JP6265364B2 - Cell identification device, cell identification method, cell identification method program, and recording medium recording the program - Google Patents

Description

  The present invention relates to a cell identification device and a cell identification method.

  In cell diagnosis (cytodiagnosis), the refractive index of a cell may be measured for the purpose of identifying normal cells and cancer cells.

  In Patent Document 1, a tissue section (cell interface) is irradiated with a terahertz wave, the intensity of the terahertz wave emitted from the tissue section is detected, and the refractive index of the biological sample (cell) is calculated using the detected result. A technique related to a sample analysis method (cell identification device) is disclosed.

  In Patent Document 2, a fluorescent agent is administered to a subject (cell) that is colorless and transparent, the fluorescence emitted by the fluorescent agent is spectrally detected, and a normal site (normal cell) and a tumor site (abnormal cell) are detected using the detection results. Discloses a technology related to an automatic tumor identification device (cell identification device).

JP 2011-112548 A JP 2010-240078 A

  In the technique disclosed in Patent Document 1, spectrum analysis is performed on the detected intensity of the terahertz wave by performing a Fourier transform process or the like, so that there are cases where normal cells and abnormal cells cannot be distinguished. That is, in the technique disclosed in Patent Document 1, an error may be included in the analysis result (for example, FIG. 6 of Patent Document 1), and normal cells and abnormal cells (for example, normal cells described later (FIG. 16 (e) )) And cancer cells (FIG. 16 (g)) may not be distinguished.

  In the technique disclosed in Patent Document 2, since cells are stained by pretreatment, it may take time to immobilize cells. Further, there are cases where cells are killed or altered by staining the cells. That is, the use of subsequent cells may be limited by staining the cells. Furthermore, although it is necessary to set a threshold according to the type of cell, Patent Document 1 and Patent Document 2 do not describe a threshold setting method. In particular, when identifying cells in an isolation system (a state floating in a liquid) (for example, FIG. 31), it is necessary to appropriately change (set) the threshold according to the cells to be identified.

  The present invention is made under such circumstances, and a cell based on an optical path length variation measured when a cell is irradiated with light using a threshold value calculated based on an optical path length variation of a normal cell. An object of the present invention is to provide a cell identification device or a cell identification method.

  According to one aspect of the present invention, there is provided a cell identification device for identifying a cell using an optical path length change amount measured when the cell is irradiated with light, wherein the light is transmitted through the cell. Measuring means for measuring the amount of change in optical path length based on the intensity of the light, and analysis means for identifying the cells using a threshold value calculated based on the amount of change in optical path length of normal cells. A cell identification device is provided. The analyzing means extracts a plurality of optical path length changes corresponding to a plurality of positions of the normal cells, calculates an average value of the extracted optical path length changes, and uses the calculated average value as the threshold value. It may be a cell identification device characterized by the above. The analysis means extracts a plurality of optical path length change amounts corresponding to a plurality of positions of the normal cells, calculates a plurality of average values for the extracted optical path length change amounts, and calculates the plurality of calculated average values The cell identification device may be characterized in that the threshold is set based on an error rate. The cell identification device characterized in that the analyzing means sets the cells having the optical path length variation amount equal to or less than the threshold as normal cells, and the cells having the optical path length variation amount exceeding the threshold as abnormal cells. Is provided. When the abnormal cell is a cancer cell, the analysis means uses a second threshold value calculated based on a plurality of optical path length change amounts corresponding to a plurality of positions of the metastatic cancer cell of the cancer cell. Thus, cancer cells with an optical path length change amount exceeding the threshold value and less than or equal to the second threshold value are metastatic cancer cells, and cancer cells with an optical path length change amount exceeding the second threshold value are non-metastatic cancer cells. A cell identification device is provided.

  According to another aspect of the present invention, there is provided a cell identification device for identifying a cell using an optical path length change amount measured when the cell is irradiated with light, wherein the light is transmitted through the cell. Measuring means for measuring the amount of change in optical path length based on the intensity of the light, and analysis means for identifying the cells using a threshold value calculated based on the amount of change in optical path length of normal cells. In the cell identification device, the analysis unit extracts a predetermined number of optical path length variation amounts from the larger one of the plurality of optical path length variation amounts of the normal cells, An identification device is provided. The measurement means changes the optical path length of the light applied to the cell by applying a voltage to the piezo element so as to be the same as the intensity of the light that does not pass through the cell, and changes the applied voltage to the applied voltage. The cell identification device may be characterized in that the optical path length change amount is measured on the basis thereof. An image generation unit that generates an image corresponding to the outer shape and refractive index difference or phase difference of the cell based on the measured change amount of the optical path length, and an output unit that displays the generated image. The cell identification device may be a feature.

  According to another aspect of the present invention, using a measurement step of irradiating a plurality of cells with light and measuring a plurality of optical path length changes, and a threshold calculated based on the optical path length change of normal cells And a step of identifying each of the plurality of cells. The identifying step extracts a plurality of optical path length changes corresponding to a plurality of positions of the normal cells, calculates an average value of the extracted optical path length changes, and uses the calculated average value as the threshold value. It may be a cell identification method characterized by that. The identifying step extracts a plurality of optical path length changes corresponding to a plurality of positions of the normal cells, calculates a plurality of average values for the extracted optical path length changes, and calculates the plurality of average values In the cell identification method, the threshold may be set based on an error rate. The measuring step includes a changing step of changing an optical path length of the light applied to the cell, and an optical path length of the light applied to the cell so as to be the same as the intensity of the light when not passing through the cell. And a feedback control step that changes the frequency of the cell identification method. The program for making a computer perform the said cell identification method, or the computer-readable recording medium which recorded the said program may be sufficient.

  According to the cell identification device or the cell identification method of the present invention, using the threshold value calculated based on the optical path length change amount of normal cells, based on the optical path length change amount measured when the cells are irradiated with light, Cells can be identified.

It is a schematic block diagram explaining an example of the cell identification device which concerns on embodiment of this invention. 1 is a schematic system diagram illustrating an example of an optical system of a cell identification device according to an embodiment of the present invention. It is explanatory drawing explaining the arrangement | positioning (example of adhesion type sample arrangement | positioning) of the cell of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining the operation | movement which measures the intensity | strength (optical path length variation | change_quantity, phase difference) of the light which permeate | transmitted the cell. It is explanatory drawing explaining operation | movement of the measurement means (voltage detection part) of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining the operation | movement which measures the phase difference of another cell identification apparatus. It is a schematic functional block diagram explaining an example of the function of the cell identification device which concerns on embodiment of this invention. It is a flowchart figure explaining an example of operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining an example (example of a phase difference image) of the operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining an example (example of extraction of a feature-value) of operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining an example (example of an error rate) of operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining an example (an example of determination of the feature-value to extract) of operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is explanatory drawing explaining an example (other example of determination of the feature-value to extract) of operation | movement of the analysis means of the cell identification device which concerns on embodiment of this invention. It is a flowchart figure explaining an example of operation of the cell discernment device concerning Example 1 of the present invention. It is explanatory drawing explaining the arrangement | positioning (adhesion type sample arrangement | positioning) of the cell of the cell identification device which concerns on Example 1 of this invention. It is explanatory drawing explaining the measurement result (phase difference image) and analysis result (optical path length variation | change_quantity image) of the cell identification device which concerns on Example 1 of this invention. It is explanatory drawing explaining the measurement result (optical path length variation | change_quantity) of the cell identification device which concerns on Example 1 of this invention. It is explanatory drawing explaining the identification result (analysis result) which the cell identification apparatus (analysis means) which concerns on Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (1% average) which the cell identification apparatus (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (2% average) which the cell identification apparatus (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (3% average) which the cell identification device (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (4% average) which the cell identification apparatus (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (5% average) which the cell identification apparatus (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is explanatory drawing explaining the result (6% average) which the cell identification apparatus (analysis means) which concerns on the modification 1 of Example 1 of this invention identified the cell. It is a flowchart figure explaining an example of operation | movement of the cell identification device which concerns on the modification 2 of Example 1 of this invention. It is a schematic system diagram explaining an example of the optical system of the cell identification device according to Example 2 of the present invention. It is a flowchart figure explaining an example of operation | movement of the cell identification device which concerns on Example 2 of this invention. It is explanatory drawing explaining an example of the measurement result of the cell identification device which concerns on Example 2 of this invention. It is explanatory drawing explaining the arrangement | positioning (isolation type sample arrangement | positioning) of the cell of the cell identification device which concerns on Example 3 of this invention. It is explanatory drawing explaining the example which has arrange | positioned the cell of the isolation system of the cell identification device which concerns on Example 3 of this invention. It is a flowchart figure explaining an example of operation | movement of the cell identification device which concerns on Example 3 of this invention. It is explanatory drawing explaining the measurement result (phase difference image) and analysis result (optical path length variation | change_quantity image) of the cell identification device which concerns on Example 3 of this invention. It is explanatory drawing explaining the result (2% average) which the cell identification apparatus (analysis means) which concerns on Example 3 of this invention identified the cell. It is explanatory drawing explaining the result (4% average) which the cell identification apparatus (analysis means) which concerns on Example 3 of this invention identified the cell. It is explanatory drawing explaining the result (6% average) which the cell identification apparatus (analysis means) based on Example 3 of this invention identified the cell. It is explanatory drawing explaining the result (8% average) which the cell identification apparatus (analysis means) which concerns on Example 3 of this invention identified the cell. It is explanatory drawing explaining the result (10% average) which the cell identification apparatus (analysis means) which concerns on Example 3 of this invention identified the cell.

  Non-limiting exemplary embodiments of the present invention will be described with reference to the accompanying drawings. In the description of all attached drawings, the same or corresponding members or parts are denoted by the same or corresponding reference numerals, and redundant description is omitted. Also, the drawings are not intended to show the relative ratio between members or parts. Accordingly, specific dimensions in the drawings can be determined by one skilled in the art in light of the following non-limiting embodiments. Furthermore, the present invention also measures the optical path length change amount when the cell is irradiated with light other than the embodiments described below, and analyzes the state of the cell based on the measured optical path length change amount (analysis, identification, Any device can be used as long as it is evaluated.

  The present invention will be described in the following order using a cell identification device according to an embodiment of the present invention.

1. 1. Configuration of cell identification device 2. Functions of the cell identification device Example of analysis means operation (data extraction and threshold setting)
4). 4. Program and recording medium Example 1 (Example of identification by adhesive sample arrangement)
6). Example 2 (Example of identification using digital holography technology)
7). Example 3 (Example of identification by sample arrangement of isolation system)

[1. Configuration of cell identification device]
FIG. 1 shows a schematic configuration diagram of a cell identification device 100 according to an embodiment of the present invention.

  As shown in FIG. 1, a cell identification device 100 according to the present embodiment is a device that measures physical properties of cells and analyzes (identifies) a cell state based on the measured physical properties. In addition, the site | part (cell) which can analyze a cell state by this invention is not limited to abnormal cells, such as a breast cancer cell, a skin cancer cell, and a leukemia cell demonstrated hereafter. That is, the present invention can be used for an apparatus for analyzing an arbitrary cell state at an arbitrary site.

  The cell identification device 100 is based on the control unit 10 that controls the operation of each component of the cell identification device 100, the measurement unit 20 that measures the optical path length change amount by irradiating the cell with light, and the measured optical path length change amount. Analyzing means 30 for analyzing cells. The cell identification device 100 also inputs the storage unit 40 that stores the measurement result of the measurement unit 20 and the analysis result of the analysis unit 30 and the measurement conditions of the measurement unit 20 from the outside of the cell identification device 100 to the cell identification device 100. And an I / F means (interface means) 50 for outputting an analysis result or the like to the outside of the cell identification device 100.

  The control means 10 is a means for instructing each component (such as a measuring unit 20 described later) of the cell identification device 100 to control the operation of each component. In this embodiment, the control means 10 controls the measurement means 20 based on the measurement conditions and the like input by the I / F means 50 (described later). Further, the control means 10 controls the analysis means 30 (described later) based on the calculation conditions and analysis conditions input by the I / F means 50 (input section 51 described later). Furthermore, the control means 10 outputs a measurement result, a calculation result, an analysis result, etc. to the I / F means 50 (the output part 52 mentioned later). The control unit 10 may control the operation of each component of the cell identification device 100 using a program (control program, application, etc.) stored in advance in the storage unit 40.

  The measuring means 20 is a means for measuring the amount of change in the optical path length (hereinafter referred to as “optical path length change amount ΔOPL”) of the light passing through the cell when the cell is irradiated with light. In this embodiment, the measuring unit 20 measures a plurality of optical path length change amounts of light respectively transmitted through a plurality of cells. Further, the measuring means 20 changes the optical path length of the light applied to the cell so as to be substantially the same as the intensity (phase) of the light (interference light) that does not pass through the cell (hereinafter “feedback control”). That said.) Thereby, the measuring means 20 can measure the change amount of the changed optical path length as the optical path length change amount ΔOPL. Further, the measurement unit 20 outputs the measurement result to the analysis unit 30 and the storage unit 40.

  In this embodiment, the measuring means 20 is an optical system 21 that irradiates light to a cell, a feedback control unit 22 that controls the optical path length of the irradiated light, and a voltage detection unit that detects a voltage when the optical path length is controlled. And an optical path length conversion unit 24 that converts the detected voltage into an optical path length change amount ΔOPL.

  The optical system 21 (FIG. 1) irradiates light to a cell to be identified (diagnosed). In this embodiment, the optical system 21 includes a light source, a mirror, an objective lens, and the like.

Specifically, as shown in FIG. 2, the optical system 21 uses a wave plate (WP) and a splitter (PBS) to emit light emitted from a light source (He-Ne Laser) and use two lights (object light and object light). (Reference light). In addition, the optical system 21 irradiates the cell (Sample) with object light via a mirror (M _PZT ) capable of moving the position. Next, the optical system 21 combines the object light and the reference light using a splitter (BS). Thereafter, the optical system 21 detects the intensity (phase) of the interference light (the combined light of the object light and the reference light) using a photodetector (PD).

  Here, the measuring means 20 of the cell identification device 100 according to the present embodiment uses a coherent laser (light that has a certain relationship in amplitude and phase and can generate interference fringes) as the light source of the optical system 21. be able to. Further, the measuring means 20 can reduce the beam diameter by using a lens by using light having a short wavelength. Thereby, the measuring means 20 can increase the spatial resolution of the optical system 21. The measuring means 20 can use, for example, a He—Ne laser having a wavelength λ of 632.8 nm as the light source of the optical system 21. The measuring means 20 may use light of other wavelengths that can pass through the cells as the light source.

  The measuring means 20 can use a half-wave plate (λ / 2) as the wave plate (WP) of the optical system 21. Here, the half-wave plate can make the light amounts of the object light and the reference light divided by the splitter the same. That is, the half-wave plate can be rotated about the optical axis, thereby adjusting the ratio of the P-polarized component and the S-polarized component of the light emitted from the light source.

  The measuring means 20 can use a polarization beam splitter as the splitter (PBS) of the optical system 21. Here, the polarization beam splitter transmits one of the orthogonal polarization directions (for example, P polarization component) and reflects the other (for example, S polarization component). Thereby, the measurement means 20 can generate | occur | produce two light (two optical paths) by dividing | segmenting the light irradiated from the light source into object light and reference light using a polarization beam splitter.

  As shown in FIG. 2, the object light is reflected by the polarization beam splitter PBS, reaches the beam splitter BS via a cell sample to be identified, and the like. The reference light passes through the polarization beam splitter PBS, reaches the beam splitter BS via the plane mirror M and the like.

The measuring means 20 can change the optical path length of the object light by moving the position of the mirror (M _PZT ) of the optical system 21. The measuring means 20 is a mirror (M _PZT ) of the optical system 21, for example, a plane mirror attached to a piezo element (PZT), a plane mirror attached to an electromagnetic movable device (speaker or the like), or an electro-optic element (Pockels). A plane mirror or the like attached to the cell) can be used.

In the present embodiment, the measuring means 20 uses a plane mirror (mirror M _PZT ) attached to the piezo element PZT (FIG. 2). Here, the measuring means 20 can move the mirror _{MPZT in} a direction of 45 degrees with respect to the optical axis of the object light by applying a voltage to the piezo element PZT. The mirror M _PZT can be arranged at any position in the optical path of the object light (the optical path between the PBS and the BS in FIG. 2).

  FIG. 3 shows a cell arrangement (preparation) of the cell identification device 100 according to the present embodiment. Here, FIG. 3B shows an example of the arrangement (sample arrangement) of the adhesion-type cells TC. However, in the cell identification device 100 according to the present invention, an isolation-type sample arrangement described later (for example, FIGS. 29 and 30). ) May be used.

  As shown in FIG. 3A, the measuring means 20 arranges the cell TC in the gap between the pair of glass plates 21g (the glass plate 21gu and the glass plate 21gd in FIG. 3B). Here, in this embodiment, the cell TC is dripped with physiological saline Wn in order to prevent drying. Moreover, the measurement means 20 can change the space | interval of a pair of glass plate 21g using the spacer (for example, shim ring) 21S, and can keep it constant. Furthermore, the measurement means 20 can irradiate light to the micro area | region of the arrange | positioned cell TC using a pair of objective lenses 21La and 21Lb. The glass plate 21gu and the glass plate 21gd may have a thickness of 500 μm. Further, the spacer 21S may have a thickness of 50 μm.

  Specifically, the measuring means 20 uses the first lens 21La to collect object light substantially parallel to the optical axis, and focus the collected light (object light) into the cell TC. Thereafter, the measuring means 20 uses the second lens 21Lb to convert the light (object light) transmitted through the cell TC into parallel light.

  Furthermore, as shown in FIG. 3C, the measuring means 20 moves a pair of glass plates 21g (21gu, 21gd) on which the cells TC are arranged, thereby moving the two-dimensional plane in the x-axis direction and the y-axis direction ( Hereinafter, the optical path length variation ΔOPL distributed in the “xy plane”) can be measured. The measuring means 20 measures the three-dimensional distribution of the optical path length change amount ΔOPL (or phase or refractive index change amount) by further moving the glass plate 21 gu or the like in the z-axis direction (optical axis direction). May be.

  The measuring means 20 is a photo sensor (PD) of the optical system 21, a CMOS sensor or CCD sensor capable of two-dimensional detection, an inexpensive and small semiconductor detector (for example, a photodiode or a pin photodiode), or a photomultiplier (photomultiplier). For example, a light detection element such as a tube (PMT)) or an avalanche photodiode (Avalanche Photo Diode) can be used.

  The feedback control unit 22 (FIG. 1) controls the optical path length of the irradiated light. In this embodiment, the feedback control unit 22 changes the optical path length of the object light so as to be substantially the same as the intensity (phase) of the light (interference light) that does not pass through the cell TC. That is, the feedback control unit 22 feedback-controls the length of the optical path length of the object light so that the intensity of the interference light detected by the photodetector PD (FIG. 2) is within a predetermined range.

Specifically, as shown in FIG. 4A, the feedback control unit 22 uses the light Lr when the phase of the light Lt (object light) transmitted through the cell TC detected by the photodetector PD does not pass through the cell TC. Using the mirror M _PZT (FIG. 2), the optical path length of the object light is changed by the change amount ΔL so as to be substantially the same as the phase of (reference light). Thereby, the measuring means 20 can detect (measure) the change amount ΔL obtained by changing the optical path length as the optical path length change amount ΔOPL.

The voltage detector 23 (FIG. 1) detects an applied voltage when the feedback controller 22 changes the optical path length. In the present embodiment, the voltage detector 23 detects an applied voltage applied to the piezo element PZT (FIG. 2) that drives the mirror M _PZT when the feedback controller 22 changes the optical path length. The voltage detector 23 can detect an applied voltage V _PZT as shown in FIG. 5, for example.

Optical path length conversion unit 24 (FIG. 1) is to convert the applied voltage V _PZT of the voltage detection unit 23 detects the optical path length change amount DerutaOPL. In this embodiment, the optical path length conversion unit 24 assumes that the applied voltage V _PZT applied to the piezo element PZT (FIG. 2) is proportional to the optical path length change amount ΔOPL, and uses the following formula 1 to apply the applied voltage V _An optical path length change amount ΔOPL is calculated from _PZT .

Here, V _λ is a change amount of the applied voltage V _PZT corresponding to one wavelength (for example, 633 nm) of the light used for measurement, as shown in FIG. 5, for example.

  Further, the optical path length conversion unit 24 can calculate the phase difference Δφ using the calculated optical path length change amount ΔOPL. Specifically, the optical path length conversion unit 24 can calculate the phase difference Δφ from the optical path length change amount ΔOPL calculated in Equation 1 using Equation 2. That is, the optical path length conversion unit 24 can calculate the phase difference Δφ from the measured optical path length change amount ΔOPL without performing phase unwrapping processing (described later).

Furthermore, the measuring means 20 (optical path length conversion unit 24) may calculate the refractive index (refractive index difference) of the cell from the phase difference Δφ using the cell thickness or the like that has been measured in advance. The measuring means 20 (the optical path length conversion unit 24) corrects the measurement result (for example, the intensity of the interference light detected by the photodetector PD) using the cell thickness, transmittance, refractive index, or the like that has been measured in advance. May be.

  The analysis means 30 (FIG. 1) is a means for analyzing a cell state. In this embodiment, the analysis unit 30 can analyze the cell state (identify the cell) using the optical path length change amount ΔOPL measured by the measurement unit 20. The analysis means 30 may be a normal cell (eg, white blood cell, human breast cell, normal human dermis-derived fibroblast) or abnormal cell (eg, cancer cell (human breast cancer cell, skin cancer cell), leukemia cell) Etc.). Moreover, the analysis means 30 can further distinguish cancer cells into, for example, metastatic cancer cells or non-metastatic cancer cells.

  In the present embodiment, as shown in FIG. 1, the analysis means 30 is identified from an image generation unit 31 that generates an image using a plurality of optical path length variation ΔOPL of the cell, and a plurality of optical path length variation ΔOPL of the cell. Data extraction unit 32 that extracts the optical path length change amount, threshold calculation unit 33 that calculates a threshold value based on the extracted specific optical path length change amount, and a cell identification unit that identifies other cells using the calculated threshold value 34.

  The image generation unit 31 (FIG. 1) generates an image (optical path length variation distribution, phase difference distribution, refractive index distribution, etc.) using the optical path length variation ΔOPL measured by the measuring means 20. In addition, when the measurement unit 20 measures the distribution of the optical path length variation, the image generation unit 31 may generate a three-dimensional image (such as a three-dimensional optical path length variation distribution) based on the measurement result.

  The data extraction unit 32 (FIG. 1) extracts a specific optical path length change amount from a plurality of optical path length change amounts ΔOPL corresponding to a plurality of positions of the cell. In this embodiment, the data extraction unit 32 extracts a predetermined number of optical path length variation amounts from the larger one of the plurality of optical path length variation amounts.

  Here, the predetermined number can be a number corresponding to the type and cell state of the cell to be analyzed (identified). Further, the predetermined number can be a number obtained by multiplying the number of the plurality of optical path length variation amounts ΔOPL by a specific ratio. Furthermore, the predetermined number can be a number determined in advance by experiment or numerical calculation. A specific example of the operation in which the data extraction unit 32 sets a predetermined number will be described later [3. An example of the operation of the analysis means (data extraction and threshold setting)] will be described.

  The threshold value calculation unit 33 (FIG. 1) calculates a threshold value of the optical path length change amount used for identifying the cell state. The threshold value calculation unit 33 calculates a threshold value for the optical path length change amount based on a plurality of optical path length change amounts extracted by the data extraction unit 32 (for example, a predetermined number of optical path length change amounts of normal cells). The threshold calculation unit 33 can calculate, for example, an average value of the plurality of optical path length change amounts ΔOPL extracted by the data extraction unit 32 as a threshold.

  In this embodiment, the threshold calculation unit 33 calculates a threshold TH for identifying whether a cell is a normal cell (normal cell) or an abnormal cell (for example, a cancer cell). Moreover, the threshold value calculation unit 33 may further calculate a second threshold value TH2 for identifying whether the cancer cell is a metastatic cancer cell or a non-metastatic cancer cell as an abnormal cell. A specific example of the operation in which the threshold calculation unit 33 calculates (sets) the threshold will be described later [3. An example of the operation of the analysis means (data extraction and threshold setting)] will be described.

  The cell identification unit 34 (FIG. 1) identifies cells using the threshold value TH calculated by the threshold value calculation unit 33. That is, when identifying an abnormal cell, the cell identification unit 34 sets a cell having an optical path length variation ΔOPL equal to or less than the calculated threshold TH as a normal cell (normal cell), and an optical path length variation ΔOPL exceeding the threshold TH. The cell can be an abnormal cell (eg, a cancer cell).

  In addition, the cell identification unit 34 may identify a cell using the second threshold TH2 calculated by the threshold calculation unit 33. That is, when identifying a cancer cell as an abnormal cell (for example, Modification 2 or Example 3 of Example 1), the cell identification unit 34 exceeds the calculated threshold TH and is equal to or less than the second threshold TH2. A cancer cell having a change amount ΔOPL can be a metastatic cancer cell, and a cancer cell having an optical path length change amount ΔOPL exceeding the second threshold TH2 can be a non-metastatic cancer cell.

  The storage means 40 (FIG. 1) is means for storing information related to the cell identification device 100 (for example, operation conditions or operation results). The storage unit 40 can store, for example, a measurement result measured by the measurement unit 20 and an analysis result analyzed by the analysis unit 30. Here, the storage means 40 can use known techniques (hard disk, memory, ROM, RAM, etc.).

  The I / F unit 50 (FIG. 1) is a unit that inputs and outputs information (for example, electric signals) between the cell identification device 100 and the outside of the cell identification device 100. The I / F unit 50 can input information on the cell identification device 100 from an external device (such as a PC), for example. Further, the I / F unit 50 can output information about the cell identification device 100 to an external device (such as a PC).

  In the present embodiment, the I / F unit 50 is configured so that a user (diagnostic doctor, device operator, device manager, etc.) sends predetermined information (for example, measurement conditions, operation conditions, An input unit 51 for inputting output conditions and the like. Further, the I / F unit 50 includes an output unit 52 that outputs information to the outside of the cell identification device 100.

  In the present embodiment, the input unit 51 (FIG. 1) can be input with information regarding the cell to be identified, for example, information regarding the cell type, thickness, and other physical properties. The output unit 52 (FIG. 1) can output (for example, display) information related to the measurement result measured by the measurement unit 20 or the analysis result analyzed by the analysis unit 30.

  In addition, the operation | movement which measures the phase difference of another cell identification apparatus is shown in FIG.4 (b) and FIG.

  Other cell identification devices directly measure the phase difference from the intensity of the interference light. Specifically, as shown in FIG. 4 (b), other cell identification devices first measure the interference light expressed by Equation 3 (object light of FIG. 4 (b)) in order to measure the phase difference Δφ. The output light intensity I (x, y) of the interference light of Lt and reference light Lr is measured.

Here, _IO is the sum of the intensity (light intensity) of the object light Lt and the reference light Lr.

  Next, the other cell identification device substitutes the measured output light intensity I (x, y) into Equation 4 to calculate the phase difference Δφ (x, y). That is, other cell identification devices calculate the phase difference Δφ (x, y) using the inverse function arccos.

Next, other cell identification devices cause phase change wrapping when the calculated phase difference Δφ (x, y) has a phase difference of 2π or more (La in FIG. 6A). I do. Specifically, the other cell identification devices approximate (select) so as to be smoothly connected to neighboring values based on a plurality of candidate values (phase differences) calculated using Equation 4. Here, other cell identification devices can approximate the phase distribution line Lb shown in FIG. 6B when, for example, the phase difference La shown in FIG. 6A is calculated.

  For this reason, in other cell identification devices, since Equation 4 is a non-linear and multivalent function, the value of the phase difference Δφ may not be determined as a single value. In other cell identification devices, the accuracy may decrease at the connection part of the phase unwrapping process due to the influence of interference light noise or cell shape.

[2. Functions of cell identification device]
FIG. 7 shows a functional block diagram of the cell identification device 100 according to the embodiment of the present invention. In addition, the function of the cell identification apparatus 100 which can use this invention is not limited to what is shown in FIG.

  As shown in FIG. 7, in this embodiment, the cell identification device 100 (FIG. 1) is information (hereinafter referred to as “identification”) about the operation of the cell identification device 100 acquired (input) using the I / F means 50. Command ") is output to the control means 10 (block B01 in the figure). The cell identification device 100 according to the present embodiment receives an identification instruction using a sensor mounted on the measurement unit 20 or the input unit 51 (or the touch panel of the output unit 52) of the I / F unit 50. May be.

  The control means 10 controls the operation of the cell identification device 100 based on the input identification instruction (block B02). Specifically, the control unit 10 outputs a measurement instruction to the measurement unit 20 based on the identification instruction. Further, the control means 10 outputs an analysis instruction to the analysis means 30 based on the identification instruction. Note that the control unit 10 or the I / F unit 50 may further output an identification instruction or the like to the storage unit 40.

  The measuring means 20 measures the optical path length change amount ΔOPL of the cell based on the input measurement instruction (block B03 to block B06).

  Specifically, the measurement means 20 uses the feedback control unit 22 to feedback control the operation (block B04) of the optical system 21 based on the input measurement instruction (block B03). Further, the measuring means 20 (feedback control unit 22) outputs a detection instruction to the voltage detection unit 23. Furthermore, the measuring means 20 (optical system 21) outputs measurement data (for example, a light intensity image) to the optical path length conversion unit 24 (block B06 described later).

  Next, the measuring means 20 (voltage detection unit 23) detects an applied voltage applied to the piezo element PZT (FIG. 2) used for feedback control of the feedback control unit 22 in this embodiment (block B05). The measuring means 20 (voltage detection unit 23) outputs detection data (applied voltage or the like) to the optical path length conversion unit 24.

  Next, the measuring means 20 (optical path length conversion unit 24) converts the input detection data into an optical path length change amount ΔOPL of the cell (block B06). Further, the measuring means 20 (optical path length conversion unit 24) outputs the conversion data (optical path length change amount ΔOPL) to the data extraction unit 32. Further, the optical path length conversion unit 24 further outputs the converted data to the image generation unit 31.

  The analysis unit 30 identifies cells based on the input conversion data (optical path length change amount ΔOPL) (block B07 to block B10).

  Specifically, the analysis unit 30 uses the data extraction unit 32 to calculate a predetermined number of optical path length variation ΔOPL from the input conversion data (a plurality of optical path length variations ΔOPL at a plurality of positions of normal cells). Is extracted (block B07). The analysis unit 30 (data extraction unit 32) outputs the extracted data (a predetermined number of optical path length change amounts ΔOPL) to the threshold value calculation unit 33.

  Next, the analysis unit 30 (threshold value calculation unit 33) calculates a threshold value based on the input extracted data (block B08). The analysis unit 30 (threshold value calculation unit 33) outputs the calculated threshold value to the cell identification unit 34. A specific example of the operation in which the threshold calculation unit 33 calculates (sets) the threshold will be described later [3. An example of the operation of the analysis means (data extraction and threshold setting)] will be described.

  Next, the analysis unit 30 (cell identification unit 34) identifies a cell using the input threshold value and optical path length change amount ΔOPL (block B09). The analysis unit 30 (cell identification unit 34) outputs identification data (identification result) to the storage unit 40.

  The analysis unit 30 (image generation unit 31) uses the optical path length change amount ΔOPL (conversion data) to generate image data (an image showing the optical path length change amount ΔOPL, phase difference and / or refractive index difference distribution). Generate (block B10). Further, the analysis unit 30 (image generation unit 31) outputs the generated image data to the storage unit 40. The image generation unit 31 and the cell identification unit 34 may have a function of inputting / outputting the generated image data or identification data.

  The storage unit 40 stores the measurement result of the measurement unit 20, the analysis result of the analysis unit 30, information regarding the operation of the cell identification device 100, and the like (block B11). The storage unit 40 can output (display) stored information using the I / F unit 50 (for example, the output unit 52).

[3. Example of operation of analysis means (data extraction and threshold setting)]
The operation for extracting data and setting the threshold value by the cell identification device 100 (analyzing means 30) according to the embodiment of the present invention will be described with reference to FIGS. Here, FIG. 8 is a flowchart explaining an example of the operation of the analysis unit 30 of the cell identification device 100 according to the embodiment of the present invention. FIG. 9 is an explanatory diagram for explaining an example of the optical path length change amount ΔOPL analyzed by the analysis unit 30. FIG. 10 is an explanatory diagram illustrating an example of the feature amount extracted by the data extraction unit 32 of the analysis unit 30. FIG. 11 is an explanatory diagram for explaining an example of an error rate calculated by the threshold value calculation unit 33 of the analysis unit 30. FIG. 12 is an explanatory diagram for explaining an example of the feature amount (predetermined number) selected by the threshold calculation unit 33 of the analysis unit 30. FIG. 13 is an explanatory diagram for explaining another example of the feature amount (predetermined number) selected by the threshold calculation unit 33 of the analysis unit 30.

  As shown in FIG. 8, the cell identification device 100 first measures the optical path length variation ΔOPL using the measuring means 20 in step S801, and uses the image generation unit 31 to distribute the optical path length variation distribution image (OPDI). (Optical Path-length Difference Image), hereinafter referred to as “optical path length change amount image”).

  Specifically, the cell identification device 100 generates an optical path length change amount image Img1 for each of a plurality of samples (normal cells and abnormal cells) as shown in FIG. 9A. Here, the optical path length change amount image Img1 shown in FIG. 9A is an optical path length change amount image Img2 having a resolution of 100 pixels × 100 pixels as shown in FIG. 9B. In this embodiment, normal cells and abnormal cells (for example, cancer cells) identified in advance are used as a plurality of samples, and optical path length change amount images of normal cells and abnormal cells are generated.

  Thereafter, the cell identification device 100 proceeds to step S802.

  In step S <b> 802, the analysis unit 30 of the cell identification device 100 uses the data extraction unit 32 and the threshold calculation unit 33 to calculate the average value AOPD of the optical path length change amount.

Specifically, first, the data extraction unit 32, in order of 10000 pixels (100 pixels × 100 pixels) of the optical path length change amount image generated in step S801, in descending order of the value of the optical path length change amount as shown in FIG. Rearrange from X (1) to X (10000) in the figure. Next, the data extraction unit 32 extracts n _th (predetermined number) as feature amounts from the higher order (X (1) in the figure) of the rearranged optical path length variation values. Next, the threshold value calculation unit 33 calculates an average value AOPD of the plurality of extracted feature values (n _th optical path length change amounts (X _i )) using the following equation. That is, in this embodiment, a plurality of average values AOPD are calculated for normal cells and abnormal cells, respectively.

Here, the data extraction unit 32 (cell identification device 100) uses, as n _th (predetermined number), for example, the top 1% of 10000 pixels is 100, 2% is 200, 3% is 300, 4 A plurality of numbers are extracted from the top, such as 400, which is%, 500, which is 5%, 600 which is 6%, and the average value of each is calculated.

  Thereafter, the cell identification device 100 proceeds to step S803.

  In step S803, the analysis unit 30 calculates the error rate ER from the plurality of average values AOPD calculated in step S802 using the threshold value calculation unit 33.

  Here, the error rate ER is a value indicating the degree of overlap of the average value AOPD calculated for normal cells and abnormal cells. The error rate ER is 0 (hereinafter referred to as “equal error rate EER”) when the average value AOPD calculated for normal cells and the average value AOPD calculated for abnormal cells do not all overlap. The error rate ER is 1 when the average value AOPD calculated for normal cells and the average value AOPD calculated for abnormal cells all overlap.

In this embodiment, for example, as shown in FIG. 11, n _th (predetermined number) is 200 (2% from the top), 300 (3% from the top), 400 (4% from the top), and 500 The error rate ER was 0 (equal error rate EER) when (5% from the top).

  After that, the cell identification device 100 proceeds to step S804 when there is one in which the calculated error rate ER is zero. In other cases, the cell identification device 100 proceeds to step S806.

In step S804, the analysis unit 30 uses the threshold calculation unit 33 to calculate the threshold width Dth from the plurality of average values AOPD calculated in step S802, and n _th (predetermined number) that maximizes the threshold width Dth. Select.

  Here, the threshold width Dth refers to the value of the difference between the average value AOPD calculated for normal cells and the average value AOPD calculated for abnormal cells. The threshold width Dth is, for example, the difference between the maximum value of ΔOPL of normal cells and the minimum value of ΔOPL of abnormal cells (for example, cancer cells).

In the present embodiment, as shown in FIG. 11, 300 (3% from the top) threshold width Dth2, 400 (4% from the top) threshold width Dth3, and 500 (5% from the top) threshold width Dth4 The threshold width Dth1 when n _th (predetermined number) is 200 (2% from the top) is the maximum. That is, the threshold value calculation unit 33 of the analysis unit 30 selects 200 (2% from the top) as n _th (predetermined number).

  Thereafter, the cell identification device 100 proceeds to step S805.

  In step S <b> 805, the analysis unit 30 sets a threshold value using the threshold value calculation unit 33. Here, the threshold value calculation unit 33 can set an intermediate value of the maximum threshold value width Dth (Dth1 in FIG. 11) selected in step S804 as the threshold value. Note that the threshold value calculation unit 33 may use any value included in the threshold value width Dth selected in step S804 as the threshold value.

  Thereafter, the cell identification device 100 proceeds to END in the figure, and ends the operation of the cell identification device 100 (analysis means 30) extracting data and setting the threshold value.

On the other hand, in step S806, the analysis unit 30 uses the threshold value calculation unit 33 to identify the minimum error rate ER from the error rate ER calculated in step S803, and n corresponding to the identified minimum value error rate ER. Select _th (predetermined number).

FIG. 12 is an explanatory diagram for explaining an example of the number of feature amounts selected by the threshold value calculation unit 33 of the analysis unit 30. FIG. 13 is an explanatory diagram for explaining another example of the number of feature amounts selected by the threshold value calculation unit 33 of the analysis unit 30. Here, the horizontal axis of FIG. 12 and FIG. 13 is n _th values, and the vertical axis is the error rate ER value. Lc1 in FIG. 12 and Ld1 in FIG. 13 are rejection ratios of normal cells (ratio of normal cells having a minimum value of ΔOPL of abnormal cells). Lc2 in FIG. 12 and Ld2 in FIG. 13 are acceptance rates of abnormal cells (ratio of abnormal cells below the maximum value of ΔOPL of normal cells).

In FIG. 12, when n _th values are 1 to 600, the error rate ER of Lc3, which is the sum of Lc1 and Lc2, is minimized. Further, in FIG. _13, if _{n th} number of values of pieces 1 to 700, the error rate ER of Ld3 summation of Ld1 and Ld2 is minimized. That is, the threshold value calculation unit 33 of the analysis unit 30 can select 600 (FIG. 12) or 700 (FIG. 13) corresponding to the minimum error rate ER as n _th (predetermined number).

  Thereafter, the cell identification device 100 proceeds to step S807.

In step S807, the analysis unit 30 sets a threshold value using the threshold value calculation unit 33. Here, the threshold value calculation unit 33 can set the average value AOPD (step S802) calculated using n _th (predetermined number) corresponding to the minimum error rate ER selected in step S806 as the threshold value. .

  Thereafter, the cell identification device 100 proceeds to END in the figure, and ends the operation of the cell identification device 100 (analysis means 30) extracting data and setting the threshold value.

[4. Cell identification method program and recording medium recording the program]
According to the program Pr of the cell identification method according to the embodiment of the present invention, a measurement step of irradiating a plurality of cells with light and measuring a plurality of optical path length variations, and an optical path length variation of a normal cell. And a step of identifying each of the plurality of cells using the threshold value calculated in this way. The identifying step extracts a plurality of optical path length changes corresponding to a plurality of positions of the normal cells, calculates an average value of the extracted optical path length changes, and uses the calculated average value as the threshold value. A cell identification method characterized by the above may be executed. The identifying step extracts a plurality of optical path length changes corresponding to a plurality of positions of the normal cells, calculates a plurality of average values for the extracted optical path length changes, and calculates the plurality of average values The cell identification method characterized in that the threshold value is set based on an error rate. The measuring step includes a changing step of changing an optical path length of the light applied to the cell, and an optical path length of the light applied to the cell so as to be the same as the intensity of the light when not passing through the cell. A cell identification method characterized by including a feedback control step of changing the frequency. According to these configurations, an effect equivalent to that of the cell identification device 100 according to the embodiment of the present invention can be obtained.

  Further, the present invention may be a recording medium Md that can be read by a computer in which the program Pr is recorded. As the recording medium Md on which the program Pr is recorded, a flexible disk, a CD-ROM, a memory card, and other computer-readable media can be used.

  As described above, according to the cell identification device 100 according to the embodiment of the present invention, the optical path length measured when the cell TC is irradiated with light using the threshold value TH calculated based on the optical path length change amount of the normal cell. Based on the change amount ΔOPL, the cells can be identified. In addition, according to the cell identification device 100 according to the present embodiment, since the cell can be identified based on the optical path length change amount ΔOPL measured when the cell TC is irradiated with light, the phase difference of the cell is measured. The cells can be easily identified without performing complicated processing (for example, phase unwrapping processing or Fourier transform processing). Furthermore, according to the cell identification device 100 according to the present embodiment, since the cell can be identified based on the optical path length change amount ΔOPL measured when the cell TC is irradiated with light, the identification result is displayed. Thus, it is possible for a user (diagnostic doctor, etc.) to easily recognize abnormal cells such as cancer cells. That is, according to the cell identification device 100 according to the present embodiment, it is possible to identify cells with high accuracy based on the measured optical path length change amount ΔOPL.

  The present invention will be described using examples including the cell identification device 100 according to the embodiment.

[Example 1]
The present invention will be described using the cell identification device 100E according to Example 1 of the present invention.

[Configuration of cell identification device], [Function of cell identification device] and [Example of operation of analysis means]
A configuration and the like of a cell identification device 100E according to the present embodiment are shown in FIGS. Here, the configuration and the like of the cell identification device 100E are the same as the configuration and the like of the cell identification device 100 according to the embodiment, and thus description thereof is omitted.

In addition, as shown in Table 1, the specification of the cell identification device 100E according to this example is a non-contact (and non-destructive) measurement, a maximum measurement area of 100 × 100 μm ² , and a measurement wavelength (light source wavelength) of 632.8 nm. Laser, optical path length resolution 0.3 nm, measurement speed 1 to 100 μm / s, spatial resolution 0.97 μm (when a 20 × objective lens is used at a wavelength of 632.8 nm). The cell identification device 100E has a width of 320 mm, a depth of 180 mm, and a height of 290 mm, and is excellent in portability. Furthermore, the cell identification device 100E can reduce the influence of gravity on the cells to be identified by setting the optical path of the light applied to the cells in the vertical direction.

[Operation to analyze cell state]
The operation | movement which the cell identification apparatus 100E which concerns on a present Example analyzes a cell state (cell identification) is demonstrated using FIGS. 14-18. Note that the cell identification device 100E performs the operations of Step S1401 to Step S1404 in FIG. 14 described later on each cell in order to identify a plurality of cells.

  As shown in FIG. 14, in step S1401, the cell identification device 100E according to the present embodiment first performs a gap between the pair of glass plates of the optical system 21 (the glass plate 21gu and the glass plate in FIG. 3B). The cell TC to be identified is placed in a gap (with a gap of 21 gd). Thereafter, the cell identification device 100E proceeds to step S1402.

  In this embodiment, the cell identification device 100E arranges the cells TC by an adhesion type arrangement method. Specifically, as shown in FIG. 15, in the cell identification device 100E, first, the cell TC is adhered on the glass plate 21gd (FIG. 15 (a)). Next, in the cell identification device 100E, a spacer 21S (a shim ring having a thickness of 50 μm in this embodiment) is disposed on the glass plate 21gd (FIG. 15B). Next, in the cell identification device 100E, physiological saline Wn is dropped onto the glass plate 21gd in order to prevent the cell TC from drying (FIG. 15 (c)). Thereafter, in the cell identification device 100E, the glass plate 21gu is disposed on the spacer 21S (glass plate 21gd) (FIG. 15 (d)). Thereby, the cell identification apparatus 100E can arrange | position the cell TC by the adhesion type arrangement | positioning method (for example, FIG.3 (b)).

  Next, in step S1402, the cell identification device 100E uses the optical system 21 and the feedback control unit 22 (FIG. 1) of the measuring unit 20 to perform feedback control of the optical path length of light (object light) irradiated to the arranged cells. To do. The operation of feedback control by the cell identification device 100E according to the present example is the same as the operation of the cell identification device 100 according to the embodiment, and thus the description thereof is omitted. Thereafter, the cell identification device 100E proceeds to step S1403.

Next, in step S1403, the cell identification device 100E drives the mirror M _PZT when the feedback control unit 22 changes the optical path length using the voltage detection unit 23 (FIG. 1) of the measuring unit 20 (step S1402). The applied voltage V _PZT applied to the piezo element PZT (FIG. 2) is detected. Thereafter, the cell identification device 100E proceeds to step S1404.

In step S <b> 1404, the cell identification device 100 </ b> E uses the optical path length conversion unit 24 (FIG. 1) of the measuring unit 20 to apply the applied voltage V _PZT detected by the voltage detection unit 23 as in the cell identification device 100 according to the embodiment. (Step S1403) is converted into an optical path length change amount ΔOPL. Thereafter, the cell identification device 100E proceeds to step S1405.

  FIG. 17 shows an example of the optical path length change amount ΔOPL measured by the cell identification device 100E. Here, FIG. 17A shows the number of pixels Np (vertical axis) with respect to the optical path length variation ΔOPL (horizontal axis) in the case of normal cells. FIG. 17B is the number of pixels Np (vertical axis) with respect to the optical path length change amount ΔOPL (horizontal axis) in the case of metastatic cancer cells. FIG. 17C shows the number of pixels Np (vertical axis) with respect to the optical path length variation ΔOPL (horizontal axis) in the case of non-metastatic cancer cells. FIG. 17 shows a distribution (histogram) of 10,000 optical path length variations ΔOPL when the cells are measured on the xy plane (FIG. 3C). That is, FIG. 17 shows the result of measuring the optical path length variation ΔOPL corresponding to 1 pixel × 1 pixel in the range of 100 pixels × 100 pixels (10,000).

  Next, in step S1405, the cell identification device 100E generates an image based on the optical path length change amount ΔOPL converted by the optical path length conversion unit 24 using the image generation unit 31 (FIG. 1) of the analysis unit 30. . Thereafter, the cell identification device 100E proceeds to step S1406. Note that the cell identification device 100E may proceed to step S1406 without performing step S1405.

  Next, in step S1406, the cell identification device 100E uses the data extraction unit 32 (FIG. 1) of the analysis unit 30 to calculate a plurality of optical path length change amounts ΔOPL (steps S1401 to S1404) measured by the measurement unit 20. A specific optical path length change amount is extracted.

  Specifically, the cell identification device 100E (data extraction unit 32) determines a plurality of optical path length changes corresponding to a plurality of positions of normal cells in order to identify whether the cells are normal cells or cancer cells. A predetermined number of optical path length variation amounts are extracted from the amount ΔOPL. Here, in this embodiment, the cell identification device 100E (data extraction unit 32) determines a predetermined number (for example, from the larger value of the plurality of optical path length change amounts ΔOPL (FIG. 17A) of normal cells (for example, It is possible to extract the optical path length change amount of the top 5% (500 pieces). The operation in which the data extraction unit 32 extracts a predetermined number of optical path length variation amounts is described in [3. This is the same as the operation (step S802 and step S803 in FIG. 8) described in the example of the operation of the analysis unit (data extraction and threshold setting), and thus the description thereof is omitted.

  Thereafter, the cell identification device 100E proceeds to step S1407.

  In step S1407, the cell identification device 100E identifies the cell based on the predetermined number of optical path length variation ΔOPL extracted by the data extraction unit 32 using the threshold value calculation unit 33 (FIG. 1) of the analysis unit 30. Calculate the threshold. Specifically, in this embodiment, the cell identification device 100E (threshold value calculation unit 33) can calculate an average value of a plurality of optical path length change amounts ΔOPL extracted by the data extraction unit 32 as the threshold value TH. The operation in which the threshold calculation unit 33 calculates (sets) the threshold is described in [3. This is the same as the operation (step S804 and step S805 in FIG. 8) described in the example of the operation of the analysis unit (data extraction and threshold setting), and thus the description thereof is omitted.

  Thereafter, the cell identification device 100E proceeds to step S1408.

  Specifically, the cell identification device 100E (image generation unit 31) can generate an optical path length variation distribution image (optical path length variation image) in this embodiment. That is, the cell identification device 100E (image generation unit 31) can generate an image that emphasizes normal cells or cancer cells.

  FIG. 16 shows a measurement result (phase difference image) of the cell identification apparatus 100E (for example, a phase contrast microscope apparatus (IX71, DP71 (Olympus Corporation)) according to the present embodiment and an analysis result (optical path) of the cell identification apparatus 100E. An example of a long variation image) is shown. Here, FIG. 16A is a phase difference image of a mammary gland (normal cell). FIG. 16B is an optical path length change amount image of the mammary gland (normal cells). FIG. 16C is a phase difference image of breast cancer cells (abnormal cells, cancer cells). FIG. 16D is an optical path length change amount image of breast cancer cells (abnormal cells, cancer cells). FIG. 16E is a phase difference image of skin cells (normal cells). FIG. 16F is an optical path length change amount image of skin cells (normal cells). FIG. 16G is a phase difference image of skin cancer cells (abnormal cells, cancer cells). FIG. 16 (h) is an optical path length change amount image of skin cancer cells (abnormal cells, cancer cells).

  The cell identification device 100E (image generation unit 31) may generate an optical path length change amount image (for example, FIGS. 16B, 16D, 16F, and 11H) that emphasizes normal cells or cancer cells. it can. For example, as shown in FIG. 16A and FIG. 16C, the image generation unit 31 has an optical path length change amount ΔOPL even when normal cells and cancer cells are approximately the same size and difficult to identify. By emphasizing the high value part (FIG. 16D), normal cells and breast cancer cells can be easily distinguished. Further, as shown in FIGS. 16 (e) and 16 (g), for example, the image generation unit 31 emphasizes a portion where the optical path length variation ΔOPL is high even in the case of skin cancer cells that are difficult to identify by shape. By doing (FIG. 16 (h)), normal cells and skin cancer cells can be easily identified.

  Thus, the cell identification device 100E displays the generated image on the display unit (the output unit 52 in FIG. 1), thereby allowing a user (diagnostic doctor or the like) who has viewed the display unit to easily recognize cancer cells. it can. In addition, the cell identification device 100E (image generation unit 31) can clearly and quantitatively confirm the difference between normal cells and cancer cells by allowing the user to check the distribution of the optical path length variation image. .

  In step S1408, the cell identification device 100E identifies a plurality of cells based on the threshold value TH calculated by the threshold value calculation unit 33 using the cell identification unit 34 (FIG. 1) of the analysis unit 30.

  Specifically, in this embodiment, the cell identification device 100E (cell identification unit 34), for example, as shown in FIG. 18, when the optical path length change amount 245nm is calculated as the threshold TH (step S1407), It is possible to distinguish between normal cells Cr and cancer cells Cc of a plurality of cells with TH as a boundary. Here, the horizontal axis of FIG. 18 represents the optical path length change amount ΔOPL. Moreover, the vertical axis | shaft of FIG. 18 is the number Ns of applicable cells.

  Similar to the operations in step S1406 and step S1407, the cell identification device 100E (cell identification unit 34) has a predetermined number (for example, the present embodiment) in the plurality of optical path length change amounts ΔOPL at a plurality of cell positions. In the example, the optical path length variation amount of the top 5%) is extracted, and the average value of the extracted predetermined number of optical path length variation amounts ΔOPL can be used as the optical path length variation amount ΔOPL of the cell. The predetermined number can be the same as the predetermined number when the threshold is calculated.

  Thereafter, the cell identification device 100E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  As described above, according to the cell identification device 100E according to Example 1 of the present invention, the same effects as those of the cell identification device 100 according to the embodiment can be obtained.

[Modification 1 of Example 1]
The present invention will be described using the cell identification device 110E according to the first modification of the first embodiment of the present invention.

[Configuration of cell identification device], [Function of cell identification device] and [Example of operation of analysis means]
The configuration and the like of the cell identification device 110E according to this modification are shown in FIG.

  As shown in FIG. 1 and the like, the configuration and the like of the cell identification device 110E according to the present modification are the same as the configuration and the like of the cell identification device 100E according to Example 1 described above, and thus the description thereof is omitted.

[Operation to analyze cell state]
The operation | movement which the cell identification apparatus 110E which concerns on this modification analyzes a cell state (cell identification) is demonstrated using FIGS. 14-17 and FIGS. 19-24. Here, the horizontal axis of FIGS. 19 to 24 represents the optical path length change amount ΔOPL. Moreover, the vertical axis | shaft of FIGS. 19-24 is the number Ns of applicable cells. The operation of analyzing the cell state by the cell identification device 110E according to the present modification is basically the same as the operation of the cell identification device 100E according to the above-described first embodiment, and thus different parts will be mainly described.

As illustrated in FIG. 14, the cell identification device 110E according to this modification example uses the optical path length conversion unit 24 of the measurement unit 20 and the like in steps S1401 to S1404 in the same manner as the cell identification device 100E according to the first embodiment. The applied voltage V _PZT detected by the voltage detector 23 is converted into an optical path length change amount ΔOPL. Thereafter, the cell identification device 110E proceeds to step S1405.

  In step S1405, the cell identification device 110E generates an image based on the optical path length change amount ΔOPL converted by the optical path length conversion unit 24 using the image generation unit 31 (FIG. 1) of the analysis unit 30. Thereafter, the cell identification device 110E proceeds to step S1406. Note that the cell identification device 110E may proceed to step S1406 without performing step S1405.

  In step S1406, in this modification, the cell identification device 110E uses a data extraction unit 32 of the analysis unit 30 or the like to group a plurality of optical path length variation amounts from a plurality of optical path length variation amounts ΔOPL measured by the measuring unit 20. To extract.

  Specifically, the cell identification device 110E (data extraction unit 32) includes a plurality of optical path length change amounts ΔOPL (FIG. 17A) of normal cells in order to increase the accuracy of the threshold value to be calculated (step S1407). A group of optical path length change amounts (for example, the top 1%, 2%, 3%, 4%, 5%, and 6%) of a plurality of ratios is extracted from the larger value of. The cell identification device 110E (data extraction unit 32) is configured as a group of optical path length change amounts at a plurality of ratios corresponding to, for example, upper 1%, 2%, 3%, 4%, 5%, and 6% as shown in FIG. 1 (for 1%), FIG. 20 (for the top 2%), FIG. 21 (for the top 3%), FIG. 22 (for the top 4%), FIG. 23 (for the top 5%), FIG. 6%) can be extracted.

  Thereafter, the cell identification device 110E proceeds to step S1407.

  In step S1407, the cell identification device 110E calculates a plurality of threshold regions corresponding to a group of optical path length change amounts at a plurality of ratios, using the threshold calculation unit 33 (FIG. 1) of the analysis unit 30.

  Specifically, in this modification, the cell identification device 110E (threshold value calculation unit 33) first changes the optical path length at a ratio corresponding to the top 1%, 2%, 3%, 4%, 5%, and 6%. As threshold regions corresponding to groups of amounts, threshold regions Rth2, Rth3, Rth4, and Rth5 are calculated as shown in FIGS. In the upper 1% and 6% cases in FIGS. 19 and 24, there is no threshold region. Next, the cell identification device 110E (threshold value calculation unit 33) selects, from the calculated threshold value region Rth2 and the like, the threshold value of the optical path length change amount corresponding to the threshold value region having the largest absolute value as the threshold value TH for identifying the cell. (calculate. Here, the cell identification device 110E (threshold value calculation unit 33) can select a threshold value of 2% or 3% (for example, an intermediate value of the threshold region Rth2 or Rth3) as the threshold value TH.

  Thereafter, the cell identification device 110E proceeds to step S1408.

  The other operations of the cell identification device 110E according to the present modification are basically the same as the operations of the cell identification device 100E according to Example 1 described above, and thus the description thereof is omitted.

  As described above, according to the cell identification device 110E according to the first modification of the first embodiment of the present invention, the same effects as those of the cell identification device 100E according to the first embodiment can be obtained. In addition, according to the cell identification device 110E according to the present modification, a plurality of threshold regions corresponding to a group of optical path length change amounts at a plurality of ratios can be calculated using the threshold calculation unit 33. The threshold value can be calculated (selected) by using a group of optical path length variation amounts corresponding to the threshold region having a wide area in the threshold region. Furthermore, according to the cell identification device 110E according to the present modification, it is possible to select a threshold corresponding to a threshold region having a wide threshold region from a plurality of thresholds corresponding to a group of optical path length change amounts at a plurality of ratios. Therefore, it is possible to improve the accuracy of identifying the cell using the selected threshold value.

[Modification 2 of Embodiment 1]
This invention is demonstrated using the cell identification apparatus 120E which concerns on the modification 2 of Example 1 of this invention.

[Configuration of cell identification device], [Function of cell identification device] and [Example of operation of analysis means]
A configuration or the like of a cell identification device 120E according to this modification is shown in FIG.

  As shown in FIG. 1 and the like, the configuration and the like of the cell identification device 120E according to this modification are the same as the configuration and the like of the cell identification device 100E according to Example 1 described above, and thus the description thereof is omitted.

[Operation to analyze cell state]
The operation | movement which the cell identification apparatus 120E which concerns on this modification analyzes a cell state (cell identification) is demonstrated using FIGS. 14-17 and FIG. The operation of analyzing the cell state by the cell identification device 120E according to the present modification is basically the same as the operation of the cell identification device 100E according to Example 1 described above, and therefore, different parts will be mainly described.

As illustrated in FIG. 14, the cell identification device 120E according to the present modification example uses the optical path length conversion unit 24 of the measuring unit 20 in steps S1401 to S1404 in the same manner as the cell identification device 100E according to the first embodiment. The applied voltage V _PZT detected by the voltage detector 23 is converted into an optical path length change amount ΔOPL. Thereafter, the cell identification device 120E proceeds to step S1405.

  In step S1405, the cell identification device 120E generates an image based on the optical path length change ΔOPL converted by the optical path length conversion unit 24 using the image generation unit 31 (FIG. 1) of the analysis unit 30. Thereafter, the cell identification device 120E proceeds to step S1406. Note that the cell identification device 120E may proceed to step S1406 without performing step S1405.

  Next, in step S1406, the cell identification device 120E uses a plurality of optical path length change amounts ΔOPL (steps S1401 to S1404) measured by the measurement unit 20 using the data extraction unit 32 (FIG. 1) of the analysis unit 30. A specific optical path length change amount is extracted.

  Specifically, in the present modification, the cell identification device 120E (data extraction unit 32) uses a normal cell in order to identify whether the cancer cell is a metastatic cancer cell or a non-metastatic cancer cell. Similarly to the case, a predetermined number of optical path length variation amounts are further extracted from a plurality of optical path length variation amounts ΔOPL (FIG. 17B) corresponding to a plurality of positions of metastatic cancer cells.

  Thereafter, the cell identification device 120E proceeds to step S1407.

  In step S1407, the cell identification device 120E identifies a cell based on the predetermined number of optical path length variation ΔOPL extracted by the data extraction unit 32 using the threshold value calculation unit 33 (FIG. 1) of the analysis unit 30. A threshold value and a second threshold value are calculated. Specifically, in this modification, the cell identification device 120E (threshold value calculation unit 33) calculates, as the threshold value TH, an average value of a plurality of optical path length change amounts ΔOPL of normal cells extracted by the data extraction unit 32. Can do. Further, the cell identification device 120E (threshold value calculation unit 33) can calculate an average value of the plurality of optical path length change amounts ΔOPL of the metastatic cancer cells extracted by the data extraction unit 32 as the second threshold value TH2.

  Thereafter, the cell identification device 120E proceeds to step S1408.

  In step S1408, the cell identification device 120E identifies a plurality of cells using the threshold TH calculated by the threshold calculation unit 33 and the second threshold TH2 using the cell identification unit 34 (FIG. 1) of the analysis unit 30. To do. Thereafter, the cell identification device 120E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  FIG. 25 specifically shows an operation in which the cell identification device 120E (cell identification unit 34) according to the present modification identifies a plurality of cells using the threshold value TH and the second threshold value TH2.

  As shown in FIG. 25, in the present modification, the cell identification device 120E (cell identification unit 34) determines whether or not the optical path length variation ΔOPL of the identified cell is equal to or less than a threshold value TH in step S2501. If the optical path length variation ΔOPL of the cell to be identified is equal to or less than the threshold value TH, the cell identification device 120E (cell identification unit 34) proceeds to step S2502. In other cases, the cell identification device 120E (cell identification unit 34) proceeds to step S2503.

  In step S2502, the cell identification device 120E (cell identification unit 34) identifies the cell as a normal cell. Thereafter, the cell identification device 120E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  In step S2503, the cell identification device 120E (cell identification unit 34) determines whether or not the optical path length change amount ΔOPL of the cell to be identified is equal to or less than the second threshold value TH2. When the optical path length variation ΔOPL of the cell to be identified is equal to or smaller than the second threshold TH2, the cell identification device 120E (cell identification unit 34) proceeds to step S2504. In other cases, the cell identification device 120E (cell identification unit 34) proceeds to step S2505.

  In step S2504, the cell identification device 120E (cell identification unit 34) identifies the cell as a metastatic cancer cell. Thereafter, the cell identification device 120E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  On the other hand, in step S2505, the cell identification device 120E (cell identification unit 34) identifies the cell as a non-metastatic cancer cell. Thereafter, the cell identification device 120E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  As described above, according to the cell identification device 120E according to the second modification of the first embodiment of the present invention, the same effects as those of the cell identification device 100E according to the first embodiment can be obtained. In addition, according to the cell identification device 120E according to the present modification, the second threshold value TH2 can be calculated using the threshold value calculation unit 33, and thus the cell is calculated using the calculated threshold value TH and the second threshold value TH2. Can be identified as normal cells, metastatic cancer cells or non-metastatic cancer cells. Further, according to the cell identification device 120E according to the present modification, the cells are normal cells, metastatic cancer cells, or non-metastatic cancer cells using the threshold TH and the second threshold TH2 calculated by the threshold calculation unit 33. Since it can be identified, the user (diagnostic doctor, etc.) can easily allow the user to recognize cells (for example, metastatic cancer cells or non-metastatic cancer cells) that cannot be determined with the naked eye. Further, according to the cell identification device 120E according to this modification, the user can clearly and quantitatively confirm the difference between the normal cell and the cancer cell by confirming the distribution of the optical path length variation image. it can.

[Example 2]
The present invention will be described using a cell identification device 200E according to Example 2 of the present invention.

[Configuration of cell identification device]
The configuration and the like of the cell identification device 200E according to the present embodiment are shown in FIGS. 1 and 26 and FIG. Since the configuration and the like of the cell identification device 200E are basically the same as the configuration of the cell identification device 100 according to the embodiment, different portions will be mainly described.

  The cell identification device 200E according to the present embodiment measures phase information (and amplitude) of a cell to be identified using a digital holography technique.

  Specifically, as shown in FIG. 26, the cell identification device 200E generates an interference fringe of the Fresnel diffracted light Ld of the cell (Sample) and the reference light Lr by the beam splitter BS, and forms an image as a hologram (interference fringe image). Obtained with sensor CCD. Next, the cell identification device 200E performs image reproduction of the acquired hologram using a computer PC or the like. Thereby, the cell identification device 200E can acquire cell phase information (and amplitude).

  Note that the optical system 21 and the like of the cell identification device 200E are not limited to those shown in FIG. That is, the cell identification device 200E can use a configuration that can measure phase information of a cell to be identified using a known technique related to digital holography.

[Operation to analyze cell state]
The operation of the cell identification device 200E according to the present embodiment analyzing the cell state (identifying the cell) will be described with reference to FIGS.

  As shown in FIG. 27, the cell identification device 200E according to the present embodiment first, in step S2701, by the user, a gap between a pair of glass plates of the optical system 21 (for example, the glass plate 21gu and the glass plate 21gu in FIG. A cell TC to be identified is arranged in a gap with the plate 21gd. Thereafter, the cell identification device 200E proceeds to step S2702.

  Next, in step S2702, the cell identification device 200E acquires the hologram (interference fringe image) of the Fresnel diffracted light Ld of the cell and the reference light Lr using the optical system (FIG. 26) by the image sensor CCD. Thereafter, the cell identification device 200E proceeds to step S2703.

  Next, in step S2703, the cell identification device 200E acquires (calculates) phase information (and amplitude) of the cell based on the acquired hologram using the digital holography technique. Further, the cell identification device 200E generates a phase difference image using the acquired phase information. The cell identification device 200E may calculate the optical path length change amount ΔOPL by substituting the acquired phase information into Equation 2, and generate an optical path length change amount image using the calculated optical path length change amount ΔOPL. .

  Thereafter, the cell identification device 200E proceeds to step S2704.

  FIG. 28 shows an example of a phase difference image generated by the cell identification device 200E according to the present embodiment. Here, FIG. 28A is a phase difference image of a plurality of cells in a 4 mm × 4 mm region. FIG. 28B is an enlarged view in which one cell Rb in the region of FIG. 28A is extracted and enlarged.

  The cell identification device 200E according to the present embodiment can simultaneously image a plurality of cells and acquire phase information of the plurality of cells in one operation using digital holography technology. For this reason, when there are a plurality of cells to be identified, the cell identification device 200E according to the present embodiment can reduce the time required for the operation of acquiring the cell phase information (optical path length change amount ΔOPL).

  Next, in step S2704, the cell identification device 200E extracts an image corresponding to the cell to be identified from the generated image (phase difference image or optical path length change amount image) (FIG. 28 (b)). Thereafter, the cell identification device 200E proceeds to step S2705.

  In step S2705, the cell identification device 200E uses the threshold TH (and the second threshold TH2) calculated by the threshold calculation unit 33 using the cell identification unit 34 (FIG. 1) of the analysis unit 30. Identify Thereafter, the cell identification device 200E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  Here, the cell identification device 200E is similar to the cell identification device 100 according to the embodiment or the cell identification device 100E according to Example 1 (for example, step S1408 in FIG. 14), and the optical path length change amount of the cell in the extracted image. Cells can be identified based on ΔOPL (or phase difference).

  As described above, according to the cell identification device 200E according to the second embodiment of the present invention, it is possible to acquire phase information (or optical path length variation ΔOPL) of a plurality of cells in one operation using the digital holography technology. Therefore, it is possible to shorten the time required for the operation of acquiring the phase information (optical path length change amount ΔOPL) of a plurality of cells. Further, according to the cell identification device 200E according to the present embodiment, the phase information (optical path length change amount ΔOPL) of a plurality of cells can be acquired by one operation, and thus the present invention is used for, for example, a CTC examination. In some cases, the time required for the inspection can be shortened. That is, according to the cell identification device 200E according to the present embodiment, when several to several tens of circulating cancer cells (CTC) are detected per 10 ml of blood, a large number of cells are identified in a short time. Therefore, it has a particularly advantageous effect in the CTC examination.

  Moreover, according to the cell identification device 200E according to Example 2 of the present invention, the same effects as those of the cell identification device 100 according to the embodiment can be obtained.

[Example 3]
The present invention will be described using a cell identification device 300E according to Example 3 of the present invention.

[Configuration of cell identification device], [Function of cell identification device] and [Example of operation of analysis means]
The configuration and the like of the cell identification device 300E according to the present example are shown in FIG. The configuration of the cell identification device 300E is basically the same as the configuration of the cell identification device 100 according to the embodiment, and a description thereof will be omitted. In this embodiment, the cell identification device 300E identifies white blood cells as normal cells, leukemia cells and breast cancer cells (non-metastatic breast cancer cells, metastatic breast cancer cells) as abnormal cells.

[Operation to analyze cell state]
An operation in which the cell identification device 300E according to the present embodiment analyzes a cell state (identifies a cell) will be described with reference to FIGS. 14 and 29 to 37. FIG.

  As shown in FIG. 14, in the cell identification device 300E according to the present embodiment, first, in step S1401, the user places a cell TC to be identified in the gap between the pair of glass plates of the optical system 21. Thereafter, the cell identification device 300E proceeds to step S1402.

  In this embodiment, the cell identification device 300E places an isolation cell TC. Specifically, as shown in FIG. 29, the cell identification device 300E first drops a cell TC floating in the liquid Ln (FIG. 29A) onto the glass plate 21gd using the pipette Pp. (FIG. 29B). Next, in the cell identification device 300E, a spacer 21S (a shim ring having a thickness of 50 μm in this embodiment) is disposed on the glass plate 21gd (FIG. 29C). Next, in the cell identification device 300E, the glass plate 21gu is disposed on the spacer 21S (glass plate 21gd) (FIG. 29 (d)). Thereby, the cell identification apparatus 300E can arrange | position the cell TC of an isolation system (FIG. 30).

  Next, in steps S1402 to S1404, the cell identification device 300E measures the optical path length change amount ΔOPL in the same manner as the operation of the cell identification device 100E according to the first embodiment. Thereafter, similarly to the operation of the cell identification device 100E according to the first embodiment, the cell identification device 300E proceeds to step S1405 or step S1406, and then proceeds to step S1407. The operation in which the data extraction unit 32 extracts a predetermined number of optical path length variation amounts is described in [3. This is the same as the operation described in “Example of operation of analysis unit (data extraction and threshold setting)”.

In step S1407, the cell identification device 300E identifies a cell based on the predetermined number of optical path length variation ΔOPL extracted by the data extraction unit 32 using the threshold value calculation unit 33 (FIG. 1) of the analysis unit 30. Calculate the threshold. Specifically, in this embodiment, the cell identification device 300E (threshold calculation unit 33) selects n _th (predetermined number) corresponding to the minimum error rate ER, and selects the selected n _th (predetermined number). ) Is set as a threshold value (steps S806 and S807 in FIG. 8).

  Here, in this embodiment, the cell identification device 300E (threshold value calculation unit 33) is the normal cell extracted by the data extraction unit 32 as the threshold value TH for identifying normal cells (white blood cells) and abnormal cells (leukemia cells, etc.). The average value of the plurality of optical path length variations ΔOPL is calculated, and the average value of the plurality of optical path length variations ΔOPL of the leukemia cells is further calculated as a second threshold TH2 for distinguishing between leukemia cells and breast cancer cells.

  Thereafter, the cell identification device 300E proceeds to step S1408.

  FIG. 32 shows a measurement result (phase difference image) of the cell identification device 300E (for example, a phase contrast microscope device (IX71, DP71 (Olympus Corporation)) and the like and an analysis result (optical path) of the cell identification device 300E according to the present embodiment. An example of a long variation image) is shown. Here, FIG. 32A is a phase difference image of normal cells (white blood cells). FIG. 32B is an optical path length change amount image of normal cells (white blood cells). FIG. 32C is a phase difference image of an abnormal cell (leukemia cell). FIG. 32 (d) is an optical path length variation image of abnormal cells (leukemia cells). FIG. 32 (e) is a phase difference image of abnormal cells (non-metastatic breast cancer cells). FIG. 32F is an optical path length change amount image of abnormal cells (non-metastatic breast cancer cells). FIG. 32 (g) is a phase difference image of abnormal cells (metastatic breast cancer cells). FIG. 32 (h) is an optical path length change amount image of abnormal cells (metastatic breast cancer cells).

  The cell identification device 300E (image generation unit 31) uses the optical path length change amount ΔOPL to emphasize an optical path length change amount image (for example, FIGS. 32B, 32D, 32F, and 32F) that emphasizes normal cells or abnormal cells. (H)) is generated. For example, as shown in FIGS. 32 (a) and 32 (c), the image generation unit 31 has the optical path length variation ΔOPL even when the normal cells and the abnormal cells are approximately the same size and difficult to identify. By emphasizing the high value portion (FIG. 32 (d)), normal cells (white blood cells) and abnormal cells (leukemia cells) can be easily distinguished. In addition, as shown in FIGS. 32 (e) and 32 (g), for example, the image generation unit 31 has a high optical path length change amount ΔOPL for non-metastatic breast cancer cells and metastatic breast cancer cells that are difficult to identify by shape. It can be easily identified by emphasizing the value portion (FIG. 32 (f)).

  In step S1408, the cell identification device 300E identifies a plurality of cells based on the threshold value TH and the second threshold value TH2 calculated by the threshold value calculation unit 33 using the cell identification unit 34 (FIG. 1) of the analysis unit 30. To do. Thereafter, the cell identification device 300E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  FIG. 31 specifically shows an operation in which the cell identification device 300E (cell identification unit 34) according to the present embodiment identifies a plurality of cells using the threshold value TH and the second threshold value TH2.

  As shown in FIG. 31, in step S3101, the cell identification device 300E (cell identification unit 34) determines whether or not the optical path length change amount ΔOPL of the cell to be identified is equal to or less than a threshold value TH. If the optical path length variation ΔOPL of the cell to be identified is equal to or less than the threshold value TH, the cell identification device 300E proceeds to step S3102. In other cases, the cell identification device 300E proceeds to step S3103.

  In step S3102, the cell identification device 300E identifies the cell as a normal cell (white blood cell). Thereafter, the cell identification device 300E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  On the other hand, in step S3103, the cell identification device 300E (cell identification unit 34) determines whether or not the optical path length variation ΔOPL of the cell to be identified is equal to or less than the second threshold value TH2. If the optical path length variation ΔOPL of the cell to be identified is equal to or smaller than the second threshold value TH2, the cell identification device 300E proceeds to step S3104. In other cases, the cell identification device 300E proceeds to step S3105.

  In step S3104, the cell identification device 300E (cell identification unit 34) identifies the cell as an abnormal cell (leukemia cell). Thereafter, the cell identification device 300E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell).

  In step S3105, the cell identification device 300E (cell identification unit 34) identifies the cells as abnormal cells (breast cancer cells). Thereafter, the cell identification device 300E proceeds to END in the figure, and ends the operation of analyzing the cell state (identifying the cell). Note that the cell identification device 300E further uses a third threshold value for identifying non-metastatic breast cancer cells and metastatic breast cancer cells in the breast cancer cells to identify the non-metastatic breast cancer cells and the metastatic breast cancer cells. Good.

  As described above, according to the cell identification device 300E according to the third embodiment of the present invention, the same effects as those of the cell identification device 100E according to the first embodiment can be obtained. In addition, according to the cell identification device 300E according to the present embodiment, using the calculated threshold value TH and the second threshold value TH2, the cells are normal cells (white blood cells) or abnormal cells (leukemia cells, non-metastatic breast cancer cells, Metastatic breast cancer cells). Furthermore, according to the cell identification device 300E according to the present embodiment, it is possible to identify whether a cell is a normal cell or an abnormal cell using the threshold value TH and the second threshold value TH2. ) Makes it easy for users to recognize cells that cannot be judged with the naked eye, and the difference between normal and abnormal cells can be clearly and quantitatively confirmed by checking the distribution of the optical path length variation image. .

  Further, according to the cell identification device 300E according to the third embodiment of the present invention, the phase information (optical path length variation ΔOPL) of a plurality of isolated cells TC can be acquired. The time required for the inspection can be shortened when using. That is, according to the cell identification device 300E according to the present embodiment, when several to several tens of circulating cancer cells (CTC) are detected per 10 ml of blood, a large number of cells can be identified in a short time. Therefore, it has a particularly advantageous effect in the CTC examination.

[Experimental result]
33 to 37, optical paths measured by the cell identification device 300E according to the present example for normal cells (20 samples of white blood cells Cr) and abnormal cells (15 samples of leukemia cells Le and 5 samples of breast cancer cells Bc). The experimental result of long variation | change_quantity (DELTA) OPL is shown. Here, FIG. 33 shows an experimental result of the average optical path length variation ΔOPL in the top 2%. FIG. 34 shows the experimental results of the average optical path length variation ΔOPL in the top 4%. FIG. 35 shows the experimental results of the average optical path length variation ΔOPL in the top 6%. FIG. 36 shows the experimental results of the average optical path length variation ΔOPL in the top 8%. FIG. 37 shows the experimental results of the average optical path length variation ΔOPL in the top 10%.

  As shown in FIG. 33 (upper 2%), the cell identification device 300E sets the threshold value (the optical path length variation ΔOPL on the horizontal axis) to a value within the range of 160 to 200 nm, thereby enabling the white blood cell Cr (normal cell). And leukemia cells Le (abnormal cells). In FIG. 34 (upper 4%) to FIG. 37 (upper 10%), it is difficult for the cell identification device 300E (analyzing means 30) to set a threshold value. That is, it is difficult for the cell identification device 300E (analysis means 30) to distinguish between normal cells and abnormal cells.

  As shown in FIG. 37 (top 10%), the cell identification device 300E sets the second threshold value (the optical path length change amount ΔOPL on the horizontal axis) to a value of 270 nm, whereby leukemia cells Le (abnormal cells) and breast cancer Cells Bc (abnormal cells) can be distinguished. In FIG. 33 (upper 2%) to FIG. 36 (upper 8%), it is difficult for the cell identification device 300E (analyzing means 30) to set the second threshold. That is, it is difficult for the cell identification device 300E (analysis means 30) to identify the type of abnormal cell (leukemia cell Le, breast cancer cell Bc).

  In addition to the above-described cell identification device, the present invention measures the optical path length change amount by irradiating an object with light in, for example, industrial, medical, bio, agricultural, security or information communication / electronics, and the measured optical path length It can be used for an object that analyzes (identifies, etc.) the properties of an object using a change amount.

100, 100E, 110E, 120E, 200E, 300E: Cell identification device 10: Control unit 20: Measuring unit 21: Optical system 22: Feedback control unit 23: Voltage detection unit 24: Optical path length conversion unit 30: Analysis unit 31: Image Generation unit 32: Data extraction unit 33: Threshold calculation unit 34: Cell identification unit 40: Storage unit 50: I / F unit 51: Input unit 52: Output unit (display unit, etc.)
Pr: Program Md: Storage medium TH: Threshold value TH2: Second threshold value Rth2 to Rth5: Threshold area

Claims (10)

A cell identification device that identifies whether the cell is a normal cell or an abnormal cell by using an optical path length change amount measured when the cell is irradiated with light,
Measuring means for measuring the amount of change in the optical path length based on the intensity of the light when transmitted through the cell;
Using a threshold value calculated based on the amount of change in the optical path length of a normal cell, and an analysis means for identifying whether the cell is a normal cell or an abnormal cell,
The analysis means extracts a plurality of optical path length changes corresponding to a plurality of positions for each of normal cells and abnormal cells in a plurality of groups, and each of the plurality of groups of normal cells and abnormal cells. When the average value of the optical path length change amount is calculated, and there is no overlap between the plurality of average values calculated for the normal cells and the plurality of average values calculated for the abnormal cells, the average value of the abnormal cells and Select the group having the largest difference in average value of the normal cells, determine an average value of the average value of the abnormal cells and the average value of the normal cells of the selected group as the threshold value, and calculate the normal cells The average of the normal cells of the group with the smallest error rate when there is an overlap between the average values calculated and the average values calculated for the abnormal cells Cells identification apparatus and determines as the threshold value.   2. The analysis unit according to claim 1, wherein the cell having the optical path length change amount equal to or less than the threshold is a normal cell, and the cell having the optical path length change amount exceeding the threshold is an abnormal cell. 2. The cell identification device according to 1. The analysis means uses a second threshold value calculated based on a plurality of optical path length changes corresponding to a plurality of positions of metastatic cancer cells of the cancer cell, and exceeds the threshold value and is equal to or less than the second threshold value. of the optical path length change amount in cancer cells and metastatic cancer cells, the optical path length change amount of cancer cells beyond the threshold of the second and non-metastatic cancer cells, characterized in that, according to claim 2 Cell identification device. Said analyzing means, said extracts a normal multiple optical path length change amount from the larger values of the predetermined number of the optical path length change amount of the cells, characterized in that, any of claims 1 to 3 The cell identification device according to claim 1. The measurement means changes the optical path length of the light applied to the cell by applying a voltage to the piezo element so as to be the same as the intensity of the light that does not pass through the cell, and changes the applied voltage to the applied voltage. The cell identification device according to any one of claims 1 to 4 , wherein the optical path length change amount is measured based on the measurement result. An image generation unit that generates an image corresponding to the outer shape and refractive index difference or phase difference of the cell based on the measured change amount of the optical path length, and an output unit that displays the generated image. The cell identification device according to any one of claims 1 to 5 , wherein the cell identification device is characterized. A measurement step of irradiating a plurality of cells with light and measuring a plurality of optical path length changes,
An identification step for identifying whether each of the plurality of cells is a normal cell or an abnormal cell using a threshold value calculated based on an optical path length change amount of a normal cell, and
The identifying step extracts a plurality of optical path length change amounts corresponding to a plurality of positions for each of the normal cells and the abnormal cells in a plurality of groups, and each of the plurality of groups of the normal cells and the abnormal cells. When the average value of the optical path length change amount is calculated, and there is no overlap between the plurality of average values calculated for the normal cells and the plurality of average values calculated for the abnormal cells, the average value of the abnormal cells and Select the group having the largest difference in average value of the normal cells, determine an average value of the average value of the abnormal cells and the average value of the normal cells of the selected group as the threshold value, and calculate the normal cells When there is an overlap between a plurality of average values calculated and a plurality of average values calculated for the abnormal cells, the normal cells of the group with the smallest error rate Cells identification method which comprises the step of determining the average value as the threshold value. The measuring step includes a changing step of changing an optical path length of the light applied to the cell, and an optical path length of the light applied to the cell so as to be the same as the intensity of the light when not passing through the cell. The cell identification method according to claim 7 , further comprising: a feedback control step of changing. The program for making a computer perform the cell identification method of Claim 7 or 8 . A computer-readable recording medium on which the program according to claim 9 is recorded.