Fluorescence polarization measurement device and polarization degree measurement method

Abstract

The invention discloses a fluorescence polarization measuring device and a polarization degree measuring method. The fluorescence polarization measurement apparatus includes a light source unit, a polarization direction modulation element, a detector, and a controller. The light source unit emits linearly polarized excitation light. The polarization direction modulation element spatially modulates the polarization direction of the excitation light at a predetermined frequency, and emits the excitation light, the polarization direction of which has been spatially modulated at the predetermined frequency, onto a measurement target solution. The detector detects a spatial distribution of fluorescence intensity of fluorescence having a polarization direction in a predetermined direction from among fluorescence emitted from the measurement target solution due to the excitation light whose polarization direction is spatially modulated at the predetermined frequency. The controller extracts a direct-current component and a component having the same frequency as the predetermined frequency from a spatial distribution of the detected fluorescence intensity, and calculates a degree of polarization of the measurement target solution based on the extracted component and the direct-current component.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from Japanese Patent Application No. 2023-136206 filed on August 24, 2023, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present application generally relates to a fluorescence polarization measurement device and a polarization degree measurement method. Background Art

[0004] Among immunoassays using fluorescence, a fluorescence polarization immunoassay (FPIA) method is known that uses an antigen-antibody reaction to detect a measurement target substance to be measured. For example, Japanese Patent Application Publication No. H03-103765 ​​describes a method for calculating the concentration of a measurement antigen (measurement target substance) based on the measured fluorescence polarization degree.

[0005] In addition, U.S. Patent No. 6970241 describes a fluorescence measuring device including a modulation unit that periodically (temporally) rotates the polarization plane of fluorescence emitted from a sample by 90°. In U.S. Patent No. 6970241, the modulation unit rotates the polarization plane of fluorescence, and thus, the fluorescence intensity of fluorescence having a polarization direction parallel to the polarization direction of excitation light and the fluorescence intensity of fluorescence having a polarization direction perpendicular to the polarization direction of excitation light can be measured without rotating the polarizer. Therefore, the fluorescence measuring device of U.S. Patent No. 6970241 suppresses the influence of polarization characteristics of detectors, filters, etc.

[0006] With the fluorescence measuring apparatus of US Pat. No. 6,970,241, the influence of polarization characteristics of detectors, filters, etc. can be suppressed, but when the fluorescence intensity is low, it is difficult to obtain the fluorescence intensity at a sufficient signal-to-noise ratio (SN ratio).

[0007] The present disclosure has been made in view of the above-described circumstances, and an object of the present disclosure is to provide a fluorescence polarization measurement device and a polarization degree measurement method having high measurement sensitivity. Summary of the invention

[0008] In order to achieve the above object, a fluorescence polarization measurement device according to a first aspect of the present disclosure includes:

[0009] a light source unit that emits linearly polarized excitation light;

[0010] a polarization direction modulation element that spatially modulates the polarization direction of the excitation light at a predetermined frequency, and emits the excitation light whose polarization direction is spatially modulated at the predetermined frequency onto a measurement target solution;

[0011] a detector that detects a spatial distribution of fluorescence intensity of fluorescence having a polarization direction in a predetermined direction from fluorescence emitted from the measurement target solution due to the excitation light whose polarization direction is spatially modulated at the predetermined frequency; and

[0012] A controller extracts a direct current component and a component having the same frequency as the predetermined frequency from the spatial distribution of the detected fluorescence intensity, and calculates a polarization degree of the measurement target solution based on the extracted components and the direct current component.

[0013] The polarization degree measurement method according to the second aspect of the present disclosure includes:

[0014] emitting excitation light whose polarization direction has been spatially modulated at a predetermined frequency onto a measurement target solution; detecting a spatial distribution of fluorescence intensity of fluorescence having a polarization direction in a predetermined direction from fluorescence emitted from the measurement target solution;

[0015] extracting a DC component and a component having the same frequency as the predetermined frequency from the detected spatial distribution of the fluorescence intensity; and

[0016] Based on the extracted components and the DC component, the polarization degree of the measurement target solution is calculated.

[0017] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the disclosure.

[0018] According to the present disclosure, a component having the same frequency as a predetermined frequency used to spatially modulate the polarization direction of excitation light is extracted from the spatial distribution of fluorescence intensity, thereby being able to improve measurement sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] A more complete understanding of the present application can be obtained when the following detailed description is considered in conjunction with the following drawings, in which:

[0020] Figure 1 is a schematic diagram showing a fluorescence polarization measuring device according to Example 1;

[0021] Figure 2 is a plan view showing a microdevice according to Embodiment 1;

[0022] Figure 3 yes Figure 2 A cross-sectional view of the microdevice shown is taken along line AA;

[0023] Figure 4 is a diagram showing the optical axis of the polarization direction modulation element and the intensity of the polarization light component of the excitation light according to Example 1;

[0024] Figure 5 is a diagram showing the alignment direction of liquid crystal molecules and the optical axis of a polarization direction modulation element according to Example 1;

[0025] Figure 6 is a block diagram showing the configuration of a controller according to Embodiment 1;

[0026] Figure 7 is a diagram showing fluorescence intensity calculated from image data according to Example 1;

[0027] Figure 8 is a diagram showing a direct current component and a component having the same frequency as a spatial frequency of a polarization direction modulation element according to Example 1;

[0028] Fig. 9 is a diagram for explaining the fluorescence intensity of fluorescence whose polarization direction is parallel to the polarization direction of the excitation light and the fluorescence intensity of fluorescence whose polarization direction is perpendicular to the polarization direction of the excitation light according to Example 1;

[0029] Fig.10 is a diagram showing a hardware configuration of a controller according to Embodiment 1;

[0030] Fig.11 is a diagram for explaining the intensity distribution of excitation light according to Example 1;

[0031] Fig.12 is a flowchart showing a detection process according to Embodiment 1;

[0032] Fig.13 is a flowchart showing polarization degree measurement processing according to Embodiment 1;

[0033] Fig.14 is a diagram showing an X-direction component, an X-direction DC component, a Z-direction component, and a Z-direction DC component according to Embodiment 2;

[0034] Fig.15 is a diagram showing the polarized light component of the excitation light according to Example 2, and the intensity of the X-direction component, the X-direction DC component, the Z-direction component, and the Z-direction DC component of the fluorescence;

[0035] Fig.16 is a flowchart showing polarization degree measurement processing according to Embodiment 2;

[0036] Fig.17 is a diagram showing the intensity of the polarized light component of the second excitation light according to Example 3;

[0037] Fig.18 is a diagram showing the polarized light component of the first excitation light, and the first direct current component and the intensity of the first component of the fluorescence according to Example 3;

[0038] Fig.19 is a diagram showing the intensities of the polarized light component of the second excitation light, and the second DC component and the second component of the fluorescence according to Example 3;

[0039] Fig. 20 is a diagram showing a first component, a first DC component, a second component, and a second DC component of fluorescence according to Example 3;

[0040] Fig.21 is a flowchart showing polarization degree measurement processing according to Embodiment 3;

[0041] Fig. 22 is a schematic diagram showing a fluorescence polarization measuring apparatus according to Example 4;

[0042] Fig.23 is a diagram showing the polarized light component of the excitation light according to Example 4, and the intensities of the X-direction component, the X-direction DC component, the Y-direction component, and the Y-direction DC component of the fluorescence; and

[0043] Fig.24 is a diagram showing the optical axis of the polarization direction modulation element, the intensity of the polarization light component of the excitation light, and the fluorescence intensity. DETAILED DESCRIPTION

[0044] Hereinafter, a fluorescence polarization measuring apparatus according to various embodiments is described with reference to the accompanying drawings.

[0045] Example 1

[0046] First, refer to Figures 1 to 12 The fluorescence polarization measurement device 100 according to the present embodiment is described. In one example, the fluorescence polarization measurement device 100 is used to detect a measurement target substance to be measured contained in a measurement target solution to be measured using fluorescence polarization immunoassay.

[0047] like Figure 1As shown, the fluorescence polarization measuring device 100 includes a light source unit 10, a polarization direction modulating element 30, a dichroic mirror 40, an objective lens 42, a detector 50, and a controller 70. The light source unit 10 emits linearly polarized excitation light EL. The polarization direction modulating element 30 spatially modulates the polarization direction of the excitation light EL emitted from the light source unit 10 at a predetermined frequency. The excitation light EL whose polarization direction is spatially modulated at a predetermined frequency passes through the dichroic mirror 40 and the objective lens 42 and is emitted on a measurement target solution introduced into a microchannel 220 of a microdevice 200 described later. The detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction from the fluorescence FL emitted from the measurement target solution. The controller 70 controls the various components of the fluorescence polarization measuring device 100. In addition, the controller 70 calculates the polarization degree P of the measurement target solution based on the spatial distribution of the detected fluorescence intensity. In addition, the controller 70 calculates the concentration of the measurement target substance based on the polarization degree P of the measurement target solution.

[0048] Note that in this specification, for ease of understanding, Figure 1 In the fluorescence polarization measurement device 100, the left direction (leftward direction on paper) is referred to as the "+Z direction", the upward direction (upward direction on paper) is referred to as the "+Y direction", and the direction perpendicular to the +Y direction and the +Z direction (front direction on paper) is referred to as the "+X direction". The excitation light EL is light that excites the fluorescent labeled derivative described later and causes the fluorescent labeled derivative to emit fluorescence. The light source unit 10, the polarization direction modulation element 30, the dichroic mirror 40 and the objective lens 42 form an illumination optical system, and the objective lens 42, the dichroic mirror 40 and the detector 50 form an observation optical system.

[0049] First, the measurement target solution and the microdevice 200 are described. The measurement target solution includes a measurement target substance, a fluorescently labeled derivative, and an antibody. The measurement target substance to be measured is the detection target of the fluorescence polarization measurement device 100. The measurement target substance to be measured can be any compound detectable in the fluorescence immunoassay. Examples of measurement target substances include antibiotics, bioactive substances, mycotoxins, etc. Specific examples of measurement target substances include prostaglandin E2, β-lactoglobulin, chloramphenicol, deoxynivalenol, etc. The fluorescently labeled derivative is a derivative obtained by fluorescently labeling the measurement target substance with a fluorescent substance. The fluorescently labeled derivative can be obtained by binding the fluorescent substance to the measurement target substance using a known method. The fluorescent substance is fluorescein (wavelength of excitation light EL: 494nm, wavelength of fluorescence FL: 521nm), rhodamine B (wavelength of excitation light EL: 550nm, wavelength of fluorescence FL: 580nm), etc. The antibody specifically binds to the measurement target substance due to the antigen-antibody reaction. In one example, the antibody is obtained by inoculating a host animal (eg, mouse or cow) with a measurement target substance, and collecting and purifying the antibody produced in the blood of the host animal. Alternatively, a commercially available antibody may be used as the antibody.

[0050] The measurement target substance and the fluorescent labeled derivative compete with the antibody due to the antigen-antibody reaction and specifically bind. In the fluorescence polarization immunoassay, the polarization degree P of the fluorescence FL emitted by the fluorescent labeled derivative included in the measurement target solution is calculated. In addition, the concentration of the measurement target substance is calculated based on the obtained polarization degree P and a calibration curve created in advance.

[0051] The fluorescent labeled derivative not bound to the antibody moves violently in the measurement target solution, and thus, when the fluorescent labeled derivative not bound to the antibody is irradiated with the polarized excitation light EL, fluorescence FL is randomly emitted. At the same time, the movement of the fluorescent labeled derivative bound to the antibody in the measurement target solution is restricted, and thus, when the fluorescent labeled derivative bound to the antibody is irradiated with the polarized excitation light EL, fluorescence FL biased in the polarization direction of the excitation light EL is emitted. The fluorescence intensity Ih of the fluorescence FL whose polarization direction is parallel to the polarization direction of the excitation light EL and the fluorescence intensity Iv of the fluorescence FL whose polarization direction is perpendicular to the polarization direction of the excitation light EL are measured, and the deviation of the fluorescence intensity is calculated as the degree of polarization P. The degree of polarization P depends on the amount of the fluorescent labeled derivative bound to the antibody, and thus, the concentration of the measurement target substance can be calculated based on the obtained degree of polarization P and a calibration curve created in advance. Note that the degree of polarization P is expressed as P = (Ih-Iv) / (Ih+Iv).

[0052] like Figure 2 and Figure 3As shown, the microdevice 200 includes a first substrate 202, a second substrate 204, a partition wall 206, and three microchannels 220. A measurement target solution is introduced into each microchannel 220. The microdevice 200 is placed on a stage ST of the fluorescence polarization measurement device 100.

[0053] The first substrate 202 of the microdevice 200 is implemented as a flat quartz glass substrate. The excitation light EL whose polarization direction is spatially modulated at a predetermined frequency enters the microdevice 200 through the first substrate 202. The excitation light EL is emitted from the -Z direction to Figure 2 The measurement region S is shown. The excitation light EL enters the main surface 202 a of the first substrate 202 vertically.

[0054] The second substrate 204 of the micro device 200 is implemented as a flat substrate. The second substrate 204 is formed of a material that has almost no autofluorescence. In one example, the second substrate 204 is formed of polydimethylsiloxane (PDMS) containing carbon black. The second substrate 204 faces the first substrate 202, and the second substrate 204 and the first substrate 202 sandwich a partition wall 206.

[0055] The partition wall 206 of the micro device 200 is sandwiched by the first substrate 202 and the second substrate 204 to form the micro channel 220. The partition wall 206 is formed of a material that has almost no autofluorescence. In addition, preferably, the partition wall 206 is formed of a material that absorbs light such as excitation light EL, fluorescence FL, etc. In the present embodiment, the partition wall 206 is formed integrally with the second substrate 204.

[0056] The microchannels 220 of the microdevice 200 extend parallel to the X direction in the measurement region S. In one example, the width of each microchannel 220 in the measurement region S is 200 μm. Each microchannel 220 includes two openings 222 penetrating the second substrate 204 and the partition wall 206. The measurement target solution is introduced and discharged through the openings 222.

[0057] Next, the components of the fluorescence polarization measuring apparatus 100 are described. Figure 1 As shown, the light source unit 10 of the fluorescence polarization measurement device 100 includes a light source 12 , a condenser lens 14 , an aperture 16 , a collimator 18 , a first polarization adjustment element 20 and an excitation light filter 22 .

[0058] The light source 12 emits light including the excitation light EL in the +Z direction. In one example, the light source 12 is composed of a light emitting diode (LED) device. The light emitted from the light source 12 is focused by a condenser lens 14 and then passes through an aperture 16. The aperture 16 reduces the influence of external light (light other than the light emitted from the light source 12). The light that has passed through the aperture 16 enters a collimator 18.

[0059] The collimator 18 converts the incident light into parallel light, and the light converted into parallel light enters the first polarization adjusting element 20 .

[0060] The first polarization adjustment element 20 selects light having a polarization direction in a predetermined direction from the incident light, and emits light having a polarization direction in the predetermined direction. The light emitted from the first polarization adjustment element 20 enters the excitation light filter 22. In the present embodiment, the predetermined direction in the first polarization adjustment element 20 is the X direction, and the first polarization adjustment element 20 is a polarizing plate. Specifically, the light including the excitation light EL entering the first polarization adjustment element 20 is emitted as linearly polarized light having a polarization direction in the X direction, and enters the excitation light filter 22. The excitation light filter 22 removes light other than the excitation light EL from the light emitted from the light source 12. In one example, the excitation light filter 22 is implemented as a bandpass filter.

[0061] Therefore, the excitation light EL having the polarization direction in the X direction is emitted in the +Z direction from the light source unit 10 . The excitation light EL having the polarization direction in the X direction enters the polarization direction modulation element 30 .

[0062] The polarization direction modulation element 30 of the fluorescence polarization measurement device 100 spatially modulates the polarization direction of the excitation light EL having a polarization direction in the X direction at a predetermined frequency (predetermined period) along the X direction. In one example, the polarization direction modulation element 30 is a half-wave plate whose optical axis changes continuously along the X direction. In the polarization direction modulation element (half-wave plate) 30 of the present embodiment, as shown in FIG. Figure 4 As shown in FIG. 1 , the optical axis rotates continuously by 180° from the -X direction to the +X direction along the X direction. As a result, the polarization direction of the excitation light EL rotates continuously from the X direction along the X direction. Figure 4 As shown, the intensities of the component with the polarization direction in the X direction and the component with the polarization direction in the Y direction in the excitation light EL emitted from the polarization direction modulation element 30 periodically increase and decrease at the same frequency as the predetermined frequency used by the polarization direction modulation element 30 to modulate the polarization direction. The intensities of the component with the polarization direction in the X direction and the component with the polarization direction in the Y direction are opposite (have opposite phases). The polarization direction modulation element 30 emits the spatially modulated excitation light EL to the dichroic mirror 40.

[0063] In one example, the polarization direction modulation element 30 is formed by a liquid crystal cell in which nematic liquid crystals are uniformly arranged. Figure 5 As shown, the alignment direction of the liquid crystal molecules rotates continuously by 180° along the X direction. In addition, the retardation value of the liquid crystal cell is 1 / 2 of the wavelength of the excitation light EL.

[0064] Back to Figure 1 , the dichroic mirror 40 of the fluorescence polarization measurement device 100 transmits the spatially modulated excitation light EL in the +Z direction. In addition, the dichroic mirror 40 reflects the fluorescence FL emitted from the microdevice 200 toward the detector 50 (in the +Y direction). The objective lens 42 of the fluorescence polarization measurement device 100 focuses the fluorescence FL and the excitation light EL that have passed through the dichroic mirror 40.

[0065] The excitation light EL spatially modulated by the polarization direction modulation element 30 is emitted through the dichroic mirror 40 and the objective lens 42, and is emitted onto the measurement area S of the microdevice 200. As a result, fluorescence FL is emitted from the measurement target solution (fluorescent labeling derivative) introduced into the microchannel 220 of the microdevice 200. The fluorescence FL travels in the +Y direction through the objective lens 42 and the dichroic mirror 40, and enters the detector 50 (absorption filter 52, described later).

[0066] The detector 50 of the fluorescence polarization measuring apparatus 100 is arranged on the +Y side of the dichroic mirror 40. The detector 50 includes an absorption filter 52, a second polarization adjusting element 54, an imaging lens 56, and a first light receptor 58A.

[0067] The absorption filter 52 separates the fluorescent light FL emitted from the microdevice 200 from scattered light, leaked light, etc., and transmits the fluorescent light FL. In one example, the absorption filter 52 is implemented as a bandpass filter. The fluorescent light FL emitted from the absorption filter 52 enters the second polarization adjusting element 54.

[0068] The second polarization adjustment element 54 selects the fluorescence FL having a polarization direction in a predetermined direction from the incoming fluorescence FL, and emits the fluorescence FL having a polarization direction in the predetermined direction. In the present embodiment, the predetermined direction in the second polarization adjustment element 54 is the X direction, and the second polarization adjustment element 54 is a polarizing plate. Therefore, the fluorescence FL having a polarization direction in the X direction is emitted from the second polarization adjustment element 54. The fluorescence FL having a polarization direction in the X direction passes through the imaging lens 56 and enters the first light receptor 58A.

[0069] In one example, the first photoreceptor 58A is implemented as a charge coupled device (CCD) image sensor. The first photoreceptor 58A detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (polarization direction in the X direction) as an image. The first photoreceptor 58A generates image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL. In addition, the first photoreceptor 58A sends the image data to the controller 70. Note that the main surface 202a of the first substrate 202 of the microdevice 200 and the imaging surface of the first photoreceptor 58A have an imaging relationship.

[0070] The controller 70 of the fluorescence polarization measuring device 100 extracts a DC component and a component having the same frequency as a predetermined frequency from the spatial distribution of the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (polarization direction in the X direction), and the predetermined frequency is the frequency used by the polarization direction modulation element 30 to spatially modulate the polarization direction of the excitation light EL having the polarization direction in the X direction along the X direction. The controller 70 calculates the polarization degree P of the measurement target solution based on the extracted DC component and the component having the same frequency. In addition, the controller 70 calculates the concentration of the measurement target substance based on the polarization degree P and the calibration curve. Figure 6 As shown, the controller 70 includes an input / output device 72, a memory 74, an extractor 76, a polarization degree calculator 78, and a concentration calculator 80. The controller 70 also includes a light source controller 82 and a detection controller 84. Note that, hereinafter, the predetermined frequency used by the polarization direction modulation element 30 to spatially modulate the polarization direction of the excitation light EL having a polarization direction in the X direction is referred to as the spatial frequency of the polarization direction modulation element 30.

[0071] The input / output device 72 inputs / outputs signals, data, etc. between the controller 70 and various components.

[0072] The memory 74 stores programs, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL, data of a calibration curve representing the degree of polarization P and the concentration of a measurement target substance, and the like.

[0073] The extractor 76 obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL. Furthermore, the extractor 76 extracts a direct current component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the obtained spatial distribution of the fluorescence intensity of the fluorescence FL.

[0074] Next, the spatial distribution of the fluorescence intensity of the fluorescence FL obtained from the image data and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 are described.

[0075] In this embodiment, the excitation light EL spatially modulated by the polarization direction modulation element 30 is emitted to the measurement target solution (microdevice 200). Figure 4As shown, the excitation light EL spatially modulated by the polarization direction modulation element 30 includes a component having a polarization direction in the X direction and a component having a polarization direction in the Y direction, and the intensities of these components periodically increase and decrease at the same frequency as the spatial frequency of the polarization direction modulation element 30. The detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction as an image. Therefore, the spatial distribution of the fluorescence intensity of the fluorescence FL obtained from the image data has the same periodicity as the intensity of the component having a polarization direction in the X direction of the excitation light EL. However, when the concentration of the measurement target substance is low, as in Figure 7 As shown, the spatial distribution of the fluorescence intensity of the fluorescent light FL obtained from the image data becomes a spatial distribution including a lot of noise caused by stray light, vibration, sensitivity change of the photoreceiver, etc. It is difficult to directly calculate the polarization degree P from such a spatial distribution of fluorescence intensity including a lot of noise.

[0076] Thus, the extractor 76 extracts the DC component and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL. The spatial frequency of the noise included in the spatial distribution of the fluorescence intensity of the obtained fluorescence FL is different from the spatial frequency of the polarization direction modulation element 30, and thus, the SN ratio of the fluorescence intensity of the fluorescence FL can be improved by extracting the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL. Note that the phrase "extracting the component having the same frequency as the spatial frequency of the polarization direction modulation element 30" can also be expressed as "obtaining a function representing the component having the same frequency as the spatial frequency of the polarization direction modulation element 30".

[0077] Specifically, the extractor 76 performs Fourier transform on the spatial distribution of the fluorescence intensity of the fluorescence FL obtained from the image data using the spatial frequency of the polarization direction modulation element 30. Figure 8 As shown, the extractor 76 performs inverse Fourier transform to obtain a direct current component (DC offset) and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 .

[0078] The polarization degree calculator 78 calculates the polarization degree P based on the DC component obtained by the extractor 76 and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30. As described above, the intensities of the component having the polarization direction in the X direction and the component having the polarization direction in the Y direction of the excitation light EL emitted to the measurement target solution have an opposite phase relationship. In addition, the detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. Therefore, as Fig. 9As shown, the maximum value of the component (including the DC component) having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL. In addition, the minimum value of the component (including the DC component) having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL. Therefore, the polarization degree calculator 78 calculates the polarization degree P based on the maximum value and the minimum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30. The SN ratio of the fluorescence intensity of the fluorescence FL is high, and thus, the fluorescence polarization measuring device 100 can detect the polarization degree P with high measurement sensitivity.

[0079] The concentration calculator 80 calculates the concentration of the measurement target substance based on the polarization degree P calculated by the polarization degree calculator 78 and a calibration curve of the polarization degree P and the concentration of the measurement target substance.

[0080] The light source controller 82 controls the light source unit 10 (light source 12 ). The detection controller 84 controls the detector 50 (first light receiver 58A).

[0081] Fig.10 : shows the hardware configuration of the controller 70. The controller 70 includes a central processing unit (CPU) 92, a read-only memory (ROM) 93, a random access memory (RAM) 94, and an input / output interface 96. The CPU 92 executes a program stored in the ROM 93. The ROM 93 stores programs, data, etc. The RAM 94 stores data. The input / output interface 96 inputs and outputs signals, data, etc. between the various components. The functions of the controller 70 are realized by the CPU 92 executing the program.

[0082] As described above, a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 is extracted from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL, and thus the fluorescence polarization measurement device 100 can suppress the influence of noise and improve the measurement sensitivity of the polarization degree P of the fluorescence polarization measurement device 100. Fig.11 Even when the intensity of the excitation light EL emitted to the measurement target solution is not uniform as shown, the fluorescence polarization measurement apparatus 100 can suppress the influence of the intensity distribution of the excitation light EL and can calculate the polarization degree P. In addition, the measurement sensitivity of the polarization degree P is high, and thus, even if the concentration of the measurement target substance is low, the fluorescence polarization measurement apparatus 100 can accurately detect the measurement target substance.

[0083] Furthermore, the maximum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Ih, and the minimum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Iv. Therefore, the fluorescence polarization measurement device 100 can calculate the polarization degree P from one piece of image data, and can shorten the measurement time without switching any one of the polarization direction of the excitation light EL emitted onto the measurement target solution and the polarization direction of the fluorescence FL to be detected.

[0084] Next, refer to Fig.12 and Fig.13 The detection process (i.e., the detection method of the measurement target substance) of the fluorescence polarization measurement device 100 is described below. Fig.12 As shown, the detection process sequentially includes a polarization degree measurement process (step S100) for calculating the polarization degree P and a concentration calculation process (step S200) for detecting the concentration of the measurement target substance.

[0085] Reference Fig.13 Describe the polarization degree measurement process (step S100). In the polarization degree measurement process (step S100), the polarization degree P of the measurement target solution is calculated based on the fluorescence FL emitted by the measurement target solution introduced into the microchannel 220 of the microdevice 200. First, the controller 70 controls the light source 12 to emit the excitation light EL whose polarization direction is spatially modulated at a predetermined frequency to the measurement target solution introduced into the microchannel 220 of the microdevice 200 placed on the stage ST (step S110). The light including the excitation light EL emitted from the light source 12 passes through the first polarization adjustment element 20, the polarization direction modulation element 30, etc., and is converted into the excitation light EL whose polarization direction is spatially modulated at a predetermined frequency (the spatial frequency of the polarization direction modulation element 30). Then, the excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency is emitted to the measurement target solution. As a result, fluorescence FL is emitted from the measurement target solution.

[0086] Next, the controller 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction (X direction) from the fluorescence FL emitted from the measurement target solution (step S120). Specifically, the fluorescence FL emitted from the measurement target solution passes through the dichroic mirror 40, the second polarization adjustment element 54, etc., and enters the first light receptor 58A as the fluorescence FL having a polarization direction in a predetermined direction (X direction). The extractor 76 of the controller 70 obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL acquired from the first light receptor 58A.

[0087] In addition, the controller 70 extracts a DC component and a component having the same frequency as a predetermined frequency (the spatial frequency of the polarization direction modulation element 30) from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL (step S130). Specifically, the extractor 76 of the controller 70 performs Fourier transform on the spatial distribution of the fluorescence intensity using the spatial frequency of the polarization direction modulation element 30, and then performs inverse Fourier transform. As a result, the DC component and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 can be extracted. In addition to the fluorescence intensity, noise is also included in the spatial distribution of the detected fluorescence intensity. The spatial frequency of the noise is different from the spatial frequency of the polarization direction modulation element 30, and thus, by extracting a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the detected fluorescence FL, the SN ratio of the fluorescence intensity of the fluorescence FL can be improved.

[0088] The controller 70 calculates the polarization degree P of the measurement target solution based on the DC component and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (step S140). Specifically, the polarization degree calculator 78 of the controller 70 calculates the polarization degree P using the maximum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 as the fluorescence intensity Ih, and the minimum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 as the fluorescence intensity Iv.

[0089] return Fig.12 , the concentration calculation process (step S200) is described next. In the concentration calculation process (step S200), the concentration of the measurement target substance included in the measurement target solution is calculated based on the polarization degree P calculated in step S140 and the calibration curve measured in advance. Specifically, the concentration calculator 80 of the controller 70 calculates the concentration of the measurement target substance based on the polarization degree P calculated by the polarization degree calculator 78, and the calibration curve of the polarization degree P and the concentration of the measurement target substance. When the concentration calculation process (step S200) ends, the detection process ends.

[0090] As described above, the fluorescence polarization measurement device 100 extracts a component having the same frequency as the predetermined frequency with which the excitation light EL is spatially modulated from the spatial distribution of the fluorescence intensity of the detected fluorescence FL, and thus can suppress noise and can detect the polarization degree P with high measurement sensitivity. In addition, the fluorescence polarization measurement device 100 can suppress the influence of the intensity distribution of the excitation light EL and can detect the polarization degree P with high measurement sensitivity. With the fluorescence polarization measurement device 100, the measurement sensitivity of the polarization degree P is high, and thus, even when the concentration of the measurement target substance is low, the measurement target substance can be accurately detected.

[0091] In addition, the maximum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Ih, and the minimum value of the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Iv, and thus, the fluorescence polarization measurement device 100 can calculate the polarization degree P based on a piece of image data.

[0092] Example 2

[0093] In Embodiment 1, the detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. However, a configuration may be adopted in which the detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction.

[0094] Like the fluorescence polarization measuring device 100 of Embodiment 1, the fluorescence polarization measuring device 100 of this embodiment includes a light source unit 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detector 50, and a controller 70. The configuration of the light source unit 10 to the objective lens 42 of this embodiment is the same as that of Embodiment 1, and thus the detector 50 and the controller 70 of this embodiment are described.

[0095] Like the detector 50 of the first embodiment, the detector 50 of the present embodiment includes an absorption filter 52, a second polarization adjustment element 54, an imaging lens 56 and a first light receiver 58A. The configurations of the absorption filter 52 and the imaging lens 56 of the present embodiment are the same as those of the first embodiment.

[0096] The second polarization adjustment element 54 of the present embodiment switches the polarization direction of the fluorescent light FL emitted from the second polarization adjustment element 54 to the X direction and the Z direction. The fluorescent light FL emitted from the second polarization adjustment element 54 of the present embodiment passes through the imaging lens 56 and enters the first light receptor 58A. The second polarization adjustment element 54 of the present embodiment is implemented as a TN liquid crystal cell, a rotatably held polarizing plate, or the like. The second polarization adjustment element 54 of the present embodiment is controlled by the detection controller 84 of the controller 70. Note that the light source unit 10 emits the excitation light EL having a polarization direction in the X direction, and the fluorescent light FL travels in the +Y direction. Thus, the second polarization adjustment element 54 of the present embodiment switches the fluorescent light FL emitted from the second polarization adjustment element 54 to the fluorescent light FL having a polarization direction parallel to the polarization direction of the excitation light EL emitted from the light source unit 10, and to the fluorescent light FL having a polarization direction perpendicular to the polarization direction of the excitation light EL emitted from the light source unit 10.

[0097] As with the first light receiver 58A of the first embodiment, the first light receiver 58A of the present embodiment detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction as an image. In addition, the first light receiver 58A of the present embodiment detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction as an image. The first light receiver 58A of the present embodiment sends image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction and image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction to the controller 70.

[0098] The controller 70 of the present embodiment extracts a DC component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. In addition, the controller 70 of the present embodiment extracts a DC component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction. The controller 70 of the present embodiment calculates the degree of polarization P of the measurement target solution based on the extracted components and the DC component. In addition, the controller 70 of the present embodiment calculates the concentration of the measurement target substance based on the degree of polarization P and the calibration curve. Like the controller 70 of Example 1, the controller 70 of the present embodiment includes an input / output device 72 to a detection controller 84. The configuration of the input / output device 72, the memory 74, the concentration calculator 80, the light source controller 82 and the detection controller 84 of the present embodiment is the same as that of Example 1.

[0099] The extractor 76 of the present embodiment obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL. In the present embodiment, the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction are obtained. As in Embodiment 1, the obtained spatial distribution of the fluorescence intensity of the fluorescence FL includes noise.

[0100] As with the extractor 76 of the first embodiment, the extractor 76 of the present embodiment extracts a direct current component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL. That is, in the present embodiment, the extractor 76 extracts a direct current component (hereinafter referred to as "X-direction direct current component") and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as "X-direction component") from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. In addition, the extractor 76 extracts a direct current component (hereinafter referred to as "Z-direction direct current component") and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as "Z-direction component") from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction. As a result, as with the first embodiment, the SN ratio of the fluorescence intensity of the fluorescence FL can be improved.

[0101] Specifically, as in Embodiment 1, the extractor 76 of this embodiment performs Fourier transform and inverse Fourier transform on the spatial distribution of the fluorescence intensity of the fluorescence FL using the spatial frequency of the polarization direction modulation element 30. As a result, the X-direction component, the X-direction DC component, the Z-direction component, and the Z-direction DC component are obtained, as shown in FIG. Fig.14 shown.

[0102] The polarization degree calculator 78 of this embodiment calculates the polarization degree P based on at least one of the X-direction component and the X-direction DC component, and the Z-direction component and the Z-direction DC component. Figure 4 The excitation light EL having the spatial distribution of the intensity of the polarized light component shown is emitted to the measurement target solution. Fig.15 As shown, the intensities of the polarized light component, the X-direction component, and the Z-direction component of the excitation light EL are synchronized, and the maximum value of the X-direction component (including the DC component) or the Z-direction component (including the DC component) corresponds to the fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL. In addition, the minimum value of the X-direction component (including the DC component) or the Z-direction component (including the DC component) corresponds to the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL. Therefore, the polarization degree calculator 78 of the present embodiment calculates the polarization degree P based on the maximum value of the X-direction component or the Z-direction component, and the minimum value of the X-direction component or the Z-direction component.

[0103] In this embodiment, if Fig.15As shown, the fluorescence intensity Ih of the fluorescent light FL and the fluorescence intensity Iv of the fluorescent light FL are obtained at the same position (same X coordinate) of the microchannel 220. Therefore, by comparing the fluorescence intensity Ih of the fluorescent light FL and the fluorescence intensity Iv of the fluorescent light FL at multiple positions, it is possible to easily find noise caused by foreign matter in the microchannel 220, bright spot noise of the first light receptor 58A, and the like.

[0104] Next, the detection process of this embodiment is described. As in Embodiment 1, the detection process of this embodiment includes polarization degree measurement processing (step S100) and concentration calculation processing (step S200) in sequence ( Fig.12 ). The concentration calculation process (step S200) of this embodiment is the same as that of the first embodiment, and therefore, refer to Fig.16 The polarization degree measurement process (step S100 ) of the present embodiment will be described.

[0105] In the polarization degree measurement process (step S100) of the present embodiment, first, the controller 70 controls the polarization direction of the fluorescence FL emitted from the second polarization adjustment element 54 to the X direction, and emits the excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency to the measurement target solution (step S112a). Next, the controller 70 acquires image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction among the fluorescence FL emitted from the measurement target solution from the first photoreceptor 58A (step S114a). In addition, the controller 70 controls the polarization direction of the fluorescence FL emitted from the second polarization adjustment element 54 to the Z direction, and emits the excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency to the measurement target solution (step S112b). Then, the controller 70 acquires image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction among the fluorescence FL emitted from the measurement target solution from the first photoreceptor 58A (step S114b).

[0106] The controller 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction (X direction and Z direction) from the fluorescence FL emitted from the measurement target solution (step S120). Specifically, the extractor 76 of the controller 70 obtains the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction. In addition, the extractor 76 of the controller 70 obtains the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction.

[0107] In addition, the controller 70 extracts a DC component and a component having the same frequency as a predetermined frequency (the spatial frequency of the polarization direction modulation element 30) from the spatial distribution of the obtained fluorescence intensity (step S130). Specifically, the extractor 76 extracts an X-direction component and an X-direction DC component from the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction. In addition, the extractor 76 extracts a Z-direction component and a Z-direction DC component from the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction. As in Embodiment 1, the X-direction component and the X-direction DC component, and the Z-direction component and the Z-direction DC component are extracted by performing Fourier transform and inverse Fourier transform on the spatial distribution of the fluorescence intensity using the spatial frequency of the polarization direction modulation element 30.

[0108] The controller 70 calculates the polarization degree P of the measurement target solution based on the DC component and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (step S140). Specifically, the polarization degree calculator 78 of the controller 70 calculates the polarization degree P based on the maximum value of the X-direction component (including the DC component) or the Z-direction component (including the DC component) and the minimum value of the X-direction component (including the DC component) or the Z-direction component (including the DC component).

[0109] As described above, the fluorescence polarization measurement device 100 of the present embodiment can suppress noise and detect the polarization degree P with high measurement sensitivity, as with the fluorescence polarization measurement device 100 of the first embodiment. In addition, the fluorescence polarization measurement device 100 of the present embodiment can suppress the influence of the intensity distribution of the excitation light EL and can detect the polarization degree P with high measurement sensitivity. Since the measurement sensitivity of the polarization degree P is high, the fluorescence polarization measurement device 100 can accurately detect the measurement target substance even if the concentration of the measurement target substance is low.

[0110] Furthermore, the fluorescence polarization measurement device 100 of the present embodiment can easily detect noise caused by foreign matter in the microchannel 220 , bright spot noise of the first light receiver 58A, and the like.

[0111] Example 3

[0112] In Embodiment 1, light source unit 10 emits excitation light EL having a polarization direction in the X direction onto polarization direction modulation element 30. However, a configuration may be adopted in which light source unit 10 switches the polarization direction of excitation light EL between two orthogonal directions (X direction and Y direction).

[0113] Like the fluorescence polarization measuring device 100 of Embodiment 1, the fluorescence polarization measuring device 100 of this embodiment includes a light source unit 10, a polarization direction modulating element 30, a dichroic mirror 40, an objective lens 42, a detector 50, and a controller 70. The configuration of the polarization direction modulating element 30 to the objective lens 42 of this embodiment is the same as that in Embodiment 1, and thus the light source unit 10, the detector 50, and the controller 70 of this embodiment are described.

[0114] Like the light source unit 10 of Embodiment 1, the light source unit 10 of this embodiment includes a light source 12, a focusing lens 14, an aperture 16, a collimator 18, a first polarization adjustment element 20, and an excitation light filter 22. The configuration of the light source 12 to the collimator 18, and the excitation light filter 22 of this embodiment is the same as that of Embodiment 1, and thus the first polarization adjustment element 20 of this embodiment is described.

[0115] The first polarization adjustment element 20 of the present embodiment switches the polarization direction of light including the excitation light EL emitted from the first polarization adjustment element 20 to the X direction and the Y direction. The first polarization adjustment element 20 of the present embodiment is implemented as a TN liquid crystal cell, a rotatably held polarizing plate, etc. The first polarization adjustment element 20 of the present embodiment is controlled by the light source controller 82 of the controller 70.

[0116] Light emitted from first polarization adjusting element 20 of the present embodiment is emitted from light source unit 10 through excitation light filter 22. Therefore, light source unit 10 of the present embodiment emits while switching the polarization direction of excitation light EL to the X direction and the Y direction.

[0117] In the present embodiment, when the excitation light EL having the polarization direction in the X direction is emitted from the light source unit 10, the excitation light EL whose intensity of the polarized light component periodically increases / decreases at the spatial frequency of the polarization direction modulation element 30 is emitted onto the measurement target solution (microdevice 200) through the polarization direction modulation element 30. When the excitation light EL having the polarization direction in the X direction is emitted from the light source unit 10, as in Embodiment 1, the intensities of the component having the polarization direction in the X direction and the component having the polarization direction in the Y direction in the excitation light EL periodically increase / decrease at the spatial frequency of the polarization direction modulation element 30 and are in opposite phases to each other ( Figure 4 ). Hereinafter, the excitation light EL emitted onto the measurement target solution when the excitation light EL having the polarization direction in the X direction is emitted from the light source unit 10 is referred to as “first excitation light EL”.

[0118] At the same time, when the excitation light EL having the polarization direction in the Y direction is emitted from the light source unit 10, such excitation light EL is emitted onto the measurement target solution: wherein the component of the first excitation light EL having the polarization direction in the X direction is switched to the component having the polarization direction in the Y direction, and the component of the first excitation light EL having the polarization direction in the Y direction is switched to the component having the polarization direction in the X direction ( Fig.17 ). Hereinafter, the excitation light EL emitted onto the measurement target solution when the excitation light EL having the polarization direction in the Y direction is emitted from the light source unit 10 is referred to as “second excitation light EL”.

[0119] Like the detector 50 of the first embodiment, the detector 50 of the present embodiment includes an absorption filter 52, a second polarization adjustment element 54, an imaging lens 56, and a first light receiver 58A. The configuration of the absorption filter 52 to the imaging lens 56 of the present embodiment is the same as that of the first embodiment.

[0120] As with the first photoreceptor 58A of Embodiment 1, the first photoreceptor 58A of this embodiment detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction as an image. In this embodiment, the first photoreceptor 58A detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction in the fluorescence FL light emitted from the measurement target solution when the first excitation light is emitted onto the measurement target solution (i.e., when the light source unit 10 emits the excitation light EL having the polarization direction in the X direction) (hereinafter referred to as "the spatial distribution of the first fluorescence intensity"). In addition, the first photoreceptor 58A detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction in the fluorescence FL light emitted from the measurement target solution when the second excitation light is emitted onto the measurement target solution (i.e., when the light source unit 10 emits the excitation light EL having the polarization direction in the Y direction) (hereinafter referred to as "the spatial distribution of the second fluorescence intensity"). The first photoreceptor 58A of this embodiment sends image data corresponding to the spatial distribution of the first fluorescence intensity and image data corresponding to the spatial distribution of the second fluorescence intensity to the controller 70.

[0121] The controller 70 of the present embodiment extracts a DC component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the first fluorescence intensity. In addition, the controller 70 of the present embodiment extracts a DC component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the second fluorescence intensity. The controller 70 of the present embodiment calculates the degree of polarization P of the measurement target solution based on the extracted components and the DC component, and calculates the concentration of the measurement target substance based on the degree of polarization P and the calibration curve. Like the controller 70 of Example 1, the controller 70 of the present embodiment includes an input / output device 72 to a detection controller 84. The configuration of the input / output device 72, the memory 74, the concentration calculator 80, the light source controller 82 and the detection controller 84 of the present embodiment is the same as that in Example 1.

[0122] The extractor 76 of this embodiment obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from the image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL. In this embodiment, the extractor 76 obtains the spatial distribution of the first fluorescence intensity and the spatial distribution of the second fluorescence intensity. As in Embodiment 1, the obtained spatial distribution of the first fluorescence intensity and the spatial distribution of the second fluorescence intensity include noise.

[0123] As with the extractor 76 of the first embodiment, the extractor 76 of the present embodiment extracts a direct current component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL. That is, in the present embodiment, the extractor 76 extracts a direct current component (hereinafter referred to as "first direct current component") and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as "first component") from the spatial distribution of the first fluorescence intensity. In addition, the extractor 76 extracts a direct current component (hereinafter referred to as "second direct current component") and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as "second component") from the spatial distribution of the second fluorescence intensity. As a result, as with the first embodiment, the SN ratio of the fluorescence intensity of the fluorescence FL can be improved.

[0124] Specifically, as in Embodiment 1, the extractor 76 of this embodiment performs Fourier transform and inverse Fourier transform on the spatial distribution of the first fluorescence intensity using the spatial frequency of the polarization direction modulation element 30. As a result, the following is obtained: Fig.18 In addition, the extractor 76 of the present embodiment uses the spatial frequency of the polarization direction modulation element 30 to perform Fourier transform and inverse Fourier transform on the spatial distribution of the second fluorescence intensity. As a result, the following is obtained: Fig.19 The second component and the second DC component are shown.

[0125] The polarization degree calculator 78 of this embodiment calculates the polarization degree P based on the first component and the first DC component, or the second component and the second DC component. In this embodiment, as in Embodiment 1, the maximum value of the first component (including the first DC component) and the second component (including the second DC component) corresponds to the fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL, as shown in FIG. Fig.18 and Fig.19 As shown. In addition, the minimum value of the first component (including the first DC component) and the second component (including the second DC component) corresponds to the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL. Therefore, in this embodiment, the polarization degree calculator 78 calculates the polarization degree P based on the maximum and minimum values ​​of the first component or the maximum and minimum values ​​of the second component.

[0126] In this embodiment, if Fig. 20 As shown, the first component and the second component have an opposite phase relationship, and the fluorescence intensity Ih of the fluorescence FL and the fluorescence intensity Iv of the fluorescence FL are obtained at the same position (same X coordinate) of the microchannel 220. Therefore, by comparing the fluorescence intensity Ih of the fluorescence FL and the fluorescence intensity Iv of the fluorescence FL of the first component and the second component, it is possible to easily find noise caused by foreign matter in the microchannel 220, bright spot noise of the first light receptor 58A, and the like.

[0127] Next, the detection process of this embodiment is described. As in Embodiment 1, the detection process of this embodiment includes polarization degree measurement processing (step S100) and concentration calculation processing (step S200) in sequence ( Fig.12 ). The concentration calculation process (step S200) of this embodiment is the same as that of the first embodiment, and therefore, refer to Fig.21 The polarization degree measurement process (step S100 ) of the present embodiment will be described.

[0128] In the polarization degree measurement process (step S100) of the present embodiment, first, the controller 70 controls the polarization direction of the light emitted from the first polarization adjustment element 20 (the excitation light EL emitted from the light source unit 10) to the X direction, and emits the first excitation light EL onto the measurement target solution (step S116a). Next, the controller 70 acquires image data corresponding to the spatial distribution of the first fluorescence intensity from the first light receptor 58A (step S118a). In addition, the controller 70 controls the light emitted from the first polarization adjustment element 20 to the Y direction, and emits the second excitation light EL onto the measurement target solution (step S116b). Next, the controller 70 acquires image data corresponding to the spatial distribution of the second fluorescence intensity from the first light receptor 58A (step S118b).

[0129] The controller 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction (X direction) from the fluorescence FL emitted from the measurement target solution (step S120). Specifically, the extractor 76 of the controller 70 obtains the spatial distribution of the first fluorescence intensity from the image data corresponding to the spatial distribution of the first fluorescence intensity. In addition, the extractor 76 of the controller 70 obtains the spatial distribution of the second fluorescence intensity from the image data corresponding to the spatial distribution of the second fluorescence intensity.

[0130] In addition, the controller 70 extracts a DC component and a component having the same frequency as a predetermined frequency (the spatial frequency of the polarization direction modulation element 30) from the spatial distribution of the obtained fluorescence intensity (step S130). Specifically, the extractor 76 extracts the first component and the first DC component from the spatial distribution of the first fluorescence intensity. In addition, the extractor 76 extracts the second component and the second DC component from the spatial distribution of the second fluorescence intensity. As in Embodiment 1, the spatial distribution of the fluorescence intensity is subjected to Fourier transform and inverse Fourier transform using the spatial frequency of the polarization direction modulation element 30, and the first component and the first DC component, and the second component and the second DC component are extracted.

[0131] The controller 70 calculates the polarization degree P of the measurement target solution based on the DC component and the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (step S140). Specifically, the polarization degree calculator 78 of the controller 70 calculates the polarization degree P based on the maximum and minimum values ​​of the first component (including the DC component) or the maximum and minimum values ​​of the second component (including the DC component).

[0132] As described above, the fluorescence polarization measurement device 100 of this embodiment can suppress noise and detect the polarization degree P with high measurement sensitivity, as with the fluorescence polarization measurement device 100 of Example 1. In addition, the fluorescence polarization measurement device 100 of this embodiment can suppress the influence of the intensity distribution of the excitation light EL and detect the polarization degree P with high measurement sensitivity. Since the measurement sensitivity of the polarization degree P is high, the fluorescence polarization measurement device 100 can accurately detect the measurement target substance even if the concentration of the measurement target substance is low. In addition, the fluorescence polarization measurement device 100 of this embodiment can easily detect noise caused by foreign matter in the microchannel 220, bright spot noise of the first light receiver 58A, and the like.

[0133] Example 4

[0134] In Embodiment 2, the detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction by switching the polarization direction of the fluorescence FL emitted from the second polarization adjusting element 54 to the X direction and the Z direction. However, a configuration may be adopted in which the detector 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Y direction by separating the polarized light components of the fluorescence FL.

[0135] Like the fluorescence polarization measuring device 100 of Embodiment 1, the fluorescence polarization measuring device 100 of this embodiment includes a light source unit 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detector 50, and a controller 70. The configuration of the light source unit 10 to the objective lens 42 of this embodiment is the same as that in Embodiment 1, and thus the detector 50 and the controller 70 of this embodiment are described.

[0136] like Fig. 22 As shown, the detector 50 of this embodiment includes an absorption filter 52, a second polarization adjustment element 54, two imaging lenses 56, a first light receiver 58A and a second light receiver 58B. The configuration of the absorption filter 52 of this embodiment is the same as that of the first embodiment.

[0137] The second polarization adjusting element 54 of the present embodiment is implemented as a polarization beam splitter. In the present embodiment, for the fluorescence FL incident from the -Y direction, the second polarization adjusting element 54 transmits the fluorescence FL having a polarization direction in the X direction, and reflects the fluorescence FL having a polarization direction in the Z direction to the +Z direction to become the fluorescence FL having a polarization direction in the Y direction.

[0138] The fluorescent light FL having the polarization direction in the X direction enters the first light receiver 58A through the imaging lens 56. In addition, the fluorescent light FL having the polarization direction in the Y direction enters the second light receiver 58B through the imaging lens 56.

[0139] The first light receiver 58A of this embodiment detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction as an image, as does the first light receiver 58A of Embodiment 1. The first light receiver 58A of this embodiment transmits image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction to the controller 70.

[0140] In one example, the second light receptor 58B is implemented as a charge coupled device (CCD) image sensor. The spatial distribution of the fluorescence intensity of the fluorescent light FL having the polarization direction in the Y direction is detected as an image. The second light receptor 58B transmits image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescent light FL having the polarization direction in the Y direction to the controller 70.

[0141] The controller 70 of the fluorescence polarization measuring device 100 extracts a direct current component (X-direction direct current component) and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (X-direction component) based on the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction detected by the first light receiver 58A. In addition, the controller 70 of the present embodiment extracts a direct current component (hereinafter referred to as "Y-direction direct current component") and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as "Y-direction component") based on the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Y direction detected by the second light receiver 58B. The controller 70 of the present embodiment calculates the degree of polarization P of the measurement target solution based on the extracted components and the direct current component, and calculates the concentration of the measurement target substance based on the degree of polarization P and the calibration curve.

[0142] Like the controller 70 of Examples 1 and 2, the controller 70 of this embodiment includes an input / output device 72 to a detection controller 84. The configuration of the various components of the controller 70 of this embodiment is the same as the configuration of the various components of the controller 70 of Example 2, except that the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Y direction is used instead of the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction of Example 2.

[0143] like Fig.23 As shown, in this embodiment, the maximum value of the X-direction component (including the DC component) or the Y-direction component (including the DC component) corresponds to the fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL. In addition, the minimum value of the X-direction component (including the DC component) or the Y-direction component (including the DC component) corresponds to the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL. The polarization degree P is calculated based on the maximum value of the X-direction component or the Y-direction component and the minimum value of the X-direction component or the Y-direction component. In addition, the concentration of the measurement target substance is calculated based on the calibration curve of the polarization degree P and the concentration of the measurement target substance.

[0144] Like the fluorescence polarization measurement device 100 of Example 2, the fluorescence polarization measurement device 100 of this embodiment can suppress noise and detect the polarization degree P with high measurement sensitivity. In addition, the fluorescence intensity Ih of the fluorescence FL and the fluorescence intensity Iv of the fluorescence FL are obtained at the same position (same X coordinate) of the microchannel 220, and thus, the fluorescence polarization measurement device 100 of this embodiment can easily find noise caused by foreign matter in the microchannel 220, bright spot noise of the first light receptor 58A, and the like. Furthermore, the fluorescence polarization measurement device 100 of this embodiment can measure the polarization degree P without switching any of the polarization direction of the excitation light EL emitted to the measurement target solution and the polarization direction of the fluorescence FL to be detected, and thus can shorten the measurement time.

[0145] Modification example

[0146] The embodiments have been described, but various modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure.

[0147] For example, in the above-described embodiment, the microdevice 200 includes three microchannels 220. It is sufficient that the microdevice 200 includes at least one microchannel 220. A configuration in which the microdevice 200 includes a plurality of microchannels 220 may be adopted.

[0148] A configuration may be adopted in which the first light receptor 58A and the second light receptor 58B are implemented as complementary metal oxide semiconductor (CMOS) image sensors. In addition, the first light receptor 58A and the second light receptor 58B are not limited to imaging elements.

[0149] In Embodiments 1 to 4, the polarization direction modulation element 30 continuously modulates the polarization direction of the excitation light EL at a predetermined frequency along the X direction. However, it is sufficient that the polarization direction modulation element 30 modulates the polarization direction of the excitation light EL at a predetermined frequency, and the polarization direction does not necessarily need to be continuously modulated. It is sufficient that the polarization direction modulation element 30 modulates the polarization direction of the excitation light EL so that the controller 70 can extract a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the fluorescence intensity.

[0150] In the detection process of Example 2, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction is acquired, and then image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction is acquired (steps S112a, S114a, and then steps S112b, S114b). However, in Example 2, a configuration may be adopted in which image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction is acquired, and then image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction is acquired (steps S112b, S114b, and then steps S112a, S114a).

[0151] In the detection process of Example 3, image data corresponding to the spatial distribution of the first fluorescence intensity is acquired, and then image data corresponding to the spatial distribution of the second fluorescence intensity is acquired (steps S116a, S118a, and then steps S116b, S118b). In Example 3, a configuration may be adopted in which image data corresponding to the spatial distribution of the second fluorescence intensity is acquired, and then image data corresponding to the spatial distribution of the first fluorescence intensity is acquired (steps S116b, S118b, and then steps S116a, S118a).

[0152] A configuration may be adopted in which the spatial distribution of fluorescence intensity is detected from a plurality of image data in the polarization degree measurement process.

[0153] Furthermore, a configuration may be adopted in which the background fluorescence intensity is detected without emitting the excitation light EL in the polarization degree measurement process. A configuration may be adopted in which the background fluorescence intensity is subtracted from the spatial distribution of the detected fluorescence intensity, and then a direct current component and a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 are extracted. As a result, dark noise of the first light receptor 58A and the second light receptor 58B, noise from the circuits of the first light receptor 58A and the second light receptor 58B, and the like can be suppressed.

[0154] A configuration may be adopted in which, in the fluorescence polarization measuring apparatus 100, the excitation light EL is emitted to the reference sample, and the reference sample emits fluorescence to position the polarization direction modulation element 30 and the first light receptor 58A (the first light receptor 58A and the second light receptor 58B) so that the polarization direction of the excitation light EL coincides with the positions of the maximum and minimum values ​​of the fluorescence intensity ( Fig.24 ).

[0155] Some example embodiments have been described above for purposes of illustration. Although specific embodiments have been given in the above discussion, those skilled in the art will recognize that changes may be made in form and detail without departing from the broader spirit and scope of the present invention. Therefore, the specification and drawings are to be regarded as illustrative rather than restrictive. Therefore, this detailed description should not be regarded as limiting, and the scope of the present invention is limited only by the claims included therein and the full range of equivalents to which such claims are entitled.

Claims

1. A fluorescence polarization measuring device, comprising: a light source unit that emits linearly polarized excitation light; a polarization direction modulation element that spatially modulates the polarization direction of the excitation light at a predetermined frequency, and emits the excitation light whose polarization direction is spatially modulated at the predetermined frequency onto a measurement target solution; a detector that detects a spatial distribution of fluorescence intensity of fluorescence having a polarization direction in a predetermined direction from fluorescence emitted from the measurement target solution due to the excitation light whose polarization direction is spatially modulated at the predetermined frequency; as well as A controller extracts a direct current component and a component having the same frequency as the predetermined frequency from the spatial distribution of the detected fluorescence intensity, and calculates a polarization degree of the measurement target solution based on the extracted components and the direct current component. 2 . The fluorescence polarization measuring device according to claim 1 , wherein the light source unit emits while switching the polarization direction of the excitation light to two orthogonal directions.

3. The fluorescence polarization measuring device according to claim 1, wherein the detector includes a polarization adjustment element and a photoreceptor, wherein the polarization adjustment element selects fluorescence having a polarization direction in the predetermined direction from the fluorescence emitted from the measurement target solution, and the photoreceptor detects the spatial distribution of the fluorescence intensity of the fluorescence having a polarization direction in the predetermined direction.

4. The fluorescence polarization measuring device according to claim 3, wherein the polarization adjusting element switches the fluorescence having a polarization direction in the predetermined direction into the fluorescence having a polarization direction parallel to the polarization direction of the excitation light emitted from the light source unit and the fluorescence having a polarization direction perpendicular to the polarization direction of the excitation light emitted from the light source unit. The fluorescence polarization measuring device according to claim 3 , wherein the polarization adjusting element is a polarization beam splitter. 6 . The fluorescence polarization measurement device according to claim 1 , wherein the measurement target solution includes a measurement target substance, a fluorescent labeled derivative obtained by fluorescently labeling the measurement target substance, and an antibody that specifically binds to the measurement target substance.

7. A method for measuring polarization degree, comprising: emitting excitation light whose polarization direction has been spatially modulated at a predetermined frequency onto a measurement target solution; detecting a spatial distribution of fluorescence intensity of fluorescence having a polarization direction in a predetermined direction from fluorescence emitted from the measurement target solution; extracting a DC component and a component having the same frequency as the predetermined frequency from the detected spatial distribution of the fluorescence intensity; as well as Based on the extracted components and the DC component, the polarization degree of the measurement target solution is calculated.

Images