Aligning component

Abstract

This aligning component comprises a base portion (101) and marks (103) formed on the base portion (101). A sample to be observed is placed on the base portion (101). The marks (103) are formed on a placement surface (102) of the base portion (101) on which the sample is to be placed. Further, the marks (103) are formed in the shape of a reentrant polygon in a plan view. Further, the marks (103) are made of a material having an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more. Further, the marks (103) are provided at a plurality of locations on the placement surface (102), and the spacings between adjacent marks (103) are known.

Description

Alignment parts

[0001] The present invention relates to an alignment component.

[0002] Ultrasonic microscopes have been developed as a technology for non-invasively observing biological tissues, and in recent years, their improved resolution has led to their application in cell observation and tissue observation at the scale of several micrometers. The physical quantity observed with an ultrasonic microscope is acoustic impedance, which is information on the mechanical properties corresponding to the hardness of the biological tissue being observed.

[0003] On the other hand, biochemical observation techniques, such as tissue composition, are required to analyze tissue changes due to tissue condition or disease. Techniques for biochemical observation of cells or tissue sections include fluorescent immunostaining and observation under a fluorescence microscope. However, observation using immunostaining is invasive, and it is necessary to be able to analyze the biochemical information obtained by immunostaining using only information from ultrasound microscope images. To do this, it is necessary to clarify in advance the correspondence between the acoustic impedance map obtained from ultrasound microscope images and the immunostained image observed by fluorescence microscope. To clarify this correspondence, a technique for aligning microscopic images is required, which allows comparison of observation results from the same position in ultrasound microscope images and fluorescence microscope images.

[0004] RM Lemor et al., "Measurements of Elastic Properties of Cells Using High-Frequency Time-Resolved Acoustic Microscopy", IEEE Symposium on Ultrasonics, vol. 1, 2003.K Raum et al., "Site-matched assessment of structural and tissue properties of cortical bone using scanning acoustic microscopy and synchrotron radiation μCT", Physics in Medicine & Biology, vol. 51, no. 3, pp. 733-746, 2006.

[0005] The following two techniques have been used to superimpose images from an ultrasonic microscope and an optical microscope such as a fluorescence microscope.

[0006] First, there is a mechanical alignment technique (Non-Patent Document 1). This technique mechanically aligns the optical axis of the fluorescence microscope image with the axis of the ultrasound microscope. This technique requires the microscope to incorporate a mechanism that enables high-precision alignment at the cellular level (a few micrometers). Furthermore, this technique aligns the axes using a single microscope device, making it impossible to compare data observed with different microscopes.

[0007] The second technique is image registration (Non-Patent Document 2). This technique identifies (segments) characteristic structures contained in the observed sample, and then registers and overlays the images so that the positions of the identified structures match between different microscopes. For example, registration is possible for biological samples with distinctive structures, such as bone, but it cannot be applied to objects without distinctive structures, such as cell sheets or spheroids made of cardiomyocytes or fibroblasts.

[0008] As mentioned above, there has been a problem in the past in that there was no technology to match the observation position in an optical microscope image with the observation position in an ultrasonic microscope image for observation objects such as sheets of cardiomyocytes or fibroblasts (two-dimensional culture systems) or three-dimensional cell populations such as organoids or spheroids.

[0009] The present invention has been made to solve the above problems, and aims to enable the observation position in an optical microscope image to correspond to the observation position in an ultrasonic microscope image when observing biological tissue.

[0010] The alignment component according to the present invention comprises a base on which a sample to be observed is placed, and a concave polygonal mark formed on the mounting surface of the base on which the sample is placed, the mark being made of a material having an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more, and provided at a plurality of locations on the mounting surface, with the spacing between adjacent marks being known.

[0011] As described above, according to the present invention, a concave polygonal mark made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more is provided on the mounting surface of the base on which the sample to be observed is placed, so that when observing biological tissue, the observation position in the optical microscope image can be matched to the observation position in the ultrasonic microscope image.

[0012] FIG. 1 is a cross-sectional view (a) and a plan view (b) showing a partial configuration of an alignment component according to an embodiment of the present invention. FIG. 2 is a plan view showing a partial configuration of another alignment component according to an embodiment of the present invention. FIG. 3 is a cross-sectional view showing a configuration of an alignment component according to an embodiment of the present invention. FIG. 4 is a cross-sectional view showing a configuration of an alignment component according to an embodiment of the present invention. FIG. 5A is an explanatory diagram for explaining an alignment method using an alignment component according to an embodiment of the present invention. FIG. 5B is an explanatory diagram for explaining an alignment method using an alignment component according to an embodiment of the present invention. FIG. 5C is an explanatory diagram for explaining an alignment method using an alignment component according to an embodiment of the present invention. FIG. 5D is an explanatory diagram for explaining an alignment method using an alignment component according to an embodiment of the present invention. FIG. 5E is an explanatory diagram for explaining an alignment method using an alignment component according to an embodiment of the present invention. FIG. 6A is a photographed image showing an actual alignment state using an alignment component according to an embodiment of the present invention. FIG. 6B is a photographed image showing an alignment state using an alignment component according to an embodiment of the present invention. FIG. 6C is a photographed image showing an alignment state using an alignment component according to an embodiment of the present invention.

[0013] An alignment component according to an embodiment of the present invention will be described below with reference to Fig. 1. Fig. 1(a) shows a cross section taken along line aa' in Fig. 1(b). The alignment component includes a base 101 and a mark 103 formed on the base 101.

[0014] The base 101 is configured to hold a sample to be observed. The base 101 can be made of a resin such as polystyrene. The sample is, for example, a biological tissue such as a cell. The mark 103 is formed on the mounting surface 102 of the base 101 on which the sample is mounted. The mark 103 is a concave polygon in plan view. The mark 103 has a shape not seen in biological tissue such as a cell, and it is required that the mark 103 and parts thereof are easily distinguishable (distinguishable) from biological tissue such as a cell. Furthermore, it is important that the position of the mark 103 can be defined and that the shape is sensitive to this positional shift during microscopic observation. Considering these factors, the planar shape of the mark 103 is required to include straight lines and corners, and it is important that the mark 103 be a concave polygon (not a convex polygon). For example, the mark 103 can be a cross-shaped mark in plan view. Furthermore, for example, the marks 103 may be in the shape of asymmetrical Arabic numerals such as "1," "2," "3," "4," "5," "6," "7," and "9" when viewed from above.

[0015] Furthermore, the mark 103 is made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more. It is important that the mark 103 produces a characteristic reflection signal distinct from that of biological tissues such as cells when observed with an ultrasonic microscope. Because the acoustic impedance of biological tissues such as cells is in the range of 1.2 to 1.8 MRayls, the mark 103 is made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more. Furthermore, when observed with an optical microscope, it is also important that the refractive index of the mark 103 is different from that of the base 101 and the cell culture medium. For example, the mark 103 can be made of a material with a refractive index outside the range of 1.34 to 1.38 (the refractive index of the culture medium) or 1.56 to 1.62 (the refractive index of polystyrene). The mark 103 can be made of a fluorescent material. By making the mark 103 of a fluorescent material, the alignment component can be compatible with a fluorescent microscope.

[0016] Furthermore, the marks 103 are provided at multiple locations on the mounting surface 102, and the interval between adjacent marks 103 is known. If the interval between adjacent marks 103 is known, the position of each mark 103 can be easily estimated during observation with a microscope. Furthermore, the marks 103 can be provided two-dimensionally at multiple locations on the mounting surface 102. If the interval between adjacent marks 103 is known, the arrangement of the marks 103 provided at multiple locations may be random. However, by arranging the marks 103 at equal intervals in a grid pattern at multiple locations, even if a specific mark 103 overlaps with a cell or the like and becomes difficult to identify, the position of the specific mark 103 can be estimated without observing all of the marks 103 provided at multiple locations.

[0017] It is desirable that the area without marks 103 is larger (more numerous). It is desirable that the area without marks 103 occupies 90% or more of the entire area of ​​the base 101. For example, the proportion of the entire area of ​​the base 101 occupied by all marks 103 can be set to about 1.5%. Furthermore, the spacing between the marks 103 arranged in multiple locations is arbitrary, but it is desirable that at least two marks 103 are always located vertically and two marks 103 are always located horizontally within the observation area (angle of view) of the microscope being used.

[0018] 2, the reference mark 104 may be further provided in the form of a concave polygon, which is different in shape from the mark 103. The size of the reference mark 104 in a plan view may be larger than that of the mark 103. The reference mark 104, like the mark 103, is formed on the mounting surface 102. For example, the reference mark 104 may be disposed at the end of a formation region of a group of marks 103 formed at multiple locations. The reference mark 104, like the mark 103, is made of a material whose acoustic impedance is 1.2 MRayls or less or 1.8 MRayls or more.

[0019] It is also important that the dimensions of the mark 103 in a plan view are observable with an ultrasonic microscope. It is also important that the mark 103 and its shape can be identified with an ultrasonic microscope using focused ultrasonic waves. It is important that a signal with a strong characteristic reflection intensity from the mark 103 is obtained for an ultrasonic beam with a wavelength of λ, and for example, the width of a portion of the shape that constitutes the mark 103 can be set to 2λ / π or more. Furthermore, in order to be able to identify the straight line portions of the mark 103, the length of the straight line portions can be set to 2x or more where x is the ultrasonic beam diameter.

[0020] Furthermore, in order to prevent the concave shape of the mark 103 from being lost in a planar view in an observation image using an ultrasonic microscope with a beam diameter of x, it is also important that the mark has a size that does not lose the concave shape when a Gaussian filter with a half-width of x / 2 is applied.

[0021] In addition, for an ultrasonic microscope with a beam diameter of x, the area of ​​the upper surface of the mark 103 is x 2 For example, the area where the reflectance to water is 1.4 or more is x 2 If the standard deviation of the signal in this region is less than 0.1, it can be considered that the mark 103 is present in this region.

[0022] The mark 103 may be, for example, a cross mark having a linear portion with a width of 10 μm and a length of 30 μm in a plan view. Furthermore, the mark 103 may be provided at multiple locations in a grid arrangement with 200 μm intervals. The thickness of the mark 103 is not limited, but the thinner the better. For example, in cell culture, conditions without marks are most preferable, and to more closely approximate this condition, the mark 103 may be approximately 1 μm thick.

[0023] It is also important that the position of the mark 103 in the observation image can be calculated with the accuracy of the position resolution required for observation. Ideally, the angle between the sidewall surface and the top surface of the mark 103 is a right angle, but in reality, this is often an obtuse angle. In this case, the edge portion constituting the planar shape of the mark 103 has a predetermined width when viewed from above. In image processing of the observation image, in order to extract the edge portion constituting the planar shape of the mark 103 as a straight line, it is also important that the width of the edge portion is equal to or less than the required positional accuracy.

[0024] The mark 103 can be formed on the mounting surface 102 of the base 101 by, for example, a well-known lithography technique. For example, the mark 103 can be formed by a known photolithography technique using photoresist OFPR (manufactured by Tokyo Ohka Kogyo Co., Ltd.). The mark 103 can also be formed by a known pattern formation technique such as electron beam lithography or nanoimprinting.

[0025] For example, as shown in Fig. 3, the alignment component according to the embodiment can be a container 111 with a base 101 as the bottom. The container 111 has a cylindrical outer shape. As shown in Fig. 4, the alignment component according to the embodiment can be used as a container 111a as a whole by attaching the plate-like base 101 to a container 112 having an opening 112a at the bottom via an annular spacer 113. These containers are generally called dishes in cell culture and biological tissue observation techniques.

[0026] Next, a method for aligning an image observed by an ultrasonic microscope with an image observed by a fluorescent microscope using an alignment component according to an embodiment will be described with reference to FIGS. 5A to 5E.

[0027] In the first step, cells cultured on the alignment component according to the embodiment are observed with both an ultrasonic microscope and a fluorescent microscope, and a microscopic image (a) is obtained by observation with the ultrasonic microscope, and a microscopic image (b) is obtained by observation with the fluorescent microscope, as shown in Fig. 5A. Observation with each microscope is performed with, for example, four marks within the field of view.

[0028] Next, in the second step, as shown in FIG. 5B , the mark is identified in the microscope image (a) and the mark is identified in the microscope image (b). For example, in an ultrasound microscope image, first, only the area where the mark exists is extracted, this image is binarized, noise is removed from the binarized image, and the outline of the mark is extracted in the noise-removed image. In addition, in a phase-contrast microscope image, the contrast of the image is adjusted, and then only the area where the mark exists is extracted, and edges within the extracted image are detected. In addition, in a fluorescence microscope image, the contrast of the image is adjusted, and then only the area where the mark exists is extracted, this image is binarized, noise is removed from the binarized image, and the outline of the mark is extracted in the noise-removed image.

[0029] Next, in the third step, the center of gravity of each mark is calculated, as shown in Figure 5C. In Figure 5C, the calculated center of gravity is indicated by a black circle within a cross mark. For example, in ultrasound microscope images and fluorescence microscope images, the position of the extracted mark's outline is calculated. In addition, in phase-contrast microscope images, the position is calculated from the detected edge.

[0030] Next, in a fourth step, an origin mark A is determined among the identified marks in each of the microscope images (a) and (b), as shown in Fig. 5D. After this, in a fifth step, the microscope images (a) and (b) are rotated so that a mark horizontal to the origin mark A, for example, mark B horizontally adjacent to the origin mark A as shown in Fig. 5E, is positioned on the horizontal axis (y axis) of a two-dimensional coordinate system (x-y coordinate) with the origin mark A as the origin (so that the y coordinate of mark B is 0), thereby aligning the images.

[0031] Next, the observation results of each microscope using the actually fabricated alignment component will be described with reference to Figures 6A, 6B, and 6C. In Figures 6A, 6B, and 6C, (a) is an ultrasound microscope image, (b) is a phase-contrast microscope image, and (c) is a fluorescence microscope image. The marks on the fabricated alignment component were designed with an alignment accuracy of 5 μm. The observation target was a cell, and a container was fabricated with the base of the alignment component as its bottom. Each microscope image was captured with this container filled with water. In Figures 6A and 6B, the confirmed marks are identified by A, B, C, D, E, and F. Note that in the ultrasound microscope image in (a), no images were captured outside the area where the marks were recognized.

[0032] First, at the stage when the microscopic images are acquired, as shown in Figure 6A, the positions of each mark in the images are different in the ultrasound microscope image (a), phase-contrast microscope image (b), and fluorescence microscope image (c), and the corresponding relationship between each microscope image is unclear.

[0033] Next, the coordinates of each mark in each microscope image are determined, and the images are corrected so that the coordinates of each mark match across the different microscope images. This allows the positions of each mark in the ultrasound microscope image (a), phase-contrast microscope image (b), and fluorescence microscope image (c) to be consistent, as shown in Figure 6B. Figure 6C shows the result of overlay in the area where cells are present. By using the alignment components according to the embodiment, the deviation in the center of gravity of each mark was kept to 5 μm or less. Note that the scale units shown in Figures 6B and 6C are μm.

[0034] As described above, according to the present invention, a concave polygonal mark made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more is provided on the mounting surface of the base on which the sample to be observed is placed, so that when observing biological tissue, the observation position in the optical microscope image can be matched to the observation position in the ultrasonic microscope image.

[0035] Some or all of the above-described embodiments may also be described as, but are not limited to, the following supplementary notes.

[0036] [Appendix 1] An alignment component comprising a base on which a sample to be observed is placed, and concave polygonal marks formed on the placement surface of the base on which the sample is placed, the marks being made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more, and provided at a plurality of locations on the placement surface, with the spacing between adjacent marks being known.

[0037] [Supplementary Note 2] In the alignment component according to Supplementary Note 1, the mark is made of a material having a refractive index outside the range of 1.34 to 1.38 or 1.56 to 1.62, or a material that emits fluorescence.

[0038] [Supplementary Note 3] In the alignment component according to Supplementary Note 1 or 2, the marks are two-dimensionally provided at a plurality of locations on the placement surface.

[0039] [Appendix 4] The alignment component according to any one of Appendices 1 to 3, further comprising a reference mark formed on the mounting surface and having a concave polygonal shape different from that of the mark, the reference mark being made of a material having an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more.

[0040] [Supplementary Note 5] The alignment component according to any one of Supplementary Notes 1 to 4, wherein the base is a container having a bottom surface.

[0041] It should be noted that the present invention is not limited to the embodiments described above, and it is clear that many modifications and combinations can be made by a person having ordinary knowledge in the art within the technical concept of the present invention.

[0042] 101...base, 102...mounting surface, 103...mark.

Claims

1. An alignment component comprising: a base on which a sample to be observed is placed; and concave polygonal marks formed on the mounting surface of the base on which the sample is placed, the marks being made of a material with an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more, and provided at multiple locations on the mounting surface, with the spacing between adjacent marks being known.

2. An alignment component according to claim 1, wherein said mark is made of a material having a refractive index outside the range of 1.34 to 1.38 or 1.56 to 1.62, or a material that emits fluorescence.

3. An alignment component according to claim 1, wherein the marks are provided two-dimensionally at a plurality of locations on the placement surface.

4. An alignment component according to claim 1, further comprising a reference mark formed on the mounting surface and having a concave polygonal shape different from that of the mark, the reference mark being made of a material having an acoustic impedance of 1.2 MRayls or less or 1.8 MRayls or more.

5. The alignment component according to claim 1, wherein the alignment component is a container having the base as a bottom surface.

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