US-12625143-B2 - X-ray contrast medium and X-ray image acquisition method

US12625143B2US 12625143 B2US12625143 B2US 12625143B2US-12625143-B2

Abstract

Obtaining an X-ray CT image of a biological sample includes a step of penetrating a contrast agent into the biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, the contrast agent comprising wax and having a density of 0.95 g/cm 3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C., a step of melting and resolidifying the solidified contrast agent, and a step of acquiring an X-ray CT image by irradiating the resolidified biological sample with an X ray having an energy of 4 to 12 keV, the shape of the biological sample being a shape with which a maximum optical path length of the X ray in the biological sample in the step of acquiring an X-ray CT image is 2 mm or less.

Inventors

Koichiro Ito
Naoki Kunishima
Raita HIROSE

Assignees

RIGAKU CORPORATION

Dates

Publication Date: 20260512
Application Date: 20220502
Priority Date: 20210506

Claims (11)

1 . A method of obtaining an X-ray CT image of a biological sample, the method comprising: penetrating a contrast agent into the biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, the contrast agent comprising wax and having a density of 0.95 g/cm 3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C.; melting and resolidifying the solidified contrast agent; and acquiring an X-ray CT image by irradiating the resolidified biological sample with an X ray having an energy of 4 to 12 keV, the shape of the biological sample being a shape with which a maximum optical path length of the X ray in the biological sample is 2 mm or less.
2 . The method according to claim 1 , wherein melting and resolidifying the contrast agent is performed in a state in which the biological sample is placed on a sample table.
3 . The method according to claim 1 , further comprising, before penetrating a contrast agent into the biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, cutting the biological sample into a shape with which a maximum optical path length of the X ray in the biological sample is 2 mm or less.
4 . The method according to claim 1 , further comprising, after penetrating a contrast agent into the biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, cutting the solidified biological sample into a shape with which a maximum optical path length of the X ray in the biological sample is 2 mm or less.
5 . The method according to claim 4 , wherein cutting the biological sample is performed with a cylindrical pipe heated to a temperature higher than the melting point of the contrast agent.
6 . The method according to claim 1 , further comprising adding a marker to the biological sample, wherein acquiring an X-ray CT image includes correcting the X-ray CT image by moving slices of the X-ray CT image according to a movement of a projected marker image obtained from an X-ray projection image of the marker.
7 . The method according to claim 1 , wherein the contrast agent has a density of 0.95 g/cm 3 or less at 25° C.
8 . The method according to claim 1 , wherein the wax is solid paraffin.
9 . The method according to claim 1 , wherein the X-ray CT image is clearly obtained with a voxel size of 5 μm or less.
10 . A method of analyzing a biological sample, comprising: penetrating a contrast agent into a biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, the contrast agent comprising wax having a density of 0.95 g/cm 3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C.; melting and resolidifying the solidified contrast agent; acquiring an X-ray CT image by irradiating the resolidified biological sample with an X ray having an energy of 4 to 12 keV to acquire an X-ray CT image, the shape of the biological sample being a shape with which a maximum optical path length of the X ray in the biological sample is 2 mm or less; specifying a site to be further observed by an optical microscope and/or an electron microscope based on the acquired X-ray CT image and cutting and exposing the site; observing the site with the optical microscope and/or the electron microscope; and combining the X-ray CT image and results of the observation by the optical microscope and/or the electron microscope to analyze the biological sample.
11 . A contrast agent for X-ray CT of a biological sample, the contrast agent comprising wax and having a density of 0.95 g/cm 3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is based on PCT filing PCT/JP2022/019493, filed May 2, 2022, which claims priority from Japanese Patent Application No. 2021-078340, filed May 6, 2021, the entire contents of each are incorporated herein by reference. BACKGROUND Technical Field The present disclosure relates to a method of obtaining an X-ray CT image of a biological sample, a biological sample analysis method, a contrast agent, and the like. Description of Related Art X-ray CT (Computed Tomography) is a technology for irradiating a subject with X rays from various directions, detecting the intensity of transmitted X rays, and combining images indicating a spatial distribution of an X-ray absorption coefficient inside the subject with a reconstruction arithmetic operation to obtain a sectional image and a three-dimensional image of the subject. With the X-ray CT, unlike an optical microscope and an electron microscope, a three-dimensional observation is possible and internal structure can be observed without breaking the subject. Therefore, the X-ray CT is used in industrial applications and various fields such as the medical field. Micro X-ray CT currently widely spread uses a hard X-ray region with a tube voltage of approximately 50 to 160 kV. Fine and clear high image quality with strong contrast can be acquired in a subject such as a mechanical component or an electronic component configured by an element having a large atomic number. On the other hand, sufficient contrast cannot be obtained for a subject constructed from a light element having weak X-ray absorption, for example, a biological sample. Therefore, for the biological sample, the X-ray CT is performed by giving contrast with a contrast agent such as heavy metal. For example, Non Patent Literature 1 describes that an X-ray CT image of a biological sample is obtained using osmium tetroxide as a contrast agent. CITATION LIST Non Patent Literature Non Patent Literature 1: MICHAEL D. BENTLEY et al., THE ANATOMICAL RECORD 290:277-283 (2007) SUMMARY The X-ray CT, which is widely spread today, obtains contrast depending on the level of X-ray absorption by a subject and creates an image. All of elements with low atomic numbers, for example, hydrogen, carbon, oxygen, and nitrogen, which mainly constitute a biological soft tissue, have low X-ray absorption and thus sufficient contrast cannot be obtained, and thus a high visualization capability cannot be expected in the structure of a soft tissue. Therefore, a contrast agent containing a heavy element such as osmium tetroxide is used to compensate for the low contrast. However, an appropriate contrast agent is not always present in many cases in which observation is required. In some cases, for example, the contrast agent does not sufficiently penetrate into a biological sample or the influence of the contrast agent is too strong, which has been a problem for an X-ray microscope in the observation of the biological sample. An object of the present disclosure is, for example, to provide a method of obtaining a clear (for example, with a voxel size of 5 μm or less) high-quality X-ray CT image, for a biological sample and a contrast agent used for the method. The present inventors have elucidated a principle of obtaining contrast in a biological sample in CT imaging and found that, by using a contrast agent comprising wax and having a density of 0.95 g/cm3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C., sufficient contrast can be obtained even if a contrast agent consisting of a heavy element is not used. In general, wax such as paraffin is only recognized as an embedding material used in a process of making a paraffin block as pretreatment for optical microscopy and electron microscopy. It has not been known that the wax can be used as a contrast agent. As recognized by the present inventors, a fine and clear high-quality X-ray CT image can be obtained by a method of obtaining an X-ray CT image of a biological sample, the method comprising a step of penetrating a contrast agent into the biological sample and solidifying the contrast agent to provide a contrast image of the biological sample, the contrast agent comprising wax and having a density of 0.95 g/cm3 or less in its solidified state after penetration into the biological sample, and having a melting point of 40° C. to 80° C., a step of melting and resolidifying the solidified contrast agent, and a step of acquiring an X-ray CT by irradiating the resolidified biological sample with an X ray having an energy of 4 to 12 keV, the shape of the biological sample being a shape with which a maximum optical path length of the X ray in the biological sample in the step of acquiring an X-ray CT image is 2 mm or less. The present disclosure comprises the following embodiments. (1) A method of obtaining an X-ray CT image of a biological sample, the method