JP7175462B2 - Density measurement method and calibration standard sample and method for preparing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring the density of an object to be measured mainly composed of calcium carbonate, a calibration standard sample used in the method for measuring density, and a method for producing the same. Specifically, for example, a density measurement method for measuring the shell density or skeleton density of marine organisms such as shelled plankton, which have calcareous shells and skeletons, a calibration standard sample used in the density measurement method, and a method for producing the same Regarding.

It is known that some of the carbon dioxide released into the atmosphere is absorbed by the ocean. When carbon dioxide, which has an acidic property, dissolves in weakly alkaline seawater, the alkalinity of the seawater weakens and approaches acidity (acidification of the ocean (pH change)). For this reason, there have been concerns about the impact on the ecosystem due to acidification of the ocean, such as the dissolution of shell plankton shells with calcareous (calcium carbonate) shells. In recent years, as shown in FIG. 7, it has been found that the shell density of, for example, shelled plankton (pteropods) decreases due to ocean acidification (pH change). FIG. 7(a) shows a sample with high shell density and FIG. 7(b) shows a sample with low shell density.
In order to understand the impact of ocean acidification on ecosystems or to predict the degree of future ocean acidification, it is necessary to accurately measure the shell density or skeleton density of shelled plankton. It is believed that there is.

In general, as one of the methods for measuring the bone density (bone mineral content) of bones and teeth of vertebrate animals such as humans, a phantom containing a reference substance such as hydroxyapatite and a measurement object (specimen) are placed in an X-ray CT scanner. A method is known in which bone density is calculated as an equivalent amount of a reference material by simultaneously scanning with . Such techniques are utilized, for example, in the diagnosis and follow-up of osteoporosis.

JP 2005-312945 A JP 2017-070453 A

In the density measurement method using the X-ray technology described in the above patent document, when the size of the object to be measured is extremely small (for example, about several hundred microns), various methods are used to measure bone density. There are technical problems, and it is difficult to measure individual bone densities of minute measurement objects.
For example, when the size of the object to be measured is very small, it is necessary to use an X-ray tube with a small focal diameter in the X-ray CT apparatus. However, the smaller the focal diameter of the X-ray tube, the smaller the amount of X-rays generated, resulting in a lower contrast of the obtained transmission image. Therefore, it is necessary to irradiate X-rays for a long period of time, increasing the influence of artifacts due to variations in X-ray dose and energy spectrum.
Also, when beam hardening occurs, the difference in the total distance through which the X-rays pass causes the calculated density value to differ from the actual one. There is the problem of uncertainty.
Furthermore, the actual situation is that there is no phantom that is used to measure the shell density of carbonate and that is similar in shape and size to a micro-sized object to be measured. In addition, phantoms for humans containing apatite and other metal elements cannot be used to measure carbonate shell densities due to differences in X-ray absorption based on size and elemental composition.

On the other hand, conventional techniques for measuring the shell density of shelled plankton, for example, measure and average the weight of multiple individuals at the same time, and estimate the change in shell density from the weight fluctuations of samples composed of multiple individuals collected in the same way. However, the reality is that there is no technology to accurately measure the shell density of shelled plankton.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a density measuring method capable of quantitatively and accurately measuring the density of an object to be measured mainly composed of calcium carbonate. do.

As a result of extensive research by the present inventors, the calcite CT value was newly defined using air and calcium carbonate (calcite) as reference materials, and the calcite CT value obtained by the X-ray CT method was A study of the relationship between the site CT value and the density value revealed that there is a specific correlation, eg, a linear relationship, between the calcite CT value and the density value.
Then, calibration curve data indicating the correlation between the calcite CT value and the density is obtained in advance using a plurality of types of calibration reference samples having different calcium carbonate densities, and by using the calibration curve data, The inventors have found that the density can be quantitatively measured based on the calcite CT value obtained for the object to be measured, and have completed the present invention.

The density measuring method of the present invention is a method for measuring the density of a measurement object mainly made of calcium carbonate,
A powder compact of calcium carbonate particles having a calcite crystal structure or a carbonate reference sample is used as a calibration reference sample , and air and calcite are used as reference substances for each of a plurality of types of calibration reference samples having different densities. Calibration curve data indicating the correlation between the calcite CT value represented by the ratio of linear attenuation coefficients and the density of each of the plurality of calibration standard samples is obtained in advance,
acquiring primary CT image data of each of at least one calibration reference sample and the measurement object by simultaneously irradiating both the calibration reference sample and the measurement object with X-rays from the same X-ray source;
The primary CT image data obtained for the calibration reference sample is subjected to reconstruction processing for correcting the pixel values to be constant, and the primary CT image data obtained for the measurement object is subjected to the above-mentioned Acquiring secondary CT image data by performing reconstruction processing under the same processing conditions as the reconstruction processing for the primary CT image data of the calibration reference sample ,
The density of the measurement object is measured by comparing the calcite CT value of the measurement object obtained based on the secondary CT image data of the measurement object with the calibration curve data.

In the density measurement method of the present invention, a low-energy component attenuation filter for absorbing low-energy components of X-rays is provided on the X-ray irradiation path between the X-ray source and the measurement object and the calibration reference sample . is preferably arranged.
In such a case, the low energy component attenuation filter is preferably made of an aluminum plate.
Furthermore, in the density measuring method of the present invention, it is preferable that the X-ray source is composed of a microfocus X-ray tube having a focus diameter of 1 μm or less.
Furthermore, in the density measuring method of the present invention, the object to be measured is preferably the shell or skeleton of a marine organism having a shell or skeleton containing calcium carbonate as a main component.

According to the density measuring method of the present invention, by acquiring the calcite CT value of the object to be measured, the density of the object to be measured can be accurately determined based on the previously acquired calibration curve data. The calibration curve data is quantitatively clarified by experimental support of the correlation between the calcite CT value and the density value for each of a plurality of calibration standard samples with different calcium carbonate densities. The density values obtained are highly reliable. For example, for marine organisms (shell plankton) several hundred microns in size with shells or skeletons mainly composed of calcium carbonate, the shell density or skeleton density of each individual can be quantitatively and accurately measured. Moreover, measurements can also be made from shell fragments.
When acquiring the calcite CT value, X-rays from the same X-ray source are simultaneously irradiated to both the calibration reference sample and the measurement object, thereby eliminating artifacts due to fluctuations in the X-ray dose and fluctuations in the energy spectrum. Since the influence can be canceled, the shell density can be quantified with high accuracy even for a very small object to be measured, such as shelly plankton, whose size is several hundred μm or less.

According to the calibration standard sample of the present invention, since it consists essentially of calcium carbonate, it can be configured to have high homogeneity, and high reproducibility can be obtained in the measurement results obtained by the above-described density measurement method. can be done.

According to the method for producing a calibration standard sample of the present invention, only calcium carbonate particles can be formed without using a binder as a binding aid, and an intended calibration standard sample can be reliably obtained.

1 is a schematic diagram showing an outline of the configuration of an example of a microfocus X-ray CT apparatus used in the density measuring method of the present invention; FIG. FIG. 4 is a diagram for explaining the effect of the X-ray low-energy component attenuation filter, showing (a) CT image data when the X-ray low-energy component attenuation filter is not used and (b) for the same homogeneous sample. It is CT image data when an X-ray low-energy component attenuation filter is used. FIG. 4 is a diagram showing the relationship between molding pressure and calcium carbonate density of the obtained calcite phantom. It is a figure which shows an example of the CT image data of an artificial carbonate phantom. It is a figure which shows an example of calibration curve data. 2 is a diagram showing the relationship between the shell density value (measured value) of planktonic foraminifera measured in Experimental Example 1 and the shell density value (reference value) obtained by chemical analysis. FIG. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure explaining the influence on shell plankton by ocean acidification. (a) is a surface-rendered image of a specimen with high shell density, where the top and bottom images show different cross-sectional layers of the same individual. (b) is a surface-rendered image of a specimen with low shell density, where the top and bottom images show different cross-sectional layers of the same individual.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.

In the density measurement method of the present invention, the calcite CT value (a value equivalent to the density of calcite in air) with newly defined air and calcium carbonate (calcite) as reference substances, and the density value Calibration curve data showing the correlation of is obtained in advance for multiple types of calibration reference samples (hereinafter referred to as "phantoms") with known calcium carbonate densities, and the measurement object is measured by the X-ray CT method using the phantom Based on the calibration curve data, the density of the object to be measured is obtained from the calcite CT value obtained for . Here, the calcite CT value is different from the medical CT value defined using air and water as reference substances, and its definition will be described later.

The object to be measured in the density measuring method of the present invention is not particularly limited as long as it is mainly composed of calcium carbonate. It is extremely useful for measuring the density of marine organisms with skeletons that are hundreds of microns in size. Examples of such marine organisms include shell plankton having shells made of calcium carbonate. Calcium carbonate produced by marine organisms has two types, one with a calcite crystal structure and one with an aragonite crystal structure. It may have

A microfocus X-ray CT apparatus, for example, is used to measure the density of the object to be measured.
FIG. 1 is a schematic diagram showing a schematic configuration of an example of a microfocus X-ray CT apparatus used in the density measuring method of the present invention.
This microfocus X-ray CT apparatus includes an X-ray source 10 that emits X-rays, a holder 20 that holds an object to be measured and a phantom, and an X-ray detector that detects X-rays transmitted through the object to be measured and the phantom. 30. Note that FIG. 1 shows a state in which only the measurement object S is held by the holding portion 20 for convenience.

The X-ray source 10 is composed of an X-ray tube whose focal diameter can be appropriately set within a range of, for example, 1 μm or less.
The holding unit 20 is configured by a rotary stage that is rotatable about an axis extending in the vertical direction, for example. Further, the holding unit 20 is provided so as to be movable in the X-ray irradiation direction, and by adjusting the distance between the holding unit 20 and the X-ray source 10, the magnification ratio of the X-ray transmission image can be changed. there is
The X-ray detector 30 is composed of, for example, a flat panel detector, and the difference in X-ray transmission (X-ray attenuation) reflecting the shape and internal structure of the measurement object S and the phantom is converted into, for example, a 16-bit gray scale. A CT image is obtained which is expressed as image density by .

In this microfocus X-ray CT apparatus, an X-ray low-energy component attenuation filter 40 that absorbs low-energy components of X-rays is placed on the X-ray irradiation path between the X-ray source 10 and the measurement object S and the phantom. are interpolated and arranged. As the X-ray low-energy component attenuation filter 40, for example, a metal filter made of pure aluminum (eg, A1012) can be used. The X-ray low-energy component attenuation filter 40 may be made of, for example, a metal plate such as copper or titanium, or a resin plate such as vinyl chloride. Also, the shape of the X-ray low-energy component attenuation filter 40 is not particularly limited.
Here, the thickness of the X-ray low energy component attenuation filter 40 can be set based on the X-ray attenuation coefficient of the constituent material of the X-ray low energy component attenuation filter 40 and the like. When the X-ray low energy component attenuation filter 40 is a metal filter made of pure aluminum, for example, its thickness is 0.2 mm, for example.

Without the X-ray low-energy component attenuation filter 40, as shown in FIG. (heterogeneity of pixel values) is confirmed, and in this example, the result shows a density distribution as if the central portion had a low density and the edge portion had a high density. On the other hand, since the X-ray low-energy component attenuation filter 40 is provided, as shown in FIG. It is shown that CT image data showing the original density distribution can be obtained with little non-uniformity).

In the above, the distance D1 between the X-ray source 10 and the X-ray detector 30 is, for example, 73 to 374 mm, and the distance D2 between the holding section 20 (measurement object S and phantom) and the X-ray detector 30 is , for example 60 to 371.5 mm, and the distance D3 between the X-ray source 10 and the X-ray low energy component attenuation filter 40 is for example 71.8 to 372.8 mm.

In the density measurement method of the present invention, first, a plurality of types of phantoms having different densities are prepared, and the correlation between the calcite CT value for each of the plurality of types of phantoms and the density of each of the plurality of types of phantoms is Calibration curve data indicating the relationship is acquired in advance. From the viewpoint of the reliability of calibration curve data, it is desirable to obtain calcite CT values for at least 3 or more, preferably 4 to 5, phantoms.

Each of the multiple types of phantoms must satisfy the following conditions.
(1) Consist of calcium carbonate with the same crystal structure as the object to be measured.
(2) Similar in shape and size to the object to be measured.
(3) Uniform internal density (homogeneous internal structure).
(4) Calcium carbonate density is known.
For example, a calcium carbonate crystal (density: 2.71) cut into an appropriate size satisfies the conditions for a phantom.

In the density measuring method of the present invention, the phantom may be, for example, a powder compact of calcium carbonate particles having a calcite crystal structure (artificial carbonate phantom, hereinafter referred to as "calcite phantom"), or "NIST NBS -19" (carbonate reference sample) is preferably used. "NIST NBS-19" is distributed worldwide by the International Atomic Energy Agency (IAEA) as an oxygen-carbon isotope ratio reference material for calcium carbonate, and its homogeneity is guaranteed.
Since the calcite phantom consists substantially only of calcium carbonate, it can be configured as a highly homogeneous one, and more accurate and quantitative measurement can be performed.

In the above calcite phantom, calcium carbonate particles having a purity of 99.9% or more are used.
Moreover, the calcium carbonate particles constituting the calcite phantom preferably have a particle size of, for example, 5 μm or less. As used herein, the term "particle size of calcium carbonate particles" refers to the minor axis of a rectangle circumscribing the maximum projection plane of calcium carbonate particles.
When the particle diameter is 5 μm or less, the nonuniformity of the voids larger than the resolution of the CT image data to be photographed can be minimized, and the desired calcite phantom can be reliably obtained. On the other hand, when the particle size of the calcium carbonate particles is too large, it becomes difficult to form the calcite phantom itself.

The above calcite phantom can be produced as follows.
That is, first, as a calibration standard sample-forming material, ultra-high-purity calcium carbonate particles having a particle diameter of 50 μm or less and a purity of 99.9% or more, for example, having a calcite crystal structure, are prepared and weighed using a microbalance. Weigh the specified amount. Here, the amount of calibration standard sample-forming material is such that the thickness of the calcite phantom to be shaped is, for example, about 1 mm. For example, when the inner diameter of the mold cavity of the powder molding die in the molding apparatus is φ13 mm, the amount of the ultra-pure calcium carbonate particles is 250-300 mg. By setting the amount of calibration standard sample-forming material in this way, density inhomogeneities in the final calcite phantom can be avoided.

Next, a predetermined amount of the calibration standard sample-forming material is filled into the mold cavity of the powder molding die under normal temperature and normal pressure, and pressure is applied by upper and lower punches to perform compression molding.
In the molding step, it is preferable to perform a vibration imparting step of vibrating the powder molding die after filling the calibrated reference sample forming material into the mold cavity of the powder molding die. As a result, the voids between the ultrapure calcium carbonate particles can be made dense, and the obtained calcite phantom can be made homogeneous. The time required for the vibration imparting step is about several minutes.
Further, the pressurization of the calibration standard sample-forming material may be performed by changing the molding pressure continuously or by changing it in multiple stages. When the molding pressure is changed in multiple stages and pressurization is performed, it is possible to reliably avoid occurrence of density non-uniformity in the finally obtained calcite phantom.
The molding pressure is set within the range of 1.5 to 10 ton/cm ² . The molding step is performed by gradually increasing the pressure on the calibration standard sample-forming material until it reaches the set molding pressure, and then applying the set molding pressure for a predetermined time. As a result, it is possible to obtain a powder compact in which the calcium carbonate particles are destroyed by compression and have a particle size of 5 μm or less.
The pressurization time is preferably 5 to 15 minutes, for example 10 minutes.

Thereafter, the disk-shaped calcite phantom precursor thus formed is gently removed from the powder molding die and cut into a predetermined size to obtain a calcite phantom for density measurement.

As shown in FIG. 3, there is a proportional relationship between the calcium carbonate density of the calcite phantom and the magnitude of the molding pressure. can be adjusted. For example, when the molding pressure is 1.5 tons/cm ² , a homogeneous calcite phantom with a calcium carbonate density of 1.93 can be obtained. Further, when the molding pressure is set to 10 tons/cm ² (upper limit), a homogeneous calcite phantom having a calcium carbonate density of 2.23 can be obtained.
The calcium carbonate density of the calcite phantom can be measured, for example, as follows. That is, first, the weight of the prepared calcite phantom precursor is measured using a balance (micro balance) capable of measuring the weight in microgram units. The volume of the calcite phantom precursor is then measured, for example, by microfocus X-ray CT. Here, the spatial resolution of imaging is assumed to be 10 μm or less. Then, the density of the calcite phantom and the density of calcium carbonate are calculated based on the measured weight and volume.
In the present invention, a plurality of types of calcite phantom precursors having different calcium carbonate densities are prepared, and the calcium carbonate density of each of the prepared plurality of types of calcite phantom precursors is measured.
FIG. 4 shows an example of CT image data of a calcite phantom precursor. The calcium carbonate density of the calcite phantom precursor shown in FIG. 4(a) is 2.30 μg/μm ³ , the calcium carbonate density of the calcite phantom precursor shown in FIG. 4(b) is 2.00 μg/μm ³ , and (c). The calcium carbonate density of the calcite phantom precursor shown in is 1.93 μg/μm ³ . The scale is 300 μm.

The calibration curve data in the present invention were obtained as follows.
First, under a stereoscopic microscope, particles having a maximum diameter of 300 μm were cut out from the central portion of the prepared calcite phantom precursor using a microsurgery scalpel. to obtain a calcite phantom for density measurement with a size of several hundred microns. Here, the size of the grain (calcite phantom) to be cut out is a size that corresponds to the maximum thickness that can be corrected for beam hardening.

CT image data for each calcite phantom is then acquired by a microfocus X-ray CT apparatus.
Referring to FIG. 1, X-rays from the same X-ray source 10 are passed through an X-ray low energy component attenuation filter 40 to both the calcite phantom and the carbonate reference sample (calcite crystal) described above. are irradiated at the same time. At this time, by rotating the holding unit 20 that holds both the calcite phantom and the carbonate reference sample, X-ray transmission images of both the calcite phantom and the carbonate reference sample are photographed from all circumferential directions. Obtain primary CT image data for each of the phantom and carbonate reference samples . Secondary CT image data for each of the calcite phantom and the carbonate reference sample is obtained by performing reconstruction processing on the thus obtained primary CT image data for each of the calcite phantom and the carbonate reference sample. do.

Therefore, the obtained primary CT image data has the effects of beam hardening artifacts reduced by the action of the X-ray low energy component attenuation filter 40, and the calcite phantom and carbonate reference Since the sample has a homogeneous internal structure, the obtained primary CT image data should have smooth pixel values. However, the energy density of the irradiated continuous X-rays inevitably fluctuates with the passage of time due to various factors. This includes the effects of artifacts caused by
The effects of such artifacts are expected to occur equally in both calcite phantoms and carbonate reference samples. Therefore, in the reconstruction process, the primary CT image data obtained for the calcite phantom is corrected so that the pixel values in the primary CT image data obtained for the carbonate reference sample are constant (smoothed). The same is done for CT image data. As a result, it is possible to reduce the influence of artifacts due to fluctuations in the X-ray energy spectrum in the primary CT image data of the calcite phantom, and to obtain high-resolution secondary CT image data. Here, constant pixel values (smooth pixel values) means that the difference in average pixel values between the central portion and the peripheral portion of the calcite phantom per unit area (20 voxels) is within a range of less than 2.5%. Say something.
Then, for each of the calcite phantom and the carbonate reference sample, X-ray transmission image acquisition and reconstruction processing are repeated in the same manner to obtain secondary CT image data of a predetermined number of cross-sectional layers in the calcite phantom. get.

Next, the calcite CT value of the calcite phantom is acquired based on the secondary CT image data obtained for the calcite phantom.
In general, if the elemental composition of the object to be measured is uniform, the density contrast of the secondary CT image data shown here has a correlation with the X-ray attenuation coefficient and is synonymous with the density of the object to be measured. In the present invention, focusing on calcium carbonate (CaCO ₃ ), which is the main component constituting the shell or skeleton of marine organisms, which are the measurement objects, the ratio of the X-ray attenuation coefficient with air and calcite as reference substances The expressed calcite CT value is newly defined.
The calcite CT value defined in the present invention is represented by the following formula (1), and is the average of the values obtained from each of the secondary CT image data (image density) of any plurality of cross-sectional layers in the calcite phantom The value is used as the calcite CT value (representative value) of the calcite phantom.
Formula (1) Calcite CT value = k × [(μ _sample - μ _air ) / (μ _std - μ _air )]

In the above formula (1), k is a constant conforming to the Hounsfield unit and is set to 1000, for example. μ _sample , μ _air , and μ _std denote the X-ray attenuation coefficients of the calcite phantom, air, and carbonate reference samples, respectively. When obtaining the calcite CT value of the object to be measured, the X-ray attenuation coefficient of the object to be measured is used as the value μ _sample , and the X-ray attenuation coefficient of the calcite phantom is used as the value μ _std .
The calcite CT value obtained by the above formula (1) is a value within the numerical range of 0 (air) to 1,000 (calcium carbonate crystal). Indicated by a value within a numeric range.

Then, the calcite CT value is obtained from each of the plurality of secondary CT image data obtained for one calcite phantom, and the average value of these is obtained as the calcite CT value (representative value) of the calcite phantom. .
By performing such processing for each of a plurality of types of calcite phantoms, the calcite CT value of each calcite phantom is obtained.

A plurality of measured values showing the relationship between the calcite CT value and the calcium carbonate density of each calcite phantom obtained as described above, for example, the calcium carbonate density [μg/μm ³ ] on the horizontal axis and the vertical axis Plot in a coordinate system with calcite CT values on the axes. Calibration curve data can be obtained by approximating these plots (measured values) with a straight line, for example. The calibration curve data may be obtained by approximating a plurality of measured values with a polynomial curve. An example of calibration curve data is shown in FIG. The plot indicated by α in FIG. 5 is the measured value for the carbonate reference sample (NBS19). The approximation formula in FIG. 5 is given by the following formula (2), where D is the density of calcium carbonate.
Formula (2) Calcite CT value = 351.64 x D + 44.123

When measuring the shell density or skeletal density of the object to be measured, at least one calcite phantom having a calcium carbonate density value equal to or greater than the expected shell density value or skeletal density value of the object to be measured is used as a reference. Selected and used.
Then, for example, using the microfocus X-ray CT apparatus shown in FIG. Reconstruction processing is performed on the primary CT image data of the measurement object under the same processing conditions as the reconstruction processing on the primary CT image data obtained for the site phantom .
Next, based on the secondary CT image data obtained for the measurement object, the calcite CT value of the measurement object is obtained, and the obtained calcite CT value is compared with the previously obtained calibration curve data. Thus, the shell density or skeleton density of the measurement object S is obtained.

Therefore, according to the above-described density measurement method, attention is paid to calcium carbonate that constitutes the shell or skeleton of marine organisms such as shell plankton, which is the measurement object, and the measurement object is newly defined air and carbonic acid. A calcite CT value using calcium (calcite) as a reference substance is obtained by an X-ray CT method using a phantom, thereby determining the shell density or skeleton density of the object to be measured based on the calibration curve data obtained in advance. can be obtained accurately. Because the calibration curve data quantitatively clarifies the correlation between the calcite CT value and the density value for each of a plurality of types of calcite phantoms with different calcium carbonate densities through experimental support. , the density values obtained from the calibration curve data have high reliability. According to the density measurement method described above, for example, for shelled plankton, it is possible to measure not only the shell density of each individual, but also the shell fragments.
In addition, when measuring the calcite CT value, both the measurement object S and the calcite phantom are irradiated with X-rays from the same X-ray source 10 at the same time, and an X-ray transmission image is taken to obtain an X-ray dose. Since the effects of artifacts due to fluctuations and fluctuations in the energy spectrum can be canceled based on the CT image data obtained for the phantom, it is possible to measure minute objects of several hundred μm or less in size, such as shelled plankton. However, it is possible to quantify the shell density with high accuracy.

Furthermore, in the microfocus X-ray T apparatus, an X-ray low energy component attenuation filter 40 is arranged on the X-ray irradiation path between the X-ray source 10 and the measurement object S and the calcite phantom. , the effects of beam hardening artifacts in the acquired CT image data can be reliably reduced, and the quantification of shell density (or skeletal density) can be performed more reliably.

Experimental examples conducted to confirm the effects of the present invention will be described below.

<Experimental example 1>
Eighteen individuals of planktonic foraminifera (about 150 μm in size), which are zooplankton inhabiting the North Pacific Ocean, were prepared. The shell containing calcium carbonate as a main component of each individual was used as an object to be measured, and the shell density of the object to be measured was determined for each individual. The shell density was measured using the microfocus X-ray CT apparatus shown in FIG. Primary CT image data was acquired. A secondary CT image obtained after performing reconstruction processing on the obtained primary CT image data of the object to be measured under the same processing conditions as the reconstruction processing on the primary CT image data obtained from the calcite phantom. A calcite CT value was obtained based on the data and the above formula (1). Based on the obtained calcite CT value, the shell density was obtained from the calibration curve data represented by the above formula (2).
Then, using the shell density determined by the chemical analysis described below as a reference value, the relationship between the shell density value (measured value) measured based on the calibration curve data and the reference value was investigated. The results are shown in FIG. Here, the horizontal axis in FIG. 6 is the shell density as a reference value, and the vertical axis is the shell density obtained based on the calibration curve data. A dashed straight line is an ideal curve in which the error between the measured value and the reference value is zero.
In obtaining reference values, first, the calcium content (weight) of each individual is quantified by high-resolution ICP-MS (inductively coupled plasma mass spectrometer), and the calcium carbonate weight ( shell weight) was calculated. In addition, the shell volume of each measurement object was determined based on the secondary CT image data of the measurement object obtained above. Based on the obtained shell weight and shell volume, the shell density of the object to be measured was calculated for each individual (shell density=shell weight/shell volume).

[Imaging conditions]
Applied voltage to X-ray tube: 80 keV
Applied current to X-ray tube: 40 μA
Focus diameter of X-ray tube: 0.8 μm
Distance between X-ray tube and X-ray detector (D1): 374 mm
Distance (D2) between holding part and X-ray detector: 371 mm
Distance between X-ray tube and X-ray low energy component attenuation filter (D3): 372.8 mm
Number of imaging (number of cross-sectional layers): 1200 Magnification: 126.94 times Imaging time (X-ray irradiation time): 66 minutes X-ray low-energy component attenuation filter: Material: pure aluminum, thickness: 0.2 mm

As shown in FIG. 6, the shell density value (measured value) obtained based on the calcite CT value using the calibration curve data is obtained by chemical analysis from the shell weight and shell volume. The error with respect to (reference value) is 0.25 μg/μm ^{3 at maximum, and about 0.07 μg/μm 3 on} average. was confirmed.

According to the density measuring method of the present invention, it becomes possible to accurately and quantitatively measure the shell density of each shelled plankton individual. It is possible to accurately capture changes in the body size and skeletal density of individual marine organisms due to changes in the marine environment, especially global warming and ocean acidification. It is hoped that you will be able to deepen your understanding.

REFERENCE SIGNS LIST 10 X-ray source 20 Holding unit 30 X-ray detector 40 Low energy component attenuation filter S Measurement object

Claims (5)

A method for measuring the density of a measurement object mainly composed of calcium carbonate, comprising:
Using a powder compact of calcium carbonate particles having a calcite crystal structure or a carbonate reference sample as a calibration reference sample, For each of multiple types of calibration standard samples with different densities, expressed as the ratio of X-ray attenuation coefficients with air and calcite as reference substancesObtaining in advance calibration curve data showing the correlation between the calcite CT value and the density of each of the plurality of calibration reference samples,
acquiring primary CT image data of each of at least one calibration reference sample and the measurement object by simultaneously irradiating both the calibration reference sample and the measurement object with X-rays from the same X-ray source;
The primary CT image data obtained for the calibration reference sample is subjected to reconstruction processing for correcting the pixel values to be constant, and the primary CT image data obtained for the measurement object is subjected to the above-mentioned By performing the reconstruction processing under the same processing conditions as the reconstruction processing for the primary CT image data of the calibration reference sample, obtaining secondary CT image data;
Density characterized by measuring the density of the measurement object by comparing the calcite CT value of the measurement object obtained based on the secondary CT image data of the measurement object with the calibration curve data. Measuring method. A low-energy component attenuation filter for absorbing low-energy components of X-rays is arranged on an X-ray irradiation path between the X-ray source and the measurement object and the calibration reference sample . Item 1. The density measurement method according to item 1. 3. The density measuring method according to claim 2, wherein said low-energy component attenuation filter is made of an aluminum plate. 4. The density measuring method according to any one of claims 1 to 3, wherein said X-ray source comprises a microfocus X-ray tube having a focal diameter of 1 [mu]m or less. 5. The density measuring method according to any one of claims 1 to 4, wherein the object to be measured is a shell or skeleton of a marine organism having a shell or skeleton mainly composed of calcium carbonate.

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