JP6908390B2 - Imaging method and imaging device - Google Patents

Description

The present invention relates to a technique of detecting an interference light component between reflected light from an imaged object and reference light to perform imaging, and particularly to a technique of imaging an imaged object through a wall portion of a container having light transmission. It is a thing.

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.

In OCT imaging technology, the factors that affect the image quality are the setting position of the focal point of the object optical system that collects the signal light reflected from the object to be imaged and the optical path length of the signal light on the optical path of the signal light. There is a set position of a reference reference plane (also called a coherence gate), which is a virtual plane equal to the optical path length of the reference light. That is, the signal light includes clear information of the imaged object in the vicinity of the focal position, and the intensity of the interference light becomes strong and the information of the imaged object can be accurately extracted in the vicinity of the reference reference plane. In order to obtain good image quality, it is necessary to adjust these set positions. However, the position of the focal point and the position of the reference reference plane at the time of imaging cannot be read from the tomographic image obtained by imaging. Therefore, it is not easy for a user who does not have detailed knowledge to make these settings properly.

As a technique for obtaining a tomographic image of the desired quality by adjusting the optical path length difference between the optical path length of the signal light and the optical path length of the reference light, for example, there is one described in Patent Document 1. In this technique, imaging is performed while changing the position of the coherence gate in multiple stages, and the optimum position of the coherence gate is obtained from the images obtained from each. This technique is intended for ophthalmic devices, and the disclosed device images the retina.

Japanese Unexamined Patent Publication No. 2016-019635

When cells or the like supported in a container are used as an image to be imaged, imaging may be performed through a wall portion (for example, the bottom portion) of the container having light transmission. In such a case, the signal light is collected through the wall of the container, and due to the refraction of the signal light in the container, the focal position in the object to be imaged is different from the focal position when not passing through the container. It becomes. As the container, various containers can be used in terms of the thickness of the wall portion and the refractive index. A method for grasping the focal position in the tomographic image in such a case has not been established, and there is a problem that the adjustment of the focal position and the reference optical path length becomes more difficult.

The present invention has been made in view of the above problems, and in a technique for imaging an imaged object in a container by utilizing the interference between the reflected light from the imaged object and the reference light, the image is imaged through the wall portion of the container. It is an object of the present invention to provide a technique capable of specifying a focal position in a tomographic image to be performed.

One aspect of the present invention is an imaging method for tomographic imaging of an imaged object in a medium supported by a container having a light-transmitting wall portion, wherein low coherence light emitted from a light source is branched. The signal light that is incident on the object to be imaged and the reflected light is focused by the object optical system through the wall portion, and the reference light that is generated from the other branched light and reflected by the reference mirror. The step of detecting the interference light generated by the interference and outputting the interference signal corresponding to the detected interference light, and the reflected light intensity distribution of the object to be imaged based on the interference signal, and the reflected light intensity distribution. It has a process of creating a tomographic image from.

Another aspect of the present invention is an imaging device for tomographic imaging of an image to be imaged in a medium supported by a container having a light-transmitting wall portion, in which low coherence light emitted from a light source is branched. A reference that is generated from the signal light generated by the object optical system and the reflected light reflected by the reference mirror and the signal light that is incident on the object to be imaged and the reflected light is condensed by the object optical system through the wall portion. A detection means that detects interference light generated by interference with light and outputs an interference signal corresponding to the detected interference light, and obtains a reflected light intensity distribution of the object to be imaged based on the interference signal, and the reflection is obtained. A signal processing means for creating a tomographic image from a light intensity distribution, a focus position adjusting means for changing the focal position of the object optical system in the optical axis direction of the object optical system, and the above in a direction along an optical path in the reference direction. The mirror position adjusting means for changing the position of the reference mirror and the focal position calculating means for calculating the focal position of the object optical system in the tomographic image are provided.

And, in these inventions, to achieve the above object, called the object beam path length of the optical length of the optical path of the signal light, respectively reference optical path length of the optical length of the optical path of the reference light, wherein Of the main surfaces of the wall portion, the main surface on the object optical system side is referred to as the first main surface, and the main surface on the image subject side is referred to as the second main surface, respectively, and the directions along the optical axis of the object optical system. Is referred to as an optical axis direction, the amount of movement of the focal position in the air in the optical axis direction due to the operation of the object optical system is referred to as a focal position adjustment amount, and the object optical path length is the object optical path length on the optical path of the signal light. when referring an imaginary plane which is equal to the reference optical path length position and the reference surface, the position adjustment amount before Kiase point when the object optical system is focused before Symbol first major surface and a first adjustment amount , the position adjustment amount before Kiase point when the object optical system is focused on the front Stories second major surface and the second adjustment amount, the time before Kiase point position adjustment amount is the first adjustment amount By positioning the reference mirror at a position where the reference optical path length is longer than the position of the reference mirror at which the object optical path length and the reference optical path length are equal to each other by a predetermined length, the reference reference plane is set to the first. set between the main surface and the second major surface, and before the Kiase point position adjustment amount in a state set in the second adjustment amount, the reflected light in the optical axis direction to obtain the interference signal obtains the intensity distribution, the first distance optical distance between the reference surface and the second main surface obtained from the reflected light intensity distribution, the predetermined length as the second distance, before Kiase point position adjustment amount In the reflected light intensity distribution in the optical axis direction obtained from the interference signal obtained by setting the object optical system to a third adjustment amount for focusing on the object to be imaged side with respect to the second main surface. optical distance of the optical axis direction from the first main surface, the difference between the second adjustment amount and the third adjustment amount, and a value obtained by multiplying the square of the refractive index of the medium, the first The position represented by the sum of one distance and the second distance is defined as the focal position of the object optical system.

Here, the "reflected light intensity distribution" represents the relationship between the position on the image subject and the reflected light intensity. Further, the "focus position adjustment amount" means the amount of movement of the focal position in the air obtained by operating the object optical system. For example, when the focus position adjustment amount is changed from the first adjustment amount to the second adjustment amount, the focus position changes by an amount corresponding to the difference between the first adjustment amount and the second adjustment amount in the air. On the other hand, the focal position when focusing in the medium inside the container through the wall portion of the container does not match the focal position and the amount of change in the air due to the influence of the refractive index of the container and the medium.

When light passes through the wall of a container, its optical path length and physical length can be converted to each other via the index of refraction of the container. In the case of the present invention, the amount of change in the focal position adjustment amount and the amount of change in the focal position can be converted to each other via the refractive index of the container, but since the thickness and the refractive index of the container wall portion vary. , It is difficult to correctly identify the focal position in the tomographic image.

In the present invention, the focal position in the tomographic image can be specified as follows. First, the focal position adjustment amount of the object optical system when the object optical system focuses on the first main surface on the object optical system side of the main surface of the wall portion is defined as the first adjustment amount. Further, the focal position adjustment amount of the object optical system when focusing on the second main surface on the image subject side, which is opposite to the first main surface of the main surface of the wall portion, is defined as the second adjustment amount. Since the first main surface and the second main surface are strong reflecting surfaces, they can be identified by searching for a position where strong reflected light is detected while changing the focal position of the object optical system, for example.

Here, since the reflected light from the first main surface does not pass through the container, the object optical path length at that time is not affected by the refractive index of the container. Therefore, the object optical path length when the object optical system is focused on the first main surface can be specified from the first adjustment amount, and the object optical path length at this time and the reference optical path length of the reference mirror for matching are matched. The position is also easily determined .

On the other hand, positioning a reference mirror, a position where the object optical system closer to the direction of the object light path length and the reference optical path length from a reference optical path length is the same position becomes longer when focusing on the first main surface, ginseng Consider a state in which the reference plane is set between the first main plane and the second main plane, that is , inside the container wall portion. The amount of increase in the reference optical path length at this time, that is, the "predetermined length" of the present invention is equal to the optical path length from the first main surface to the reference reference surface, and therefore the reference reference surface virtually set in the container wall portion. Is equal to the value obtained by multiplying the physical distance between and the first main surface by the refractive index of the wall portion.

Further, in the profile of the reflected light intensity distribution obtained based on the interference signal acquired in the same state, the component corresponding to the reflected light from the reflecting surface existing on the optical path of the incident light is based on the position of the reference reference surface. Appears at a position corresponding to the depth of the reflective surface. When the reference reference plane is set inside the container wall, the strong reflective plane that first appears in the direction deeper than the reference reference plane, that is, in the direction in which the object optical path length is longer than the reference optical path length, is the second main surface. be. The depth is equal to the optical path length between the reference reference plane and the second main plane virtually set in the container wall, and is equal to the value obtained by multiplying the physical distance between the two by the refractive index of the wall. .. This value is the first distance of the present invention.

Therefore, the sum of the first distance and the second distance corresponds to the optical path length when light passes through the wall portion of the container, so to speak, the optical thickness of the wall portion. In this way, even if the refractive index and physical thickness of the wall of the container are not known, the refractive index and physical thickness of the wall of the container can be determined by detecting the signal obtained by actually injecting light. It is possible to specify the optical thickness represented by the product of.

Further, the difference between the first adjustment amount for focusing the object optical system on the first main surface and the second adjustment amount for focusing on the second main surface is the physical thickness of the container wall portion. Equal to the value divided by the index of refraction. In this way, if both the value obtained by multiplying the physical thickness of the wall portion by the refractive index and the value obtained by dividing the physical thickness of the wall portion by the refractive index are obtained, the physical thickness and the refractive index are obtained, respectively. Can also be obtained individually.

From this information, it is considered to identify where the actual focal position was in the tomographic image acquired with the focal position adjustment amount set to the third adjustment amount. The position of the object to be imaged in the tomographic image in the depth direction varies depending on the setting of the reference optical path length at the time of imaging. More generally, in the reflected light intensity distribution that is the basis of the tomographic image, it suffices if the focal position is specified as a relative position with respect to the position of the component corresponding to the reference object whose physical position is known. This is because these relative positions are determined by the difference in the object optical path lengths between the two, and do not depend on the setting of the reference optical path length.

Here, the first main surface of the container is used as a reference. The amount of position adjustment of the focal position for focusing the object optical system on the first main surface and the position of the reference mirror for matching the object optical path length and the reference optical path length at that time are already known. Therefore, if the focal position can be expressed with reference to the first main surface, the absolute focal position in the tomographic image should be specified from the relationship between the position of the reference mirror at this time and the position of the reference mirror at the time of imaging. Is possible.

First, consider the distance between the second main surface and the focal position in the reflected light intensity distribution. Since there is no wall portion of the container between the second main surface and the focal position, only the light passing through the medium supported on the container needs to be considered. When the position adjustment amount is changed from the second adjustment amount focusing on the second main surface to the third adjustment amount, the physical change amount of the focal position in the medium is the second adjustment amount and the third adjustment amount. Is multiplied by the refractive index of the medium.

On the other hand, in the reflected light intensity distribution, the distance between the position where the component corresponding to the second main surface appears and the focal position is represented by the product of the physical distance between the two and the refractive index of the medium. As described above, the physical distance between the two is the difference between the second adjustment amount and the third adjustment amount multiplied by the refractive index of the medium. Therefore, the difference between the second adjustment amount and the third adjustment amount is multiplied by the refractive index of the medium. The value obtained by multiplying the square of the refractive index represents the distance between the second main surface and the focal position in the reflected light intensity distribution. When the distance between the first main surface and the second main surface in the profile of the reflected light intensity distribution, that is, the optical thickness of the container wall is added to this value, and the first main surface is used as a reference. The focal position of is required. After all, the focal position when the first main surface is used as a reference in the reflected light intensity distribution is the value obtained by multiplying the difference between the second adjustment amount and the third adjustment amount by the square of the refractive index of the medium and the first distance. Can be expressed by the sum of and the second distance.

If the focal position in the reflected light intensity distribution is specified in this way, it becomes possible to clearly indicate, for example, which position is in focus in the tomographic image. This enables, for example, an operation in which the user moves the focal position to a desired position in the image to be imaged while viewing the tomographic image. Also, where the focal position is displayed in the tomographic image depends on the reference optical path length at the time of imaging, so the display position of the focal position in the tomographic image can be arbitrarily set by changing the reference optical path length. It will be possible.

As described above, according to the present invention, the focal position in the reflected light intensity distribution can be specified as a position relative to the position of the first main surface. Since the focal position is specified based on the information obtained from the actually detected light, this method is valid even if the thickness and refractive index of the container wall portion are unknown.

It is a figure which shows one Embodiment of the image pickup apparatus 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 positional relationship between the focal depth of an object optical system, and a reference reference plane. It is a figure which shows the relationship between the position of a reference reference plane, and the reflected light intensity distribution. It is a figure which shows typically the optical path at the time of condensing through a container. It is a figure which shows the principle of a focal position specifying process. It is a figure which shows the process which specifies the condition which the object optical system focuses on the bottom surface of a container. It is a figure which shows the principle of the process for determining the optical thickness of the bottom of a container. It is a figure which shows the relationship between the reference optical path length and a focal position. It is a flowchart which shows the process for specifying the relationship between a focal position and a reference optical path length. It is a flowchart which shows the image pickup processing in this image pickup apparatus.

FIG. 1 is a diagram showing an embodiment of an imaging device according to the present invention. This imaging device 1 uses a tomographic image of cells cultured in medium M, a spheroid (cell agglomerate) composed of a large number of cells, a tissue-like structure, etc. (hereinafter, collectively referred to as “cells, etc.”). An image is taken and the obtained tomographic image is image-processed to create a stereoscopic image of the imaged object. Although an example in which the spheroid in the medium is used as the imaged object will be described here, the imaged 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 a container 11 called a shallow dish-shaped dish having a transparent and homogeneous flat bottom made of glass or resin in a substantially horizontal posture with its opening surface facing upward. A predetermined amount of the appropriate medium M is injected into the container 11 in advance, and the spheroid Sp is cultured in the medium near the bottom 111 of the container 11. Although only one spheroid Sp is shown in FIG. 1, a plurality of spheroid Sps may be cultured in one container 11.

The imaging unit 20 is arranged below the container 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 imaged object 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 object to be imaged, 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 image to be imaged. The 3D restoration unit 34 has a function of creating a three-dimensional image (3D image) of the captured cell clumps based on the image data of the 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 imaging unit 20 executed by the drive control unit 40 and the detection of the amount of received light by the photodetector 26, a tomography of the spheroid (cell agglomerate) to be imaged is obtained. The 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 the container 11 as shown by the dashed arrow, and a part of the light L3 is directed toward the reference mirror 24 as shown by the alternate long and short dash arrow.

The light L2 directed toward the container 11 enters the container 11 via the object optical system 23. More specifically, the light L2 emitted from the beam splitter 22 enters the container bottom 111 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 container 11 onto the object to be imaged (in this case, the spheroid Sp) in the container 11, and condensing the reflected light emitted from the object to be imaged. 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 “focal depth”. The optical axis of the object optical system 23 is parallel to the vertical direction and therefore perpendicular to the flat container bottom 111. 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 focus depth setting information via the input device 351, the focus adjustment mechanism 41 changes the focal position of the object optical system 23 according to the setting information. Specifically, as a setting input, the user inputs a focal position adjustment amount, that is, how much the focal position of the object optical system 23 is moved from a predetermined initial position. 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. In this case, the amount of movement of the objective lens can be used as the amount of focus position adjustment. 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 bottom 111 of the container is reflected by 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 condensed 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.

A shutter 27 that can be opened and closed is provided on the optical path of the reference lights L3 and L5. More specifically, the shutter 27 is controlled by the drive control unit 40, and when the shutter 27 is closed by a control command from the drive control unit 40, the shutter 27 is on the optical path of the reference light L3. Shield. Therefore, the reflected light L5 is not incident on the beam splitter 22 by the reference mirror 24. On the other hand, when the shutter 27 is opened, the reference light L3 is reflected by the reference mirror 24 and is incident on the beam splitter 22 as the reflected light L5.

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 object to be imaged. Therefore, the reflected light intensity distribution in the depth direction of the object to be imaged 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 of this embodiment, the 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 container 11 in the X direction, the reflected light intensity distribution in a plane parallel to the XZ plane is obtained, and from the result, a tomographic image of a spheroid Sp having the plane as a cross section is 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 performed by a method of changing the beam incident position in the XY direction by using an optical component that changes the optical path such as a galvanometer mirror (not shown), a container 11 carrying a spheroid Sp, and an imaging unit 20. It can be realized by a method of moving any of the above in the XY direction 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 image subject 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 optical detector 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 subject 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 container 11 to adjust the focal depth of the object optical system 23 with respect to the object to be imaged. .. 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, the image pickup operation by the image processing device 1 will be described. The same imaging operation can be performed regardless of whether the imaging unit uses the above-mentioned beam splitter or an optical fiber coupler.

FIG. 4 is a diagram schematically showing the positional relationship between the focal depth of the object optical system and the reference reference plane. Further, FIG. 5 is a diagram showing the relationship between the position of the reference reference plane and the reflected light intensity distribution. In the OCT imaging device, in principle, the position where the optical path length of the signal light becomes the same as the optical path length of the reference light is the reference position in the depth direction in the image.

In the following description, as shown in FIG. 4, in the object system optical path in which the illumination light L2 and the signal light L4 propagate through the object optical system 23, they correspond to the reflecting surfaces of the reference mirror 24 in the reference system optical path, that is, each other. A virtual plane at a position where the optical path lengths of the above are equal is referred to as a reference reference plane Sr. Further, the surface of the bottom 111 of the container 11 on the side close to the object optical system 23 is the lower bottom surface Sa, and the surface on the opposite side, that is, the inner bottom surface in contact with the medium on the side close to the spheroid Sp, which is the object to be imaged. It is referred to as an upper bottom surface Sb. Further, the focal plane of the object optical system 23, that is, the plane including the object-side focal point FP of the object optical system 23 and perpendicular to the optical axis AX of the object optical system 23 is represented by the reference numeral Sf. In the following, the optical path length in the object system optical path will be referred to as "object optical path length", and the optical path length in the reference system optical path will be referred to as "reference optical path length".

Further, for later description, the distance between the lower bottom surface Sa and the upper bottom surface Sb, that is, the thickness of the container bottom portion 111 is represented by reference numeral T. Further, the distance between the upper bottom surface Sb and the focal plane Sf, that is, the distance from the inner bottom surface of the container 11 to the focal point FP is represented by reference numeral D. It can also be said that this distance D is the focal depth of the object optical system 23 when the inner bottom surface (upper bottom surface Sb) of the container 11 is the starting point.

When the reference reference surface Sr has a virtual reflection surface in the object system optical path, the optical path length of the light reflected by the reflection surface (object optical path length) is the optical path length of the light reflected by the reflection surface of the reference mirror 24. Equal to (reference optical path length). The position in the depth direction of each reflecting surface in the vicinity of the object to be imaged is represented by the distance in the Z direction from the reference reference surface Sr.

When the object to be imaged (Spheroid Sp) has a reflecting surface on the focal plane Sf, in the reflected light intensity distribution after the Fourier transform, a signal having a magnitude corresponding to the reflected light intensity from the reflecting surface is the reference reference plane Sr. Appears at a depth position corresponding to the distance from the reflecting surface (that is, the focal plane Sf). In an actual object to be imaged, signals corresponding to the reflected light from the reflecting surfaces at various depths appear at each position, and the reflected light intensity distribution is a superposition of these signals. However, in order to facilitate understanding, only the signal from the reflecting surface on the focal plane Sf will be considered here.

The flat surfaces Sa and Sb of the bottom 111 of the container are also strong reflecting surfaces, and in the reflected light intensity distribution, signals corresponding to these appear at positions corresponding to the distances from the reference reference surface Sr. For example, as shown in FIG. 5, in the reflected light intensity distribution, the signal Pf corresponding to the focal plane Sf, the signal Pa corresponding to the lower bottom surface Sa, and the signal Pb corresponding to the upper bottom surface Sb are the distances from the reference reference surface Sr, respectively. Appears at the position according to.

Strictly speaking, the "distance" in this case is the difference in the optical path length. Therefore, the physical distance of each surface on the object system optical path passing through the container 11 and the medium M having a refractive index different from the refractive index of air and the optical distance of each surface shown in the reflected light intensity distribution are not necessarily the same. not the same. The refractive index in air is approximately 1. On the other hand, the refractive index of glass, acrylic resin, etc. widely used as the material of the container 11 is larger than this, about 1.5, and the refractive index of water, which is the main component of the general medium M, is about 1.33.

In the following, the refractive index of air is approximated to 1, the refractive index of the container 11 is represented by the symbol Nc, and the refractive index of the medium M is represented by the symbol Ns. Further, it is known that when the object to be imaged is a cell or the like, its refractive index is almost the same as that of water. Therefore, in the following, it is assumed that the refractive index of the spheroid Sp, which is the image to be imaged, is also equal to that of the medium M.

FIG. 6 is a diagram schematically showing an optical path when condensing light through a container. As described above, the refractive index Nc of the container 11 is higher than that in air, and the refractive index Ns of the medium M and the spheroid Sp is smaller than the refractive index Nc of the container 11. Therefore, as shown in FIG. 6, light is refracted at the interface between the lower bottom surface Sa of the container 11 and air and the interface between the upper bottom surface Sb and the medium M, respectively, and the object optical system when viewed from the object optical system 23. The depth of the focal plane Sf of 23 is different from the focal length FD in the air.

In this way, the focal position fluctuates due to the refraction on the surface of the container 11, and the mode of the fluctuation changes depending on the material and thickness of the container bottom 111. Therefore, the user cannot correctly know which position is in focus in the tomographic image obtained by imaging. Further, since the adjustment amount of the focal position in the object optical system 23 and the change amount of the focal position in the object to be imaged do not match, the user can give the object optical system 23 what setting input to focus on the desired position. I don't know what to do.

When it becomes clear at which position the tomographic image is focused and captured, it is convenient for the user to adjust the focal position and the reference optical path length. For this purpose, it is necessary to specify the focal position in the tomographic image in consideration of the influence of refraction in the imaging through the container 11. Hereinafter, the principle and specific processing contents of the focus position specifying process of the present embodiment that make this possible will be described.

FIG. 7 is a diagram showing the principle of the focus position specifying process. As shown in FIG. 7, the case where the object optical system 23 focuses on the point F1 in the lower bottom surface Sa of the container 11, the case where the object optical system 23 focuses on the point F2 in the upper bottom surface Sb of the container 11, and the case in the medium M. Virtually consider the case of focusing on an arbitrary point F3. Corresponds to the focal plane Sf when the horizontal plane containing the point F3 is in focus in the medium. Then, the focal position adjustment amounts of the object optical system 23 for focusing on the points F1, F2, and F3 are set to OP1, OP2, and OP3, respectively.

As shown in FIG. 4, the physical thickness of the bottom portion 111 of the container is represented by the reference numeral T, and the physical distance between the top bottom surface Sb of the container 11 and the focal plane Sf is represented by the reference numeral D. The refractive index of the container 11 is represented by the symbol Nc, and the refractive index of the medium M (and the spheroid Sp) is represented by the symbol Ns.

As described above, in the light traveling through substances having different refractive indexes, the physical length of the optical path and the optical path length do not always match. Therefore, in the following, if necessary, the coordinate axis representing the physical distance in the Z direction will be distinguished as Zo, and the coordinate axis representing the optical distance corresponding to the optical path length will be distinguished as Zr.

Considering the case where the object optical system 23 changes the focal position from the state of being in focus on the lower bottom surface Sa of the container 11 and focusing on the upper bottom surface Sb, the amount of movement of the focal position at this time is the thickness of the container bottom portion 111. Is equal to T. On the other hand, the amount of focus position adjustment of the object optical system 23 for realizing this movement is calculated by the following equation using the refractive index Nc of the substance that fills this space.
| OP2-OP1 | = T / Nc ... (1)
Represented by.

Similarly, considering the case where the object optical system 23 is changed from the state where the object optical system 23 is focused on the point F2 of the upper bottom surface Sb of the container 11 to the state where the object optical system 23 is focused on the point F3, the amount of movement of the focal position at this time is the container 11. Is equal to the distance D between the upper bottom surface Sb and the focal plane Sf. On the other hand, the amount of focus position adjustment of the object optical system 23 for realizing this movement is calculated by the following equation using the refractive index Ns of the substance that fills the space.
｜ OP3-OP2 ｜ ＝ D / Ns… (2)
Represented by.

Next, consider the position where the signal component corresponding to each surface Sa, Sb, Sf appears in the profile of the reflected light intensity distribution obtained from the detected interference light. Since the distance between each component corresponds to the optical path length between each surface, the distance between the signal Pa corresponding to the lower bottom surface Sa and the signal Pb corresponding to the upper bottom surface Sb is the physical distance T between the two and the refractive index between them. It becomes the value T · Nc multiplied by Nc. Similarly, the distance between the signal Pb corresponding to the upper bottom surface Sb and the signal Pf corresponding to the focal plane Sf is a value DNs obtained by multiplying the physical distance D between the two by the refractive index Ns between them.

The absolute position where each signal component appears on the coordinate axis Zr representing the reflected light intensity distribution changes depending on the position of the reference reference plane, but the distance between each signal component does not change. Therefore, it is sufficient that the focal position in the profile of the reflected light intensity distribution can be expressed as a relative position with respect to a specific signal component appearing in the profile. For example, it can be expressed with reference to the position of the lower bottom surface Sa of the container 11. With this position as the origin, as is clear from FIG. 7, the focal position Zrf is expressed by the following equation:
Zrf = D ・ Ns + T ・ Nc… (3)
Can be represented by. Substituting equation (2) into this, the following equation:
Zrf = | OP3-OP2 | ・ Ns ² + T ・ Nc… (4)
Is obtained. The first term on the right side of the equation (4) describes the amount of focus position adjustment and the medium M at the time of imaging when the distance from the upper bottom surface Sb of the container 11 to the focal plane Sf is in focus on the upper bottom surface Sb of the container 11. It means that it is represented by the refractive index Ns. The second term on the right side represents the optical thickness of the bottom portion 111 of the container.

The refractive index Ns is determined by the type of medium M, and its value is known if medium M is specified. Therefore, for any container 11, if the focus position adjustment amount OP2 for focusing on the upper bottom surface Sb and the optical thickness T / Nc of the container bottom portion 111 are specified, the image is taken with the arbitrary focus position adjustment amount OP3. The focal position Zrf in the tomographic image can be obtained by Eq. (4). Hereinafter, the flow of specific processing contents for specifying these values will be described in order.

FIG. 8 is a diagram showing a process of specifying a condition for the object optical system to focus on the bottom surface of the container. As shown in FIG. 8, when the distance between the object optical system 23 and the lower bottom surface Sa is equal to the focal length FD, the object optical system 23 focuses on the lower bottom surface Sa. The focal position adjustment amount at this time is represented by the reference numeral OP1. The focal position adjustment amount OP1 at this time is hereinafter referred to as a "first adjustment amount". By further bringing the object optical system 23 closer to the container bottom portion 111, the object optical system 23 is focused on the upper bottom surface Sb. The focal position adjustment amount at this time is represented by the reference numeral OP2. The focal position adjustment amount OP2 at this time is hereinafter referred to as a "second adjustment amount".

As shown by the dotted arrow in FIG. 8, the object optical system 23 is brought closer to the bottom portion 111 of the container, and the intensity of the signal light incident on the photodetector 26 is detected. When the focal point of the object optical liquid 23 is outside the container bottom 111, only the propagation of light in the air needs to be considered. Therefore, if the focal length FD of the object optical system 23 is known, the initial position of the focal position can be determined. It is possible to make adjustments relatively easily.

At this time, the shutter 27 that shields the optical path of the reference light is in a closed state. Therefore, the light incident on the photodetector 26 is not the interference light between the signal light and the reference light, but only the signal light. The intensity of the signal light can be evaluated by the intensity of a specific wavelength component of the light incident on the photodetector 26 from the spectroscope 25, or the total intensity of each component, an integral value, or the like.

The intensity of the detected signal light becomes particularly large when the object optical system 23 focuses on the reflecting surface on the object system optical path. That is, as shown in the lower part of FIG. 8, the focal position adjustment amount is the first adjustment amount OP1, that is, when the object optical system 23 is in focus on the lower bottom surface Sa, and the focus position adjustment amount is the second adjustment amount OP2. It is considered that a strong peak is detected at each time, that is, when the object optical system 23 is in focus on the upper bottom surface Sb. From these peak positions, the focal position adjustment amounts OP1 and OP2 can be specified. Specifically, of the two maximum values of the signal light intensity acquired while changing the focal depth, the focal position adjustment amount when the maximum value far from the container bottom 111 is taken corresponds to the first adjustment amount OP1. It can be considered that the focal position adjustment amount when the maximum value closer to the container bottom 111 is taken corresponds to the second adjustment amount OP2. As described above, the distance between the first adjustment amount OP1 and the second adjustment amount OP2 is equal to the value obtained by dividing the thickness T of the bottom portion 111 of the container by the refractive index Nc.

By the above processing, the first adjustment amount OP1 which is the focus position adjustment amount when the object optical system 23 focuses on the lower bottom surface Sa, and the focus position adjustment amount when the object optical system 23 focuses on the upper bottom surface Sb. A certain second adjustment amount OP2 and a value (T / Nc) obtained by dividing the thickness of the bottom portion 111 of the container by the refractive index of the container material are specified. The process for determining the optical thickness of the container bottom 111 using this information will be described below.

FIG. 9 is a diagram showing the principle of processing for determining the optical thickness of the bottom of the container. As shown by the dotted line in FIG. 9, when the focal position adjustment amount of the object optical system 23 is set to the first adjustment amount OP1, the object optical system 23 is focused on the lower bottom surface Sa of the container 11. The object optical path length up to the lower bottom surface Sa at this time is represented by the symbol Lo. Since the container 11 is not included in the object system optical path at this time, the object optical path length Lo is uniquely expressed by using the mechanical dimensions of each part of the device, the first adjustment amount OP1, and the focal length FD of the object optical system 23. It is possible. Further, the position P1 of the reference mirror 24 for the reference optical path length Lr1 to be equal to the object optical path length Lo at this time can also be uniquely obtained from the mechanical dimensions of each part of the apparatus if the first adjustment amount OP1 is known. Is. This position P1 is hereinafter referred to as a "first position".

Here, the position of the reference mirror 24 is set to the position P2 in which the reference optical path length is longer than that of the first position P1, so that the reference reference surface Sr on the object-based optical path is set inside the container bottom 111. Consider the case. This position P2 is hereinafter referred to as a "second position". In the figure, the reference optical path length at this time is shown as the symbol Lr2, and this value Lr2 is larger than the previous value Lr1. In the object-based optical path, the distance between the upper bottom surface Sb of the container 11 and the reference reference surface Sr is represented by the reference numeral T1, and the distance between the lower bottom surface Sa and the reference reference surface Sr is represented by the reference numeral T2. As is clear from the figure, the sum of these values T1 and T2 corresponds to the physical thickness T of the bottom 111 of the container.

Regarding the value T2, the following equation is used using the difference in the reference optical path length between the two positions P1 and P2 of the reference mirror 24 and the refractive index of the container material.
Lr2-Lr1 = T2 ・ Nc… (5)
Can be represented by. The left side of the equation (5) corresponds to the amount of movement required to move the reference mirror 24 from the first position P1 to the second position P2. Since the first position P1 can be specified by using the first adjustment amount OP1 as described above, the distance from the first position P1 is specified from the set value of the second position P2 which is arbitrarily set. It is possible.

Further, the value T1 can be obtained as follows. The value T1 is the physical distance between the reference reference surface Sr and the upper bottom surface Sb in the optical path of the object system, and the optical path length between the reference reference surface Sr and the upper bottom surface Sb is the physical distance between the two and the refractive index of the container material. It can be expressed by the product T1 · Nc with. That is, in the reflected light intensity distribution profile in the depth direction obtained from the interference signal output by the photodetector 26, the signal Pb corresponding to the reflected light from the upper bottom surface Sb has a reference reference surface Sr of 0 depth. Appears at depths T1 and Nc.

Hereinafter, the values T1 and Nc may be referred to as "first distance" in the sense of the optical distance between the reference reference surface Sr and the upper bottom surface Sb. Similarly, the values T2 and Nc representing the optical distance between the reference reference surface Sr and the lower bottom surface Sa may be referred to as a "second distance".

Therefore, the value T1 · Nc is obtained by receiving the interference light to the photodetector 26 with the shutter 27 open and obtaining the depth of the upper bottom surface Sb in the reflected light intensity distribution profile obtained from the output interference signal. Can be identified. In this case, it is desirable to pay attention to the following two points in order to specify the signal corresponding to the upper bottom surface Sb without being affected by the signal corresponding to the other reflecting surface in the reflected light intensity distribution.

First, it is desirable that the object optical system 23 is in focus on the upper bottom surface Sb so that the object optical system 23 can collect the reflected light from the upper bottom surface Sb most efficiently. At this time, the level of the signal Pb corresponding to the upper bottom surface Sb is maximized in the reflected light intensity distribution profile. This object is achieved by setting the focus position adjustment amount of the object optical system 23 by the focus adjustment mechanism 41 to the second adjustment amount OP2 or a value close to the already obtained second adjustment amount OP2.

Secondly, it is necessary to prevent the complex conjugate signal due to the reflection from the other reflecting surface from appearing in the vicinity of the signal Pb corresponding to the upper bottom surface Sb. This is because the complex conjugate signal is superimposed on the signal Pb, so that the signal may be shielded or the peak position may shift. In particular, the complex conjugate signal caused by the strong reflected light from the lower bottom surface Sa can be a problem. The complex conjugate signal Pac corresponding to the lower bottom surface Sa appears at a depth corresponding to the optical path length from the reference reference plane Sr to the lower bottom surface Sa in the reference system optical path, that is, at the position of the depth T2 · Nc. On the other hand, since the signal Pb corresponding to the upper bottom surface Sb appears at the positions of the depths T1 and Nc, it is desirable to make the difference between the first distance T1 and the second distance T2 as large as possible in order to separate them.

From this, it is desirable that the reference reference surface Sr is set at a position as close as possible to the upper bottom surface Sb. Thereby, the signal appearing at the position closest to the reference reference plane Sr, that is, at the shallow position in the reflected light intensity distribution profile can be regarded as corresponding to the upper bottom surface Sb. However, if the reference reference surface Sr is set closer to the object to be imaged than the upper bottom surface Sb, it becomes necessary to consider the optical path length in the medium M, which complicates the process. Therefore, it is desirable that the reference reference surface Sr is set inside the container wall portion 111.

Since the optical thickness T / Nc of the container bottom 111 is not specified at this point, the position corresponding to the upper bottom surface Sb in the reference system optical path, that is, the object optical path length to the upper bottom surface Sb and the reference optical path length become equal. It is also not possible to accurately grasp the position of the reference mirror 24. Therefore, it is necessary to introduce some assumptions in order to set the reference reference surface Sr at an appropriate position inside the container wall portion 111.

Regarding the focal position adjustment amount of the object optical system 23, the first adjustment amount OP1 and the second adjustment amount OP2 have already been specified, and the value of T / Nc can be obtained from the equation (1). Therefore, for example, the container bottom 111 is configured. There is a method of estimating the approximate value of the first distance T · Nc by assuming an appropriate value of the refractive index Nc of the material to be used.

Materials that can be used as the material for the bottom 111 of the container include glass, quartz, and various resins, and their refractive indexes are generally in the range of about 1.45 to 1.6. Therefore, the value of the refractive index Nc possessed by the bottom portion 111 of the container is tentatively set to a value equal to or less than the minimum value of the assumed refractive index, more preferably a value smaller than this, for example, about 1.4, and the value of T · Nc is set. If obtained, a value slightly smaller than the actual optical thickness of the bottom 111 can be obtained. By specifying the second position P2 such that the distance from the first position P1 is this value and positioning the reference mirror 24 at this position, the reference reference surface Sr can be reliably set at the bottom of the container regardless of the material. It is possible to set it inside 111. As is clear from the relationship of equation (1), the required value is obtained by multiplying the absolute value of the difference between the first adjustment amount OP1 and the second adjustment amount OP2 by the square of the temporarily set refractive index value. Be done.

Further, for example, since the refractive index Nc of the container bottom 111 is larger than 1, as described above, the difference between the first adjustment amount OP1 and the second adjustment amount OP2 is the physics of the container bottom 111 as shown in the equation (1). It is smaller than the target thickness T. Therefore, the reference reference plane Sr can be ensured even by setting the second position P2 so that the distance between the first position P1 and the second position P2 is the difference between the first adjustment amount OP1 and the second adjustment amount OP2. It is possible to set the inside of the container bottom 111.

Further, for example, when the refractive index of the material of the container bottom 111 is known, the second position P2 can be appropriately set by using the value. Further, for example, the second position P2 (or a temporary setting value of the refractive index Nc for determining the second position P2) may be determined by a user's setting input.

The mirror drive mechanism 42 positions the reference mirror 24 at the second position P2 thus set, the focus adjustment mechanism 41 sets the focus position adjustment amount of the object optical system 23 to the second adjustment amount OP2, and the shutter 27 further releases the shutter 27. In the open state, the photodetector 26 detects the interference light. From the reflected light intensity distribution based on the interference signal thus obtained, it is possible to obtain the first distance T1 · Nc as the depth of the signal Pb corresponding to the upper bottom surface Sb. Since the second distance T2 · Nc is known from the set position of the reference mirror 24, the optical thickness T · Nc of the container bottom 111 is obtained as the sum of these values.

If the values of OP1, OP2 and T / Nc are obtained in this way, the thickness T of the container bottom 111 and the refractive index Nc can be uniquely obtained from these values and the formula (1). .. However, for the purpose of specifying the focal position represented by the equation (4), it is sufficient to obtain the optical thickness T / Nc of the container bottom 111, and it is necessary to individually specify the thickness T and the refractive index Nc. Is not always.

The focus position adjustment amount OP3 at the time of imaging is a value appropriately set by the user. Hereinafter, this value OP3 is referred to as a “third adjustment amount”. Since the values OP2, T · Nc, and Ns are already known by the processing so far, the focal position Zrf in the reflected light intensity distribution is specified from the equation (4) for the focal position adjustment amount OP3 set arbitrarily. be able to. At the time of imaging, the position of the reference reference surface Sr, that is, the reference optical path length can be set independently of the focal position. The general formula that holds for the arbitrary focal position adjustment amount (third adjustment amount) OP3 and the set value of the reference optical path length can be obtained as follows.

FIG. 10 is a diagram showing the relationship between the reference optical path length and the focal position. The basic idea is the same as above, but the position of the reference reference plane Sr, that is, the reference optical path length Lr can be arbitrarily set. In the reflected light intensity distribution, the distance Zf to the focal position when the reference reference plane Sr is used as a reference is obtained. As is clear from the relationship shown in FIG. 10A, where Lo is the object optical path length to the lower bottom surface Sa of the container 11 in the object system optical path.
Lr + Zf = Lo + Zrf = Lo + D ・ Ns + T ・ Nc
= Lo + | OP3-OP2 | ・ Ns ² + T ・ Nc… (6)
Is. The significance of this equation (6) is as follows.

As shown in FIG. 10B, the tomographic image It obtained from the reflected light intensity distribution includes an image of an imaged object within a predetermined imaging range with the reference reference plane Sr as one end of the image. In this tomographic image It, the distance from the image edge corresponding to the reference reference plane Sr to the focal position, that is, the focal depth in the image is represented by the value Zf in the equation (6). As described above, according to the equation (6), the focal depth Zf of the object optical system 23 in the tomographic image It, the focal position adjustment amount OP3 at the time of imaging, and the reference optical path length Lr are related.

Therefore, for example, the in-focus position in the tomographic image can be specified from the conditions at the time of imaging, and information indicating the focal position can be added to the displayed tomographic image. In the FD-OCT imaging device, the time delay from optical scanning of the image to be imaged to tomographic imaging can be reduced, and the display can be performed in substantially real time. By displaying a line or a marker indicating the in-focus position on the tomographic image displayed in real time, the user can adjust the focal position while looking at the image as in the case of microscopic observation.

Further, for example, even if the reference optical path length is changed for the purpose of changing the imaging range, the relationship between the image edge corresponding to the reference reference plane Sr and the focal position is required, so that the tomographic image displayed after the change is also focused. It is possible to point out the position correctly. That is, even if the range included in the tomographic image changes due to the change in the reference optical path length, the display position of the focal position can be changed accordingly.

Conversely, it is also possible to move the focal position while maintaining a constant distance between the image edge and the focal position on the display. That is, when the position adjustment amount OP3 of the object optical system 23 is changed, the reference optical path length Lr is changed so that the focal position Zf in the image becomes constant based on the equation (6) in the tomographic image. The focal position in is always constant. This makes it possible to meet the demand that, for example, a specific position (for example, the center) in the image is always the focal position.

Specifically, it is possible to determine where in the tomographic image the focal position is set to be captured, for example, by user setting. That is, if the user sets the distance from the image edge to the focal position, the focal position in the image can be kept unchanged even if the focal position is moved. In this case, when the user changes the focal position, the in-focus position with respect to the imaged object moves, but the focal position in the tomographic image does not change. Therefore, the image of the imaged object changes as the focal position changes. Will scroll in the depth direction.

Further, in OCT imaging, the image quality is the best in the vicinity of the reference reference plane. By clarifying the relationship between the focal position and the position of the reference reference plane and managing the distance between them, deterioration of image quality due to these deviations can be suppressed. In particular, when an object optical system having a large numerical aperture is used to increase the resolution, the image may become unclear even in the vicinity of the reference reference plane due to the deviation from the range of the depth of field. By clarifying the focal position and the position of the reference reference plane by the equation (6), such a problem can be avoided in advance. For example, in an imaging device in which the focal position and the reference optical path length are changed according to the user's operation, it is possible to set an upper limit of the distance between the focal position and the reference reference plane and prohibit the setting beyond this. Is.

In this way, even if the thickness, refractive index, etc. of the container bottom 111 are not known, the focal position in the tomographic image can be specified by injecting illumination light into the container 11 to be imaged and performing various measurements. It is possible to obtain information to do so. Hereinafter, the specific contents of the process for this purpose will be described.

FIG. 11 is a flowchart showing a process for specifying the relationship between the focal position and the reference optical path length. As will be described later, this process is executed as a pretreatment for imaging after the container 11 on which the medium M containing the image to be imaged is supported is set in the imaging device 1. First, the focal position of the object optical system 23 is initially set (step S101). The focal position in the initial state may be automatically set to an appropriate position, or may be set by a rough adjustment operation by the user. In the case of automatic setting, it is desirable to set the focus to be further below the lower bottom surface Sa of the container 11. Further, the shutter 27 is positioned on the reference system optical path at a shielding position that shields the reference light (step S102).

From this state, the photodetector 26 detects each time while the focus adjusting mechanism 41 drives the object optical system 23 to change and set the focal position in multiple stages (step S103). At this time, since the light in the reference system optical path is blocked, only the reflected light from the reflecting surface on the object system optical path, that is, the signal light is detected by the photodetector 26. It is the lower bottom surface Sa of the container 11 that generates strong reflected light at the position closest to the object optical system 23 in the object system optical path, and when the object optical system 23 focuses on the lower bottom surface Sa, the reflected light intensity is maximum. Become. Therefore, it is considered that the first peak appearing at the position closest to the object optical system 23 indicates the focusing state on the lower bottom surface Sa, and the focal position adjustment amount at that time is stored as the first adjustment amount OP1 (step S104). ).

Further, in the object system optical path, it is the upper bottom surface Sb of the container 11 that generates strong reflected light at a position closest to the object optical system 23 next to the lower bottom surface Sa, and when the object optical system 23 focuses on the upper bottom surface Sb, The reflected light intensity is maximized. Therefore, it is considered that the second peak that appears after the first peak indicates the focusing state on the upper bottom surface Sb, and the focus position adjustment amount at that time is stored as the second adjustment amount OP2 (step S105).

Then, the first position P1 and the second position P2 regarding the reference mirror 24 are specified based on this information (step S106). Specifically, the position of the reference mirror 24 in which the object optical path length and the reference optical path length are equal to each other when the focal position adjustment amount of the object optical system 23 is set to the first adjustment amount OP1 is specified as the first position P1. .. Further, the second position P2 is specified as a position where the reference optical path length is longer than the first position P1 and the distance from the first position P1 is smaller than the optical thickness of the container bottom 111. Then, the second distance T2 · Nc is specified as the distance between the first position P1 and the second position P2. The values of these positions and the second distance are stored in the memory 37.

Next, the focus adjustment mechanism 41 sets the focus position adjustment amount of the object optical system 23 to the second adjustment amount OP2 (step S107), and the mirror drive mechanism 42 positions the reference mirror 24 at the second position P2 (step S108). .. Further, the shutter 27 is positioned at a passing position through which the reference light is passed without being shielded, and is opened (step S109). These processing orders are not limited to the above and are arbitrary.

In this state, the photodetector 26 performs detection. What is detected at this time is the interference light obtained by combining the signal light and the reference light, and the signal processing unit 33 calculates the reflected light intensity distribution in the depth direction from the spectrum of the detected interference light (step S110). ). The signal appearing at the shallowest position in the obtained reflected light intensity distribution, that is, at the position closest to the reference reference plane Sr, corresponds to the upper bottom surface Sb of the container. The depth at which this signal Pb appears is calculated as the first distance T1 · Nc (step S111). The first distance T1 · Nc is stored in the memory 37.

From each value obtained so far, the optical thickness T / Nc of the container bottom 111 can be obtained (step S112). By the processing up to this point, the parameters OP2, Lo, and T / Nc included in the equation (6) are obtained, respectively. The other parameters Lr, Zf, OP3 are determined at the time of imaging, and the refractive index Ns has a value specific to the medium M used. Therefore, all the parameters that should be acquired before imaging are in the acquired state.

The following is not an essential process for imaging, but since the values OP1, OP2, and T / Nc have been specified, the thickness T and the refractive index Nc of the container 11 can be determined using these values and the formula (1). Each can be calculated (step S113).

FIG. 12 is a flowchart showing an imaging process in this imaging apparatus. When the container 11 containing the object to be imaged is set in the holding unit 10 in the medium M (step S201), the CPU 31 executes a control program stored in advance and causes each unit of the apparatus to perform the following operations for imaging. Processing is realized. First, a setting input of medium information regarding the medium M in the container 11 is received from the user (step S202). The medium information is information necessary for specifying the refractive index of the medium M used, and may be the numerical value of the refractive index itself or may specify the type of medium. If a table in which the type of medium and the refractive index are associated with each other is prepared, it is possible to specify the refractive index from the input type of medium by referring to the table.

Further, the refractive index of a medium generally used for culturing cells and tissues is about 1.33 to 1.37, and the difference depending on the type of medium is not so large. Therefore, a predetermined predetermined value may be used for the refractive index of the medium M.

Next, pre-imaging processing for specifying the relationship between the focal position and the reference optical path length is executed (step S203). The specific flow of the pre-imaging process is as described above. By the imaging pretreatment, each parameter, that is, the first adjustment amount OP1, the second adjustment amount OP2, and the optical thickness T / Nc of the container 11 is specified according to the container 11 used.

When the pre-imaging process is completed, the object to be imaged in the container 11 can be imaged. First, a setting input from the user regarding the conditions at the time of imaging is accepted (step S204). The imaging conditions to be set are mainly the focal position adjustment amount of the object optical system 23, that is, the third adjustment amount OP3 and the position of the reference mirror 24, that is, the reference optical path length Lr. However, at this point, it may be a provisional set value, and in particular, a predetermined initial value may be used for the reference optical path length Lr. For example, if the focal depth Zf in the image is set to half the height of the image, the tomographic image displays a range having the same extent in the vertical direction with the focal position as the center. It becomes easier to fine-tune the position. 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 S205).

Then, the image pickup unit 20 acquires the spectral information of the interference light while light scanning in the X direction (step S206), and the signal processing unit 33 calculates the reflected light intensity distribution based on the spectrum information (step S207). Create a tomographic image of one XZ cross section where light is scanned. The created tomographic image is presented to the user by displaying it on the display unit 352 together with the marker indicating the focal position (step S208).

The user can adjust the imaging range and the focal position by looking at the displayed image. That is, in the tomographic image, an image of a part of the image to be imaged that is included in the imaging range appears, and the focal position is clearly indicated by a marker. When the user wants to change the focal position or the imaging range in view of this, he / she can input the setting to that effect via the input device 351. Further, when the display mode in which the focal position is fixed in the image is selected by the user, the position of the reference mirror 24 is automatically adjusted according to the setting of the focal position based on the equation (5). When the user wishes to change these imaging conditions, the process returns to step S104 (YES in step S209), and the imaging conditions are reset.

If it is not necessary to repeatedly change the adjustment of the imaging conditions (NO in step S209), the final imaging is continuously performed (step S210). 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.

As described above, this embodiment is an imaging device 1 that performs OCT imaging of an image to be imaged in a medium M supported on a container 11 having a wall portion (bottom 111) that is transparent to illumination light. In this imaging device 1, by acquiring various information about the container 11 by pre-imaging processing, it is possible to eliminate the influence of refraction by the container 11 and specify the focusing position of the imaging optical system 23 in the tomographic image. be.

Therefore, the user can know the position where the obtained tomographic image is focused and captured, and the image can be accurately evaluated. Further, since the in-focus position is indicated by the marker in the tomographic image displayed in real time, it is possible to easily adjust the focal position with respect to the imaged object and set the focal position in the tomographic image. Further, if the focal position displayed in the tomographic image is set in advance, the reference optical path length can be automatically set according to the adjustment of the focal position with respect to the image to be imaged by the user. As described above, the image pickup apparatus 1 of the present embodiment can provide an appropriate tomographic image according to the desire even if the user does not have detailed knowledge about the imaging principle.

As described above, in the image pickup apparatus 1 of the above embodiment, the image pickup unit 20 functions as the "detection means" of the present invention, and the focus adjustment mechanism 41 and the mirror drive mechanism 42 respectively have the "focus position" of the present invention. It functions as "adjustment means" and "mirror position adjustment means". Further, the signal processing unit 33 functions as the "signal processing means" of the present invention, and the CPU 31 functions as the "focus position calculating means" of the present invention. Further, the display unit 352 functions as the "display means" and the "display device" of the present invention, while the input device 351 functions as the "reception means" of the present invention.

Further, the shutter 27 functions as the "shutter member" of the present invention. Further, in the above embodiment, the lower bottom surface Sa of the container 11 corresponds to the "first main surface" of the present invention, while the upper bottom surface Sb corresponds to the "second main surface". Further, the medium M of the above embodiment corresponds to the "medium" 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, in the description of the above embodiment, the focal position is derived with the lower bottom surface Sa of the container 11 as a position reference, and equations (3) and (4) are obtained. This is because the position of the lower bottom surface Sa of the container 11 can be specified without being affected by the thickness and the refractive index of the container bottom portion 111, and is therefore considered to be appropriate as a direct position reference. However, as described above, since the position of the upper bottom surface Sb of the container 11 can also be indirectly specified by the pre-imaging process, the focal position may be specified using the upper bottom surface Sb as a position reference. In this case, the focal position can be represented by the first term on the right side of the equation (4). Which position is used as a reference to represent the focal position is arbitrary, and by appropriately modifying each equation according to the reference position, it is possible to realize a process substantially equivalent to the above technical idea.

Further, in the above embodiment, the image pickup unit 20 takes an image from below via the bottom 111 of the container 11 that carries the object to be imaged, but the direction of the image pickup is not limited to the above and is arbitrary. For example, the above method can be applied even when the image is taken through the side wall of the container.

Further, the image pickup apparatus 1 of the above embodiment is an image pickup apparatus based on the FD-OCT imaging principle, but is not limited to the FD-OCT, and the technical idea of the present invention is applied to various tomographic imaging techniques using optical interference. It is possible.

Further, for example, the object to be imaged in the above embodiment is a spheroid Sp supported on a container 11 called a shallow dish, but the type of the object to be imaged and the container on which it is supported are not limited to this. For example, a cell or the like cultured in a well plate in which a plurality of wells capable of supporting a sample are provided on one plate may be used as an image subject.

Further, in the above embodiment, the description has been made on the premise that the entire spheroid Sp, which is the image to be imaged, is within the imaging range in the depth direction of the imaging unit 20. On the other hand, there is also a method of obtaining a wide range of tomographic images by synthesizing the captured partial images by changing the imaging range in the depth direction. In particular, if the object optical system 23 is required to have high magnification and high resolution, the depth of field of the object optical system becomes shallow. The range of the image obtained by imaging in the depth of field is limited. Even in such a case, by synthesizing the partial images captured by the above methods, it is possible to obtain a tomographic image that covers a wide area and has good image quality.

Further, as the control unit 30 of the above embodiment, it is also possible to use a general-purpose processing device having a general configuration such as a personal computer or a workstation. That is, an image pickup unit 20, a drive control unit 40, an image pickup device having a minimum control function for operating these, a personal computer that functions as a control unit 30 by executing a control program that describes the above processing contents, and the like. The image processing device 1 may be configured in combination with the above.

As described above by exemplifying a specific embodiment, the imaging method according to the present invention detects, for example, signal light while changing the amount of focus position adjustment in the optical axis direction of the object optical system. The configuration may be such that two reflecting surfaces having the maximum signal light intensity are searched for, and one of the two reflecting surfaces near the object optical system is regarded as the first main surface and the other is regarded as the second main surface. .. According to such a configuration, it is possible to surely find the first main surface which becomes a strong reflection surface at the boundary with the outside of the container and the second main surface which becomes a strong reflection surface at the boundary with the medium.

Further, for example, the second distance may be set to be equal to or less than the value obtained by multiplying the difference between the first adjustment amount and the second adjustment amount by the square of the assumed value as the refractive index of the wall portion. According to such a configuration, the reference reference plane can be set at an appropriate position inside the wall portion even for a container whose refractive index is unknown.

Further, for example, the image pickup apparatus according to the present invention may have a configuration in which a shutter member that can be opened and closed is provided in the optical path of the reference light. According to such a configuration, it is possible to generate interference light with the signal light in the open state in which the shutter member allows the reference light to pass through. Further, in the closed state where the shutter member shields the reference light, it is possible to detect only the signal light, and for example, it is possible to easily find the condition in which the object optical system is in focus on the first main surface of the container.

Further, for example, the imaging method according to the present invention may have a configuration in which a tomographic image to which information indicating a focal position is added is displayed on a display device. Further, the imaging apparatus according to the present invention may be configured to include, for example, a display means for displaying a tomographic image to which information indicating a focal position is added. According to such a configuration, the focusing position at the time of imaging can be presented to the user.

Further, for example, the configuration may be such that a setting input relating to the third adjustment amount is received and the focal position adjustment amount of the object optical system is set according to the setting input. According to such a configuration, the user can set the focal position at the time of imaging to a desired position and perform imaging.

Further, for example, the third adjustment amount may be set according to the setting input received from the user, and the position of the reference mirror may be changed according to the set value of the third adjustment amount. According to such a configuration, the reference optical path length is automatically set by the user adjusting the focal position, so that the adjustment operation by the user can be effectively supported.

The present invention can be applied to all OCT imaging techniques. In particular, it can be suitably applied in the fields of medicine, biochemistry, and drug discovery for imaging cells and cell agglomerates cultured in a container such as a dish.

1 Image processing device (imaging device)
11 Container 20 Imaging unit (detection means)
27 Shutter (shutter member)
31 CPU (focus position calculation means)
33 Signal processing unit (signal processing means)
40 Drive control unit 41 Focus adjustment mechanism (focus position adjustment means)
42 Mirror drive mechanism (mirror position adjusting means)
111 Bottom (wall of container 11)
Sa (container 11) lower bottom surface (first main surface)
Upper bottom surface (second main surface) of Sb (container 11)
Sf focal plane Sp spheroid (object to be imaged)
Sr reference reference plane

Claims (10)

In an imaging method for tomographic imaging of an image to be imaged in a medium supported on a container having a light-transparent wall portion.
One branch light in which low coherence light emitted from a light source is branched is incident on the object to be imaged, and the reflected light is condensed by an object optical system via the wall portion, and another branch. A step of detecting interference light generated by interference with reference light generated from light and reflected by a reference mirror, and outputting an interference signal corresponding to the detected interference light.
A step of obtaining a reflected light intensity distribution representing the relationship between the position of the object to be imaged and the reflected light intensity based on the interference signal and creating a tomographic image from the reflected light intensity distribution is provided.
The optical length of the optical path of the signal light is referred to as an object optical path length, and the optical length of the optical path of the reference light is referred to as a reference optical path length.
Of the main surfaces of the wall portion, the main surface on the object optical system side is referred to as the first main surface, and the main surface on the image subject side is referred to as the second main surface.
The direction along the optical axis of the object optical system is referred to as the optical axis direction.
The amount of movement of the focal position in the air in the optical axis direction due to the operation of the object optical system is referred to as a focal position adjustment amount.
When a virtual plane in which the object optical path length is equal to the reference optical path length on the optical path of the signal light is referred to as a reference reference plane.
The position adjustment amount before Kiase point when the object optical system is focused before Symbol first major surface and a first adjustment amount,
The position adjustment amount before Kiase point when the object optical system is focused on the front Stories second major surface and the second adjustment amount,
Than said position of the reference mirror is object light path length and said reference optical path length equal, the reference optical path length is longer by a predetermined length position when front Kiase point position adjustment amount is the first adjustment amount by the reference positioning the mirror to set the reference surface between the first major surface and said second major surface, and setting the pre Kiase point position adjustment amount in the second adjustment amount state, acquires the interference signal determined the reflected light intensity distribution in the optical axis direction, the first distance optical distance between the reference surface and the second main surface obtained from the reflected light intensity distribution , With the predetermined length as the second distance
The optical axis direction front Kiase point position adjustment amount is calculated from the interference signal the acquired is set to the third adjustment amount focus it object to be imaged side of the second main surface of the object optical system in the reflected light intensity distribution, the optical path length from the optical axis direction from the first main surface,
A value obtained by multiplying the difference between the third adjustment amount and the second adjustment amount by the square of the refractive index of the medium .
Said first distance,
Said second distance
An imaging method in which the position represented by the sum of is the focal position of the object optical system. The signal light is detected while changing the focal position of the object optical system in the optical axis direction , two reflecting surfaces having the maximum intensity of the signal light are searched, and the object of the two reflecting surfaces is searched for. The imaging method according to claim 1, wherein one of the sides close to the optical system is regarded as the first main surface and the other is regarded as the second main surface. According to claim 1 or 2, the predetermined length is set to be equal to or less than a value obtained by multiplying the difference between the first adjustment amount and the second adjustment amount by the square of an assumed value as the refractive index of the wall portion. The imaging method described. The imaging method according to any one of claims 1 to 3, wherein the tomographic image to which information indicating the focal position is added is displayed on a display device. The imaging method according to any one of claims 1 to 4, wherein the third adjustment amount is set according to a setting input received from the user, and the position of the reference mirror is changed according to the set value of the third adjustment amount. .. In an imaging device that tomographically images an image to be imaged in a medium supported on a container having a light-transmitting wall portion.
One branch light in which low coherence light emitted from a light source is branched is incident on the object to be imaged, and the reflected light is condensed by an object optical system via the wall portion, and another branch. A detection means that detects interference light generated by interference with reference light generated from light and reflected by a reference mirror, and outputs an interference signal corresponding to the detected interference light.
A signal processing means that obtains a reflected light intensity distribution that represents the relationship between the position of the object to be imaged and the reflected light intensity based on the interference signal and creates a tomographic image from the reflected light intensity distribution.
A focal position adjusting means for changing the focal position of the object optical system in the optical axis direction of the object optical system, and
A mirror position adjusting means for changing the position of the reference mirror in a direction along the optical path of the reference light, and
A focal position calculation means for calculating the focal position of the object optical system in the tomographic image is provided.
The optical length of the optical path of the signal light is referred to as an object optical path length, and the optical length of the optical path of the reference light is referred to as a reference optical path length.
Of the main surfaces of the wall portion, the main surface on the object optical system side is referred to as a first main surface, and the main surface on the image subject side is referred to as a second main surface.
The direction along the optical axis of the object optical system is referred to as the optical axis direction.
The amount of movement of the focal position in the air in the optical axis direction due to the operation of the focal position adjusting means is referred to as a focal position adjusting amount.
When a virtual plane in which the object optical path length is equal to the reference optical path length on the optical path of the signal light is referred to as a reference reference plane.
The position adjustment amount before Kiase point when the object optical system is focused before Symbol first major surface and a first adjustment amount,
The position adjustment amount before Kiase point when the object optical system is focused on the front Stories second major surface and the second adjustment amount,
Previous SL mirror position adjustment means, from the position of the reference mirror before the object light path length when an Kiase point position adjustment amount of the first adjustment amount and said reference optical path length are equal, the reference optical path length By positioning the reference mirror at a position where is longer by a predetermined length, the reference reference surface is set between the first main surface and the second main surface , and the focal position adjusting means is used. the pre Kiase point position adjustment amount in a state set in the second adjustment amount, the detection means acquires the interference signal, the interference signal based-out to the signal processing means the reflected light in the direction of the optical axis obtains the intensity distribution, the first distance optical distance between the reference surface and the second main surface obtained from the reflected light intensity distribution, the predetermined length as the second distance,
The focal position calculating means, the focal position adjusting means is obtained by setting the third adjustment amount for focusing at the object to be imaged side of the focal position adjustment amount the object optical system and the second main surface In the reflected light intensity distribution in the optical axis direction obtained from the interference signal, the optical distance in the optical axis direction from the first main surface is determined .
A value obtained by multiplying the difference between the third adjustment amount and the second adjustment amount by the square of the refractive index of the medium .
Said first distance,
Said second distance
An imaging device in which the position represented by the sum of the above is the focal position of the object optical system. The imaging device according to claim 6, wherein a shutter member that can be opened and closed is provided in the optical path of the reference light. The imaging device according to claim 6 or 7, further comprising a display means for displaying the tomographic image to which information indicating the focal position is added. A reception means for receiving a setting input regarding the third adjustment amount is provided.
Depending on the setting input, the imaging device according to any one of to the focal position adjusting means claims 6 Before moving the focal position location of the object optical system to the optical axis 8. The imaging device according to claim 9, wherein the receiving means receives a setting input for changing the position of the reference mirror, and the mirror position adjusting means sets the position of the reference mirror in response to the setting input.

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