from the lower bottom surface Wb is strong, the influence on the tomographic image can be suppressed relatively slightly.

On the other hand, it may be difficult to separate the reflected light from the upper bottom surface Wa from the reflected light from the spheroid Sp which is a short distance in the depth direction. Further, interference may occur when the reflected light from the upper bottom surface Wa is mixed with the reflected light from the spheroid Sp with a short optical path length difference. That is, the reflected light from the upper bottom surface Wa behaves as pseudo reference light in OCT imaging.

Due to these, the following effects appear on the tomographic image. That is, as shown in the tomographic image Ib1 illustrated on the left side of FIG. 4B, the image of the upper bottom surface Wa strongly appears at the position of the depth corresponding to the upper bottom surface Wa in the image. Further, the reflected light from the upper bottom surface Wa acts as a pseudo reference light, so that the ghost-like image G appears at a depth different from the original spheroid Sp image. Such ghosts are called autocorrelation noise.

Further, when the imaged object is very small, the image of the imaged object is masked by the image of the upper bottom surface Wa and does not appear clearly as shown in the tomographic image Ic1 illustrated on the left side of FIG. 4C. It can happen.

Therefore, in the well plate 11, the bottom surface of the well W has a structure shown in the left figure of FIG. 4 (d). That is, the bottom surface 110 of the well W is a transparent base material 111 having light transmission to illumination light, and a antireflection layer formed on the upper side of the main surface of the base material 111, that is, the surface on the side in contact with the medium M. It has a structure having 112 and. The antireflection layer 112 is a layer having an intermediate refractive index between the refractive index of the base material 111 and the refractive index of the medium M (typically water). Specifically, it is formed by coating the inner surface facing the storage space inside the well W. Therefore, the reflectance at the upper bottom Wa of the illumination light incident from below the well W via the well bottom surface 110, and the upper bottom surface of the reflected light from the spheroid Sp emitted downward via the well bottom surface 110. The reflectance in Wa is reduced.

Therefore, as shown in the right figure of FIG. 4D, the intensity of the reflected light from the upper bottom surface Wa of the well W is greatly reduced. As a result, as shown in the tomographic image Ib2 illustrated on the right side of FIG. 4B, it is possible to greatly reduce the influence of the ghost due to the image of the upper bottom Wa appearing in the tomographic image and the autocorrelation noise. Further, as shown in the tomographic image Ic2 illustrated on the right side of FIG. 4C, it is possible to more clearly show an image of a small imaging object that is masked by the image of the upper bottom Wa in the absence of the antireflection layer. It becomes.

As described above, the refractive index of the medium M is about 1.33, which is almost the same as that of water. On the other hand, commonly used as the base material 111 are, for example, polyethylene (refractive index 1.53), polypropylene (refractive index 1.48), polystyrene (refractive index 1.59), and non-alkali glass (refractive index 1). .51) etc., and these refractive indexes are about 1.5, which is larger than the refractive index of the medium M. The hyporeflective layer 112 preferably has an intermediate refractive index between the medium M and the base material 111. That is, it is desirable that the refractive index of the antireflection layer 112 is larger than the refractive index of the medium M (typically the refractive index of water) and smaller than the refractive index of the material used as the base material 111.

In order to maximize the effect of lowering the reflectance, the following equation is used between the refractive index Nm of the medium M, the refractive index Ns of the base material 111 and the refractive index Na of the reduced reflectance layer 112.
Na = (Nm · Ns) ^1/2 ... (Equation 1)
It is more preferable that the relationship of When the values of the refractive indexes Nm and Ns are 1.33 and 1.50, respectively, the preferable value of the refractive index Na of the reduced reflection layer 112 is 1.41.

Magnesium fluoride (refractive index 1.38) and calcium fluoride (refractive index 1) are examples of solid materials having a refractive index close to this and capable of forming the antireflection layer 112 by coating the base material 111. There are metal fluorides such as .39) and silicon dioxide (white carbon, refractive index 1.44 to 1.50). If the materials of the medium M and the base material 111 are specified, it is possible to more specifically select the material having a preferable refractive index.

FIG. 5 is a diagram showing another configuration example of the antireflection layer. The antireflection layer 112a shown in FIG. 5A is a laminate of a plurality of thin films formed of materials having different refractive indexes from each other. Although it is shown as two layers here, it may be three or more layers. In the single-layer antireflection layer 112 shown in FIG. 4D described as a principle diagram, there may be a case where the antireflection effect is insufficient or the wavelength range in which the antireflection effect is exhibited is limited. .. To compensate for this, by combining a plurality of layers having different refractive indexes and thicknesses, it is possible to obtain an excellent reflectance lowering effect over a wide band.

Further, the same effect can be obtained by using the antireflection layer 112b having a porous structure shown in FIG. 5B and the antireflection layer 112c having a fine uneven structure shown in FIG. 5C. it can. In these structures, light scattering within the layer can be prevented by making the size of the structure smaller than the wavelength range of the illumination light used. Further, the effective refractive index of the layer is an intermediate value between the refractive index of the substance constituting the layer and the refractive index of the substance (in this case, medium M) that fills the gap around the layer. The refractive index changes depending on their space occupancy. Therefore, it is possible to realize a reduced-reflection layer having a desired refractive index according to the purpose. For example, it is possible to construct an ideal antireflection layer that meets the conditions of (Equation 1) by using a material having a refractive index different from that of Na having a refractive index represented by (Equation 1).

Regarding the reduced reflection layer 112b shown in FIG. 5 (b) and the reduced reflection layer 112c shown in FIG. 5 (c), a layer of a material having such a fine structure is laminated on the base material 111. May be good. Further, by applying surface processing to one surface of the base material 111, the surface itself of the base material 111 may have such a fine structure. That is, the antireflection layer 112 may be made of the same material as the base material 111.

When the term "reduced reflection layer 112" is used below, it is used as a general term including not only the configuration shown in FIG. 4 (d) but also the configurations shown in FIGS. 5 (a) to 5 (c). And.

FIG. 6 is a diagram showing the overall structure of the well plate. More specifically, FIG. 6A is an exploded perspective view of the well plate 11, and FIG. 6B is a partial cross-sectional view of the well plate 11. The well plate 11 has an upper portion in which through holes 113 having a substantially cylindrical side surface shape (more strictly, a taper whose cross-sectional area gradually decreases toward the bottom surface) are regularly arranged in a two-dimensional matrix at a constant pitch. It has a plate 114 and a lower surface sheet 115 attached to the lower surface of the upper plate 114 so as to close each through hole 113.

As shown in FIG. 6B, the lower surface sheet 115 is closely adhered to the lower surface of the upper plate 114 by, for example, adhesion, and is a space surrounded by the side surface of the through hole 113 of the upper plate 114 and the lower surface sheet 115. However, it is a storage space that can hold the liquid. That is, this storage space functions as a well W for holding the liquid, and the side surface of the through hole 113 forms a side wall surface of the well W. Further, the portion of the lower surface sheet 115 facing the lower end side opening of the through hole 113 forms the bottom surface portion 110 of the well W.

That is, the lower surface sheet 115 is provided with a surface layer that functions as the antireflection layer 112 on the upper surface of the sheet-like base material 111 made of transparent resin or glass, that is, the surface that is in close contact with the lower surface of the upper plate 114. It was made. By attaching the lower surface sheet 115 formed in advance as such a structure to the lower surface of the upper plate 114, each of the wells W formed by the through hole 113 and the lower surface sheet 115 has the above-mentioned antireflection layer 112. It can be a structure.

The structure of the well plate is not limited to this. For example, even if the side wall surface and the bottom surface of the well are integrally formed in advance, and the antireflection layer is formed on the inner surface of the bottom surface facing the storage space in the well by post-processing. Good.

Next, imaging of a biochemical sample using the imaging device 1 and the well plate 11 described above will be described. The imaging device 1 can perform OCT imaging on various imaging objects, but when cells or the like supported on the well plate 11 described above are objects to be imaged, it is particularly near the bottom surface of the well. It is possible to clearly image an existing imaging object.

Since OCT imaging can image cells and the like non-invasively, it is also suitable for observing changes with time of cells and the like. In the following, a process flow will be described when a biochemical sample prepared by culturing cells under predetermined culture conditions is periodically imaged (time-lapse image) for observation over time.

FIG. 7 is a flowchart showing the contents of the imaging process suitable for cell observation. Here, a series of processes for observation including preparation and culture of the biochemical sample will be described, but the “imaging process in a narrow sense” realized by operating the imaging device 1 is one of the steps in FIG. Steps S103 to S109.

First, a biochemical sample to be observed is prepared. Specifically, an appropriate type and amount of medium M is injected into each well W of the well plate 11 having the above-mentioned structure, and cells to be observed are seeded (step S101). Then, the cells are cultured under predetermined culture conditions (step S102). For example, cells are cultured by holding the well plate 11 for a predetermined time in an incubator (not shown) in which temperature and humidity and atmosphere are controlled. As a result, a biochemical sample to be imaged (for example, a spheroid formed by a plurality of cells) is prepared.

When the well plate 11 is carried into the imaging device 1 and set in the holding unit 10 (step S103), the CPU 31 executes a control program stored in advance and causes each unit of the device to perform the following operations to perform the imaging process. Is realized. Specifically, a setting input from the user regarding the conditions at the time of imaging is accepted (step S104). The imaging conditions to be set are mainly the focal position of the object optical system 23 and the position of the reference mirror 24, that is, the reference optical path length. In addition, for example, setting input regarding image resolution may be made. The focus adjustment mechanism 41 drives the object optical system 23 to set the focus position according to these settings, and the mirror drive mechanism 42 sets the position of the reference mirror 24 (step S105).

Then, the image pickup unit 20 acquires the spectral information of the interference light while light scanning in the X direction (step S106), and the signal processing unit 33 calculates the reflected light intensity distribution based on the spectrum information (step S107). Create a tomographic image of one XZ cross section where light is scanned. The created tomographic image is displayed on the display unit 352 (step S108) and presented to the user.

The user can adjust the imaging range, the focal position, and the like by looking at the displayed image. When the user wants to change the focal position or the imaging range, he / she can input the setting to that effect via the input device 351. When the user wishes to change these imaging conditions, the process returns to step S104 (YES in step S109), and the imaging conditions are reset.

If it is not necessary to repeatedly change the adjustment of the imaging conditions (NO in step S109), the final imaging is continuously performed (step S110). The imaging at this time may be an imaging of one XZ cross section, or may be an imaging of a plurality of XZ cross sections in which the incident positions of the illumination light are different in the Y direction. The tomographic image data obtained by imaging is stored in the image memory 36. When imaging is performed on a plurality of XZ cross sections, the 3D restoration unit 34 creates a stereoscopic image of the imaged object as necessary from the tomographic image data. Various data obtained by imaging are appropriately presented to the user and used for observation work by the user.

If cell culture is still required (YES in step S111), the well plate 11 is returned to the culture environment and culture is continued. After that, the well plate 11 is taken out again at a necessary timing and is used for imaging. When there is no need to continue culturing (NO in step S111), all the processes are completed.

As described above, in the above imaging process, the bottom surface of each well W has the following configuration in the well plate 11 that supports the biochemical sample such as cells as the imaging target together with the liquid. That is, the bottom surface of the well W has a structure in which the antireflection layer 112 is provided on the inner surface of the base material 111 which is transparent to the illumination light used for imaging and is in contact with the liquid.

The antireflection layer 112 has an intermediate refractive index between the refractive index of the base material 111 and the refractive index of the liquid. Therefore, the reflectance of light at the interface between the bottom surface of the well W (specifically, the upper bottom surface Wa) and the liquid in the well W is suppressed to be lower than in the case where the base material 111 and the liquid are in direct contact with each other. .. As a result, it is possible to obtain a tomographic image with good image quality by suppressing autocorrelation noise caused by the reflected light on the upper bottom surface Wa and masking of a small imaging object.

As described above, in the above embodiment, the well plate 11 functions as the "sample container" of the present invention. Further, the upper plate 113 constituting the well plate 11 corresponds to the "plate-shaped member" of the present invention.

The present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, the material of each member described above shows an example thereof, and various materials can be used within a range suitable for the gist of the present invention. For example, a gel-like substance such as soft agar may be used as the medium M, and such a gel-like substance also falls under the “liquid” referred to in the present invention.

Further, although each of the well plates 11 has a plurality of wells W capable of storing a liquid, the "sample container" of the present invention is not limited to such a form. For example, even in a sample container called a "dish", a "petri dish", etc., which has only one storage space for storing a liquid, the same effect as described above can be obtained by providing a antireflection layer on the inner surface thereof. Is possible. Further, the opening shape of the storage space is not limited to a circular shape and is arbitrary.

Further, in the well plate 11 of the above embodiment, the antireflection layer 112 is provided only on the inner surface (upper bottom surface Wa) of the bottom surface portion. In addition to this, a reduced reflection layer may also be provided on the lower bottom surface Wb. In this case, it is reduced by a single or multiple materials having an index of refraction intermediate between the index of refraction of the substrate 111 and the index of refraction of air, as has already been done as a antireflective coating in optics. A reflective layer may be formed.

As described above by exemplifying a specific embodiment, in the sample container according to the present invention, the antireflection layer has, for example, an intermediate refractive index between the refractive index of the base material and the refractive index of the liquid. It may be a thin film formed by covering the inner surface of the base material with a transparent material. By covering the inner surface of the base material with such a thin film, it is possible to reduce the reflectance at the interface between the inner surface of the base material and the liquid.

In this case, the antireflection layer may have, for example, a structure in which a plurality of thin films made of different materials are laminated. The reduced reflectance layer of a single thin film is constrained by the effect of reducing reflectance and the characteristics of the thin film material in the effective wavelength range, but by stacking multiple types of thin films, high reflectance is reduced in a wider wavelength range. It is possible to obtain the effect.

Further, for example, the antireflection layer is a layer in which the effective refractive index is lowered from the material-specific refractive index due to the porous fine structure, or the effective refractive index is set to the material-specific refractive index due to the uneven structure of the surface. It may be a lower layer than. By adopting such a fine structure, it is possible to realize a layer having an intermediate refractive index between the refractive index peculiar to the material constituting the antireflection layer and the refractive index of the liquid that fills the gap of the fine structure. Can be done. Such a layer also functions effectively as a reduced reflection layer.

In these cases, the antireflection layer may be formed by surface processing of the inner surface of the base material. The antireflection layer may be formed of a material different from that of the base material, or may be formed of the same material as the base material. In the configuration in which the refractive index is adjusted by the surface structure of the base material, the bottom surface of the sample container can be formed of a single material by processing the surface of the base material.

Further, the refractive index of the antireflection layer may be an intermediate value between the refractive index of the base material and the refractive index of water. The refractive index of the liquid varies depending on its composition, and it is desirable that the refractive index of the antireflection layer is optimized according to the liquid originally used. However, in a biochemical sample such as a cell to be imaged, the main component of the liquid is water, and its refractive index is not significantly different from that of water. Therefore, the refractive index of water can be approximately applied as the refractive index of liquid.

Further, the sample container according to the present invention may have a structure in which a plate-shaped member provided with a plurality of through holes is provided and one end of the through holes is closed by a sheet-like base material. According to such a configuration, the space surrounded by the inner wall surface of each through hole and the base material can function as a storage space.

The present invention can be applied to all sample containers for supporting biochemical samples such as cells. It is particularly suitable for the purpose of OCT imaging a biochemical sample. Therefore, it can be suitably applied in the fields of medicine, biochemistry, and drug discovery for imaging cells and cell agglomerates cultured in a container.

1 Imaging device 11 Well plate (sample container)
20 Imaging unit 111 Base material 112, 112a, 112b, 112c Anti-reflection layer 113 Through hole 114 Upper plate (plate-like member)
115 Bottom sheet (base material, anti-reflection layer)
Sp spheroid (biochemical sample)
W well Wa upper bottom Wb lower bottom

Claims (9)

A sample container having a storage space capable of storing a liquid containing a biochemical sample to be imaged, and a bottom surface portion forming the bottom surface of the storage space is
With a light-transmitting base material,
It has a reduced-reflection layer provided on the inner surface of the surface of the base material facing the storage space and having a refractive index intermediate between the refractive index of the base material and the refractive index of the liquid.
The sample container, the end surfaces of the plurality of through holes are arranged plate-like member, by closing one end of the through-hole in close contact sheet member having said antireflection layer and the substrate, wherein the reservoir A sample container that forms a space. The reduced reflection layer is a thin film formed by covering the inner surface of the base material with a transparent material having a refractive index intermediate between the refractive index of the base material and the refractive index of the liquid according to claim 1. The sample container described. The sample container according to claim 2, wherein the antireflection layer has a structure in which a plurality of the thin films made of different materials are laminated. The sample container according to claim 1, wherein the antireflection layer is a layer in which the effective refractive index is lower than the refractive index peculiar to the material due to the porous fine structure. The sample container according to claim 1, wherein the antireflection layer is a layer in which the effective refractive index is lower than the refractive index peculiar to the material due to the uneven structure of the surface. The sample container according to claim 4 or 5, wherein the antireflection layer is formed by surface processing of the inner surface of the base material. The sample container according to any one of claims 1 to 6, wherein the refractive index of the reduced-reflection layer is an intermediate value between the refractive index of the base material and the refractive index of water. The sample container according to any one of claims 1 to 7, wherein the sheet member has a structure in which a surface layer functioning as the antireflection layer is provided on one surface of the sheet-shaped base material. Illumination light is incident on the biochemical sample to be imaged, which is stored together with the liquid in the storage space of the sample container according to any one of claims 1 to 8, through the bottom surface portion, and the biochemical sample is subjected to. The step of receiving the interference light generated by the interference between the reflected light reflected by the light and the reference light and the reflected light emitted through the bottom surface portion.
An imaging method including a step of creating a tomographic image of the biochemical sample based on the amount of received light of the interference light.

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