WO2017175811A1

WO2017175811A1 - Cancer detection method, cancer detection device, and cancer detection program

Info

Publication number: WO2017175811A1
Application number: PCT/JP2017/014298
Authority: WO
Inventors: 宗彰匹田; 瀧　優介; 福武　直樹; 楓横山; 寛晃伊藤
Original assignee: 株式会社ニコン; 学校法人昭和大学
Priority date: 2016-04-05
Filing date: 2017-04-05
Publication date: 2017-10-12

Abstract

Provided is a cancer detection method, wherein: an amount of amino acids or proteins in a detection region, and an amount of nucleic acids are acquired; the ratio of the acquired amount of amino acids or proteins with respect to the acquired amount of nucleic acids is calculated; and the presence of cancer cells is determined on the basis of a comparison of the calculated ratio and a preset first threshold value. In the cancer detection method, a configuration may be employed such that the amount of amino acids in the detection region is detected as an indicator of the amount of proteins, the amount of nucleobases in the detection region is detected as an indicator of the amount of nucleic acids, and the ratio of the detected amount of amino acids with respect to the detected amount of nucleobases is used as the ratio.

Description

Cancer examination method, cancer examination apparatus, and cancer examination program

The present invention relates to a cancer test method, a cancer test apparatus, and a cancer test program.

The examination of the presence or absence of cancer cells is performed by visual observation by a doctor or using a tumor marker (for example, see Patent Document 1).
Patent Document 1 Japanese Patent Laid-Open No. 2015-180212

The accuracy of visual inspection varies depending on the experience of the doctor. Tumor markers have problems with sensitivity and organ specificity.

In the first aspect of the present invention, the amount of amino acid or protein in the examination region and the amount of nucleic acid are obtained, the ratio between the amount of amino acid or protein obtained and the amount of nucleic acid is calculated, and the calculated ratio And a cancer test method for determining the presence or absence of cancer cells based on a comparison with a predetermined first threshold. In the second aspect of the present invention, the acquisition unit for acquiring the amount of amino acid or protein and the amount of nucleic acid detected in the examination region, the amount of amino acid or protein acquired by the acquisition unit, and the acquisition unit A cancer test apparatus is provided that includes an information output unit that outputs information according to a ratio to the amount of nucleic acid obtained. In the third aspect of the present invention, the acquisition step of acquiring the amount of amino acid or protein in the examination region and the amount of nucleic acid, the amount of amino acid or protein acquired in the acquisition step, and the nucleic acid acquired in the acquisition step There is provided a cancer test program for causing a processing device to execute an output step of outputting information corresponding to a ratio to a quantity.

The above summary of the invention does not enumerate all the features of the present invention. Sub-combinations of these feature groups can also be an invention.

2 is a schematic diagram illustrating the structure of an inspection apparatus 100. FIG. 3 is a block diagram of a control unit 170. FIG. 3 is a flowchart showing an inspection procedure in the inspection apparatus 100. It is a figure which illustrates the display in the display image. It is a figure which illustrates the display in the display image. It is a figure which illustrates the display in the display image. It is a figure which illustrates the display in the display image. It is a figure which illustrates the display in the display image. FIG. 6 is a diagram illustrating a display in a display image 306. It is a figure which illustrates the display in the display image 307. FIG. It is a figure which illustrates the display in the display image. It is an example of the display image 309 which shows a detection position. It is an example of the display image 310 which shows a detection position. 5 is a flowchart showing another inspection procedure in the inspection apparatus 100.

Hereinafter, the present invention will be described through embodiments of the invention. The following embodiments do not limit the claimed invention. Not all combinations of features described in the embodiments are essential for the solution of the invention.

The inventors obtained knowledge that there is a difference between the ratio in cancer cells and the ratio in normal cells when focusing on the ratio between the amount of amino acid or protein in the cell and the amount of base. Therefore, in the present embodiment, the amount of amino acid or protein and the amount of base are detected in the cell to be examined, the ratio of the detected amount of amino acid or protein and the amount of base is calculated and compared with a prepared threshold value. In this case, a cancer test method has been developed for determining that cancer cells are included in a region having a ratio exceeding the threshold.

FIG. 1 is a schematic diagram showing an overall configuration of an inspection apparatus 100 that can be used in the above-described cancer inspection method. The inspection apparatus 100 includes a stage 110, an objective optical system 120, a light source device 130, an irradiation optical system 140, a front detection system 150, a rear detection system 160, and a control unit 170.

The stage 110 supports the sample 101 to be inspected by the inspection apparatus 100 at the peripheral edge of the container in which the sample 101 is accommodated. The stage 110 has an opening that exposes the lower surface of the sample 101 in the drawing. Thereby, the sample 101 placed on the stage 110 can be observed from the lower side in the figure.

Stage 110 is coupled to stage scanner 111. The stage scanner 111 drives the stage 110 in parallel and perpendicular to the surface on which the sample 101 is placed, as indicated by arrows xyz in the drawing. Thereby, in the inspection apparatus 100, the three-dimensional area | region in the sample 101 can be made into the object area | region of observation or inspection, fixing the optical axis of an optical system, and the optical axis of excitation light. In the following description, a region to be observed or examined may be referred to as a region of interest.

The objective optical system 120 includes a front objective lens 121 and a rear objective lens 122 that are arranged symmetrically with respect to the sample 101 on opposite sides of the stage 110. In the illustrated inspection apparatus 100, the front objective lens 121 also plays a role of collecting excitation light, illumination light, and the like irradiated on the sample 101.

The light source device 130 includes a plurality of

light sources

131 and 132 that generate different types of irradiation light, and a combiner 139. At least one of the

light sources

131 and 132 may be a laser light source that generates excitation light used when measuring the Raman spectrum of the sample 101, for example, laser light having a wavelength of 532 nm. Further, one of the plurality of

light sources

131 and 132 may be a light source of illumination light used when observing a microscopic image of the sample 101.

In addition, it is preferable that the irradiation light with which the sample 101 is irradiated is long-wavelength light that hardly invades cells, whether it is excitation light or illumination light. More specifically, a wavelength range of 400 nm to 800 nm can be exemplified.

The irradiation light emitted from the

light sources

131 and 132 becomes a beam that passes through a single optical path by the combiner 139. Thereby, the irradiation light generated by the plurality of

light sources

131 and 132 can be irradiated to the same position of the sample 101.

The irradiation optical system 140 includes a galvano scanner 141 and a scan lens 142. The galvano scanner 141 includes a pair of reflecting mirrors that swing around two swing axes that are not parallel to each other. As a result, the optical path of the excitation light incident on the galvano scanner is displaced two-dimensionally in the direction intersecting the optical axis.

The scan lens 142 focuses the excitation light emitted from the galvano scanner 141 onto a predetermined primary image plane 143. Thereby, the excitation light emitted from the light source device 130 can be applied to any region of interest set in the sample 101.

The front detection system 150 includes a dichroic mirror 151,

relay lenses

152 and 153, a band pass filter 154, and a spectroscope 155. The dichroic mirror 151 transmits the excitation light irradiated toward the sample 101 with high efficiency.

The dichroic mirror 151 reflects the Raman scattered light generated in the sample 101 and guides it to the

relay lenses

152 and 153. The band pass filter 154 transmits the Raman scattered light generated from the sample 101 and enters the spectroscope 155 while absorbing or reflecting the excitation light and the Rayleigh scattered light. Thereby, the spectroscope 155 efficiently detects the Raman scattered light reflected by the sample 101 and outputs a spectral image.

The rear detection system 160 includes a reflecting mirror 161,

relay lenses

162 and 163, a band pass filter 164, and a spectroscope 165. The reflecting mirror 161 reflects the Raman scattered light generated in the sample 101 and guides it to the

relay lenses

162 and 163, the band pass filter 164, and the spectroscope 165. Instead of the reflecting mirror 161, a dichroic mirror that selectively reflects the wavelength of the Raman scattered light may be provided.

The band-pass filter 164 transmits Raman scattered light generated from the sample 101 and makes it enter the spectroscope 165 while absorbing or reflecting Rayleigh scattered light and excitation light. Thereby, the spectroscope 165 efficiently detects Raman spectroscopy by the transmitted light of the sample 101.

The control unit 170 includes a processing device 171, a mouse 172, a keyboard 173, and a display unit 174. The mouse 172 and the keyboard 173 are connected to the processing device 171 and are operated when a user instruction is input to the processing device 171. The display unit 174 returns feedback to the user's operation using the mouse 172 and the keyboard 173, and displays the image or character string generated by the processing device 171 toward the user. Further, in the illustrated inspection apparatus, the display unit 174 displays an observation image obtained by optically observing the sample 101, characters or images representing the inspection result, and the like.

The processing device 171 controls the operations of the light source device 130, the stage scanner 111, and the galvano scanner 141. Further, the processing device 171 determines the state of the sample 101 based on the spectral images acquired from the

spectroscopes

155 and 165 of the front detection system 150 or the rear detection system 160, that is, whether or not the sample 101 contains cancer cells. inspect. Furthermore, the processing device 171 can process the spectra acquired from the

spectroscopes

155 and 165 to improve the detection accuracy of amino acids or proteins and nucleic acids.

Note that the Raman scattered light detected by the front detection system 150 disposed on the same side as the irradiation optical system 140 with respect to the sample 101 is backward Raman scattered light reflected by the sample 101. On the other hand, the Raman scattered light detected by the rear detection system 160 disposed on the opposite side of the irradiation optical system 140 with respect to the sample 101 is forward Raman scattered light that has passed through the sample 101.

FIG. 2 is a block diagram schematically showing the internal structure of the processing device 171 of the control unit 170 in the inspection device 100. As illustrated, the processing device 171 includes a detection unit 271 and an information output unit 276. Furthermore, the information output unit 276 includes a calculation unit 272, a comparison unit 273, a storage unit 274, and an output unit 275.

The detection unit 271 detects the amount of protein and the amount of nucleic acid present in one region of interest in the sample 101 based on the spectra acquired from the

spectroscopes

155 and 165, respectively. The amount of protein and the amount of nucleic acid can each be detected as the height of a particular peak in the spectrum of sample 101.

In the inspection apparatus 100, the amount of amino acids in the region of interest may be detected as an index of the amount of protein in the region of interest of the sample 101. Further, as will be described later, an aromatic amino acid among amino acids, more specifically, at least one of tryptophan and phenylalanine may be detected as an indicator of the amount of protein. In the test apparatus 100, the amount of nucleobase may be detected as an index of the amount of nucleic acid in the sample 101. Further, as will be described later, at least one of cytosine and adenine may be detected in the nucleic acid.

The calculating unit 272 calculates the ratio between the detected amount of protein detected by the detecting unit 271 and the detected amount of nucleic acid. The ratio calculated here has a value reflecting the state of the cell in the region of interest. That is, the ratio of the amount of protein and the amount of nucleic acid in the cell nucleus differs between normal cells and cancer cells.

Therefore, by comparing the ratio value calculated by the calculation unit 272 with a predetermined threshold value, it is possible to easily and reliably inspect whether or not the sample 101 to be inspected contains cancer cells. In addition, when the amount of protein and the amount of nucleic acid are detected by the peak value in the spectrum, the calculated ratio can be calculated as the ratio of the peak value in the spectrum.

Note that a plurality of regions of interest included in a target region wider than one region of interest may be set for one sample 101, and the sample 101 may be inspected in each region of interest. This reduces the probability of missing locally generated cancer cells.

The comparison unit 273 executes a test for comparing the ratio value calculated by the calculation unit 272 with a threshold value stored in the threshold value storage unit 274 in advance. The result of the inspection is transmitted to the user through the output unit 275. Specifically, for example, the inspection result may be displayed on the display unit 174 as a character string or an image. Further, the inspection result may be output by voice or the like. Further, the inspection result may be accumulated and the user may be referred to at an arbitrary timing. Thus, the information output unit 276 outputs information corresponding to the ratio of the amount of nucleic acid acquired by the detection unit 271 to the amount of protein acquired by the detection unit 271.

FIG. 3 is a flowchart showing an inspection procedure using the inspection apparatus 100. First, a threshold value is stored in the threshold value storage unit 274 of the inspection apparatus 100 (step S101). The threshold value to be stored is obtained, for example, by inspecting a cell sample whose state is known with the inspection apparatus 100.

Next, a sample 101 to be inspected is prepared and loaded into the inspection apparatus 100 (step S102). Subsequently, a microscopic image obtained by optically enlarging the sample 101 is observed, and a region of interest to be inspected is set (step S103). Note that a microscopic image of the sample 101 including the region of interest observed in step S103 may be captured and stored (step S104).

Also, a plurality of regions of interest in the sample 101 may be set for one sample 101. This reduces the probability that locally generated cancer cells are missed in the test.

Next, in the region of interest, the region of interest is irradiated with excitation light, and the spectrum of the sample 101 is obtained by the spectrometers 155 and 165 (step S105). Furthermore, the spectrum detected by the

spectroscopes

155 and 165 is processed by the processing device 171 to detect the amount of protein and the amount of nucleic acid in the region of interest as, for example, the light intensity of the corresponding peak in the spectrum (step S106).

Next, in the processing device 171, the ratio between the detected protein amount and the nucleic acid amount is calculated, for example, as the ratio of peak values (step S107). Next, the calculated ratio value is compared with the threshold value stored in the threshold value storage unit 274 (step S108). Thereby, the test result whether a cancer cell is contained in the corresponding region of interest is obtained depending on whether or not the calculated ratio value exceeds a threshold value. The obtained inspection result is recorded in the inspection apparatus 100 or output to the user (step S109). Further, the control unit 170 of the inspection apparatus 100 checks whether there remains a region of interest that has been set for the sample 101 in step S103 but has not yet been inspected (step S110).

If there is a region of interest that has not been inspected yet in step S110 (step S110: YES), the control unit 170 returns the processing of the inspection apparatus 100 to step S105 and executes the inspection again. Further, in this way, the inspection apparatus 100 inspects whether cancer cells are present in the region of interest of the sample 101. If it is determined in step S110 that there is no region of interest that has not been inspected (step S110: NO), the inspection in the inspection apparatus 100 ends.

Note that the spectrum of the sample 101 can be obtained without staining the sample 101. However, operations such as setting a region of interest may be facilitated by staining the nucleus of the cells contained in the sample 101 to make the microscopic image clearer.

[Example]
As an example for determining the threshold value, the sample 101 that is known to contain cancer cells was inspected by the above-described inspection apparatus 100, and the threshold value stored in the threshold value storage unit 274 was examined. Sample 101 was made from an unstained section of the human stomach.

First, a microscopic image of sample 101 was obtained using an objective lens having a magnification of 100 times. Next, the region of interest was irradiated with laser light having a wavelength of 532 nm as excitation light, and a spectrum due to Raman scattering was detected.

For the purpose of improving the efficiency of Raman scattering, the beam diameter of the excitation light was made smaller than the cell, for example, 1 μm or less. For this reason, in each of the 10 μm × 10 μm regions of interest in the

sample

101, 121 points of detection spots in each region of interest were irradiated with excitation light for 10 seconds to detect 121 points of spectra. Furthermore, the detected values detected from the spectrum of 121 points were averaged to obtain a detected value of the amount of protein or nucleic acid in the region of interest. Thereby, the detected value is a value having a spatial spread.

Further, image processing was executed in the processing device 171 to remove a known glass peak from the detected spectrum in advance, thereby eliminating the influence of the container of the sample 101. In addition, the effect of autofluorescence was eliminated by approximating with a fifth order polynomial. From the spectra thus obtained, the light intensity of the peaks corresponding to the nucleic acids shown in Table 1 below and the proteins shown in Table 2 below were detected as the amounts of nucleic acids and proteins.

[Example 1]
FIG. 4 is a diagram illustrating an example of a graph included in the display image 301 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown in the figure, the value on the horizontal axis indicates the amount of adenine detected from the peak light intensity of Raman shift 725 cm ⁻¹ in the Raman spectroscopic image as an example of nucleic acid, and the peak light intensity of Raman shift 756 cm ⁻¹ as an example of protein. It is a ratio to the amount of tryptophan. The value on the vertical axis is the ratio between the amount of adenine and the amount of protein using the α helix structure as an index detected from the peak light intensity of Raman shift 1263 cm ⁻¹ .

Focusing on the distribution of normal cells and cancer cells plotted in the graph, it can be seen that only cancer cells are distributed in the range where the ratio of adenine and tryptophan is 0.85 or more. Therefore, in step S101 shown in FIG. 3, by setting a threshold value of 0.85 for the ratio between the amount of adenine and the amount of tryptophan, the cancer is determined based on the ratio of the amount of adenine and tryptophan detected from the Raman spectroscopic image. The presence of cells can be examined. In FIG. 4, normal cells and cancer cells are distinguished by black and white dots, but the display unit 174 can improve visibility by distinguishing the colors and shapes of the dots.

[Example 2]
FIG. 5 is a diagram illustrating an example of a graph included in another display image 302 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the illustrated graph, the value on the horizontal axis is the ratio of the amount of adenine, which is an example of nucleic acid, to the amount of tryptophan, which is an example of amino acid. The value on the vertical axis is the ratio between the amount of adenine and the amount of phenylalanine detected from the peak light intensity of Raman shift of 1002 cm ⁻¹ as an example of protein.

Focusing on the distribution of normal cells and cancer cells plotted in the graph, it can be seen that only cancer cells are distributed in the range where the ratio of adenine and tryptophan is 0.85 or more. Therefore, in step S101 shown in FIG. 3, by setting a threshold value of 0.85 for the ratio between the amount of adenine and the amount of tryptophan, the cancer is determined based on the ratio of the amount of adenine and tryptophan detected from the Raman spectroscopic image. The presence of cells can be examined.

[Example 3]
FIG. 6 is a diagram illustrating an example of a graph included in the display image 303 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown, the value on the horizontal axis is the ratio between the amount of cytosine detected from the peak light intensity of Raman shift 782 cm ⁻¹ in the Raman spectroscopic image as an example of nucleic acid and the amount of tryptophan, which is an example of amino acid. The value on the vertical axis is the ratio between the amount of cytosine, which is an example of nucleic acid, and the amount of phenylalanine, which is an example of protein.

Focusing on the distribution of normal cells and cancer cells plotted in the graph, on the horizontal axis, only the cancer cells are distributed in the range where the ratio of cytosine and tryptophan is 1.2 or more. I understand. Moreover, about a vertical axis | shaft, in the range where the ratio of cytosine and phenylalanine exceeds 0.3, it turns out that only a cancer cell is distributed.

Therefore, in step S101 shown in FIG. 3, the threshold value 1.2 is set for the ratio between the amount of cytosine and the amount of tryptophan, and the threshold value 0.3 is set for the ratio between the amount of cytosine and the amount of phenylalanine. Thus, the presence of cancer cells can be examined based on the ratio between the amount of each nucleic acid detected from the Raman spectroscopic image and the amount of protein.

[Example 4]
FIG. 7 is a diagram illustrating an example of a graph included in the display image 304 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown, the value on the horizontal axis is the ratio of the amount of cytosine, which is an example of nucleic acid, to the amount of tryptophan, which is an example of protein. The value on the vertical axis indicates the amount of cytosine, which is an example of nucleic acid, and the amount of amide bond derived from a protein having a secondary structure of β sheet detected from the peak light intensity of Raman shift 1250 cm ⁻¹ (in the following description). Is simply referred to as “amount of amide bond”). The amount of amide bond is an indicator of the amount of protein.

Focusing on the distribution of normal cells and cancer cells plotted in the graph, cancer cells can be discriminated by the threshold 1.2 on the horizontal axis, but the threshold at which cancer cells can be discriminated on the vertical axis. It can be seen that it has risen to 0.43. Therefore, in step S101 shown in FIG. 3, by setting the threshold value 0.43, the presence of cancer cells can be examined based on the ratio between the amount of cytosine and the amount of amide bond.

[Example 5]
FIG. 8 is a diagram illustrating an example of a graph included in the display image 305 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown in the figure, the value on the horizontal axis represents the ratio between the amount of cytosine, which is an example of nucleic acid, and the amount of symmetric tryptophan, which is an example of protein. The value on the vertical axis is the ratio between the amount of adenine, which is an example of nucleic acid, and the amount of symmetric tryptophan, which is an example of protein.

Focusing on the distribution of normal cells and cancer cells plotted in the graph, the horizontal axis can distinguish cancer cells with a threshold of 1.3, and the vertical axis can distinguish cancer cells with a threshold of 0.9. I understand that I can do it. Therefore, in step S101 shown in FIG. 3, by setting a threshold value of 1.3, the presence of cancer cells can be examined based on the ratio of cytosine to the amount of symmetric tryptophan. The presence of cancer cells can be examined on the basis of the ratio of the amount of glycine to the amount of symmetric tryptophan.

[Example 6]
FIG. 9 is a diagram illustrating an example of a graph included in the display image 306 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown in the figure, the value on the horizontal axis is the ratio between the amount of cytosine in the sample 101, which is an example of a nucleic acid, and the amount of protein using the α helix structure as an index. The value on the vertical axis is the ratio between the amount of adenine, which is an example of a nucleic acid, and the amount of protein using the α helix structure as an index.

Focusing on the distribution of normal cells and cancer cells plotted in the graph, cancer cells can be distinguished by a threshold of 0.5 on the horizontal axis, but cancer cells are distributed alone on the vertical axis. It can be seen that the area does not exist.

Therefore, in examining the presence of cancer cells in step S101 shown in FIG. 3, the threshold value is 0 rather than calculating the ratio between the amount of adenine, which is a nucleic acid, and the amount of protein using α-helix structure as an index. .5 is preferably set, and examination is preferably performed based on the ratio between the amount of cytosine, which is a nucleic acid, and the amount of protein using α-helix structure as an index.

[Example 7]
FIG. 10 is a diagram illustrating an example of a graph included in the display image 307 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown in the figure, the value on the horizontal axis is the ratio between the amount of cytosine in the sample 101 as an example of nucleic acid and the amount of amide bond as an example of protein. The value on the vertical axis is the ratio between the amount of adenine, which is an example of nucleic acid, and the amount of amide bond, which is an example of protein. Focusing on the distribution of normal cells and cancer cells plotted in the graph, cancer cells can be distinguished by a threshold of 0.45 on the horizontal axis, but cancer cells are distributed alone on the vertical axis. It can be seen that the area does not exist.

Therefore, in step S101 shown in FIG. 3, by setting a threshold value of 0.45, the presence of cancer cells can be examined based on the ratio between the amount of cytosine and the amount of amide bond, but the amount of adenine, Even if the ratio with the amount of amide bond is calculated, the presence of cancer cells cannot be examined.

[Example 8]
FIG. 11 is a diagram illustrating an example of a graph included in the display image 308 displayed on the display unit 174 as a result of the inspection. In the illustrated graph, the ratio between the peak value of the nucleic acid and the peak value of the protein output from the calculation unit 272 is plotted.

In the graph shown in the figure, the value on the horizontal axis represents the ratio between the amount of cytosine in the sample 101 which is an example of a nucleic acid and the amount of phenylalanine which is an example of a protein. The value on the vertical axis is the ratio between the amount of adenine, which is an example of nucleic acid, and the amount of phenylalanine, which is an example of protein. Focusing on the distribution of normal cells and cancer cells plotted in the graph, cancer cells can be distinguished by a threshold of 0.28 on the horizontal axis, but cancer cells are distributed alone on the vertical axis. It can be seen that the area does not exist.

Therefore, in step S101 shown in FIG. 3, by setting a threshold value of 0.28, the presence of cancer cells can be examined based on the ratio between the amount of cytosine and the amount of amide bond, but the amount of adenine, Even if the ratio with the amount of phenylalanine is calculated, the presence of cancer cells cannot be examined.

From the examination of the test results shown in FIG. 4 to FIG. 11, in the sample 101 used in the above example, in the ratio between the amount of cytosine that is a nucleic acid and the amount of phenylalanine that is a protein, normal cells and cancer It turned out that the difference with a cell appears notably. Also, as shown in each graph, when different ratios are assigned to the vertical and horizontal axes, the peak ratio of adenine and phenylalanine is assigned to one axis, and the peak ratio of cytosine and phenylalanine is assigned to the other axis. In addition, it was found that the difference between normal cells and cancer cells appears remarkably.

[Example 9]
FIG. 12 is a diagram showing a display image 309 showing a result of performing an inspection by the inspection apparatus 100 on the sample 101 containing cancer cells. The microscopic image of the cell in the figure is an image that was captured and stored in step S104 of FIG.

Numeral values shown in the figure correspond to the order in which the inspections are performed, and the numerical positions indicate the positions of the regions of interest in the inspection. In addition, the numbers shown in white indicate that the test result at the relevant location was normal cells. On the other hand, the numbers shown in black indicate that the test result at the relevant location was cancer cells. As shown, in the sample 101 that is known to contain cancer cells, it can be seen that a peak ratio peculiar to the cancer cells is detected in a specific region of interest.

[Example 10]
FIG. 13 is a diagram showing a display image 310 that shows a result of performing an inspection by the inspection apparatus 100 on a sample 101 that does not contain cancer cells. The microscopic image of the cell in the figure is an image that was captured and stored in step S104 of FIG. The meanings of the numbers shown in the figure are the same as those in FIG.

As shown in the figure, in this sample 101, the peak ratio peculiar to cancer cells is not detected, and only the peak ratio of normal cells is detected. Therefore, it can be seen that the sample 101 does not contain cancer cells.

It should be noted that the numbers in italics in FIGS. 12 and 13 indicate that the test result at the relevant location could not be determined as being a normal cell or a cancer cell. When all the regions of interest in the entire sample 101 have such a test result, it means that the selection of the nucleic acid and protein types for detecting the peak value was not appropriate.

Therefore, in that case, other types of nucleic acid and protein may be selected to detect the peak value of the spectral waveform, and a test may be performed by setting a threshold value suitable for such nucleic acid and protein. In addition, a significant detection result may be obtained by changing the detection conditions such as the wavelength of the excitation light applied to the sample 101 and executing the inspection.

FIG. 14 is a flowchart showing another inspection procedure. In FIG. 14, the same procedures as those in FIG. 3 are denoted by the same reference numerals, and redundant description is omitted.

In the illustrated procedure, in step S111, in addition to the threshold value to be compared in step S108, a second threshold value regarding the width of deviation between the calculation result and the threshold value is set. Further, after step S108, a step of checking whether or not the detection result exceeds the second threshold set in step S101 is provided (step S113). And in order to improve the certainty of cancer cell detection, when the initial threshold value is exceeded, but the second threshold value is not exceeded (step S113: NO), the same region of interest is examined. A step (step S112) of executing another re-inspection whose conditions have been changed is added.

This makes it possible to reduce the region of interest for which the determination based on the inspection result is suspended, and to further improve the inspection accuracy. In addition, as a test condition changed in step S112, the change etc. of the kind of nucleic acid and protein used as the object which calculates peak ratio can be illustrated, for example. Further, the wavelength of the excitation light for detecting the spectrum may be changed by further changing the procedure.

Since the above inspection method can be executed if a spectrum can be obtained, it may be incorporated into an existing spectroscopic measurement apparatus as a program for executing the above inspection method. In the examples described so far, the amounts of nucleic acids and proteins were detected from the Raman scattering spectrum. However, as a specific measurement method, an amino acid or protein amount can be detected, and any method can be selected from measurement methods that can be calculated as a ratio of the nucleic acid amount to the amino acid or protein amount. For example, the spectrum may be detected from CARS light generated by the CARS process (Coherent Anti-Stokes Raman Scattering). Infrared spectroscopy may be used instead of or in addition to using Raman spectroscopy or CARS light.

In addition, as already described, when calculating the ratio between the amount of nucleic acid used for the test and the amount of protein, the ease of determination may differ depending on the type of nucleic acid to be detected and the type of protein. . Therefore, the test | inspection apparatus 100 may be made to switch each kind of nucleic acid and protein which detect quantity by a user's operation.

Furthermore, in the inspection apparatus 100, the display method of the inspection result on the display unit 174 may be switched. For example, the display format may be graphed as shown in FIGS. 4 to 11, or may be displayed superimposed on the microscopic image as shown in FIGS. Moreover, you may make it display combining a character string, a color, a sound, etc. Furthermore, these display formats may be selected by a user operation.

Furthermore, although the above-described inspection apparatus 100 is formed by combining an optical microscope and a spectroscope, the spectrum can be detected non-invasively by, for example, an endoscope. Therefore, a cancer test can be performed non-invasively by combining an endoscope capable of spectroscopic measurement with the processing device 171.

Furthermore, in the above example, an example of inspecting each region of interest one by one was given. However, the inspection apparatus 100 may be configured so that a plurality of regions of interest are simultaneously irradiated with excitation light and the plurality of regions of interest are inspected in parallel. This can reduce the time required for cancer testing.

As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

The execution order of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior”. It should be noted that they can be implemented in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for the sake of convenience, it means that it is essential to carry out in this order. is not.

100 inspection device, 101 sample, 110 stage, 111 stage scanner, 120 objective optical system, 121 front objective lens, 122 rear objective lens, 130 light source device, 131, 132 light source, 139 combiner, 140 irradiation optical system, 141 galvano scanner , 142 scan lens, 143 primary image plane, 150 front detection system, 151 dichroic mirror, 152, 153, 162, 163 relay lens, 154, 164 band pass filter, 155, 165 spectroscope, 160 rear detection system, 161 reflection Mirror, 170 control unit, 171 processing device, 172 mouse, 173 keyboard, 174 display unit, 271 detection unit, 272 calculation unit, 273 comparison unit, 274 storage unit, 275 output unit, 276 information output Part, 301,302,303,304,305,306,307,308,309,310 display image

Claims

Obtain the amount of amino acid or protein in the test area and the amount of nucleic acid,
Calculate the ratio of the amount of the obtained amino acid or protein and the amount of the nucleic acid,
A cancer test method for determining the presence or absence of cancer cells based on a comparison between the calculated ratio and a predetermined first threshold.
The amount of amino acid in the test region is detected as an indicator of the amount of protein, the amount of nucleobase in the test region is detected as an indicator of the amount of nucleic acid, and the ratio is detected as the amount of amino acid detected and the amount of nucleobase detected The cancer test method according to claim 1, wherein the method is a ratio to the amount.
3. The cancer testing method according to claim 2, wherein the amino acid includes an aromatic amino acid, the nucleobase includes a cyclic amine, and the ratio is a ratio of a detected amount of the aromatic amino acid and a detected amount of the cyclic amine.
The cancer test method according to claim 3, wherein the amino acid includes at least one of tryptophan and phenylalanine, and the cyclic amine includes at least one of cytosine and adenine.
The amount of the nucleic acid is the amount of the first type of nucleic acid,
The ratio is a first ratio that is a ratio between the amount of the amino acid or protein and the amount of the first type of nucleic acid;
Calculating a second ratio that is a ratio of the amount of the amino acid or protein and the amount of a second type of nucleic acid different from the first type;
The cancer test method according to any one of claims 2 to 4, wherein the first ratio and the second ratio are compared with the first threshold value.
The detection of the amount of the second type of nucleic acid and the calculation of the second ratio are omitted when the calculated ratio is greater than a second threshold value that is greater than the first threshold value. 5. The cancer test method according to 5.
The amount of the amino acid or protein is the amount of the first type of amino acid or protein;
The ratio is a first ratio that is a ratio of the amount of the first type of amino acid or protein and the amount of the nucleic acid;
Calculating a second ratio which is a ratio of the amount of the second type of amino acid or protein different from the first type and the amount of the nucleic acid;
The cancer test method according to any one of claims 2 to 4, wherein the first ratio and the second ratio are compared with the first threshold value.
6. The relationship between the first ratio and the second ratio in a plurality of regions of a sample known to contain cancer cells is determined, and the first threshold value is set based on the relationship. The cancer test method according to any one of 1 to 7.
The cancer testing method according to any one of claims 1 to 8, wherein at least one of the amount of the amino acid or protein and the amount of the nucleic acid is detected by Raman spectroscopy.
10. The cancer test method according to claim 9, wherein at least one of an amount of the amino acid egg or protein and an amount of the nucleic acid is detected by a CARS microscopic method.
The cancer examination method according to any one of claims 1 to 10, comprising a step of optically observing the examination region with light in an infrared band.
At least one of the amount of the amino acid or protein and the amount of the nucleic acid is detected by an average value of measured values in a plurality of small regions located inside the inspection region and narrower than the inspection region and separated from each other. The cancer test method according to any one of claims 1 to 11.
The amount of the amino acid or protein and the amount of the nucleic acid in each of a plurality of test regions included in a target region wider than the test region,
Calculating the ratio of the amount of nucleic acid detected to the amount of amino acid or protein detected;
The cancer test method according to any one of claims 1 to 12, wherein the presence or absence of cancer cells is determined based on a comparison between the calculated ratio and a predetermined first threshold value.
An acquisition unit for acquiring the amount of amino acid or protein and the amount of nucleic acid detected in the examination region;
A cancer test apparatus comprising: an information output unit that outputs information according to a ratio between the amount of amino acid or protein acquired by the acquisition unit and the amount of nucleic acid acquired by the acquisition unit.
A threshold storage unit for storing a first threshold value determined in advance;
The information output unit includes a comparison unit that compares the ratio with the first threshold, and the cancer cell based on whether the ratio exceeds the first threshold by the comparison by the comparison unit. The cancer test apparatus according to claim 14, wherein presence / absence is output as the information.
The acquisition unit acquires the amount of amino acid in the test region as an index of the amount of protein, acquires the amount of nucleobase as an index of the amount of nucleic acid,
The cancer test apparatus according to claim 14, wherein the information output unit outputs information according to a ratio between a detected amount of amino acid and a detected amount of nucleobase.
The said acquisition part irradiates several places of a biological body with excitation light, and acquires the quantity of several amino acids or proteins based on several spectra, and the quantity of several nucleic acids. Cancer testing equipment.
The cancer testing apparatus according to any one of claims 14 to 17, further comprising a display unit that displays information output by the information output unit.
The cancer test apparatus according to claim 18, further comprising a display unit that displays a biaxial graph indicating two types of ratios in which at least one of the nucleic acid and the amino acid or protein is different.
The cancer examination apparatus according to claim 17 or 19, further comprising a display unit that displays a display image for identifying whether or not the value of the ratio is larger than a threshold value prepared in advance.
The cancer test apparatus according to any one of claims 17 to 20, further comprising a display unit that displays a plurality of output values of the information output unit together in a state where the output values can be distinguished.
An obtaining step for obtaining the amount of amino acid or protein in the examination region and the amount of nucleic acid;
A cancer test program that causes a processing device to execute an output step of outputting information according to a ratio between the amount of amino acid or protein acquired in the acquisition step and the amount of nucleic acid acquired in the acquisition step.