JP2011112548A - Biosample analysis method, biosample analyzer, and biosample analysis program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosample analysis method, a sample analyzer, and a biosample analysis program capable of shortening a time to diagnosis. <P>SOLUTION: A spectrum of a time waveform of terahertz wave intensity emitted from an interface with which each measuring point assigned to an interface between a slide and a medium of a terahertz wave is irradiated is acquired, and a refractive index in each frequency on each measuring point is calculated by using the spectrum, and an image showing a statistical value of a refractive index having a specific frequency on each measuring point or a refractive index in a specific frequency band in a position relation of the measuring point is generated. On the other hand, a figure of the biosample arranged on the slide is acquired, and the figure and the image are displayed in the comparable state each other. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a technique using electromagnetic waves (terahertz waves) in a band of approximately 0.1 × 10 ¹² [Hz] to 100 × 10 ¹² [Hz].

  Conventionally, there is terahertz time-domain spectroscopy (THz-TDS) as a method for generating or detecting terahertz waves. This terahertz time domain spectroscopy is attracting attention in various technical fields such as industrial, medical, bio, agricultural or security.

  In this terahertz time-domain spectroscopy, pulse light of about 100 fetom seconds from an ultrashort laser light source is divided into pump light and probe light, and the pump light is condensed on a terahertz wave generating element. Thereby, in the terahertz wave generating element, a current of about sub-picosecond is generated, and a terahertz wave proportional to the time differentiation is generated.

  This terahertz wave is applied to the terahertz wave detection element via the sample. At this time, when the terahertz wave detection element is irradiated with the probe light, carriers are generated, and accelerated by the electric field of the terahertz wave to generate an electric signal. Become.

  By shifting the timing at which the probe light reaches the terahertz wave detection element, the time waveform of the amplitude electric field of the terahertz wave is measured, and the spectrum of the terahertz wave can be obtained by Fourier transforming the time waveform.

  By the way, imaging using terahertz time-domain spectroscopy is being examined in order to inspect luggage such as airport customs, human clothes, and prohibited drugs in the body. In addition, since the sample can be inspected in a non-destructive state, application to pathological diagnosis is being studied.

  In general, in a pathological diagnosis, the quality of a biological sample is determined from a morphological viewpoint such as the morphology and color tone of cells in the biological sample. The production of this biological sample generally has a difference depending on the experience and skill of a professional engineer, and there is a problem that it is difficult to determine the biological sample due to this difference.

  As one method for solving this problem, an image processing method for clarifying the color difference of the cell type in the blood cell sample image has been proposed (see, for example, Patent Document 1).

JP 2009-152868 A

  This Patent Document 1 only emphasizes one morphological element in the blood cell sample image, although the variation in the judgment of the diagnostician due to the indistinct color difference between the cell types is reduced. Therefore, even if the color difference between the cell types is clear, it may be difficult to identify the cell type or it may be difficult to determine the quality of the cell.

  In such a case, generally, using another sample that has been subjected to fluorescent staining, the identification of the cell type, the presence or absence of malignant tumor, or the degree of progression is secondarily determined from a molecular biological viewpoint. However, in this case, not only a preparation period of another sample is required, but also it does not completely coincide with the first sample, so that it takes an excessive amount of time for diagnosis.

  The present invention has been made in consideration of the above points, and intends to propose a biological sample analysis method, a biological sample analysis apparatus, and a biological sample analysis program that can shorten the time to diagnosis.

  In order to solve such a problem, the present invention is a biological sample analysis method, wherein an acquisition step of acquiring an image of a biological sample arranged on a slide, and each measurement point assigned to the interface between the slide and the terahertz wave medium are provided. A spectrum acquisition step for acquiring a time waveform spectrum of the terahertz wave intensity emitted from each of the interfaces, a calculation step for calculating a refractive index for each frequency at each measurement point using the spectrum, and at each measurement point A generation step for generating an image indicating a refractive index of a specific frequency or a refractive index of a specific frequency band in a positional relationship between the measurement points, and a display control step for displaying the image in a state where the images can be compared with each other. Have.

  Further, the present invention is a biological sample analyzer, wherein each of the measurement points assigned to the interface between the acquisition means for acquiring the image of the biological sample arranged on the slide and the slide and the medium of the terahertz wave is irradiated, Spectrum acquisition means for acquiring the time waveform spectrum of the terahertz wave intensity emitted from the interface, calculation means for calculating the refractive index for each frequency at each measurement point using the spectrum, and refractive index for a specific frequency at each measurement point Alternatively, the image forming apparatus includes a generating unit that generates an image indicating a refractive index statistical value in a specific frequency band by a positional relationship between the measurement points, and a display control unit that displays the image in a state where the images can be compared.

  The present invention is also a biological sample analysis program for obtaining an image of a biological sample arranged on a slide with respect to a computer, and irradiating each measurement point assigned to the interface between the slide and the terahertz wave medium. Obtaining a spectrum of a time waveform of the terahertz wave intensity emitted from the interface, calculating a refractive index for each frequency at each measurement point using the spectrum, a refractive index or a specific frequency at a specific frequency at each measurement point An image indicating the refractive index of the frequency band in terms of the positional relationship between the measurement points is generated, and the image is displayed in a state where the images can be compared.

In the present invention, in addition to the visible image of the biological sample, the biological sample is classified based on information from a physical point of view such as a refractive index, and is further classified based on the same positional relationship as that of the visible image. For this reason, it is possible to simultaneously grasp the state of a biological sample from a known morphological aspect and a physical science aspect without requiring preparation of another preparation.
Thus, it is possible to realize a biological sample analysis method, a sample analysis apparatus, and a biological sample analysis program that can shorten the time until diagnosis.

It is the schematic which shows the structure of a sample analysis system. It is the schematic where it uses for description of the sampling of a terahertz wave. It is the schematic which shows the structure of a sample analyzer. It is the schematic where it uses for description of the measurement of a terahertz waveform. It is the schematic which shows the model at the time of calculating the theoretical refractive index of a THz waveform. It is a photograph which shows a refractive index image. It is the photograph and graph which show an experimental result. It is a graph which shows the relationship between dyeing | staining and a refractive index. It is a graph which shows the relationship between the kind of cell and a refractive index. It is a flowchart which shows an analysis process procedure. It is a photograph which shows the refractive index image with the case where an amorphous film is formed, and the case where it is not formed.

Hereinafter, an embodiment for carrying out the present invention will be described. The description will be in the following order.
<1. Embodiment>
[1-1. Configuration of sample analysis system]
[1-2. Configuration of terahertz wave spectrometer]
[1-3. Configuration of sample imaging device]
[1-4. Configuration of sample analyzer]
[1-5. Contents of analysis process]
[1-6. Analysis processing procedure]
[1-7. Effect]
<2. Other embodiments>

<1. Embodiment>
[1-1. Configuration of sample analysis system]
In FIG. 1, the structure of the sample analysis system 1 is shown. The sample analysis system 1 includes a terahertz wave spectroscopic device 10, a sample imaging device 20, and a sample analysis device 30.

[1-2. Configuration of terahertz wave spectrometer]
The terahertz wave spectrometer 10 includes an ultrashort laser 11, a beam splitter 12, an optical delay unit 13, a terahertz wave generation element 14, a preparation stage 15, and a terahertz wave detection element 16.

  For example, the ultrashort laser 11 emits pulsed light having a pulse width of about 100 [fs], a repetition period of about several tens [MHz], and a center wavelength of about 780 [nm]. Specific examples of the ultrashort laser 11 include a femtosecond pulse laser titanium and a sapphire laser.

  The beam splitter 12 separates the pulsed light emitted from the ultrashort laser 11 into pump light and probe light.

  The optical delay unit 13 includes a mirror 13A that guides the probe light coming from the beam splitter 12 to the reflecting mirror ROP. The optical delay unit 13 moves the mirror 13A at a speed specified by the sample analyzer 30 in a direction approaching or leaving the beam splitter 12 and the reflecting mirror ROP. As a result, the optical path length of the probe light with respect to the terahertz wave detection element 16 is varied, and the arrival timing is delayed.

  The terahertz wave generating element 14 generates a terahertz wave based on carriers (electrons and holes) excited by pump light that arrives through the beam splitter 12. Specific examples of the terahertz wave generating element 14 include a photoconductive antenna and a nonlinear optical crystal such as ZnTe.

  Incidentally, the photoconductive antenna is composed of a semiconductor substrate made of semi-insulating gallium arsenide, an electrode disposed on one surface of the semiconductor substrate, and an application unit for applying a voltage between gaps formed in the electrode. It is what. In this photoconductive antenna, carriers generated in the semiconductor substrate by the irradiation of pump light are accelerated by the gap gap voltage, and at this time, current flows between the gaps to generate terahertz waves.

  The terahertz wave radiated from the terahertz wave generating element 14 is collected by the condensing optical system OP1 arranged on the back surface 15B side of the surface (hereinafter also referred to as a preparation arrangement surface) 15A on which the preparation PRT is arranged in the preparation stage 15. , Collected in a state where the incident angle is acute with respect to the back surface 15B.

  For the condensing optical system OP1, for example, a hemispherical lens or a combination of a hemispherical lens and a parabolic mirror is used. When a hemispherical lens is used, the aspherical surface thereof is opposed to one surface of the semiconductor substrate that is a component of the terahertz wave generating element 14 (the surface opposite to the surface on which the pulsed light is collected).

  The preparation stage 15 is movable in the direction of the preparation arrangement surface (x-axis direction and y-axis direction) and in the normal direction (z-axis direction) of the preparation arrangement surface. In the preparation stage 15, the focal point of the terahertz wave is positioned at a specific part of the preparation PRT under the control of the sample analysis device 30.

  The preparation PRT is obtained by fixing a tissue section TR such as a connective tissue, an epithelial tissue, or both of them to a slide SD made of a glass material, and the tissue section is stained as necessary. This staining is called not only general staining represented by HE (hematoxylin and eosin) staining, Giemsa staining or Papanicolaou staining, but also special staining such as FISH (Fluorescence In-Situ Hybridization) and enzyme antibody method. Staining is included.

  The terahertz wave reflected by the preparation PRT is guided to the terahertz wave detecting element 16 as a bundle of parallel rays by the condensing optical system OP2. The condensing optical system OP2 may have the same configuration as the condensing optical system OP1 or a different configuration.

  As shown in FIG. 2, the terahertz wave detecting element 16 detects the intensity of the terahertz wave oscillating electric field using the arrival timing of the probe light varied by the optical delay unit 13 as a sampling point.

  Specific examples of the terahertz wave detecting element 16 include a photoconductive antenna and a nonlinear optical crystal such as ZnTe as in the case of the terahertz wave generating element 14. However, in the case of a photoconductive antenna employed as the terahertz wave detection element 16, the above-described application unit is changed to an ammeter.

  In this terahertz wave spectrometer 10, in order to improve the signal-to-noise ratio (S / N ratio), the pump light is modulated by an optical chopper, and the reference signal is input to the lock-in amplifier to perform synchronous detection. Done.

[1-3. Configuration of sample imaging device]
The sample imaging device 20 includes a light source 21, a polarizing beam splitter 22, a quarter wavelength plate 23, an imaging lens 24, and an imaging element 25.

  The light source 21 illuminates the unstained tissue section TR or the tissue section TR subjected to general staining (hereinafter also referred to as bright field illumination light) and the tissue section TR subjected to special staining. It is possible to switch and irradiate light (hereinafter also referred to as dark field illumination light).

  However, only one of bright field illumination light and dark field illumination light may be radiated. Note that bright field illumination light is generally visible light, and dark field illumination light is light including a wavelength that excites a fluorescent marker used in special staining. In the dark field illumination light, the background portion with respect to the fluorescent marker is cut out.

  The polarization beam splitter 22 is disposed behind the illumination light source 21, transmits light incident from the light source 21, and reflects light incident from a quarter-wave plate 23 disposed behind the transmission side of the polarization beam splitter 22. .

  The quarter wavelength plate 23 converts the linearly polarized light incident from the polarization beam splitter 22 into circularly polarized light, and circularly polarized light (including elliptically polarized light) incident from the photographing lens 24 disposed behind the quarter wavelength plate 23. Is linearly polarized light.

  The imaging lens 24 collects the light incident from the quarter wavelength plate 23 on the preparation arrangement surface of the preparation stage 15, and uses the light reflected by the preparation PRT arranged on the preparation arrangement surface as a bundle of parallel rays. Guide to the wave plate 23. The imaging lens 24 is selected by a lens switching mechanism or manually from a plurality of imaging lenses having different magnifications.

  The imaging element 25 is disposed on the rear side of the reflection side of the polarization beam splitter 22 as a imaging plane with a plane planned as a projection destination of an image formed on the lens surface of the imaging lens 24. On this imaging surface, an enlarged image of the condensing part condensed by the imaging lens 24 is projected. The imaging element 25 generates an image projected on the imaging surface as data (hereinafter also referred to as imaging data).

[1-4. Configuration of sample analyzer]
As shown in FIG. 3, the sample analysis apparatus 30 is configured by connecting various hardware to a CPU (Central Processing Unit) 31 that controls the control.

  Specifically, a ROM (Read Only Memory) 32, a RAM (Random Access Memory) 33 serving as a work memory of the CPU 31, an operation input unit 34 for inputting a command according to a user operation, an interface unit 35, a display unit 36, and a storage The unit 37 is connected via the bus 38.

  The ROM 32 stores programs for executing various processes. The interface unit 35 is connected to the ultrashort laser 11, the optical delay unit 13, the preparation stage 15, the terahertz detection element 16, and the imaging element 25 via transmission paths.

  A liquid crystal display, an EL (Electro Luminescence) display, a plasma display, or the like is applied to the display unit 36. For the storage unit 37, a magnetic disk represented by HD (Hard Disk), a semiconductor memory, an optical disk, or the like is applied. A portable memory such as a USB (Universal Serial Bus) memory or a CF (Compact Flash) memory may be applied.

  The CPU 31 expands a program corresponding to a command given from the operation input unit 34 among the plurality of programs stored in the ROM 32, and appropriately controls the display unit 36 and the storage unit 37 according to the expanded program. .

  The CPU 31 appropriately controls the ultrashort laser 11, the optical delay unit 13, the preparation stage 15, the terahertz detection element 16, or the imaging element 25 via the interface unit 35 in accordance with the developed program.

[1-5. Contents of analysis process]
When the CPU 31 receives an execution instruction for analysis processing from the operation input unit 34, the CPU 31 expands a program associated with the execution instruction in the RAM 33.

  In this case, the CPU 31 follows the program as shown in FIG. 3 as an identifier assigning unit 41, an interface scanning unit 42, a time waveform obtaining unit 43, a spectrum waveform obtaining unit 44, a refractive index calculating unit 45, and a refractive index image generation. Functions as a unit 46, a sample image generation unit 47, a presentation unit 48, and a determination unit 49.

  The identifier assigning unit 41 assigns a unique identifier such as a number to the preparation PRT arranged on the preparation arrangement surface of the preparation stage 15. The identifier assigning unit 41 acquires personal information regarding the tissue section TR in the preparation PRT, and stores the personal information in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  The personal information is, for example, information such as the name or assigned number of the subject (patient) of the tissue section, sex, age, collection date, and the like. In addition, as a method for acquiring personal information, for example, a method of displaying an input screen on the display unit 36 and inputting it from the operation input unit 34 or a method of acquiring from a barcode pasted on the preparation PRT can be applied. It is.

  The interface scanning unit 42 scans the interface between the terahertz wave medium and the slide SD (hereinafter also referred to as a reference interface). Specifically, the preparation stage 15 is set in a predetermined direction from the initial position to the preparation arrangement plane (x-axis direction or y-axis direction) with a region (hereinafter also referred to as a measurement point) where terahertz waves are to be measured as a unit. Scanned in order. As a result, measurement points are allocated to the reference interface.

  The time waveform acquisition unit 43 converts a time waveform (hereinafter also referred to as a Tz waveform) with respect to the intensity of the oscillating electric field of the terahertz wave to the terahertz wave every time the measurement point to be measured is scanned by the interface scanning unit 42. Measurement is performed using the detection element 16.

  As shown in FIG. 4, the preparation PRT has a structure in which a tissue section TR is arranged on a slide SD (FIG. 4A). For this reason, a Tz waveform reflected at the reference interface B1 (hereinafter also referred to as an Rf-Tz waveform) REF and a Tz waveform reflected at the interface B2 between the slide SD and the tissue section TR (hereinafter referred to as an Sm-Tz waveform). (Also referred to as SAM) is measured with a sufficiently separable time difference (FIG. 4B).

  In FIG. 4B, the Rf-Tz waveform REF is indicated by a solid line, and the Sm-Tz waveform SAM is indicated by a broken line. For convenience, the Sm-Tz waveform SAM is shifted upward from the Rf-Tz waveform REF. Is shown. “Cut point” is a division point between the Rf-Tz waveform REF and the Sm-Tz waveform SAM.

  The time waveform acquisition unit 43 extracts the Rf-Tz waveform and the Sm-Tz waveform from the Tz waveform at the measurement point to be measured. Then, the time waveform acquisition unit 43 stores the extracted Tz waveforms and the measurement point position information in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  When an Rf-Tz waveform at a measurement point to be measured is extracted, the spectrum waveform acquisition unit 44 performs a Fourier transform process on the Rf-Tz waveform, and a phase spectrum of each frequency in the Rf-Tz waveform. And a power spectrum (hereinafter also referred to as Rf-spectrum). Then, the spectrum waveform acquisition unit 44 stores the Rf-spectrum in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  On the other hand, when the Sm-Tz waveform at the measurement point to be measured is extracted, the spectrum waveform acquisition unit 44 performs a Fourier transform process on the Sm-Tz waveform, and each frequency in the Sm-Tz waveform is obtained. A phase spectrum and a power spectrum (hereinafter also referred to as Sm-spectrum) are acquired. Then, the spectrum waveform acquisition unit 44 stores the Sm-spectrum in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  Further, when both the Rf-spectrum and Sm-spectrum of the Tz waveform at the measurement point to be measured are acquired, the spectrum waveform acquisition unit 44 calculates a ratio of these spectra (hereinafter also referred to as a spectrum ratio). . The spectrum waveform acquisition unit 44 stores the spectrum ratio in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  When the Rf-spectrum and Sm-spectrum of the Tz waveform at the measurement point to be measured are calculated, the refractive index calculation unit 45 is guided to the terahertz wave detection element 16 according to the Rf-spectrum as the first step. A theoretical spectrum of the Tz waveform to be measured (hereinafter also referred to as a model spectrum) is calculated.

Specifically, for example, the following calculation is performed by setting the frequency as “f” and the complex amplitude at the frequency as “E (f)”. That is, using the Rf-spectrum (“E ^ex _ref.detector ” in FIG. 5), the frequency spectrum of the terahertz wave (“E _emitter (f)” in FIG. 5) incident on the preparation PRT is

Is calculated by “Θ ₀ ” in the equation (1) means an incident angle. In the equation (1), “r ^S ” is the reflectance of the S-polarized component, and “r ^P ” is the reflectance of the P-polarized component.

Is defined as In this equation (2), “i” and “t” mean layers that sandwich an interface where reflection occurs, and “θ _i ” and “θ _t ” mean incident angles in the respective layers. "N ^~" Also in (2), means the complex part of the refractive index in the layer (also referred to as the imaginary part).

Using the calculation result of equation (1), the S-polarized component and the P-polarized component (“E _imput (f)” in FIG. 5) in the frequency spectrum of the terahertz wave incident on the preparation PRT are _expressed by the following equation:

Is calculated by

Using the calculation result of equation (3), the frequency spectrum of the terahertz wave that passes through the first interface of the first layer (“E ^th _inputsam (f)” in FIG. 5) is _expressed by the following equation:

Is calculated by In this equation (4), “P ()” is the following equation:

Is defined as "N ^~" means a complex part of the refractive index in the layer, "n" means the real part of the refractive index in the layer. “K” means the attenuation coefficient, “d” means the layer thickness, and “c” means the speed of light in vacuum. These parameters are preset. “T” is a transmission coefficient at the interface between the air and the first layer, and is given by equation (7).

Using the calculation result of the equation (4), the frequency spectrum of the terahertz wave that reflects the second interface of the first layer (“E ^th _outputsam (f)” in FIG. 5) is _expressed by the following equation:

Is calculated by “R _1-2 ” in the equation (6) is the reflectance of the S-polarized component and the reflectance of the P-polarized component guided from the first interface to the second interface. “T _1-2 ” is the transmittance of the S-polarized component and the transmittance of the P-polarized component guided from the first interface to the second interface, and “t _2-1 ” is the first interface from the second interface. Are the transmittance of the S-polarized component and the transmittance of the P-polarized component. The transmittance of the S-polarized component and the transmittance of the P-polarized component are

Is defined as In addition, “FP (f, 2)” in the equation (6) means the Fabry-Perot reflection coefficient for the frequency f and the second layer (that is, the layer corresponding to the tissue section TR).

Is defined as

Using the calculation result of Equation (6), the frequency spectrum of the terahertz wave emitted from the first interface of the first layer (“E ^th _sam (f)” in FIG. 5) is _expressed by the following equation:

Is calculated by

Using the calculation result of Equation (9), the model spectrum (“E ^th _sam.detector (f)” in FIG. 5) is

Is calculated by

  As described above, the refractive index calculation unit 45 calculates the model spectrum corresponding to the Rf-spectrum in consideration of the P-polarized light and S-polarized light of the Tz waveform guided to the terahertz wave detection element 16.

  When calculating the model spectrum, the refractive index calculation unit 45 calculates the refractive index (real part, complex part and those at the measurement point to be measured from the comparison result between the model spectrum and the Sm-spectrum as the second stage. Both).

Specifically, when the _Sm- spectrum acquired by the spectrum waveform acquisition unit 44 is “E ^ex _sam.detector (f)” and the model spectrum is “E ^th _sam.detector (f)”,

As described above, the refractive index when the sum of the absolute value of the ratio of the Sm-spectrum and the model spectrum and the square value of the declination ratio is minimized is calculated for each frequency. Incidentally, “abs” represents an absolute value, and “arg” represents an argument. Note that the optimization function M for calculating the refractive index for each frequency by minimizing the difference between the Sm-spectrum and the model spectrum is not limited to the expression (11).

  As described above, the refractive index calculation unit 45 detects the attenuation and the phase delay caused by the sample for each measurement point scanned by the interface scanning unit 42 for each frequency, so that the refractive index (the real part, the complex part, and both of them). ) Is calculated.

  The refractive index image generation unit 46, when the refractive index for each frequency at each measurement point is calculated by the refractive index calculation unit 45, an image indicating the refractive index (real part, complex part, and both) (hereinafter referred to as the refractive index image generation part 46). This is also called a refractive index image).

  Specifically, the refractive index of a specific frequency or the average refractive index of a specific frequency band at each measurement point is classified by color according to a plurality of stages (gradations) to be set, and is indicated by the positional relationship between the measurement points. It is. The specific frequency or specific frequency band, the number of gradations, the color to be associated with the gradation, and the resolution to be expressed as a refractive index image are set via the operation input unit 34 and can be changed as necessary. It is said.

  When the refractive index image generation unit 46 finishes generating the refractive index image, the refractive index image generation unit 46 stores the refractive index image in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  A reclassification command for reclassifying the refractive index image based on the designated position is given to the refractive index image generation unit 46 from the operation input unit 34 as necessary. The designated position is designated by a pointer or the like that can move the refractive index image in accordance with the operation of the operation input unit 34.

  When this reclassification command is given, the refractive index image generation unit 46 generates an image (hereinafter also referred to as a refractive index correlation image) indicating a difference in refractive index from another position with reference to the designated position. .

  Specifically, as shown in FIG. 6A, the difference between the refractive index of the pixel corresponding to the designated position and the autocorrelation coefficient of the refractive index corresponding to the pixel other than the designated position is set. Are classified by color according to the stage (gradation) and indicated by the positional relationship of the measurement points. The number of gradations, the color to be associated with the gradation, and the resolution to be expressed as a refractive index correlation image are set via the operation input unit 34 and can be changed as necessary.

  When the refractive index image generation unit 46 has generated the refractive index correlation image, the refractive index correlation image is stored in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  The refraction index image generation unit 46 is provided with a reclassification command for reclassifying the refraction index image based on the value input as the refraction index of the specific frequency band from the operation input unit 34 as necessary. Yes.

  When this reclassification command is given, the refractive index image generation unit 46 performs cluster analysis based on the similarity of refractive indexes in a specific frequency band. Then, the refractive index image generation unit 46 generates a refractive index image as shown in FIG. Specifically, for example, the difference in refractive index at each measurement point with respect to a specified value is classified by color according to a plurality of stages (gradations) to be set, and is indicated by the positional relationship of each measurement point. Even when the refractive index image generation unit 46 has generated the refractive index correlation image, the refractive index image generation unit 46 stores the refractive index image generation unit 46 in the storage unit 37 in association with the identifier assigned to the preparation PRT.

  Incidentally, in FIG. 6, a tissue section having a thickness of 10 [μm] is used, and 0.8-1.2 [THz] is targeted.

  The sample image generation unit 47 controls the imaging device 25 for each measurement point scanned by the interface scanning unit 42, and an enlarged image of the prepared portion projected on the imaging surface of the imaging device 25 (hereinafter referred to as PT region enlargement). (Also called an image).

  When the sample image generation unit 47 acquires the PT site enlarged images corresponding to the respective measurement points, the sample image generation unit 47 connects these PT site enlarged images, and extracts a tissue section (hereinafter also referred to as a tissue sample image) from the connected image. To do. Then, the sample image generation unit 47 generates a tissue sample image for each of a plurality of resolutions, and stores the tissue sample images of these resolutions in the storage unit 37 in association with identifiers assigned to the preparations PRT.

  When the tissue sample image is extracted, the sample image generation unit 47 generates a reduced image for selection (hereinafter also referred to as a thumbnail image) from the tissue sample image, and uses this as an identifier assigned to the preparation PRT. The information is also stored in the storage unit 37 in association with each other. This thumbnail image is used when a refractive index image or tissue sample image associated with a specific identifier is displayed from a refractive index image or tissue sample image associated with a plurality of identifiers.

  When the refractive index image and the tissue sample image are generated, the presentation unit 48 displays the refractive index image and the tissue sample image corresponding to the resolution that is initially set or specified by the operation input unit 34 in a state in which they can be compared. 36. The refractive index image to be displayed is any one of an image based on the refractive index of the real part, the refractive index of the complex part, or both, and can be appropriately set by the operation input unit 34.

  In addition, when the refractive index correlation image is generated, the presentation unit 48 displays the refractive index correlation image on the display unit 36 in a state where it can be compared with the refractive index image and the tissue sample image.

  Here, the experimental results are shown in FIG. FIG. 7A is a photograph showing a microscopic image (visible image) of a section of lung tissue subjected to silver staining. 7B is a refractive index image of the real part of the refractive index, FIG. 7D is a refractive index image of the complex part of the refractive index, and FIG. 7F is obtained from both the real part and the complex part of the refractive index. It is a photograph which shows the refractive index image obtained. FIG. 7C shows the real part of the normalized refractive index, and FIG. 7E shows the complex part of the normalized refractive index. In FIG. 7, similarly to FIG. 6, a tissue section having a thickness of 10 [μm] is used, and the target is 0.8-1.2 [THz].

  As can be seen from FIG. 7, the tissue sections TR are classified and expressed from an approach called refractive index, which is different from the morphological approach. As can be seen from the experimental results shown in FIG. 8, the refractive index can be obtained as an equal value regardless of the difference or presence of staining, and a refractive index image can be obtained without special data processing depending on the difference or presence of the staining. This is convenient.

  When the refractive index image is generated by the refractive index image presentation unit 46, the determination unit 49 determines whether or not the refractive index image becomes a characteristic part for each predetermined search range.

  Specifically, a feature portion that is matched with a plurality of refractive index patterns managed as a database and has a matching result larger than a threshold set for the similarity to the refractive index pattern is searched from the refractive index image. Is done.

  In general, since terahertz waves are absorbed by moisture, the refractive index is low in the cell-free portion, and increases as the cell density increases. For example, as shown in FIG. 9, it is known that the refractive index varies depending on the state of the cell such as the type of cell and malignancy. Therefore, in the storage unit 37 in this embodiment, the association between the cell state such as the cell type and the malignancy and the refractive index pattern is stored and managed as a database.

  Incidentally, the photograph shown in the upper part of FIG. 9 is a visible image, and the photograph shown in the middle part is an image obtained by cluster analysis based on the similarity of refractive indexes in a specific frequency band. The graph shown in the lower part of FIG. 9 is a cluster analysis result based on the similarity in refractive index for the second lung tissue section from the left.

  The determination unit 49, in the case where a characteristic part that results in a matching result larger than the threshold is searched, is a process for distinguishing the characteristic part from other parts for the refractive index image and the tissue sample image displayed on the display unit 36. Apply. Specifically, for example, the refractive index image and the tissue sample image are subjected to a process of surrounding a region corresponding to the characteristic portion with a single color frame. Then, the determination unit 49 notifies the display unit 36 of the state of the cell associated with the refractive index pattern whose similarity with the feature portion is equal to or greater than the threshold.

  In addition, when a feature portion to be registered as a refractive index pattern is specified in the refractive index image, the determination unit 49 presents an input screen for a cell state such as the cell type and malignancy of the feature portion. When the cell state input via this input screen is input, the determination unit 49 associates the cell state with the feature portion to be specified and stores the associated cell in the storage unit 37 and updates the database.

  As described above, when the refractive index image generated by the refractive index image presenting unit 46 includes a part exhibiting a characteristic refractive index pattern, the determination unit 49 notifies the part, and the refractive index pattern is not stored in the database. If there is a part that is registered, the part can be registered.

[1-6. Analysis processing procedure]
Next, the analysis processing procedure will be described with reference to the flowchart shown in FIG. That is, for example, when the CPU 31 receives an execution instruction of the analysis process from the operation input unit 34, the CPU 31 starts this analysis process procedure and proceeds to step SP1.

  In step SP1, the CPU 31 assigns a unique identifier to the preparation PRT arranged on the preparation arrangement surface of the preparation stage 15, and proceeds to step SP2. In step SP2, the CPU 31 scans the preparation stage 15 so that the measurement point is located at the target portion of the interface with the terahertz wave medium, and proceeds to step SP3.

  In step SP3, the CPU 31 obtains an enlarged image of the preparation site at the measurement point (PT site enlarged image), and proceeds to step SP4. In step SP4, the CPU 31 starts measuring the Tz waveform and extracts the Rf-Tz waveform and the Sm-Tz waveform (FIG. 4). The spectrum of these Tz waveforms (Rf-spectrum Sm-spectrum) and its The spectrum ratio is calculated, and the process proceeds to step SP5.

  In step SP5, the CPU 31 calculates the refractive index using the Rf-Tz waveform and Sm-Tz waveform calculated in step SP4 and the above-described equations (1) to (11), and then proceeds to step SP6. In step SP6, the CPU 31 determines whether or not measurement points are distributed over the entire interface with the terahertz wave medium. If a negative result is obtained, the CPU 31 returns to step SP2 and repeats the above processing.

  When the measurement points are distributed over the entire interface with the terahertz wave medium through the processing loop of step SP2 to step SP6, the CPU 31 proceeds to step SP7 and uses the refractive index of each measurement point to generate a refractive index image. (FIG. 6) is generated. Further, the CPU 31 generates a tissue sample image using the PT region enlarged image of each measurement point, and proceeds to step SP8.

  In step SP8, the CPU 31 displays the refractive index image generated in step SP7 on the display unit 36 in a state in which the tissue sample image can be compared, and proceeds to step SP9. In step SP9, the CPU 31 determines whether or not a characteristic portion is included in the refractive index image generated in step SP7.

  When the CPU 31 determines in step SP9 that the characteristic portion is included in the refractive index image, the CPU 31 proceeds to step SP10 and performs a predetermined process on the refractive index image and the tissue sample image displayed in step SP8 to display the characteristic portion. After highlighting, the analysis processing procedure is terminated.

  On the other hand, when the CPU 31 determines in step SP9 that the characteristic portion is not included in the refractive index image, the CPU 31 ends the analysis processing procedure without performing the highlighting process in step SP10.

[1-7. Effect]
In the above configuration, the sample analyzer 30 irradiates each measurement point assigned to the interface between the slide SD and the terahertz wave medium, and acquires the spectrum of the Tz waveform emitted from the interface.

  Then, the sample analyzer 30 uses this spectrum to calculate the refractive index for each frequency at each measurement point, and calculates the refractive index of the specific frequency or the average refractive index of the specific frequency band at each measurement point. An image (refractive index image) indicated by the positional relationship is generated.

  The sample analyzer 30 acquires an enlarged image (tissue sample image) of the tissue section TR arranged on the slide SD, and displays the enlarged image on the display unit 36 in a state where the tissue sample image and the refractive index image can be compared.

  Therefore, in this sample analysis apparatus 30, the tissue slice TR is classified based on information from the viewpoint of physical properties such as refractive index in addition to the visible image, and further classified based on the same positional relationship as that of the visible image. The For this reason, the state of the tissue section TR from a known morphological aspect and a physical science aspect can be grasped simultaneously without requiring preparation of another preparation PRT.

  The spectrum of the Tz waveform in this embodiment includes the spectrum (Rf-spectrum) of the Rf-Tz waveform REF (FIG. 4) reflected at the reference interface B1, and Sm− reflected at the interface B2 between the slide SD and the tissue section TR. Obtained as the spectrum (Sm-spectrum) of the Tz waveform SAM (FIG. 4).

  A model spectrum corresponding to the Rf-spectrum is calculated in consideration of the P-polarized light and the S-polarized light of the Tz waveform guided to the terahertz wave detecting element 16 (equation (1) to (10)). -The refractive index is calculated using the spectrum (equation (11)).

  Therefore, since the sample analysis apparatus 30 can obtain a refractive index closer to the true value, the refractive index image showing the state of the tissue section TR from the physical aspect can be shown finely.

  Further, the refractive index image in this embodiment is classified as a color by a plurality of stages (gradations) that are set based on the refractive index of a specific frequency or the average refractive index of a specific frequency band, and the positional relationship between the measurement points. Indicated. Therefore, for example, the state of the tissue section TR from the physical aspect can be classified more finely than the case indicated by the bar graph, and the classification ability can be changed.

  In addition, when the refractive index at a certain position in the refractive index image is designated, the sample analyzing apparatus 30 determines the difference in autocorrelation of refractive material other than the designated position with respect to the refractive index at the designated position at a plurality of stages (steps). The image is classified as a color according to the tone, and an image (refractive index correlation image) indicated by the positional relationship of the measurement point is generated. Then, the sample analyzer 30 displays the refractive index correlation image on the display unit 36 in a state where it can be compared with the tissue sample image and the refractive index image.

  Therefore, in this sample analyzer 30, for example, the tissue slice TR is considered from the viewpoint of the refractive index so that the distribution of the presence or absence of cells with high or low malignancy with respect to the malignant cells is grasped on the basis of the malignant cells to be noted. The cell distribution in can be grasped.

  According to the above configuration, it is possible to simultaneously grasp the state of the tissue section TR from a known morphological aspect and a physical science aspect without requiring preparation of another preparation PRT. As a result, the sample analysis device 30 capable of shortening the time until diagnosis is realized.

<2. Other embodiments>
In the above-described embodiment, the tissue section TR is applied as the biological sample. However, the biological sample is not limited to this embodiment. For example, smear cells, chromosomes, and the like can be applied, and all or part of various organisms other than those illustrated can be widely applied as biological samples.

  In the above-described embodiment, a glass slide SD is used. However, any material other than glass can be used as the material of the slide as long as it has a small optical influence on the terahertz band.

  In addition, since the glass material has high hydrophobicity, a gap is easily generated between the slide SD surface and the tissue section TR, and the adhesiveness is poor. Therefore, in general, the surface of the slide SD is coated with a highly hydrophilic silane to enhance the adhesion between the slide SD and the tissue section TR.

  However, since the silane coating is relatively easily peeled off by cleaning the preparation PRT, the use of the preparation PRT is effectively only once. In recent years, there has been a demand for a coating technique that enhances the adhesion between the slide SD and the tissue section TR and satisfies the condition that it can be used a plurality of times.

  In this regard, the present inventors have found that a slide SD coated with an amorphous film in which both diamond bonds (SP3 bonds) and graphite bonds (SP2 bonds) are mixed is transparent in the terahertz band while satisfying such conditions. Has been confirmed.

  This amorphous film is called amorphous carbon or DLC (Diamond Like Carbon), and has such properties as being difficult to peel off, being able to be obtained as a smooth film, and having high hydrophilicity. Therefore, as in the above-described embodiment, when measuring the time waveform of the reflection intensity of the terahertz wave irradiated from the surface opposite to the surface on which the biological sample is arranged, or calculating the refractive index from the time waveform of the terahertz wave intensity. When calculating, it is particularly preferable to use a slide SD coated with an amorphous film.

  Here, FIG. 11 shows a photograph of a refractive index image of 1 [THz] when uncoated (upper side of the broken line) and when an amorphous film is coated (lower side of the broken line). In FIG. 11, a tissue section TR having a thickness of 10 [μm] is arranged on a slide SD having a thickness of 3 [mm], and 2 [ A preparation PRT with a cover glass having a thickness of mm] was used. FIG. 11 shows that the refractive index images are the same regardless of whether or not the amorphous film is coated, and as a result, the amorphous film has no influence on the telehertz band.

Note that the amorphous film can be formed using plasma CVT (Chemical Vapor Deposition). Incidentally, 1-propanol (CH ₃ CH ₂ CH ₂ OH) is adopted as the source in the plasma CVT coating process in the experiment shown in FIG. 11, the source flow rate is 10 [SCCM], the source pressure is 10 [Pa], RF The power is 10 [W] and the coating time is 180 [sec]. In the plasma treatment process, oxygen is used as a source, the flow rate of the source is 30 [SCCM], the source pressure is 20 [Pa], the RF power is 100 [W], and the plasma treatment time is 30 [sec].

  In the above-described embodiment, the medium MD (FIG. 4A) on the interface side on which the tissue section TR is fixed in the slide SD is air. However, the medium MD is not limited to air. As the medium MD, a substance that causes strong reflection of terahertz waves at the interface between the tissue section TR and the medium MD, such as a solution layer or a deposited metal layer, can be applied. When this substance is applied, since the amplitude of the reflected wave SAM is enhanced, the reproducibility of the reflected intensity in the time waveform of the terahertz wave irradiated from the surface opposite to the surface on which the biological sample is arranged is greatly improved. .

  In general, the medium MD is often an atmosphere substituted with an inert gas such as nitrogen or argon in order to prevent absorption of terahertz waves due to air or moisture in the air. Moreover, generally used slide SD has insufficient flatness and parallelism. For this reason, it has been confirmed that the obtained terahertz image has an incorrect image contrast that is not caused by the tissue section TR. In addition, when the tissue slice TR is produced, thickness nonuniformity cannot be avoided. Furthermore, it has been confirmed that the slide glass absorbs the terahertz wave and the amplitude of the reflected wave SAM is reduced.

  The flatness and parallelism of the slide SD can be improved by using, for example, a high-quality quartz plate used for optical experiments or the like for the slide SD and fixing the tissue section TR on the slide SD. Alternatively, there is a method in which the tissue section TR is fixed to a normal slide glass and the side of the tissue section TR is pressure-bonded to a quartz plate which is a slide SD. In this case, the medium MD is a slide glass.

  The thickness error of the tissue section TR and the problem of the amplitude of the reflected wave SAM can be solved simultaneously as follows. The tissue slice TR is fixed on the slide SD, and the medium MD uses water and other solvents (cell culture solution, glycerol, various alcohols, fats and oils). It is important that the refractive indexes in the terahertz region of the medium MD and the tissue section TR are greatly different. The same effect can be obtained when the medium MD uses not only a solution but also a layer on which a metal such as gold is deposited. In short, as described above, a substance that causes strong reflection of terahertz waves at the interface between the tissue slice TR and the medium MD is preferable.

  As the tissue section TR, either a paraffin-embedded tissue section or a tissue section obtained by melting and staining paraffin can be used. In the case of the paraffin-embedded tissue section TR, since the difference in refractive index from the medium MD is large, the amplitude of the reflected wave SAM is remarkably enhanced. On the other hand, in the case of the stained tissue section TR, since the difference in refractive index from the medium MD is small, the increase in the amplitude of the reflected wave SAM is not expected. However, errors due to thickness non-uniformity are reduced and measurement reproducibility is greatly improved. In any case, since it is possible to use specimens obtained in the process of preparing pathological specimens that are widely adopted without special treatment as pathological specimens for terahertz, in terms of shortening the time to diagnosis Useful.

  In the above-described embodiment, the Rf-Tz waveform, the Sm-Tz waveform, the Rf-spectrum, the Sm-spectrum, and the spectrum ratio are acquired from the measurement results in the terahertz spectrometer 10. However, the acquisition method is not limited to this embodiment.

  For example, Rf-Tz waveform, Sm-Tz waveform, Rf-spectrum, Sm-spectrum, and spectral ratio all or part of the spectrum ratio is stored in advance from the ROM 32, the storage unit 37, or a storage medium of another device via a transmission line. The acquisition method is applicable.

  In the above-described embodiment, the refractive index is calculated using the equations (1) to (11). However, the refractive index calculation method is not limited to this embodiment. For example, the refractive index may be calculated from the Rf-Tz waveform and the Sm-Tz waveform using Kramers-Kronig transformation. In addition, the type of the biological sample may be input from the operation input unit 37 and may be acquired by referring to the database in which the type and the refractive index are associated with each other, and the refractive index may be input from the operation input unit 37. Also good.

  In the above-described embodiment, the refractive index image has a plurality of stages (gradation levels) in which the refractive index of the specific frequency at the measurement point or the average of the refractive index of the specific frequency band is set in the positional relationship of each measurement point. ) And classified as a color. However, the way of showing as a refractive index image is not limited to this embodiment.

  For example, a refractive index image indicating the refractive index of a specific frequency or the average refractive index of a specific frequency band by a bar graph as a positional relationship of measurement points may be applied. In short, any image may be used as long as the refractive index of the specific frequency or the average refractive index of the specific frequency band at each measurement point is indicated by the positional relationship between the measurement points.

  The refractive index in the specific frequency band is not limited to the average, and may be a value (statistical value) obtained by a predetermined statistical method such as dispersion.

  In the above-described embodiment, the refractive index image for each frequency at each measurement point is used for display. In addition to this, it can also be used as a parameter for gain adjustment.

  In the analysis processing in the above-described embodiment, the refractive index image and the tissue sample image (visible image) are displayed in a state where they can be compared. Information to be displayed in a state where the refractive index image can be compared is not limited to the tissue sample image (visible image).

  For example, a form in which all or part of the Rf-Tz waveform, Sm-Tz waveform, Rf-spectrum, Sm-spectrum, and spectrum ratio is displayed together with the tissue sample image (visible image) may be applied. By applying this form, the state of the biological sample can be grasped in more detail.

  In the above-described embodiment, the focal position is changed by the movement of the preparation stage 15. However, the variable focus method is not limited to this embodiment. For example, a focus variable method that changes the focus position by moving the stage with respect to the condensing optical system OP1 or the imaging lens 24 is applicable. As another example, it is possible to apply a variable focus method that changes the focal position by moving both the preparation stage 15 and the condensing optical system OP1 or the stage with respect to the imaging lens 24.

  Incidentally, the stage driving means includes an electromagnetic linear motor, a stepping motor, a piezo actuator, a MEMS (Micro Electro Mechanical Systems), and the like.

  The present invention can be used in industries such as industry, medicine, biotechnology, agriculture, security, information communication, and electronics.

  DESCRIPTION OF SYMBOLS 1 ... Sample analysis system, 10 ... Terahertz wave spectrometer, 11 ... Ultra-short laser, 12 ... Beam splitter, 13 ... Optical delay part, 14 ... Terahertz wave generating element, 15 ... Preparation stage, 16 …… Terahertz wave detecting element 20 …… Sample imaging device 21 …… Light source 22 ≦ Polarizing beam splitter 23 ¼ wavelength plate 24 …… Image lens 25 ‘Image sensor 30 …… Sample analysis device, 31 ... CPU, 32 ... ROM, 33 ... RAM, 34 ... operation input unit, 35 ... interface unit, 36 ... display unit, 37 ... storage unit, 41 ... identifier assignment unit , 42 ... interface scanning unit, 43 ... time waveform unit, 44 ... spectral waveform acquisition unit, 45 ... refractive index calculation unit, 46 ... refractive index image generation unit, 47 ... sample image generation unit, 4 ...... presentation section, 49 ...... determination unit, OP1, OP2 ...... focusing optical system, PRT ...... preparation, SD ...... slide, TR ...... tissue sections.

Claims (4)

An acquisition step of acquiring an image of a biological sample arranged on the slide;
A spectrum acquisition step of acquiring a spectrum of a time waveform of the terahertz wave intensity emitted to each measurement point assigned to the interface between the slide and the medium of the terahertz wave, and emitted from the interface;
A calculation step of calculating a refractive index for each frequency at each measurement point using the spectrum,
A generation step of generating an image indicating a refractive index of a specific frequency at each measurement point or a statistical value of a refractive index of a specific frequency band in a positional relationship between the measurement points;
A biological sample analysis method comprising: a display control step of displaying the image and the image in a state where the image can be compared. In the above generation step,
When the refractive index in the image is specified, a correlation image indicating the difference in refractive quality outside the specification with respect to the specified refractive index is generated by the positional relationship of the measurement points,
In the display control step,
The biological sample analysis method according to claim 1, wherein the correlation image is displayed instead of the image or in a state in which the image and the image can be compared. An acquisition means for acquiring an image of a biological sample arranged on the slide;
A spectrum acquisition means for acquiring a spectrum of a time waveform of the terahertz wave intensity emitted to each measurement point assigned to the interface between the slide and the medium of the terahertz wave, and emitted from the interface;
A calculating means for calculating a refractive index for each frequency at each measurement point using the spectrum;
Generating means for generating an image indicating a refractive index of a specific frequency at each measurement point or a statistical value of a refractive index of a specific frequency band in a positional relationship between the measurement points;
A biological sample analyzer having display control means for displaying the image and the image in a state where the image and the image can be compared. Against the computer,
Obtaining an image of a biological sample placed on a slide;
Obtaining a spectrum of the time waveform of the terahertz wave intensity emitted to each measurement point assigned to the interface between the slide and the medium of the terahertz wave, and emitted from the interface;
Calculating the refractive index for each frequency at each measurement point using the spectrum;
Generating an image indicating a refractive index of a specific frequency at each measurement point or a statistical value of a refractive index of a specific frequency band in a positional relationship of the measurement point;
A biological sample analysis program for executing display of the image and the image in a state where the image can be compared.