JP5682124B2 - Biological substance measuring device, biological substance measuring method and in vivo circulating tumor cell measuring method - Google Patents

Description

  The present invention relates to a biological material measuring apparatus, a biological material measuring method, and an in vivo circulating tumor cell measuring method, for example, acquired by an image element.

  In the medical field, in particular, when patients are treated with anticancer drugs, it is required to obtain information on the therapeutic effects in the patient's body in order to minimize the side effects caused by the drugs and to obtain sufficient therapeutic effects. ing. Conventionally, as a most general method, blood is collected from a patient's vein, the blood concentration of a tumor marker is measured, and the follow-up method is analyzed by analyzing the data. However, there are many exceptions, such as when the tumor marker value is rising despite the absence of cancer, or when the tumor marker value does not increase despite the presence of cancer. Does not reflect movement. Therefore, there is a need for a method that can accurately grasp the effects of anticancer drug treatment and the like. In addition, knowing circulating tumor cells in the body plays an important role not only in the therapeutic field but also in fields such as early detection of cancer.

JP-T-2002-503814 Special table 2009-525468 gazette

ADAMS Andre A. Et al., “Highly efficient circulating tumor cell separation and whole label counting from whole blood using microfluidics based on polymers with built-in conductivity sensors”, (USA), J Am Chem Soc. 2008 July 9; 130 (27): 8633-8641. doi: 10.1021 / ja801502 HE Wei. Et al., “In vivo quantification of rare circulating tumor cells by multiphoton biological flow cytometry,” (USA), PNAS July 10, 2007 vol. 104 no. 28 11760-11765

However, none of the techniques disclosed in Patent Documents 1 and 2 and Non-Patent Documents 1 and 2 satisfy the above requirements.
In the method of Patent Document 1, it is necessary to puncture a human body with a puncture device and collect blood as a sample when measuring. That is, the method of Patent Document 1 cannot measure circulating tumor cells non-invasively. Similarly, in the method of Patent Document 2, it is necessary to collect blood as a sample. In the device of Patent Document 3, it is necessary to collect blood as a sample. Sampling errors due to blood sampling in the order of 5 liters from a blood volume of 5 liters in adults, and biological materials resulting from processing taken out of the body The accuracy was low due to errors caused by denaturation. Non-Patent Document 2 has been proposed as a means for solving the precision degradation due to such sampling, but there is a problem in practicality due to a large-scale apparatus called a two-photon microscope.

  In addition, when a blood sample collected from the human body is measured outside the body, it is called in vitro measurement, and when the measurement is performed without taking the blood in the living body outside the body, it is called in vivo (in vivo) called measurement.

  The present invention has been made to solve at least a part of the above-described problems, and is a non-invasive biological material measuring apparatus and biological material measuring device that can accurately and quickly measure biological materials such as circulating tumor cells in a living body. In order to provide a method and a method for measuring circulating tumor cells in vivo, it can be realized as the following forms or application examples.

[Application Example 1] A biological material measuring apparatus according to this application example is an apparatus for detecting an object by a fluorescent labeling agent that binds to the object,
A light source for irradiating the measurement target with light for exciting the fluorescent labeling agent;
Light emitted from the fluorescent labeling agent when the fluorescent labeling agent is irradiated with the light is incident, and transmits light in a predetermined wavelength range of the entire wavelength range of incident light. A tunable filter that can be changed,
A light detection means for detecting the intensity of light incident through the wavelength tunable filter and obtaining intensity distribution of light emitted from a plurality of positions of the measurement target;
Concentration calculating means for calculating the concentration at which the fluorescent labeling agent is present based on the intensity distribution of light at the plurality of positions of the measurement object acquired by the light detecting means;
A binding degree calculating means for calculating a binding degree to which the fluorescent labeling agent is bound, based on light intensity distribution at a plurality of positions of the measurement target acquired by the light detecting means;
It is provided with.
According to the biological material measuring apparatus described in this application example, the concentration of the fluorescent labeling agent and the degree of binding between the fluorescent labeling agent and the biological material that is the binding target can be calculated, and the biological material such as circulating tumor cells can be detected. It can be performed with high accuracy.

[Application Example 2] In the biological material measurement apparatus according to the application example described above, light having a wavelength of 600 nm to 1200 nm is applied to a wavelength range of a light source that irradiates light of the light detection unit and a wavelength range of a wavelength tunable filter. It is characterized by using.
According to this application example, since it penetrates the living body efficiently, it can be accurately measured from outside the living body without invading the living body.

Application Example 3 In the biological material measurement apparatus according to the application example, the binding degree calculation unit calculates a binding degree using a calculation result obtained by the concentration calculation unit as mask data.
According to this application example, only the coupled portion can be extracted to remove noise, and measurement can be performed with high accuracy.

Application Example 4 In addition, the biological material measuring apparatus according to the application example described above is characterized in that a vector calculation is performed on the calculation result obtained by the concentration calculation unit and the coupling degree obtained by the coupling degree calculation unit.
According to this application example, noise can be removed by vector addition similar to additive color mixing, and measurement can be performed with high accuracy.

Application Example 5 A biological material measurement method according to this application example is a biological material measurement method for detecting an object with a fluorescent labeling agent that binds to the object,
A light irradiation step of irradiating the measurement target with light for exciting the fluorescent labeling agent;
Detecting the intensity of light in a specific wavelength range emitted from a plurality of positions of the measurement target among the light emitted from the fluorescent labeling agent when the fluorescent labeling agent is irradiated with the light, and the wavelength An intensity distribution acquisition step of acquiring an intensity distribution of light emitted from a plurality of positions of the measurement target by repeating the detection of the intensity a plurality of times while changing the area;
A concentration calculating step for calculating the concentration of the fluorescent labeling agent based on the intensity distribution of light at a plurality of positions of the measurement object;
A degree-of-binding calculation step of calculating the degree of binding of the fluorescent labeling agent based on the intensity distribution of light at a plurality of positions of the measurement object;
It is provided with.
According to the biological material measurement method described in this application example, the concentration and binding degree of the fluorescent labeling agent can be calculated, and the biological material between the fluorescent labeling agent and the biological substance to be bound can be detected with high accuracy. Can do.

Application Example 5 In addition, the in-vivo circulating tumor cell measurement method according to this application example is an in-vivo circulating tumor cell measurement method that detects an in-vivo target with a fluorescent labeling agent that binds to the target. ,
An administration step of administering the fluorescent labeling agent to a living body;
A light irradiation step of irradiating the living body to be measured with light that excites the fluorescent labeling agent;
Among the light emitted from the fluorescent labeling agent to the outside of the body when the fluorescent labeling agent is irradiated with the light from outside the living body, the intensity of light in a specific wavelength range emitted from a plurality of positions of the measurement target Intensity distribution acquisition step of acquiring intensity distribution of light emitted from a plurality of positions of the measurement object by repeating the detection of the intensity a plurality of times while changing the wavelength range, and
A concentration calculating step for calculating the concentration of the fluorescent labeling agent based on the intensity distribution of light at a plurality of positions of the measurement object;
A degree-of-binding calculation step of calculating the degree of binding of the fluorescent labeling agent based on the intensity distribution of light at a plurality of positions of the measurement object;
It is provided with.
According to the in vivo circulating tumor cell measurement method described in this application example, the concentration and the degree of binding can be calculated, and the circulating tumor cells can be detected in vivo with high accuracy.

It is a block diagram of the fluorescence distribution measuring apparatus of the embodiment of the present invention. It is a perspective view which shows the image of the use condition of the fluorescence distribution measuring apparatus in vivo. It is a perspective view shown in the state which decomposed | disassembled the wavelength variable filter (liquid crystal filter) of the 1st structural example. It is sectional drawing of one liquid crystal cell which comprises a liquid crystal filter. It is a graph which shows the relationship between the voltage applied to each liquid crystal cell, and a transmission peak wavelength. It is a figure which shows the spectral characteristic of a liquid crystal filter. It is a graph which shows the relationship between the fluorescence intensity and the detection wavelength which calculates | requires a density | concentration of a labeling agent. It is a graph which shows the relationship between the fluorescence intensity of a labeling agent, the density | concentration, and the detection wavelength which calculates | requires a bond degree. It is explanatory drawing which shows the image layer which operates image data. It is a flowchart which shows the procedure which calculates binding degree distribution from the fluorescence intensity distribution of a labeling agent. It is a flowchart which shows another procedure which calculates binding degree distribution from the fluorescence intensity distribution of a labeling agent. It is a figure explaining the detection in vitro. It is a figure explaining the detection in vitro. It is a perspective view of a small-sized embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The biological material measuring apparatus of the present embodiment is a circulating tumor cell measuring apparatus (hereinafter simply referred to as “CTC measuring apparatus”), which is a CTC (in vivo) in which a labeling agent that binds to circulating tumor cells is administered as a biological substance. It is an example of the apparatus which measures a circulating tumor cell: Circulating Tumor Cell). The subject to be measured may be a human or an animal such as a rat or a mouse that is often used in pharmacological experiments, but in the present embodiment, description will be made assuming a human (patient).
FIG. 1 is a block diagram showing a schematic configuration of a circulating tumor cell measuring apparatus according to the present embodiment.

[Device configuration]
As shown in FIG. 1, a CTC measuring apparatus 1 of this embodiment includes a light source 3, a wavelength tunable filter 4, an area sensor 5 (light detection means), and a signal processor (Digital Signal Processor, DSP) 6 (concentration). A calculation means), an image memory 7, and a band-pass filter 8. The light source 3 irradiates light (excitation light) to the living body 100 that is a measurement target. The wavelength tunable filter 4 receives light emitted from the labeling agent when the labeling agent in the living body 100 is irradiated with light, and transmits light in a specific wavelength region in the entire wavelength region of incident light. . The area sensor 5 detects the intensity of the radiated light incident through the wavelength tunable filter 4. The DSP 6 calculates the concentration and the degree of binding of the labeling agent based on the light intensity distribution at the plurality of positions of the measurement target acquired by the area sensor 5. The image memory 7 stores image data obtained by the DSP 6 and stores data after image processing.

  Prior to the measurement of CTC, the labeling agent is administered to the human body by the drug administration device 2 (administration step). The labeling agent has a function of specifically binding to an object such as a ligand or an antibody and a fluorescence function. That is, the labeling agent is composed of a fluorescent agent and a binder. Of course, the fluorescent agent and the binder may be the same. The object (CTC) is detected by capturing the object (CTC) and emitting fluorescence there. In order to obtain information in the living body 100 using light, it is necessary to use light having a wavelength other than the wavelength absorbed by the components of the living body 100. Light having a wavelength of about 600 nm or less is absorbed by hemoglobin, and light having a wavelength of about 1500 nm or more is absorbed by water. Therefore, light in these wavelength ranges cannot be used, and light having a wavelength of about 600 to 1500 nm is a living tissue. It penetrates relatively well. From this viewpoint, a labeling agent having a characteristic of receiving light in a wavelength region of about 600 to 1500 nm and emitting light in this wavelength region can be used for this measurement. Furthermore, when a silicon light receiving element such as a Si photodiode is used as the light receiving element, since the upper limit of the wavelength range of sensitivity of the light receiving element is about 1200 nm, light in this wavelength range is received by receiving light in a wavelength range of about 600 nm to 1200 nm. It is desirable to perform this measurement using a labeling agent that releases.

  Specifically, indocyanine green, fluorescent contrast agent SF64 (trade name, manufactured by Fuji Film) or the like can be used as the fluorescent agent. Indocyanine green is a substance that is excited by irradiation with light having a wavelength of 750 to 780 nm and emits near infrared light having a wavelength of about 800 to 1200 nm. Examples of substances that can replace indocyanine green include patent blue and indigo carmine. The fluorescent contrast agent SF64 has a characteristic of generating strong fluorescence in the near infrared region. The antibody or ligand that specifically binds is appropriately selected according to the target cancer cell.

  The labeling agent can be administered by intradermal injection, subcutaneous injection, intramuscular injection, (peripheral infusion) intravenous injection, central intravenous injection, rectal administration, transdermal administration, pulmonary administration, nasal administration, buccal administration. Any of invasive and non-invasive methods may be used. More specifically, for example, in the case of intravenous injection using the drip method, a drug administration micropump capable of adjusting the dose and the rate of administration of the drug can be used.

  The light source 3 may be a single-wavelength light source such as a laser. Here, an example in which a xenon lamp, for example, that selects a wavelength from a wide range is used will be described. Light having a continuous spectral spectrum from the visible region (blue region) to the infrared region is emitted from the light source 3 of the xenon lamp, and irradiated to the human body that is the measurement target (light irradiation step). At this time, since the light passes through the bandpass filter 8, when light having a part of the light from the light source 3 passes through the human body and is irradiated onto the labeling agent, fluorescence is emitted from the labeling agent. . For example, when indocyanine green is used as the fluorescent agent, the fluorescent agent is excited by light having a wavelength of 750 to 780 nm out of the light emitted from the light source 3, and has a wavelength of about 800 to 1200 nm depending on the concentration of the fluorescent agent. Near infrared light is emitted. Indocyanine green has the property of being excited by near infrared light and emitting near infrared light.

  The wavelength tunable filter 4 may be a device that allows a part of wavelengths such as a grating and an etalon to pass through, but here, an example using a liquid crystal cell will be described. Although the detailed configuration will be described later, it is composed of a plurality of sets of liquid crystal cells including a pair of electrodes for applying a voltage to the liquid crystal layer. In the case of the present invention, in the area sensor 5 described later, it is necessary to detect the light amounts at a plurality of wavelengths and not to detect the light amount at a certain wavelength, and to acquire the spectrum of the emitted light. A wavelength tunable filter that can vary the transmission wavelength region on the incident side is required. The wavelength variable filter 4 functions as such a wavelength variable filter. Some configuration examples of the wavelength tunable filter 4 will be described later.

  As the area sensor 5, a two-dimensional imaging sensor composed of a CCD, a CMOS sensor or the like is used. In general, an imaging sensor such as a CCD or CMOS sensor has a sensitivity region including near-infrared light having a wavelength of about 800 to 1200 nm. Since light is incident on the area sensor 5 through the wavelength tunable filter 4, only light in a predetermined wavelength band is received at a certain time, but the wavelength sensor has a different wavelength band according to the change in the transmission wavelength range of the wavelength tunable filter 4. Light is received, and the light intensity distribution (spectrum) for each selected wavelength can be obtained. The area sensor 5 transmits the intensity of the received near-infrared light as an electrical signal to the DSP 6 described later. In this example, a two-dimensional imaging sensor is used, but a one-dimensional imaging sensor, a so-called linear sensor may be used. In that case, for example, a linear sensor may be scanned over the measurement region to detect the light intensity in the two-dimensional measurement region.

Further, although not shown in FIG. 1, an optical filter that blocks light unnecessary for the main concentration distribution measurement within the sensitivity region of the area sensor 5, for example, light having a wavelength of 700 nm or less, is arranged on the incident side of the area sensor 5. You may do it. As this type of optical filter, an ultraviolet / visible light cut filter R-72 (trade name: manufactured by Edmund) can be used.
The DSP 6 receives the electrical signal from the area sensor 5 and calculates the concentration and binding degree of the labeling agent based on the light intensity distribution at each point on the measurement target. Thus, by calculating the concentration of the labeling agent and the degree of binding at each point on the measurement target, the drug concentration distribution in the living body 100 can be known as a whole. As shown in FIG. 2, the measurement data of the obtained drug concentration distribution is sent from the DSP 6 to an arbitrary output device 9 such as a liquid crystal monitor, and the magnitude of the object concentration is expressed by the color or its density. Users such as doctors and nurses can visually confirm.

[Wavelength tunable filter]
Here, a first configuration example of the wavelength tunable filter 4 will be described with reference to FIGS.
FIG. 3 is a perspective view showing the wavelength tunable filter 4 of this configuration example in an exploded state. FIG. 4 is a cross-sectional view of one liquid crystal cell constituting the wavelength tunable filter 4. FIG. 5 is a graph showing the relationship between the voltage applied to each liquid crystal cell and the transmission peak wavelength. FIG. 6 is a diagram showing the spectral characteristics of the wavelength tunable filter 4.
The specific numerical values such as the voltage applied to the liquid crystal cell and the transmission wavelength shown below are based on the simulation results performed by the present inventors.

  As shown in FIGS. 3 and 4, the wavelength tunable filter 4 of this configuration example is disposed on a pair of glass substrates 12 and 13 bonded together with a sealant 11 and on the outer surfaces of the pair of glass substrates 12 and 13. A pair of polarizing plates 14 and 15, a liquid crystal layer 16 sandwiched between the pair of glass substrates 12 and 13, and a pair of electrodes that are disposed on the inner surfaces of the pair of glass substrates 12 and 13 and apply a voltage to the liquid crystal layer 16. The liquid crystal cells 21a, 21b, and 21c each having the electrodes 17 and 18 and the alignment films 19 and 20 on the electrodes 17 and 18 are laminated. However, for the polarizing plate located between adjacent pairs of liquid crystal cells, one polarizing plate is shared by these two liquid crystal cells. In the following description, for the sake of convenience, in FIG. 3, they are referred to as “first liquid crystal cell 21a”, “second liquid crystal cell 21b”, and “third liquid crystal cell 21c” from the upper side to the lower side of the drawing. .

  In the first to third liquid crystal cells 21a, 21b, and 21c, the pair of glass substrates 12 and 13 are parallel to each other and have opposite orientation directions, and exhibit so-called antiparallel orientation. As a result, the liquid crystal molecules constituting the liquid crystal layer 16 of each liquid crystal cell 21a, 21b, 21c are in a homogeneous alignment state. In addition, the pair of polarizing plates 14 and 15 disposed with the pair of glass substrates 12 and 13 interposed therebetween have their transmission axes parallel to each other, and the direction of the transmission axis is 45 ° with respect to the orientation direction of one substrate. They are arranged at an angle of ± 5 °. As the polarizing plates 14 and 15, an iodine-based polarizing film is not preferable because it does not have a polarizing property in the near infrared region, and a laminated reflective polarizer or a wire grid in which retardation layers and isotropic layers are alternately stacked. It is preferable to use a type reflective polarizer. Further, a drive circuit 22 for applying a voltage to these electrodes 17 and 18 is connected between the pair of electrodes 17 and 18 of each liquid crystal cell 21a, 21b and 21c.

  Over all of the first to third liquid crystal cells 21a, 21b, and 21c, the value of the optical anisotropy Δn with respect to light having a wavelength of 590 nm of the liquid crystal layer 16 of each liquid crystal cell is set to 0.201. On the other hand, the layer thickness of the liquid crystal layer 16 (cell gap between the pair of glass substrates 12 and 13) is different in each of the first to third liquid crystal cells 21a, 21b, and 21c, and the liquid crystal layer thickness of the first liquid crystal cell 21a is 6. The liquid crystal layer thickness of the second liquid crystal cell 21b is 12.9 μm, and the liquid crystal layer thickness of the third liquid crystal cell 21c is 25.8 μm. In the wavelength tunable filter 4 of this configuration example, 15 levels of spectral spectra are selected in the wavelength range of 830 to 1098 nm. Therefore, the DSP 6 controls the drive circuit 22 so as to supply any one of the 15 levels of applied voltages to the liquid crystal cells 21 a, 21 b, and 21 c of the wavelength tunable filter 4. The DSP 6 applies substantially the same voltage to the first to third liquid crystal cells 21a, 21b, and 21c, but corrects a minute voltage with reference to correction data stored in advance in the memory.

  When capturing the intensity of light emitted from the measurement object as image data, first, the applied voltage to the first to third liquid crystal cells 21a, 21b, and 21c is set to 1.3V. Then, the wavelength tunable filter 4 exhibits a transmission characteristic having a peak at 910 nm, transmits light with a wavelength of 910 nm, and this light enters the arrayed pixel surface of the area sensor 5. Here, the area sensor 5 sequentially detects the intensity of incident light for each pixel, reads the entire image for one screen in 30 msec, converts it into an electrical signal, and outputs it to the DSP 6. The DSP 6 stores image data for one screen at a wavelength of 910 nm in the image memory 7. Thus, the selected image data capturing process in the first cycle is completed.

  Next, the applied voltage to the first to third liquid crystal cells 21a, 21b, and 21c is set to 1.21V. Then, the wavelength tunable filter 4 exhibits a transmission characteristic having a peak at 980 nm, and light having a wavelength of 980 nm enters the area sensor 5. The area sensor 5 reads an image for one screen in 30 msec, converts it into an electrical signal, and then outputs it to the DSP 6. The DSP 6 stores image data for one screen at a wavelength of 980 nm in the image memory 7. Thus, the selected image data capturing process in the second cycle is completed.

  Next, the applied voltage to the first to third liquid crystal cells 21a, 21b, and 21c is set to 1.10V. Then, the wavelength tunable filter 4 exhibits a transmission characteristic having a peak at 1057 nm, and light having a wavelength of 1057 nm enters the area sensor 5. The area sensor 5 reads an image for one screen in 30 msec, converts it into an electrical signal, and then outputs it to the DSP 6. The DSP 6 stores image data for one screen at a wavelength of 1057 nm in the image memory 7. Thus, the selected image data capturing process in the third cycle is completed.

  In this embodiment, the three wavelengths of 910 nm, 1980 nm, and 1057 nm are taken in. However, as shown in Table 1, the applied voltage to the first to third liquid crystal cells 21a, 21b, and 21c is 1.10V. 1.15V, 1.18V, 1.21V, 1.23V, 1.26V, 1.28V, 1.30V, 1.32V, 1.34V, 1.35V, 1.37V, 1.39V (last In this cycle, only the first liquid crystal cell 21a is corrected to 1.42 V). Then, the transmission peak wavelength changes to 1057 nm, 1018 nm, 999 nm, 980 nm, 962 nm, 944 nm, 927 nm, 910 nm, 893 nm, 877 nm, 861 nm, 845 nm, and 830 nm according to this voltage change. The method of selecting these first to third wavelengths is appropriately selected according to the wavelength-intensity characteristic of the fluorescence wavelength of the labeling agent and the absorption wavelength of the binding portion. Here, one-wavelength excitation and two-wavelength fluorescence detection (also referred to as a ratio method) is performed at the first wavelength and the second wavelength, and wavelength absorption indicating a binding state with the CTC is detected at the third wavelength. This will be described in detail in a CTC detection method described later (intensity distribution acquisition step).

  FIG. 5 is a graph showing the data in Table 1. That is, FIG. 5 is a graph showing the relationship between the voltage applied to each of the liquid crystal cells 21a, 21b, and 21c and the transmission peak wavelength. The vertical axis represents the applied voltage (V), and the horizontal axis represents the transmission peak wavelength (nm). Show. FIG. 6 shows the spectral characteristics of the wavelength tunable filter 4 based on these transmission peak wavelength values. That is, according to this configuration example, it is possible to realize a wavelength tunable filter having a spectral wavelength characteristic shown in FIG. 6 and having a wavelength range of 800 to 1100 nm. In the spectrum of FIG. 6, the wavelength component of 700 nm or less becomes a noise component that is unnecessary for this concentration distribution measurement. Therefore, it is desirable to remove it using the above-described optical filter.

[CTC detection method]
Next, the CTC detection method by the DSP 6 will be described in detail.
An analysis method (one-wavelength excitation and two-wavelength fluorescence detection) based on calculation of the ratio of fluorescence intensity with two wavelengths of light is known and is not essential, but it is desirable to combine with this ratio method also in this embodiment. In the embodiment in combination with the ratio method, the ratio of the image data by the first wavelength and the second wavelength is calculated. FIG. 7 shows the relationship between the fluorescence intensity and the detection wavelengths λ1 and λ2. (Details of this technique called the ratio method are omitted because they are described in “RB. Silver, Methods Cell Biol, 2003, 72, 369”.) This calculation can cancel the difference in measurement conditions. That is, excitation light power, fluorescent dye concentration, etc. are cancelled. The first wavelength and the second wavelength need not overlap with the third wavelength for detecting the absorption wavelength shift due to the coupling.

  The DSP 6 reads out the image data at the same position detected by the first wavelength and the second wavelength stored in the image memory 7, performs division, and stores the calculation result in the image memory 7. Furthermore, based on the intensity ratio of the fluorescence at the first wavelength and the second wavelength, it may be calculated so that the predetermined intensity ratio is the maximum concentration. In this calculation, the concentration of the labeling agent is obtained by comparing the light intensity distribution of each pixel constituting the image data with the correlation between the light intensity and the labeling agent concentration measured in advance. This calculation result is information for detecting the concentration of the labeling agent. (This calculation result is hereinafter referred to as a density layer.) This density layer is position data (X, Y) and array data of the concentration of the labeling agent there. The data word length (concentration resolution) may be 8 bits, 10 bits, or 64 bits. The correlation between the light intensity and the wavelength is obtained by a preliminary experiment, and a reference data table is created. For example, if the light intensity of the first wavelength is 1, the light intensity of the second wavelength is 0.9, the light intensity of the uncoupled state of the third wavelength is 0.8, and the coupled state of the third wavelength A correlation reference data table (Table 2) having a light intensity of 0.5 is prepared in advance.

  Various algorithms for such processing can be considered, but the following calculation is given as an example for explanation as the simplest calculation. FIG. 9 illustrates and describes the intensity pixel data and the image layers (D0 (i, j) to D5 (i, j)) based on the data calculated based on the intensity pixel data. That is, the data represented by the array corresponds to each image layer.

  Let D1 (i, j) be pixel data indicating the detection intensity of the image sensor measured by the first wavelength. This array indicates that the array data is assigned to the first wavelength. This arrangement means image data stored in the image memory 7 and detected by the first wavelength. i indicates the position in the horizontal direction (X-axis), and j indicates the position in the vertical direction (Y-axis). Next, let D2 (i, j) be pixel data of the image sensor measured by the second wavelength. The ratio (ratio) of the fluorescence intensity of the first wavelength and the second wavelength, which is known in advance, is r and stored in a part of the reference data table. From this r, the density data D0 (i, j) is obtained by the following equation (1). In an embodiment, r from Table 2 is 0.9. The closest match is 0, and the pixel data of the non-matching portion has a negative value.

  Next, the third wavelength data is read, and the ratio of the third wavelength is calculated from the data of the first wavelength or the second wavelength. If the calculation capability is high, the accuracy may be increased by calculating both the first wavelength data and the second wavelength data. In this calculation, the light intensity distribution of each pixel constituting the image data is collated with the correlation between the light intensity and the binding state in the data table to obtain the binding state of the labeling agent. Generally, since fluorescence of a normal labeling agent is weakened when it is taken up by a cancer cell or the like, this weakness is stored in the reference data table at the third wavelength. The ratio of the portion where the dotted concave curve and the solid curve in FIG. 8 intersect with the two-dot chain line of λ3. (Alternatively, when the fluorescence of the labeling agent increases as it binds to the cancer cells, the intensity of the labeling agent is stored in the reference data table. Although it becomes a dashed line of a convex curve at λ3, it is not shown. The calculation result is information for detecting the binding state of the labeling agent. This calculation result is hereinafter referred to as a connectivity layer D5 (i, j).

Similar to the density processing algorithm, various calculations can be considered here, but the simplest calculation is as follows.
Let D3 (i, j) be pixel data of the image sensor measured by the third wavelength. D3 (i, j) indicates that the array data is assigned to the third wavelength.
A density threshold value Dth of the density data D0 (i, j) is determined, and mapping data of 1 is created if the array data D4 (i, j) exceeds the threshold value Dth, and 0 if it falls below. When expressed by a conditional statement in a general programming language, the processing is as shown in the following equation (2).

In this way, binary mapping data of D4 (i, j) indicating whether or not the labeling agent is present there is completed. Using this binarized mapping data as a mask condition, the combined state at each pixel is then calculated.

  The calculation method of the combined state is as shown in the following equation (3). The fluorescence intensity ratio (r_bind) that distinguishes the binding state is measured in advance. The fluorescence intensity ratio (r_bind) is defined as a value obtained by dividing the average Iλ3_mid of the fluorescence intensity Iλ3_on when the labeling agent is bound and the fluorescence intensity Iλ3_off when the labeling agent is not bound by the fluorescence intensity Iλ1 of the first wavelength.

In the present embodiment, when this ratio (r_bind) is calculated from the intensity ratio of the reference data table, the following equation (4) is obtained.

Using this ratio (r_bind), the following equation (5) is calculated.

The target may be only the coordinates (i, j) where the labeling agent exists in the binarized mapping data D4 (i, j). The procedure so far is shown as a flowchart in FIG.

  Since the density layer D0 (i, j) and the binding degree layer D5 (i, j) thus obtained indicate the presence and binding state of the labeling agent, the labeling agent bound to the CTC has two It can be extracted with high accuracy by layering. The method of overlapping the two layers is selected as appropriate, such as logical sum, logical product, and vector sum. Here, the method has been described in which the connectivity layer is also obtained by the logical product overlap based on the binarized mapping data. Another way of superimposing visual images is additive color mixing and vector summation. Next, the method of overlaying with additive color mixing on the display will be described in detail.

  In Table 3, the coupling degree layer does not include a procedure based on the mask condition of the density layer, and is configured as a condition in which each is independently parallel. It is obtained from the flowchart shown in FIG. That is, these layers in the previous embodiment, for example, on the display, if the density layer is expressed in green and the coupling layer is expressed in red, the two high-level places are yellow. It can be easily read that it is a CTC. This is an example of processing that is easy for humans to recognize, but when processing with a computer, by calculating the vector sum of the pixels of two layers and extracting data that exceeds a certain threshold, The presence of CTC can be automatically recognized by a computer.

  FIG. 12 shows an explanatory diagram thereof. In the green density layer, all of the labeling agent is visible, so the cells to be detected are buried in the noise. In the Red coupling degree layer, a portion causing a predetermined intensity change (λ3 / λ1) is extracted in order to detect the coupling degree. Only the information of the connectivity layer includes a portion without a labeling agent in which the intensity change happens to be the value. In the Yellow layer, a portion where a labeling agent is present and a predetermined intensity change is extracted, and an object such as a desired cell can be accurately detected. Parts that cause noise in the green density layer and red connectivity layer can be eliminated by superposition.

  Although FIG. 12 has been described with an image of a preparation under a microscope for cell counting, in vivo detection is performed using a capillary or the like called microcirculation as shown in FIG. Target cells are flowing in a cylindrical blood vessel. Unlabeled labeling agent also flows into the blood. In other words, the density layer of Green is calculated from the density data of λ1 and λ2, and the presence portion of the labeling agent is detected. (This means the ratio method as described above.) Next, in the Red coupling degree layer, a portion causing a predetermined intensity change (λ3 / λ1) is extracted in order to detect the coupling degree. The part without the labeling agent whose intensity change coincides with that value is also mixed. Here, a noise component that happens to have the same intensity change as the labeling agent in the coupling degree layer is indicated by a cross mark. In vivo, similar to the method described so far, only the portion of the bound labeling agent can be extracted by superimposing the concentration layer and the binding degree layer, and even rare cells such as CTC can be detected with high accuracy. .

By performing image processing in this way, the number of CTCs can be counted with high accuracy, and information on the therapeutic effect in the patient's body can be obtained.
In the CTC measuring apparatus 1 of the present embodiment, the CTC is not calculated only by the intensity value of light of a specific wavelength, but the label is coupled from the intensity distribution of light in a predetermined wavelength band such as 910, 980, 1057 nm. Therefore, the CTC can be calculated with high accuracy. Thus, according to the CTC measuring apparatus 1 of this embodiment, CTC can be measured accurately and rapidly by detecting the emitted light from the living body 100 by a non-invasive method without collecting blood or the like.

  In this example, an example in which three wavelengths of λ1 to λ3 are used is shown, but multiple wavelengths such as four wavelengths and five wavelengths may be used for higher accuracy. In addition, if the labeling agent concentration of a specific part (such as the position of a blood vessel) can be estimated in advance, two wavelengths by the ratio method are not required, so the labeling concentration is calculated at one wavelength (λ1 = λ2) and the binding of λ3 Variations of the embodiment that only require degree detection and two wavelengths are also possible.

  A medical technique called therapeutic drag monitoring (TDM) is known in which dosage and dosage are designed so as to be within a desired effective therapeutic concentration by measuring the blood drug concentration of an individual patient. Since the CTC measuring apparatus 1 of this embodiment can measure CTC in real time, it is thought that it is useful also at the time of the administration treatment of an anticancer agent. Normal TDM is an open-loop process because the drug concentration is managed by grasping the blood concentration of the drug. In other words, since only the concentration of the anticancer drug is controlled, there are individual differences in whether or not it works. Although it was not effective, it was also treated with anticancer drugs that only caused pain to the patient. However, according to the present invention, whether or not a drug is effective can be evolved into a closed-loop TDM that grasps the behavior of CTC. That is, since the amount of CTC released is monitored by attacking the cancer tissue, the effect can be constantly monitored. In addition, the conventional blood sampling methods listed in Patent Documents 1 and 2 can obtain only one point of concentration data in the vein as two-dimensional concentration distribution data of the time axis and the location. As a result, the range of information that can be obtained spreads three-dimensionally from the point, so that the measurement accuracy is greatly improved and the range of application is greatly expanded.

  In addition, even if there is no genetic diagnosis database (race, gender, individual difference, etc.), it is possible to immediately feedback the effect, so you can try out a lot of anticancer drugs. In this way, the side effects can be minimized and the effects of various drugs can be greatly extracted. Therefore, an anticancer agent that is difficult to use because of its large pharmacological effect but large side effects and narrow effective concentration range is also an option, and effective treatment can be performed while maintaining QOL. .

As an effect, the focus is on preventing the recurrence of cancer treatment (prognosis), but it can of course also be used for testing and prevention purposes. Thus, according to this embodiment, the quality of medical treatment can be improved and the medical cost can be reduced, which can greatly contribute to society.
The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, in the above embodiment, an example in which a labeling agent is administered to the living body 100 has been described. However, if a labeling agent (vitamins) having a supplement function can be administered to the living body 100, a labeling agent for testing is used. There is no need. In this case, if you drink without being conscious of it every day, you will be able to perform a medical checkup once a year and a cancer screening at a medical checkup.

  Moreover, although FIG. 2 of the said embodiment is drawn with the image which arrange | positions the comparatively large CTC measuring apparatus which drip-treats an anticancer agent, even if it is a small apparatus which measures only the terminal of a body For example, as shown in FIG. 14, a device equipped with a light source, a wavelength filter, a CCD, etc. in a form like a wristwatch may be directly attached to the skin. It is also possible to have functions such as displaying the CTC count value and wirelessly transferring the data stored in the memory to the host computer.

  DESCRIPTION OF SYMBOLS 1 ... Circulating tumor cell measuring device, 3 ... Light source, 4 ... Wavelength variable filter, 5 ... Area sensor (light detection means), 6 ... Signal processor (calculation means), 7 ... Image memory, 21a, 21b, 21c ... Liquid crystal Cell, D0 (i, j) ... concentration data, D1 (i, j) ... detected data by λ1, D2 (i, j) ... detected data by λ2, D3 (i, j) ... detected data by λ3, D4 ( i, j)... binarized mapping data (mask data), D5 (i, j)... connectivity data, 100.

Claims (6)

A biological substance measuring apparatus for detecting the biological material by fluorescence labeling agent that binds to the biological material,
A light source that emits light that excites the fluorescent labeling agent;
Light emitted from the fluorescent labeling agent when the fluorescent labeling agent is irradiated with the light is incident, and transmits light in a predetermined wavelength range of the entire wavelength range of incident light. A tunable filter that can be changed,
A light detection means for detecting the intensity of light incident through the wavelength tunable filter and obtaining an intensity distribution of light emitted from the biological material ;
A concentration calculating means for calculating a concentration at which the fluorescent labeling agent is present based on an intensity distribution of light emitted from the biological material acquired by the light detecting means;
A binding degree calculating means for calculating a binding degree to which the fluorescent labeling agent is bound, based on light intensity distribution at a plurality of positions of the measurement target acquired by the light detecting means;
A biological material measuring apparatus comprising:   2. The biological material measuring apparatus according to claim 1, wherein light having a wavelength of 600 nm to 1200 nm is used in a wavelength range of a light source that irradiates light of the light detection means and a wavelength range of a wavelength tunable filter.   The biological substance measuring apparatus according to claim 1, wherein the binding degree calculating unit calculates a binding degree using a calculation result obtained by the concentration calculating unit as mask data. The biological material measuring apparatus according to claim 1 or 2, wherein a calculation result by the concentration calculating means and a binding degree by the binding degree calculating means are calculated. A fluorescent labeling agent that binds to the biological material a biological substance measurement method for detecting the biological material,
A light irradiation step of irradiating light for exciting the fluorescent labeling agent;
Detecting the intensity of light in a specific wavelength range emitted from the biological material among the light emitted from the fluorescent labeling agent when the fluorescent labeling agent is irradiated with the light, and changing the wavelength range by detecting the intensity, the intensity distribution acquisition step of acquiring an intensity distribution of light emitted from the biological material,
A concentration calculating step for calculating a concentration of the fluorescent labeling agent based on an intensity distribution of light emitted from the biological material ;
A degree-of-binding calculation step for calculating the degree of binding of the fluorescent labeling agent based on the intensity distribution of light emitted from the biological material ;
A biological material measuring method comprising: In vivo circulating tumor cell measurement method for detecting the biological material in the living body by a fluorescent labeling agent that binds to the biological material ,
A light irradiation step of irradiating the living body with light that excites the fluorescent labeling agent;
Wherein among the ex vivo fluorescent labeling agent of the light which the light is emitted in vitro from the fluorescent labeling agent upon irradiation, detecting the intensity of light of a specific wavelength range emitted from the biological material, by the detection of the intensity by changing the wavelength range, and intensity distribution acquisition step of acquiring an intensity distribution of light emitted from the biological material,
A concentration calculating step for calculating a concentration of the fluorescent labeling agent based on an intensity distribution of light emitted from the biological material ;
A degree-of-binding calculation step for calculating the degree of binding of the fluorescent labeling agent based on the intensity distribution of light emitted from the biological material ;
A method for measuring circulating tumor cells in vivo, comprising: