X-ray contrast agent and method for obtaining X-ray image

Abstract

The present invention relates to a method for obtaining an x-ray CT image of a biological sample, and the like, said method including: a step for contrasting by causing a contrast medium that includes wax, that has a density of 0.95 g / cm3 or less in a state of having permeated a biological sample and hardened, and that has a melting point of 40-80°C, to permeate a biological sample and hardening said contrast medium; a step for melting and resolidifying the hardened contrast medium; and a step for irradiating the resolidified biological sample with x-rays having 4-12 keV of energy and acquiring an x-ray CT image. Moreover, said method is such that the shape of the biological sample is a shape for which the maximum optical path length of x-rays within the biological sample in the step for acquiring the x-ray CT image is 2 mm or less.

Description

Technical Field

[0001] The present invention relates to a method for obtaining an X-ray CT image of a biological sample, a method for analyzing a biological sample, a contrast agent, and the like.

Background Art

[0002] X-ray CT (Computed Tomography) irradiates a subject with X-rays from various directions, detects the intensity of the transmitted X-rays, and synthesizes an image showing the spatial distribution of the internal X-ray absorption coefficient by reconstruction calculation to obtain a cross-sectional image or a three-dimensional image of the subject. Different from optical microscopes and electron microscopes, X-ray CT enables three-dimensional observation and can observe the internal structure without destroying the subject, so it is used in various fields such as industrial applications and the medical field.

[0003] Currently widely used micro X-ray CT utilizes the hard X-ray region of about 50 - 160 kV tube voltage. For subjects such as machine parts and electronic components composed of elements with a large atomic number, high-quality images with strong contrast, fineness, and clarity can be obtained. On the other hand, for a subject constructed from light elements with weak X-ray absorption, such as a biological sample, sufficient contrast cannot be obtained. Therefore, for biological samples, X-ray CT is performed with contrast enhanced by a contrast agent such as a heavy metal. For example, Non-Patent Document 1 describes obtaining an X-ray CT image of a biological sample using osmium tetroxide as a contrast agent.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

[0005] Currently, X-ray CT, which is widely used, obtains contrast and forms images based on the magnitude of X-ray absorption by the subject. However, elements with low atomic numbers, such as hydrogen, carbon, oxygen, and nitrogen, which mainly constitute biological soft tissues, all have low X-ray absorption, so sufficient contrast cannot be obtained, and high depiction ability cannot be expected in the structure of soft tissues. Therefore, contrast agents containing heavy elements such as osmium tetroxide are used to supplement low contrast. However, in many cases required for observation, there is no appropriate contrast agent. Also, there are cases where the penetration of the contrast agent into the biological sample is insufficient, or the influence of the contrast agent becomes too strong, which has been an issue for X-ray microscopes in the observation of biological samples.

[0006] An object of the present invention is to provide a method for obtaining a clear (for example, with a voxel size of 5 μm or less) high-quality X-ray CT image of a biological sample, and a contrast agent used in the method.

[0007] The inventor of the present invention has elucidated the principle of generating contrast in a biological sample during CT image acquisition, and found that sufficient contrast can be obtained by using a contrast agent containing wax, which penetrates into the biological sample and solidifies, and has a density of 0.95 g / cm 3 as follows, and having a melting point of 40°C to 80°C. Generally, waxes such as paraffin have only been recognized as embedding materials used in the process of making paraffin blocks as a pretreatment for optical microscopy and electron microscopy measurement, and it has not been known until now that they can be used as contrast agents.

[0008] In addition, the inventor of the present invention has provided a method for obtaining an X-ray CT image of a biological sample, wherein the density of the wax-containing state that penetrates into the biological sample and solidifies is 0.95 g / cm 3The method includes the steps of infiltrating a contrast agent having a melting point of 40°C to 80°C into a biological sample, solidifying it, and performing imaging; melting the solidified contrast agent and re-solidifying it; and irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image. The shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less. By this method, a high-quality X-ray CT image that is fine and clear is obtained, and the present invention is completed.

[0009] The present invention includes the following embodiments. (1) A method for obtaining an X-ray CT image of a biological sample, comprising: a contrast agent containing wax, the density of which in the state of infiltrating and solidifying into the biological sample is 0.95 g / cm 3 The steps of infiltrating a contrast agent having a melting point of 40°C to 80°C into a biological sample, solidifying it, and performing imaging; melting the solidified contrast agent and re-solidifying it; irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image and the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less. (2) The method according to (1), wherein the step of melting and re-solidifying the contrast agent is performed with the biological sample placed on the sample stage. (3) The method according to (1) or (2), further comprising, before the step of infiltrating the contrast agent into the biological sample, solidifying it, and performing imaging, a step of cutting the biological sample into a shape such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less. (4) The method according to (1) or (2), further comprising, after the step of infiltrating the contrast agent into the biological sample, solidifying it, and performing imaging, a step of cutting the solidified biological sample into a shape such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less. (5) The method according to (4), wherein the step of cutting the biological sample is performed with a cylindrical pipe heated to a temperature higher than the melting point of the contrast agent. Further comprising the step of adding a marker to the biological sample, In the step of acquiring an X-ray CT image, the X-ray CT image is corrected by moving each slice of the X-ray CT image in accordance with the movement of the projection image obtained from the X-ray projection image of the marker, according to the method of any one of (1) to (5). (7) The contrast agent has a density of 0.95 g / cm at 25 °C, 3 According to the method of any one of (1) to (6), having the following density. (8) The wax is solid paraffin, according to the method of any one of (1) to (7). (9) The X-ray CT image is clearly obtained with a voxel size of 5 μm or less, according to the method of any one of (1) to (8). (10) A method for analyzing a biological sample, The density of the wax-containing biological sample in a state of being infiltrated and solidified is 0.95 g / cm 3 Below, and a contrast agent having a melting point of 40 °C to 80 °C is infiltrated into the biological sample and solidified for imaging; Melting and re-solidifying the solidified contrast agent; Irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to acquire an X-ray CT image, where the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of acquiring the X-ray CT image is 2 mm or less; Furthermore, identifying a site to be further observed by an optical microscope and / or an electron microscope based on the acquired X-ray CT image, cutting out and exposing the site; Observing the site by an optical microscope and / or an electron microscope; Analyzing the biological sample by combining the X-ray CT image and the observation results by an optical microscope and / or an electron microscope; Including, the method. (11) A contrast agent for X-ray CT of a biological sample, which contains wax and has a density of 0.95 g / cm 3 Below, and a melting point of 40 °C to 80 °C.

[0010] According to the present invention, high-quality X-ray CT images that are detailed and clear can be obtained for biological samples.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] In one embodiment, the present invention is a method for obtaining an X-ray CT image of a biological sample, comprising a contrast agent containing wax, having a density of 0.95 g / cm 3 or less in a state of being infiltrated into and solidified in the biological sample, and having a melting point of 40°C to 80°C, the steps of infiltrating and solidifying the biological sample for imaging, melting and re-solidifying the solidified contrast agent, and irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image, wherein the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less.

[0013] The method of the present invention can obtain a high-quality X-ray CT image with high clarity (for example, a voxel size of 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less). The voxel size of the X-ray CT image refers to the size of a three-dimensional pixel (a cube with the voxel size as one side). The voxel size can be calculated by dividing the size of the field of view of the CT image by the number of pixels included in that field of view.

[0014] In this specification, wax means a lipophilic compound that is solid at normal temperature (20°C to 30°C) and has a melting point of 40°C to 80°C. The wax may be either natural wax or synthetic wax, and examples thereof include petroleum wax (natural wax), such as paraffin wax, microcrystalline wax, and petrolatum. The main components of these petroleum waxes are saturated hydrocarbons, which are linear normal paraffins, branched isoparaffins, and cyclic cycloparaffins. In one embodiment, the petroleum wax is solid paraffin, which means a mixture of alkanes having 20 or more carbon atoms. The petroleum wax may contain any one of the above waxes alone, or a combination of the above waxes.

[0015] The contrast agent described in this specification containing wax has a density of 0.95 g / cm 3The following is true. In one embodiment, the density is a value at normal temperature, for example, 20°C to 30°C or 25°C. The density of the contrast agent can be measured by a pycnometer method or a gravimetric method using Archimedes' principle, etc.

[0016] The melting point of the contrast agent described in this specification may be 40°C to 80°C, for example, 45°C or higher, 50°C or higher, or 55°C or higher, and / or 80°C or lower, 70°C or lower, or 60°C or lower. The melting point of the contrast agent can be measured by a differential scanning calorimeter (DSC), etc.

[0017] The contrast agent described in this specification contains wax such as paraffin as a main component, but may also contain other components (for example, the contrast agent contains 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% of wax such as paraffin based on the weight of the contrast agent).

[0018] In this specification, the "contrast agent" means a drug used to provide contrast to an image in the observation of a biological sample by X-ray CT (in this specification, the contrast agent is also described as a "low-density solidified contrast agent"). When the contrast agent described in this specification penetrates into a biological sample, the density of the solidified contrast agent is 0.95 g / cm 3Since it is lower than the density of water, by infiltrating a biological sample (for example, soft tissue having a density similar to that of water for most of it), contrast can be given to the image in observation by X-ray CT due to the density difference. Further, by infiltrating and solidifying the contrast agent described in the present specification into the cells and / or tissue spaces of the biological sample, it is possible to suppress changes in the state of the biological sample (for example, movement during measurement of a soft and brittle biological sample) during observation by X-ray CT. Further, since the X-ray absorption of wax is between that of air and water, metal artifacts (image noise that may occur when substances with significantly different X-ray transmittances are included in one imaging target) such as those that occur when heavy metals are used as contrast agents do not occur. However, since wax has poor compatibility with water, the presence of moisture in the tissue hinders wax penetration. Therefore, before infiltrating and solidifying a contrast agent containing wax, it is preferable to sufficiently perform dehydration treatment with alcohol or acetone.

[0019] In this specification, in the step of "penetrating a contrast agent into a biological sample, solidifying it, and performing imaging", the contrast agent described in this specification is penetrated into the biological sample and solidified, thereby providing contrast to the image in the subsequent observation of the biological sample by X-ray CT. Without limitation, the step of "penetrating a contrast agent into a biological sample, solidifying it, and performing imaging" can be performed, for example, by heating a biological sample collected from a subject to a temperature above the melting point of the contrast agent to liquefy the contrast agent, penetrating the liquefied contrast agent into the biological sample, and cooling it to a temperature below the melting point to solidify the contrast agent. By using a mold, the contrast agent can be solidified into any shape. When using a mold, imaging can be performed with a smooth sample surface, so there is no need to melt and re-solidify the solidified contrast agent. However, the solidified contrast agent may be melted and re-solidified to fix it to the sample stage or remove excess paraffin. A contrast agent with a low viscosity in the liquid state can be used to more easily perform penetration. Before penetrating the contrast agent into the biological sample, one or more of the following steps may be performed: immersing the biological sample in a fixing solution such as formalin or Bouin's solution for chemical fixation, dehydrating the fixed biological sample with an alcohol such as ethanol or acetone, and substituting the dehydrated sample with an intermediate agent such as xylene, propylene oxide, or chloroform to make it compatible with wax. Fixation methods that can be used include formalin fixation (using formaldehyde, paraformaldehyde, glutaraldehyde, etc.), alcohol fixation (using ethanol, methanol, acetone, chloroform, etc.), picric acid fixation (using Bouin's solution, alcoholic Bouin's solution, etc.), and other fixation methods (HOPE, PAXgene). By performing these steps, it is possible to reduce tissue shrinkage, cracking, changes in cell structure, etc. associated with the evaporation of moisture when the contrast agent penetrates and solidifies in the biological sample. The step of penetrating a contrast agent into a biological sample, solidifying it, and performing imaging may further include, in addition to the above steps, a step of degreasing the biological sample with acetone or the like and a step of decalcifying the biological sample with an acidic solution as necessary.

[0020] The step of melting and re-solidifying the solidified contrast agent can be performed, for example, by heating a biological sample or the space around it and then cooling it. In an embodiment where a heat conduction part (e.g., a metallic sample stage) is in contact with the biological sample, the melting of the solidified contrast agent may be performed by heating the heat conduction part. When heating the heat conduction part (e.g., a metallic sample stage), for example, it can be performed by immersing the heat conduction part in boiling water at 40 °C or higher, 50 °C or higher, 60 °C or higher, 70 °C or higher, or 80 °C or higher for several minutes, for example, 1 to 30 minutes, 1 to 20 minutes, or 1 to 5 minutes. Cooling can be performed, for example, by removing the heat conduction part from the boiling water and returning it to room temperature. By melting and re-solidifying the solidified contrast agent, excess contrast agent around the biological sample can be removed and processed into a smooth surface. Thereby, it is possible to prevent or reduce the mixing of artifacts due to sharp irregularities on the surface of the biological sample solidified with the contrast agent into the image, and / or prevent a decrease in the contrast of the X-ray absorption image that may occur when a large amount of contrast agent adheres to the biological sample. Note that considering that if the heating time is too long, there is a risk that the necessary contrast agent will flow out or the biological sample itself will denature, the heating time can be set appropriately. For example, the heating time can be set to such an extent that the surface of the biological sample does not dry out and / or, when the biological sample is directly fixed to the sample stage, the paraffin required for fixation remains. The heating time can be, for example, 30 seconds to 30 minutes, 1 minute to 10 minutes, 3 minutes to 8 minutes, or 5 minutes.

[0021] In one embodiment, the step of melting and re-solidifying the contrast agent is performed with the biological sample placed on the sample stage. Thereby, excess contrast agent around the biological sample is removed by melting and processed into a smooth surface. Furthermore, since the biological sample is directly fixed to the sample stage by the contrast agent during re-solidification, noise that may occur in the image due to the use of another fixture (such as a tube or film) can be reduced.

[0022] The step of irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image can be carried out by a conventional method using, for example, an X-ray microscope. The type of X-ray microscope is not limited, but for example, nano3DX (Rigaku Corporation) can be used.

[0023] As X-ray generation sources, X-ray tubes and synchrotron radiation are known. An X-ray tube is a device that accelerates and focuses thermoelectrons emitted from a cathode (filament) by the potential difference between the cathode and the anode and collides them with an anode (target) to generate X-rays. The X-rays generated from an X-ray tube include continuous X-rays due to bremsstrahlung of the accelerated electrons and characteristic X-rays (showing a line spectrum) associated with the excitation and transition of extranuclear electrons of the atoms constituting the target.

[0024] In one embodiment, the X-rays described in this specification include characteristic X-rays derived from targets such as Ti, Cr, Cu, and Ga. By using Ti, Cr, Cu, Ga, etc. as target materials, it becomes possible to utilize low-energy characteristic X-rays with good sensitivity. In one embodiment, the energy of the X-rays is 5 to 9 keV or 5.41 to 8.04 keV.

[0025] Synchrotron radiation is X-rays generated when the orbit of an electron beam accelerated to a speed close to the speed of light is changed by a magnet or the like in a synchrotron (a type of circular accelerator). Since the diameter of a synchrotron reaches, for example, several tens of meters or more, a large facility is usually required to use synchrotron radiation. In one embodiment, the X-rays described in this specification are not synchrotron radiation, and in this embodiment, the method described in this specification does not require a large facility.

[0026] In one embodiment, the method described herein does not use contrast agents other than the contrast agent (e.g., osmium, iodine, barium, etc.). In this embodiment, there is no need for a contrast process using other contrast agents, and metal artifacts and image degradation caused by the use of other contrast agents can be reduced (e.g., osmium tetroxide used as a contrast agent reacts chemically with biological components having double bonds and binds, so unsaturated fatty acids containing many double bonds may be emphasized in X-ray CT images).

[0027] In this specification, the types of biological samples are not limited, but may be cells, tissues, organs, or organ systems of mammals including humans, birds, reptiles, amphibians, insects, fish, benthic animals, or plants. Examples of organs or organ systems include the kidney, liver, heart, pancreas, intestinal tract, stomach, lung, brain / nerves, bone / muscle, blood vessels, sensory organs, genital organs, and organoids, and further include scaffold materials of bioabsorbable polymers that can be used for tissue / organ regeneration.

[0028] In order to obtain an X-ray CT image with a spatial resolution of about several tens of μm or less, it is necessary for more than 10% of the irradiated X-rays to pass through the sample. The X-ray attenuation length of water having an X-ray absorption coefficient equivalent to that of a biological substance is about 1 mm when the energy of the X-rays is 8 keV, which means that the X-rays are attenuated by approximately one digit when passing through a biological sample with a thickness of 2 mm. Therefore, when the energy of the X-rays is 8 keV, if the biological sample has a maximum optical path length of the X-rays of 2 mm or less (e.g., a shape with a maximum width in the X-ray transmission direction such as a cylinder with a diameter of about 2 mm or less), it can be observed with an X-ray transmittance of 10% or more. Examples of biological samples with a maximum optical path length of the X-rays of 2 mm or less include shapes with a diameter of 2 mm or less or 1 mm or less, or a maximum width of 2 mm or 1 mm or less (e.g., a prismatic shape). In this specification, a shape with a maximum width of 2 mm may be a prismatic shape, such as a triangular prism, a quadrangular prism (e.g., a cuboid), a pentagonal prism, or a hexagonal prism. In this specification, a cylindrical or prismatic shape includes a substantially cylindrical or substantially prismatic shape. Note that the biological sample solidified with the contrast agent described in this specification is preferably cylindrical for compatibility with CT reconstruction.

[0029] In this specification, the "optical path length" of X-rays in a biological sample in the process of acquiring an X-ray CT image means the length of the path through which X-rays pass through the biological sample assuming the refractive index of the X-rays of the biological sample is 1. In the process of acquiring an X-ray CT image, the sample is rotated to take a number of X-ray projection images from different directions. The maximum X-ray optical path length in these projection images is defined as the "maximum X-ray optical path length in the biological sample in the process of acquiring an X-ray CT image". The maximum X-ray optical path length can be obtained by measuring the maximum length of the biological sample in the direction perpendicular to the sample rotation axis during X-ray CT data acquisition. For example, one X-ray projection image can be taken in an orientation where the side of the maximum length is perpendicular to the X-ray irradiation direction, and the maximum length of the biological sample in the direction perpendicular to the sample rotation axis can be measured using image analysis software such as ImageJ. More precisely, the maximum length of the biological sample in the direction perpendicular to the sample rotation axis can be measured from the projection image data of X-ray CT or from the sinogram in the direction of the sample rotation axis created by ImageJ or the like.

[0030] In this specification, the maximum width of a shape means the maximum length of the shape in the direction perpendicular to the sample rotation axis during X-ray CT data acquisition. Although the lower limit of the diameter or maximum width of the biological sample is not limited, due to processing technology problems, it is preferably 0.1 mm or more, 0.3 mm or more, or 0.5 mm or more.

[0031] The height of the cylinder or shape described in this specification (the length in the direction of the sample rotation axis during X-ray CT data acquisition) is not limited.

[0032] In one embodiment, the method for obtaining an X-ray CT image described herein further includes a step of cutting a biological sample into a shape such that the maximum optical path length of X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less before the step of infiltrating and solidifying a contrast agent into the biological sample for imaging. The contrast agent is infiltrated into the biological sample obtained by this step, solidified, and imaged (in this case, the solidification and imaging may be performed using a mold so that the solidified biological sample has the above shape). In another embodiment, the method for obtaining an X-ray CT image described herein includes a step of cutting the solidified biological sample into a shape such that the maximum optical path length of X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less after the step of infiltrating and solidifying a contrast agent into the biological sample for imaging and before the step of melting and re-solidifying the solidified contrast agent. The step of cutting the biological sample can be performed using a cylindrical pipe, for example, a cylindrical pipe with a sharp blade tip having a diameter of 2 mm or less and no seam, such as a biopsy punch. The step of cutting the biological sample may be performed using a cylindrical pipe heated to a temperature higher than the melting point of the contrast agent. Thereby, since the biological sample can be collected while melting the contrast agent, it is possible to collect the biological sample with less cracking and a smooth surface.

[0033] In one embodiment, the method described herein further includes a step of adding a marker to the biological sample, and in the step of obtaining the X-ray CT image, the X-ray CT image is corrected by moving each slice of the X-ray CT image in accordance with the movement of the projection image obtained from the X-ray projection image of the marker. Observation by X-ray CT may take several hours to 48 hours or several hours to 24 hours, and the image quality may deteriorate due to drift (the observation sample moves in the order of μm during image acquisition). In this case, a clearer image can be obtained by performing the above correction.

[0034] As used herein, the marker means a landmark used for the above drift correction. As the marker, materials having a sufficiently higher density than the biological sample, such as diamond, graphite, silicon, titanium, and aluminum, can be used.

[0035] The step of adding a marker to the biological sample can be performed, for example, by heat-melting and then re-solidifying a wax described in this specification containing the marker (for example, when using diamond as the marker, obtained by cutting the solidified wax with a diamond wire saw) together with the biological sample solidified by the contrast agent described in this specification, so as to disperse the marker in the contrast agent. The marker concentration in the contrast agent is preferably such that it provides a situation where drift correction is possible (that is, there is one or more single-particle markers at a desirable position in the X-ray imaging image and it is distinguishable from other markers). Also, the marker is preferably added at a position close to the sample rotation axis on the surface of the biological sample. If the marker is added at a position far from the axis, the marker may move out of the field of view due to the rotation of the sample during X-ray image acquisition, and in this case, drift correction becomes difficult.

[0036] Drift correction can be performed by correcting the X-ray CT image by moving each slice of the X-ray projection image in accordance with the movement of the marker in the projection image obtained from the X-ray projection image of the biological sample to which the marker is added. The correction can be performed, for example, as follows according to the description of the examples in this specification. That is, the X-ray projection image of the biological sample to which the marker is added (a large number of continuous image data obtained by sequentially and systematically changing the sample orientation while rotating the sample) is read by software such as the program ImageJ, and a projection image (Linogram) of the marker's trajectory in the direction perpendicular to the sample rotation axis is created. Subsequently, the above projection image is further processed to extract one trajectory. Note that the drift amount in the sample rotation axis direction is the numerical value of the position of this line. Subsequently, the X-ray CT image can be corrected by moving each slice of the X-ray projection image of the biological sample up and down so as to cancel out the drift amount.

[0037] In one embodiment of the step of infiltrating and solidifying a contrast agent into a biological sample in the method for obtaining an X-ray CT image of a biological sample of the present invention, the contrast agent is shown in FIG. 8. First, a sample is cut out from the living body. Subsequently, chemical fixation is performed to protect the biological sample from deterioration due to self-decomposition or decay. Chemical fixation may be any of formalin fixation (using formaldehyde, paraformaldehyde, glutaraldehyde, etc.), alcohol fixation (using ethanol, methanol, acetone, chloroform, etc.), picric acid fixation (using Bouin's solution, alcoholic Bouin's solution, etc.), and other fixation methods (HOPE, PAXgene, etc.). Subsequently, a dehydration step is performed to replace the water in the tissue with alcohol or the like and remove the water (preferably completely) from the biological tissue. The dehydration step can be performed using an ethanol series, an acetone series, or other solutions. After the dehydration step, an intermediate agent treatment step, which is a treatment for replacing alcohol or the like in the biological tissue with an intermediate agent, is performed. The intermediate agent treatment step can be performed using xylene, propylene oxide, chloroform, etc. Subsequently, a contrast agent infiltration step is performed. In the contrast agent infiltration step, a contrast agent containing wax, having a density of 0.95 g / cm 3 or less and a melting point of 40°C to 80°C, is infiltrated into the biological sample. Subsequently, excess contrast agent around the biological sample may be removed using a diamond wire saw or a razor. In the case of a larger biological sample, a part may be collected with a cylindrical pipe with a sharp blade tip having a diameter of 2 mm or less, such as a biopsy punch (in the collection step, the cylindrical pipe may be heated until it becomes higher than the melting point of the contrast agent). Also, for the purpose of drift correction, markers such as diamond, graphite, silicon, titanium, and aluminum may be added to the biological sample. In the step of adding a marker to the biological sample, the marker can be obtained, for example, when using diamond, by cutting the solidified contrast agent with a diamond wire saw. Subsequently, melting and re-solidification treatment may be performed. By removing the excess contrast agent around the biological sample in this way, the maximum optical path length of the X-ray can be finally made 2 mm or less, the sample surface can be smoothed, and the sample can be fixed to the sample holder of the X-ray microscope.

[0038] In one embodiment, the present invention relates to a method for analyzing a biological sample, comprising: a step of infiltrating a contrast agent containing wax into the biological sample and solidifying it so that the density in the solidified state is 0.95 g / cm 3 or less and having a melting point of 40°C to 80°C, infiltrating the biological sample, solidifying it, and performing imaging; a step of melting and re-solidifying the solidified contrast agent; a step of irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image, wherein the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less; further, a step of specifying a site to be further observed by an optical microscope and / or an electron microscope based on the obtained X-ray CT image, cutting out and exposing the site; a step of observing the site with an optical microscope and / or an electron microscope; and a step of analyzing the biological sample by combining the X-ray CT image and the observation results by the optical microscope and / or the electron microscope.

[0039] In this analysis method, for the steps of infiltrating and solidifying the contrast agent into the biological sample, melting and re-solidifying the solidified contrast agent, and obtaining the X-ray CT image, each step of the method for obtaining the X-ray CT image is as described herein.

[0040] Based on the obtained X-ray CT image, the step of identifying the site to be further observed by an optical microscope and / or an electron microscope and cutting out and exposing the site can be performed, for example, as follows. First, as shown in FIG. 1D, the step of melting and re-solidifying the contrast agent on the sample stage and acquiring the above X-ray CT image in a state where the sample is fixed on the sample stage by the contrast agent is performed. Subsequently, in a commercially available cutting device (for example, a diamond wire saw of Musashino Electronics or WELL Diamond Wire Saws), if necessary, a commercially available unit is removed, a goniometer is newly installed as shown in FIG. 4, and the sample stage in a state where the biological sample is fixed is transferred to the sample stage unit of the X-ray microscope attached to the goniometer. The sample stage unit is rotatable and movable in the X-Y axes, preferably provided with a tilting mechanism (a mechanism for adjusting the direction shown in FIG. 4A), and the goniometer is movable in a range of 90° or more, so that the biological sample can be cut according to the purpose. Here, a mark (reference) for determining the angle is provided on the sample stage, and the sample stage can be installed on the sample stage unit according to the reference. Cutting can be performed based on the information (numerical values) taken by X-ray CT while confirming with a microscope provided in the cutting machine. When performing optical microscope observation after cutting with a diamond wire saw, the biological sample is removed from the sample stage, infiltrated and solidified in a paraffin block conforming to the specifications of a microtome, sliced with a microtome using the cutting surface by the diamond wire saw as a mark, and an observation sample for an optical microscope is prepared according to a conventional method. On the other hand, when performing electron microscope observation after cutting with a diamond wire saw, the biological sample is removed from the sample stage, deparaffinized, embedded in an epoxy resin block conforming to the specifications of a microtome, ultrathin sliced with a microtome using the cutting surface by the diamond wire saw as a mark, and an observation sample for an electron microscope is prepared according to a conventional method. If necessary, the surface of the sample may be smoothed by ion milling, a cross-section polisher TM etc. If it is determined from the results of X-ray CT observation that cutting with a diamond wire saw is unnecessary, the cutting may be omitted and the observation may proceed with an optical microscope or an electron microscope.

[0041] In one embodiment, the step of "penetrating a contrast agent into a biological sample, solidifying it, and performing imaging" can be carried out by the same procedure as the paraffin embedding step.

[0042] Since the analysis method can observe a biological sample by combining an X-ray CT image with an optical microscope and / or an electron microscope, more detailed information can be obtained than when observing with any one of the X-ray CT image, the optical microscope, and the electron microscope alone. First, in X-ray CT, unlike an optical microscope or an electron microscope, three-dimensional observation is possible, and the subject can be inspected non-destructively, so that the biological sample can be observed in a form closer to its original state. On the other hand, generally, X-ray CT has inferior spatial resolution compared to an optical microscope or an electron microscope. Therefore, by specifying the site to be observed based on the X-ray CT image and observing the site with an optical microscope or an electron microscope, the site can be observed in more detail.

[0043] The method for obtaining an X-ray CT image of a biological sample and the analysis method of a biological sample described in this specification can be used for the diagnosis or assistance of a disease or disorder, or for research purposes of cells and / or tissues.

[0044] In one embodiment, the present invention relates to a contrast agent for X-ray CT of a biological sample that contains the wax described in this specification, has a density of 0.95 g / cm 3 or less in the state of penetrating and solidifying into the biological sample, and has a melting point of 40°C to 80°C. Details of the contrast agent are as described in this specification regarding the method for obtaining an X-ray CT image of a biological sample.

Example

[0045] <Example 1: Preparation of Sample> A biological sample (mouse kidney section chemically fixed with 4% paraformaldehyde at room temperature for 16 hours, dehydrated with ethanol, and having a melting point of 56 to 58°C and a density of about 0.90 g / cm 3The paraffin (paraffin stick CT-PARA-ST manufactured by Genostaff Co., Ltd.) was infiltrated and solidified; the temperature in Fig. 1A was set higher than the melting point of the paraffin to melt the paraffin. Specifically, first, a part of the mouse kidney in the paraffin block was cut out in a 0.5-mm diameter cylindrical shape using a dermatological biopsy punch (Kai Industries Co., Ltd., disposable biopsy punch 0.5 mm, BP-A05F) (Figs. 1A and B). This cylindrical sample was placed on the upper surface of a 3-mm diameter cylindrical metal rod, which is a standard sample mounting jig for an X-ray microscope (Rigaku Corporation, nano3DX) (Fig. 1C). In this state, the lower part of the metal rod was heated with a hot water bath to melt and drain the excess paraffin around the sample (heating temperature about 90 °C, heating time about 5 minutes). At the same time, a disposable pipette tip (Molecular Bioproducts, 0.1 - 10 μl, cat#103) and hair were used to adjust the orientation of the sample so that the long axis of the sample was generally parallel to the long axis of the metal rod. Then, when the hot water bath was removed, the paraffin in the sample solidified while maintaining the surface smoothness upon cooling at room temperature, and the sample in a nearly naked state was fixed standing on the metal rod (Fig. 1D). A biological sample with paraffin-based contrast was prepared by the above method.

[0046] <Example 2: Application of Marker to Biological Sample> The following steps were added to the method for preparing a biological sample with paraffin-based contrast described in Example 1. As the marker, diamond wire saw dust (presumed diamond particles) was used. Specifically, when placing a part of the sample block on the 3-mm diameter metal rod and performing the hot water bath in Example 1, an additional 1 mg or so of a paraffin piece containing the marker (the black part of the cut surface obtained by cutting the paraffin block with a diamond wire saw (Musashino Electronics) and scraping it off with a scalpel) was placed, and the marker was dispersed in the paraffin by heat melting. As a result, the marker could be added at a position close to the sample rotation axis (Fig. 2). If the marker is added at a position far from the sample rotation axis, the marker may move out of the field of view due to the rotation of the sample during imaging, and in this case, drift correction becomes difficult.

[0047] <Example 3: Drift Correction> Drift correction was performed by processing X-ray projection images of a biological sample subjected to contrast imaging with paraffin (several hundred to several thousand consecutive image data obtained by sequentially and systematically changing the sample orientation by rotation around the sample rotation axis). The image in Fig. 2 in Example 2 is one of the above X-ray projection images. 1) A series of X-ray projection images were read using the program ImageJ (free software), and a linogram of the trajectory of a marker in the direction perpendicular to the sample rotation axis was created (Fig. 3A). 2) The above linogram was further processed to extract one trajectory (Fig. 3B). Here, a single line connecting from end to end, located approximately 1 / 4 from the top of the image in Fig. 3A, was used (arrow in Fig. 3A). The digitized position of this line is the vertical drift amount. Particularly large displacements were observed at two locations marked with asterisks in Fig. 3B. 3) Each slice of the projection image was translated vertically to cancel out the drift amount. Comparing the CT images before and after drift correction, the radial noise etc. that appears around the object was significantly reduced (Fig. 3C).

[0048] <Example 4: Cutting out a point of interest observed by X-ray microscopy with a cutting machine> The standard jig of a commercially available diamond wire saw (Musashino Electronics) was removed, and a goniometer was newly installed as shown in Fig. 4, and the sample stage unit of the X-ray microscope was attached to the goniometer. 1) For an epon-embedded sample of an Os-contrasted mouse kidney (the sample in Fig. 5A of Example 5), resin trimming was performed using a diamond wire saw. As a result, the contrast of the CT image was slightly improved (Fig. 4B). 2) For a sample (mouse kidney) subjected to contrast imaging with paraffin, an overly long cylindrical sample in the paraffin block was cut into an appropriate length using a diamond wire saw (Fig. 4C). There is a high risk of damaging the sample when cutting with scissors.

[0049] <Example 5: Comparison with the prior art> Using the renal corpuscles of mouse kidneys as samples, a comparison was made between an observation example of the prior art stained with osmium tetroxide and an embodiment of the present invention using paraffin as a low-density solidifying contrast agent. The observation was carried out using an X-ray microscope nano3DX (Rigaku Corporation) (CCD detector) with the 8.0 keV characteristic X-rays of a Cu-target. Using the rotating anode type high-intensity X-rays employed in nano3DX is effective for shortening the total exposure time required for data recording (for example, for shortening it to within approximately 24 hours). Also, reducing the influence of light source drift by the proximity imaging method employed in nano3DX is effective for increasing the spatial resolution (for example, for performing the observation with a sub-micron spatial resolution).

[0050] In the observation example of the prior art, mouse kidney sections were chemically fixed with 2.4% glutaraldehyde at 4°C for 2 hours, stained with 1% osmium tetroxide at 4°C overnight, dehydrated with acetone, and then embedded in epoxy resin to obtain resin blocks. The tip portion of this resin block containing the kidney sections was trimmed isotropically to approximately 0.7 to 0.8 mm, and the trimmed portion was observed with an X-ray microscope (upper part of Fig. 4B). The sample was attached to an X-ray microscope nano3DX (Rigaku Corporation) (CCD detector), and X-ray projection image data was taken under the conditions of Cu-target (40 kV / 30 mA), L0270-bin2-XD5 (0.53 μm / voxel), Step scan (1600 sheets with 30 s exposure per sheet) (required time: 13.8 hours). After processing this projection image data with general noise reduction filters (median 1 and Gaussian 1), CT reconstruction was performed using software based on a general FBP algorithm.

[0051] When preparing samples using paraffin as a low-density solidified contrast agent, the procedure followed Example 1. The samples were attached to an X-ray microscope nano3DX (Rigaku Corporation) (CCD detector), and X-ray projection image data was taken under the conditions of Cu-target (40 kV / 30 mA), L0270-bin2-XD2 (0.54 μm / voxel), Step scan (1700 exposures of 40 s each) (total time required: 19.4 hours). After processing this projection image data with general noise reduction filters (median 1 and Gaussian 1), CT reconstruction was performed using the nano3DX standard software based on the general FBP algorithm.

[0052] The results are shown in Fig. 5. Fig. 5A shows the results of epoxy resin-embedded Os contrast. Since the X-ray absorption by the sample was large, sufficient contrast could not be obtained with an exposure time of 13.8 hours, and the substantial spatial resolution was about 10 μm. Fig. 5B shows the results of paraffin contrast. Sufficient contrast was obtained with an exposure time of 19.4 hours, and the substantial spatial resolution was about 0.9 μm. The fine structure inside the renal corpuscle could also be distinguished. Note that Fig. 5B shows the results when drift correction was performed using a marker. The spatial resolution was measured from the luminance value line profile of the substance boundary in the CT slice image according to the method described in, for example, the following reference: Kunishima et al. Plant Methods (2020) 16:7

[0053] <Example 6: Sampling of Samples Using a Heated Instrument> Paraffin was sampled from a paraffin block (PARAPLASTPLUS 502004 from McCormick Scientific, melting point 56°C) using a dermatological biopsy punch (Kai Industries Co., Ltd., disposable biopsy punch 1.0 mm, BPP-10F) immersed in 90°C hot water for 20 seconds for heat treatment and an identical biopsy punch without heat treatment. After that, the sampled samples were photographed in contact to obtain optical photographs and observed with an optical microscope (zoom stereomicroscope (with LED illumination) CP745, AS ONE Corporation). The results are shown in Fig. 6 (Fig. 6A: Optical photograph when paraffin was collected using a heated biopsy punch. Fig. 6B: Optical microscope photograph when paraffin was collected using a heated biopsy punch. Fig. 6C: Optical photograph when paraffin was collected using a biopsy punch without heat treatment. Fig. 6D: Optical microscope photograph when paraffin was collected using a biopsy punch without heat treatment.) As shown in Fig. 6, when compared with the case where heat treatment was performed, when heat treatment was not performed, cracks occurred in the center of the paraffin or the contour of the paraffin was unclear. This indicates that by using a heated biopsy punch, a biological sample can be collected while dissolving the contrast agent, and the biological sample can be collected in a state where cracks are less likely to occur and the surface is smooth.

[0054] <Example 7: Test Using a Polyimide Tube> Since the X-ray attenuation length of polyimide at 8 keV (mm) is 1.2, which is close to 1.0, the X-ray attenuation length under the same conditions of water (considered to be almost equivalent to a living body), it was considered that the contrast effect of wax could be evaluated using polyimide instead of a biological sample. Therefore, the contrast effect of wax was evaluated by the following method. A polyimide tube with an inner diameter of 0.5 mm (Furukawa Electric Co., Ltd.'s polyimide tube PIT-S (inner diameter 0.50 mm, wall thickness 0.06 mm)) was heated and melted with boiling water at about 90 °C, and the wax described in Table 1 was inhaled by capillary action to a length of about 5 - 10 mm and then solidified by cooling at room temperature. The polyimide tube filled with this wax was adhered to the tip of the sample stage for an X-ray microscope with an adhesive (Aron Alpha manufactured by Toagosei Co., Ltd.) (Figure 7A). At this time, the sample rotation axis and the long axis of the polyimide tube were made to be approximately coincident (adjusted so as not to protrude from the field of view of the X-ray microscope in the direction perpendicular to the sample rotation axis). This sample stage was attached to an X-ray microscope nano3DX (Rigaku Corporation) (CCD detector), and X-ray projection image data was taken under the conditions of Cu-target (40 kV / 30 mA), L0270-bin4-XD2 (1.07 μm / voxel), Step scan (700 sheets with an exposure of 10 s per sheet) (required time: 2.2 hours). After processing this projection image data with general noise reduction filters (median 1 and Gaussian 1), CT reconstruction was performed using the nano3DX standard software based on the general FBP algorithm. Since orthogonal CT slices from three directions are output as a report file in tiff format, this report was input into the free software ImageJ for image analysis (Figure 7B). Square portions of 15×15 pixels were arbitrarily taken in each of the wax region and the polyimide region, and the average luminance value and standard deviation within that portion were measured. Then, the value obtained by dividing the difference in the average luminance values of the two regions by its standard deviation (the square root of the sum of the variances of the two regions) was defined as the SN ratio (Reference: KUNISHIMA N. et. al., PLANT METHODS 16:7 (2020)). This measurement was performed at 5 locations, and the average value and its 95% confidence interval were calculated (Table 1). The measurement locations were selected so as not to be biased from the three directions of the report. Also, as a negative example, polyethylene glycol (dried 50% PEG3350 aqueous solution manufactured by Hampton Research) and, as an example of an actual biological sample, the results of similar measurements for the mouse kidney sample described in Example 5B were added and shown. For the mouse kidney sample, the SN ratio between the wax (paraffin) and the biological tissue (renal tubule) was measured.The SN ratios were about 18 for all five commercially available paraffins, about 2.5 for polyethylene glycol, and about 8 for biological samples. Based on the above results, a wax showing an SN ratio of 8 or more in comparison with polyimide by the above method is recognized as a wax with a contrast effect. These results indicate that, instead of biological samples, a polyimide tube can be used to evaluate whether a candidate substance has an effect as a contrast agent.

[0055]

Table 1

Claims

1. A method for obtaining an X-ray CT image of a biological sample, comprising: The density of the state in which it has penetrated and solidified in a biological sample containing wax is 0.95 g / cm ３ equal to or less than the following, and a step of allowing a contrast agent having a melting point of 40°C to 80°C to penetrate and solidify in a biological sample for imaging; a step of melting and re-solidifying a solidified contrast agent; a step of irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image, wherein the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less.

2. The method according to claim 1, wherein the step of melting and re-solidifying the contrast agent is performed with the biological sample placed on a sample stage.

3. The method according to claim 1 or 2, further comprising a step of cutting the biological sample into a shape such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less, before the step of infiltrating the biological sample with the contrast agent, solidifying it, and performing contrast imaging.

4. The method according to claim 1 or 2, further comprising a step of cutting the solidified biological sample into a shape such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less, after the step of infiltrating the biological sample with the contrast agent, solidifying it, and performing contrast imaging.

5. The method according to claim 4, wherein the step of 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 further comprising a step of adding a marker to the biological sample, wherein in the step of obtaining the X-ray CT image, the X-ray CT image is corrected by moving each slice of the X-ray CT image in accordance with the movement of the projection image obtained from the X-ray projection image of the marker.

7. The contrast agent has a density of 0.95 g / cm at 25°C ３ The method according to claim 1 or 2, having the following density.

8. The method according to claim 1 or 2, wherein the wax is solid paraffin.

9. The method according to claim 1 or 2, wherein the X-ray CT image is clearly obtained with a voxel size of 5 μm or less.

10. A method for analyzing a biological sample, comprising: The density of the state in which it has penetrated and solidified into a biological sample containing wax is 0.95 g / cm ３ The following, and a step of allowing a contrast agent having a melting point of 40°C to 80°C to penetrate into a biological sample, solidify, and perform imaging; a step of melting and re-solidifying a solidified contrast agent; a step of irradiating the re-solidified biological sample with X-rays having an energy of 4 to 12 keV to obtain an X-ray CT image, wherein the shape of the biological sample is such that the maximum optical path length of the X-rays in the biological sample in the step of obtaining the X-ray CT image is 2 mm or less; further, based on the obtained X-ray CT image, identifying a site to be further observed by an optical microscope and / or an electron microscope, cutting out and exposing the site; observing the site with an optical microscope and / or an electron microscope. A method comprising a step of analyzing a biological sample by combining an X-ray CT image and an observation result by an optical microscope and / or an electron microscope.

11. A contrast agent for X-ray CT of a biological sample, which contains wax, has a density in the solidified state penetrating the biological sample of 0.95 g / cm ３ or less, and has a melting point of 40°C to 80°C.

Images