Fluorescence polarization measuring device and method for measuring polarization degree

Abstract

To provide a fluorescence polarization measuring device having high sensitivity of measurement, and a method for measuring the degree of polarization.SOLUTION: A fluorescence polarization measuring device 100 comprises a light source unit 10, a polarization direction modulation element 30, a detection unit 50, and a control unit 70. The light source unit 10 emits linearly polarized exciting light EL. The polarization direction modulation element 30 spatially modulates the polarization direction of the exciting light EL with a prescribed frequency and emits the exciting light EL having had its polarization direction spatially modulated with the prescribed frequency. The detection unit 50 detects, by the exciting light EL having had its polarization direction spatially modulated with the prescribed frequency, a spatial distribution of fluorescence intensity of fluorescent light FL having been polarized in a prescribed direction from among fluorescent lights FL emitted from a solution as the object to be measured. The control unit 70 extracts a component having the same frequency as the prescribed frequency and a direct-current component from the spatial distribution of the detected fluorescence intensity, and determines the degree of polarization of the solution to be measured, on the basis of the extracted component and direct-current component.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a fluorescence polarization measurement device and a method for measuring the degree of polarization. [Background technology]

[0002] As an immunoassay method using fluorescence, a fluorescence polarization immunoassay (FPIA) is known that detects a substance to be measured by utilizing an antigen-antibody reaction. For example, Patent Document 1 discloses a method for determining the concentration of a measured antigen (substance to be measured) from the polarization degree of the measured fluorescence.

[0003] Furthermore, Patent Document 2 discloses a fluorescence measuring device equipped with a modulation means for periodically (temporally) rotating the polarization plane of the fluorescence emitted from a sample by 90°. In Patent Document 2, the modulation means rotates the polarization plane of the fluorescence, so that it is possible to directly measure the fluorescence intensity of the fluorescence having a polarization direction parallel to the polarization direction of the excitation light and the fluorescence intensity of the fluorescence having a polarization direction perpendicular to the polarization direction of the excitation light without rotating a polarizer. As a result, the fluorescence measuring device of Patent Document 2 suppresses the influence of the polarization characteristics of the detector, filter, etc. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 3-103765 [Patent Document 2] U.S. Patent No. 6,970,241 Summary of the Invention [Problem to be solved by the invention]

[0005] In the fluorescence measuring device of Patent Document 2, the influence of the polarization characteristics of the detector, filter, etc. can be suppressed, but when the fluorescence intensity is low, it is difficult to obtain a fluorescence intensity with a sufficient SN ratio (signal-to-noise ratio).

[0006] The present disclosure has been made in consideration of the above circumstances, and has an object to provide a fluorescence polarization measurement device and a method for measuring the degree of polarization with high measurement sensitivity. [Means for solving the problem]

[0007] In order to achieve the above object, a fluorescence polarization measurement device according to a first aspect of the present disclosure comprises: 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, the polarization direction of which has been spatially modulated at the predetermined frequency, to a measurement target solution; a detection unit that detects a spatial distribution of fluorescence intensity of the fluorescence having a polarization direction in a predetermined direction among the fluorescence emitted from the measurement target solution by the excitation light whose polarization direction has been spatially modulated at the predetermined frequency; and and a control unit that extracts a component having the same frequency as the predetermined frequency and a DC component from the spatial distribution of the detected fluorescence intensity, and determines the degree of polarization of the measurement target solution based on the extracted component and the DC component.

[0008] A method for measuring a degree of polarization according to a second aspect of the present disclosure includes: Irradiating a solution to be measured with excitation light whose polarization direction has been spatially modulated at a predetermined frequency; detecting a spatial distribution of the fluorescence intensity of the fluorescence having a polarization direction in a predetermined direction among the fluorescence emitted from the solution to be measured; extracting a component having the same frequency as the predetermined frequency and a direct current component from the spatial distribution of the detected fluorescence intensity; and determining the degree of polarization of the measurement target solution based on the extracted component and the DC component. Effect of the Invention

[0009] According to the present disclosure, a component having the same frequency as a predetermined frequency for spatially modulating the polarization direction of excitation light is extracted from the spatial distribution of fluorescence intensity, thereby improving measurement sensitivity. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing a fluorescence measuring device according to a first embodiment. [Diagram 2] FIG. 1 is a plan view showing a microdevice according to a first embodiment. [Diagram 3] 3 is a cross-sectional view of the microdevice shown in FIG. 2 taken along line AA. [Figure 4] 4 is a diagram showing the optical axis of the polarization direction modulation element and the intensity of the polarized component of the excitation light in the first embodiment. FIG. [Diagram 5] 3 is a diagram showing the alignment direction of liquid crystal molecules and the optical axis of a polarization direction modulation element in accordance with the first embodiment. FIG. [Figure 6] 2 is a block diagram showing the configuration of a control unit according to the first embodiment. FIG. [Figure 7] FIG. 4 is a diagram showing fluorescence intensity determined from image data according to the first embodiment. [Figure 8] 4 is a diagram showing a component having the same frequency as the spatial frequency of the polarization direction modulation element and a direct current component according to the first embodiment. FIG. [Figure 9] FIG. 2 is a diagram for explaining 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 in the first embodiment. [Figure 10] FIG. 2 is a diagram illustrating a hardware configuration of a control unit according to the first embodiment. [Figure 11] FIG. 2 is a diagram illustrating the intensity distribution of excitation light according to the first embodiment. [Figure 12] 4 is a flowchart showing a detection process according to the first embodiment. [Figure 13] 5 is a flowchart showing a polarization degree measurement process according to the first embodiment. [Figure 14] 10 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 the second embodiment. FIG. [Figure 15] FIG. 10 is a diagram showing the intensity of a polarized component of excitation light, and the X-direction component, X-direction DC component, Z-direction component, and Z-direction DC component of fluorescence according to the second embodiment. [Figure 16] 10 is a flowchart showing a polarization degree measurement process according to the second embodiment. [Figure 17] FIG. 11 is a diagram illustrating the intensities of polarized components of second excitation light according to the third embodiment. [Figure 18] FIG. 11 is a diagram showing the intensity of the polarized component of the first excitation light, and the first component and first DC component of the fluorescence in the third embodiment. [Figure 19] FIG. 13 is a diagram showing the intensity of the polarized component of the second excitation light, the second component of the fluorescence, and the second DC component in the third embodiment. [Figure 20] FIG. 11 is a diagram showing a first component, a first DC component, a second component, and a second DC component of fluorescence according to the third embodiment. [Figure 21] 11 is a flowchart showing a polarization degree measurement process according to the third embodiment. [Figure 22] FIG. 11 is a schematic diagram showing a fluorescence measuring device according to a fourth embodiment. [Diagram 23] FIG. 11 is a diagram showing the intensity of a polarized component of excitation light, and the X-direction component, X-direction DC component, Y-direction component, and Y-direction DC component of fluorescence according to the fourth embodiment. [Figure 24] 13A and 13B are diagrams illustrating the optical axis of a polarization direction modulation element, the intensity of the polarized component of excitation light, and the intensity of fluorescence according to a modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, a fluorescence polarization measurement device according to an embodiment will be described with reference to the drawings.

[0012] <Embodiment 1> 1 to 12, a fluorescence polarization measurement device 100 according to this embodiment will be described. The fluorescence polarization measurement device 100 is used for detecting a measurement target substance contained in a measurement target solution, for example, by using a fluorescence polarization immunoassay method.

[0013] As shown in FIG. 1, the fluorescence polarization measurement device 100 includes a light source unit 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detection unit 50, and a control unit 70. The light source unit 10 emits linearly polarized excitation light EL. The polarization direction modulation 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, the polarization direction of which is spatially modulated at a predetermined frequency, is irradiated onto a measurement target solution introduced into a microchannel 220 of a microdevice 200 described later via the dichroic mirror 40 and the objective lens 42. The detection unit 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction among the fluorescence FL emitted from the measurement target solution. The control unit 70 controls each unit of the fluorescence polarization measurement device 100. The control unit 70 also determines the polarization degree P of the measurement target solution from the spatial distribution of the detected fluorescence intensity. Furthermore, the control unit 70 determines the concentration of the substance to be measured from the degree of polarization P of the solution to be measured.

[0014] For ease of understanding, in this specification, the left direction (left direction on the paper) of the fluorescence polarization measurement device 100 in Fig. 1 is described as the +Z direction, the upward direction (upward on the paper) as the +Y direction, and the direction perpendicular to the +Y and +Z directions (toward the viewer on the paper) as the +X direction. The excitation light EL is light that excites a fluorescent labeling derivative (described later) and causes the fluorescent labeling 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 detection unit 50 form an observation optical system.

[0015] First, the measurement target solution and the microdevice 200 will be described. The measurement target solution includes a measurement target substance, a fluorescently labeled derivative, and an antibody. The measurement target substance is a detection target of the fluorescence polarization measurement device 100. The measurement target substance may be any compound that can be detected by a fluorescent immunoassay. Examples of the measurement target substance include antibiotics, physiologically active substances, and mycotoxins. Specific examples of the measurement target substance include prostaglandin E2, β-lactoglobulin, chloramphenicol, and deoxynilebanol. The fluorescently labeled derivative is a derivative in which the measurement target substance is fluorescently labeled with a fluorescent substance. The fluorescently labeled derivative is obtained by binding the measurement target substance to a fluorescent substance using a known method. The fluorescent substance is fluorescein (wavelength of excitation light EL: 494 nm, wavelength of fluorescence FL: 521 nm), rhodamine B (wavelength of excitation light EL: 550 nm, wavelength of fluorescence FL: 580 nm), and the like. The antibody specifically binds to the measurement target substance by an antigen-antibody reaction. The antibody can be obtained, for example, by injecting the substance to be measured into a host animal (e.g., a mouse or a cow) and then collecting and purifying the antibody produced in the blood of the host animal. Alternatively, commercially available antibodies can be used.

[0016] The substance to be measured and the fluorescent labeling derivative bind specifically to the antibody through an antigen-antibody reaction. In the fluorescence polarization immunoassay, the polarization degree P of the fluorescence FL emitted by the fluorescent labeling derivative contained in the solution to be measured is obtained, and the concentration of the substance to be measured is calculated from the polarization degree P and a calibration curve that has been prepared in advance.

[0017] Since the fluorescent label derivative not bound to the antibody moves violently in the measurement solution, when the polarized excitation light EL is irradiated to the fluorescent label derivative not bound to the antibody, the fluorescence FL is emitted randomly. On the other hand, the fluorescent label derivative bound to the antibody is restricted in its movement in the measurement solution, so when the polarized excitation light EL is irradiated to the fluorescent label derivative bound to the antibody, the fluorescence FL is emitted polarized in the polarization direction of the excitation light EL. The fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL and the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL are measured, and the polarization of the fluorescence intensity is calculated as the polarization degree P. Since the polarization degree P depends on the amount of the fluorescent label derivative bound to the antibody, the concentration of the substance to be measured can be obtained from the obtained polarization degree P and a calibration curve created in advance. The polarization degree P is expressed as P = (Ih-Iv) / (Ih+Iv).

[0018] 2 and 3, 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 of the microchannels 220. The microdevice 200 is placed on a stage ST of the fluorescence polarization measurement apparatus 100.

[0019] The first substrate 202 of the microdevice 200 is a flat quartz glass substrate. Excitation light EL, whose polarization direction is spatially modulated at a predetermined frequency, enters the microdevice 200 from the first substrate 202. The excitation light EL is irradiated onto the measurement region S shown in FIG. 2 from the -Z direction, and is perpendicularly incident on the main surface 202a of the first substrate 202.

[0020] The second substrate 204 of the microdevice 200 is a flat substrate. The second substrate 204 is made of a material with low autofluorescence. The second substrate 204 is made of, for example, 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 therebetween.

[0021] The partition 206 of the microdevice 200 is sandwiched between the first substrate 202 and the second substrate 204 to form a microchannel 220. The partition 206 is made of a material with low autofluorescence. The partition 206 is preferably made of a material that absorbs light such as excitation light EL and fluorescence FL. In this embodiment, the partition 206 is formed integrally with the second substrate 204.

[0022] The microchannel 220 of the microdevice 200 extends parallel to the X direction in the measurement region S. The width of the microchannel 220 in the measurement region S is, for example, 200 μm. Each of the microchannels 220 has two openings 222 penetrating the second substrate 204 and the partition wall 206. A solution to be measured is introduced or discharged through the openings 222.

[0023] Next, we will explain each part of the fluorescence polarization measurement device 100. The light source part 10 of the fluorescence polarization measurement device 100 has a light source 12, a condenser lens 14, an iris 16, a collimator 18, a first polarization adjustment element 20, and an excitation light filter 22, as shown in FIG.

[0024] The light source 12 emits light including excitation light EL in the +Z direction. The light source 12 is composed of, for example, an LED (Light Emitting Diode) element. The light emitted from the light source 12 is condensed by a condenser lens 14 and then passes through an iris 16. The iris 16 reduces the influence of external light (light other than the light emitted from the light source 12). The light that passes through the iris 16 is incident on a collimator 18.

[0025] The collimator 18 converts the incident light into parallel light. The parallel light is incident on the first polarization adjusting element 20.

[0026] The first polarization adjustment element 20 selects light having a predetermined polarization direction from among the incident light, and emits light having the predetermined polarization direction. The light emitted from the first polarization adjustment element 20 is incident on an excitation light filter 22. In this 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. That is, the light including the excitation light EL that is incident on the first polarization adjustment element 20 is emitted as linearly polarized light having a polarization direction in the X direction, and is incident on 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. The excitation light filter 22 is, for example, a band-pass filter.

[0027] Therefore, the excitation light EL having the polarization direction in the X direction is emitted in the +Z direction from the light source section 10. The excitation light EL having the polarization direction in the X direction is incident on the polarization direction modulation element 30.

[0028] 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. The polarization direction modulation element 30 is, for example, a half-wave plate whose optical axis changes continuously along the X direction. In the polarization direction modulation element (half-wave plate) 30 of this embodiment, as shown in FIG. 4, 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, and 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 output from the polarization direction modulation element 30 increase and decrease periodically at the same frequency as the predetermined frequency at which the polarization direction is modulated by the polarization direction modulation element 30, as shown in FIG. 4. The intensities of the component having the polarization direction in the X direction and the component having the polarization direction in the Y direction are in opposite phase (opposite phase). The polarization direction modulation element 30 emits the spatially modulated excitation light EL to a dichroic mirror 40 .

[0029] The polarization direction modulation element 30 is formed, for example, from a liquid crystal cell in which nematic liquid crystal is homogeneously aligned. In this liquid crystal cell, the alignment direction of the liquid crystal molecules is continuously rotated by 180° along the X direction as shown in Fig. 5. The retardation value of the liquid crystal cell is 1 / 2 the wavelength of the excitation light EL.

[0030] 1, the dichroic mirror 40 of the fluorescence polarization measurement device 100 transmits the spatially modulated excitation light EL in the +Z direction, and reflects the fluorescence FL emitted from the microdevice 200 to the detection unit 50 (+Y direction). The objective lens 42 of the fluorescence polarization measurement device 100 collects the excitation light EL and the fluorescence FL that have transmitted through the dichroic mirror 40.

[0031] The excitation light EL spatially modulated by the polarization direction modulation element 30 is irradiated onto the measurement region S of the microdevice 200 via the dichroic mirror 40 and the objective lens 42. This causes fluorescence FL to be emitted from the measurement target solution (fluorescence labeled derivative) introduced into the microchannel 220 of the microdevice 200. The fluorescence FL travels in the +Y direction via the objective lens 42 and the dichroic mirror 40, and enters the detection unit 50 (an absorption filter 52, described later).

[0032] The detection section 50 of the fluorescence polarization measurement device 100 is disposed on the +Y side of the dichroic mirror 40. The detection section 50 has an absorption filter 52, a second polarization adjustment element 54, an imaging lens 56, and a first light receiving section 58A.

[0033] The absorption filter 52 separates the fluorescence FL emitted from the microdevice 200 from scattered light, leaked light, and the like, and transmits the fluorescence FL. The absorption filter 52 is, for example, a band-pass filter. The fluorescence FL emitted from the absorption filter 52 is incident on the second polarization adjustment element 54.

[0034] The second polarization adjusting element 54 selects fluorescence FL having a predetermined polarization direction from the incident fluorescence FL light, and emits fluorescence FL having the predetermined polarization direction. In this embodiment, the predetermined direction in the second polarization adjusting element 54 is the X direction, and the second polarization adjusting element 54 is a polarizing plate. Therefore, fluorescence FL having the X direction polarization direction is emitted from the second polarization adjusting element 54. The fluorescence FL having the X direction polarization direction is incident on the first light receiving unit 58A via the imaging lens 56.

[0035] The first light receiving unit 58A is, for example, a CCD (Charge Coupled Device) image sensor. The first light receiving unit 58A detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (polarization direction in the X direction). The first light receiving unit 58A generates image data according to the spatial distribution of the fluorescence intensity of the fluorescence FL, and transmits the image data to the control unit 70. Note that the main surface 202a of the first substrate 202 of the microdevice 200 and the imaging surface of the first light receiving unit 58A are in an imaging relationship.

[0036] The control unit 70 of the fluorescence polarization measurement device 100 extracts a predetermined frequency at which the polarization direction modulation element 30 spatially modulates the polarization direction of the excitation light EL having the polarization direction in the X direction along the X direction, a component having the same frequency, and a direct current component, from the spatial distribution of the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (polarization direction in the X direction). The control unit 70 calculates the polarization degree P of the measurement target solution based on the extracted component and the direct current component, and further calculates the concentration of the measurement target substance from the polarization degree P and the calibration curve. As shown in FIG. 6, the control unit 70 has an input / output unit 72, a storage unit 74, an extraction unit 76, a polarization degree calculation unit 78, and a concentration calculation unit 80. The control unit 70 further has a light source control unit 82 and a detection control unit 84. In the following, the predetermined frequency at which the polarization direction modulation element 30 spatially modulates the polarization direction of the excitation light EL having the polarization direction in the X direction is referred to as the spatial frequency of the polarization direction modulation element 30.

[0037] The input / output unit 72 inputs and outputs signals, data, etc. between the control unit 70 and each unit.

[0038] The storage unit 74 stores programs, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL, data representing a calibration curve of the degree of polarization P and the concentration of the substance to be measured, and the like.

[0039] The extraction unit 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 extraction unit 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the obtained spatial distribution of the fluorescence intensity of the fluorescence FL.

[0040] Here, 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 will be described.

[0041] In this embodiment, the excitation light EL spatially modulated by the polarization direction modulation element 30 is irradiated onto the measurement target solution (microdevice 200). As shown in FIG. 4, the excitation light EL spatially modulated by the polarization direction modulation element 30 has a component having a polarization direction in the X direction and a component having a polarization direction in the Y direction, whose intensity increases and decreases periodically at the same frequency as the spatial frequency of the polarization direction modulation element 30. Meanwhile, the detection unit 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 a periodicity similar to that of 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, the spatial distribution of the fluorescence intensity of the fluorescence FL obtained from the image data is a spatial distribution containing a lot of noise due to stray light, vibration, and variations in the sensitivity of the light receiving unit, as shown in FIG. 7. It is difficult to directly obtain the polarization degree P from such a spatial distribution of the fluorescence intensity with a large amount of noise.

[0042] Therefore, the extraction unit 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the spatial distribution of the fluorescence intensity of the fluorescence FL that has been found. Since the spatial frequency of the noise contained in the found spatial distribution of the fluorescence intensity of the fluorescence FL is different from the spatial frequency of the polarization direction modulation element 30, the S / N ratio of the fluorescence intensity of the fluorescence FL can be improved by extracting a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 from the found spatial distribution of the fluorescence intensity of the fluorescence FL. Note that extracting a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component can also be said to be determining a function that represents a component having the same frequency as the spatial frequency of the polarization direction modulation element 30.

[0043] Specifically, the extraction unit 76 performs a 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, and then performs an inverse Fourier transform to obtain a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a direct current component (DC offset), as shown in FIG. 8.

[0044] The polarization degree calculation unit 78 obtains the polarization degree P from the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and the DC component obtained by the extraction unit 76. 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 irradiated to the measurement target solution are in an inverse phase relationship. In addition, the detection unit 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. Therefore, 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 the polarization direction parallel to the polarization direction of the excitation light EL, as shown in FIG. 9. 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 the polarization direction perpendicular to the polarization direction of the excitation light EL. Therefore, the polarization degree calculation unit 78 obtains the polarization degree P from 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. Since the signal-to-noise ratio of the fluorescence intensity of the fluorescence FL is high, the fluorescence polarization measurement device 100 can detect the degree of polarization P with high measurement sensitivity.

[0045] Concentration calculation section 80 determines the concentration of the substance to be measured from the degree of polarization P determined by degree of polarization calculation section 78 and a calibration curve of the degree of polarization P and the concentration of the substance to be measured.

[0046] The light source control unit 82 controls the light source unit 10 (light source 12). The detection control unit 84 controls the detection unit 50 (first light receiving unit 58A).

[0047] 10 shows the hardware configuration of the control unit 70. The control unit 70 is composed of a CPU (Central Processing Unit) 92, a ROM (Read Only Memory) 93, a RAM (Random Access Memory) 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 each unit. The functions of the control unit 70 are realized by the execution of the program by the CPU 92.

[0048] As described above, because components having the same frequency as the spatial frequency of the polarization direction modulation element 30 are extracted from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL, the fluorescence polarization measurement device 100 can suppress the influence of noise and improve the measurement sensitivity of the degree of polarization P of the fluorescence polarization measurement device 100. Furthermore, even when the intensity of the excitation light EL irradiated to the measurement target solution is non-uniform as shown in Fig. 11, the fluorescence polarization measurement device 100 can obtain the degree of polarization P while suppressing the influence of the intensity distribution of the excitation light EL. Furthermore, because the measurement sensitivity of the degree of polarization 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.

[0049] Furthermore, the maximum value among the components 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 among the components 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 obtain the degree of polarization P from one image data without switching either the polarization direction of the excitation light EL irradiated to the measurement target solution or the polarization direction of the fluorescence FL to be detected, thereby shortening the measurement time.

[0050] 12 and 13, the detection process (i.e., the method of detecting a substance to be measured) of the fluorescence polarization measurement device 100 will be described. As shown in Fig. 11, the detection process is performed in the order of a polarization degree measurement process (step S100) for determining the polarization degree P, and a concentration calculation process (step S200) for detecting the concentration of the substance to be measured.

[0051] The polarization degree measurement process (step S100) will be described with reference to FIG. 13. In the polarization degree measurement process (step S100), the polarization degree P of the measurement target solution is obtained from the fluorescence FL emitted by the measurement target solution introduced into the microchannel 220 of the microdevice 200. First, the control unit 70 controls the light source 12 to irradiate the measurement target solution introduced into the microchannel 220 of the microdevice 200 placed on the stage ST with the excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency (step S110). The light including the excitation light EL emitted from the light source 12 is converted into the excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency (spatial frequency of the polarization direction modulation element 30) via the first polarization adjustment element 20, the polarization direction modulation element 30, etc. Then, the excitation light EL whose polarization direction has been spatially modulated at the predetermined frequency is irradiated onto the measurement target solution. As a result, the fluorescence FL is emitted from the measurement target solution.

[0052] Next, the control unit 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction (X direction) among the fluorescence FL emitted from the solution to be measured (step S120). Specifically, the fluorescence FL emitted from the solution to be measured is incident on the first light receiving unit 58A as the fluorescence FL having a polarization direction in a predetermined direction (X direction) via the dichroic mirror 40, the second polarization adjustment element 54, etc. The extraction unit 76 of the control unit 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 receiving unit 58A.

[0053] Furthermore, the control unit 70 extracts a component having the same frequency as a predetermined frequency (spatial frequency of the polarization direction modulation element 30) and a DC component from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL (step S130). Specifically, the extraction unit 76 of the control unit 70 performs a Fourier transform on the spatial distribution of the fluorescence intensity using the spatial frequency of the polarization direction modulation element 30, and then performs an inverse Fourier transform. This makes it possible to extract a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component. The detected spatial distribution of the fluorescence intensity contains noise in addition to the fluorescence intensity. Since the spatial frequency of the noise and the spatial frequency of the polarization direction modulation element 30 are different, the signal-to-noise ratio of the fluorescence intensity of the fluorescence FL can be improved 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.

[0054] The control unit 70 determines the degree of polarization P of the measurement target solution based on the component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and the DC component (step S140). Specifically, the polarization degree calculation unit 78 of the control unit 70 determines the degree of polarization P by taking 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.

[0055] Returning to Fig. 12, the concentration calculation process (step S200) will be described. In the concentration calculation process (step S200), the concentration of the target substance contained in the target solution is calculated from the degree of polarization P calculated in step S140 and a calibration curve measured in advance. Specifically, the concentration calculation section 80 of the control section 70 calculates the concentration of the target substance from the degree of polarization P calculated by the degree of polarization calculation section 78 and a calibration curve of the degree of polarization P and the concentration of the target substance. When the concentration calculation process (step S200) ends, the detection process ends.

[0056] As described above, the fluorescence polarization measurement device 100 extracts components having the same frequency as a predetermined frequency for spatially modulating the excitation light EL from the spatial distribution of the fluorescence intensity of the detected fluorescence FL, and can therefore detect the degree of polarization P with high measurement sensitivity while suppressing noise. Furthermore, the fluorescence polarization measurement device 100 can detect the degree of polarization P with high measurement sensitivity while suppressing the influence of the intensity distribution of the excitation light EL. Because the fluorescence polarization measurement device 100 has high measurement sensitivity for the degree of polarization P, it can accurately detect the substance to be measured even if the concentration of the substance to be measured is low.

[0057] Furthermore, since the maximum value among the components 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 among the components having the same frequency as the spatial frequency of the polarization direction modulation element 30 corresponds to the fluorescence intensity Iv, the fluorescence polarization measuring device 100 can obtain the degree of polarization P from one image data.

[0058] <Embodiment 2> In the first embodiment, the detection unit 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction. The detection unit 50 may detect the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction.

[0059] Similar to the fluorescence polarization measurement device 100 of embodiment 1, the fluorescence polarization measurement device 100 of this embodiment includes a light source section 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detection section 50, and a control section 70. Since the configurations from the light source section 10 to the objective lens 42 of this embodiment are similar to those of embodiment 1, only the detection section 50 and control section 70 of this embodiment will be described.

[0060] The detection unit 50 of this embodiment has an absorption filter 52, a second polarization adjustment element 54, an imaging lens 56, and a first light receiving unit 58A, similar to the detection unit 50 of embodiment 1. The configurations of the absorption filter 52 and the imaging lens 56 of this embodiment are similar to those of embodiment 1.

[0061] The second polarization adjustment element 54 of this embodiment switches the polarization direction of the fluorescence FL emitted from the second polarization adjustment element 54 between the X direction and the Z direction. The fluorescence FL emitted from the second polarization adjustment element 54 of this embodiment is incident on the first light receiving unit 58A through the imaging lens 56. The second polarization adjustment element 54 of this embodiment is a TN liquid crystal cell, a rotatably held polarizing plate, or the like. The second polarization adjustment element 54 of this embodiment is controlled by the detection control unit 84 of the control unit 70. Note that the light source unit 10 emits the excitation light EL having a polarization direction in the X direction, and the fluorescence FL travels in the +Y direction, so the second polarization adjustment element 54 of this embodiment switches the fluorescence FL emitted from the second polarization adjustment element 54 to the fluorescence FL having a polarization direction horizontal to the polarization direction of the excitation light EL emitted from the light source unit 10, and the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL emitted from the light source unit 10.

[0062] The first light receiving unit 58A of this embodiment detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction, similar to the first light receiving unit 58A of embodiment 1. Moreover, the first light receiving unit 58A of this embodiment detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction. The first light receiving unit 58A of this embodiment transmits, to the control unit 70, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction and image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction.

[0063] The control unit 70 of this embodiment extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a direct current component from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. The control unit 70 of this embodiment also extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a direct current component from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction. The control unit 70 of this embodiment calculates the degree of polarization P of the measurement target solution based on the extracted component and the direct current component. Furthermore, the control unit 70 of this embodiment calculates the concentration of the measurement target substance from the degree of polarization P and the calibration curve. The control unit 70 of this embodiment has an input / output unit 72 to a detection control unit 84, similar to the control unit 70 of the first embodiment. The configurations of the input / output unit 72, the storage unit 74, the concentration calculation unit 80, the light source control unit 82, and the detection control unit 84 of this embodiment are similar to those of the first embodiment.

[0064] The extraction unit 76 of the present embodiment obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from 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. The obtained spatial distribution of the fluorescence intensity of the fluorescence FL contains noise, as in the first embodiment.

[0065] The extraction unit 76 of this embodiment, like the extraction unit 76 of the first embodiment, extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the spatial distribution of the fluorescence intensity of the obtained fluorescence FL. That is, in this embodiment, the extraction unit 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as the X-direction component) and a DC component (hereinafter referred to as the X-direction DC component) from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. In addition, the extraction unit 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as the Z-direction component) and a DC component (hereinafter referred to as the Z-direction DC component) from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction. This makes it possible to improve the S / N ratio of the fluorescence intensity of the fluorescence FL, like the first embodiment.

[0066] Specifically, the extraction unit 76 of this embodiment performs a Fourier transform and an inverse Fourier transform on the spatial distribution of the fluorescence intensity of the fluorescence FL, similarly to the first embodiment, by using the spatial frequency of the polarization direction modulation element 30. As a result, an X-direction component, an X-direction DC component, a Z-direction component, and a Z-direction DC component are obtained as shown in FIG.

[0067] The polarization degree calculation unit 78 of this embodiment obtains the polarization degree P from 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. In this embodiment, as in the first embodiment, the excitation light EL having the spatial distribution of the intensity of the polarization component shown in FIG. 4 is irradiated onto the measurement target solution. Therefore, as shown in FIG. 15, the intensity of the polarization component of the excitation light EL is synchronized with the X-direction component and the Z-direction component, 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 calculation unit 78 of this embodiment obtains the polarization degree P from 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.

[0068] 15, in this embodiment, 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 micro-channel 220. Therefore, by comparing the fluorescence intensity Ih of the fluorescence FL and the fluorescence intensity Iv of the fluorescence FL at a plurality of positions, noise due to foreign matter in the micro-channel 220, bright spot noise of the first light receiving unit 58A, and the like can be easily found.

[0069] Next, the detection process of this embodiment will be described. As in the first embodiment, the detection process of this embodiment is performed in the order of a degree of polarization measurement process (step S100) and a concentration calculation process (step S200) (FIG. 12). Since the concentration calculation process (step S200) of this embodiment is the same as in the first embodiment, the degree of polarization measurement process (step S100) of this embodiment will be described with reference to FIG. 16.

[0070] In the polarization degree measurement process (step S100) of this embodiment, the control unit 70 first controls the polarization direction of the fluorescence FL emitted from the second polarization adjustment element 54 to the X direction, and irradiates the measurement target solution with excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency (step S112a). Next, the control unit 70 acquires image data from the first light receiving unit 58A according 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 (step S114a). Furthermore, the control unit 70 controls the polarization direction of the fluorescence FL emitted from the second polarization adjustment element 54 to the Z direction, and irradiates the measurement target solution with excitation light EL whose polarization direction has been spatially modulated at a predetermined frequency (step S112b). Then, the control unit 70 acquires image data from the first light receiving unit 58A according 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 (step S114b).

[0071] The control unit 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in predetermined directions (X direction and Z direction) among the fluorescence FL emitted from the solution to be measured (step S120). Specifically, the extraction unit 76 of the control unit 70 obtains the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction from image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction. The extraction unit 76 of the control unit 70 also obtains the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction from image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction.

[0072] Furthermore, the control unit 70 extracts a component having the same frequency as a predetermined frequency (spatial frequency of the polarization direction modulation element 30) and a DC component from the spatial distribution of the obtained fluorescence intensity (step S130). Specifically, the extraction unit 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 the polarization direction in the X direction. The extraction unit 76 also extracts a Z-direction component and a Z-direction DC component from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction. The X-direction component, the X-direction DC component, and the Z-direction component and the Z-direction DC component are extracted by performing a Fourier transform and an inverse Fourier transform on the spatial distribution of the fluorescence intensity using the spatial frequency of the polarization direction modulation element 30, as in the first embodiment.

[0073] The control unit 70 determines the degree of polarization 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 degree of polarization calculation unit 78 of the control unit 70 determines the degree of polarization P from the maximum value of the X direction component (including DC component) or the Z direction component (including DC component) and the minimum value of the X direction component (including DC component) or the Z direction component (including DC component).

[0074] As described above, the fluorescence polarization measurement apparatus 100 of this embodiment, like the fluorescence polarization measurement apparatus 100 of embodiment 1, can detect the degree of polarization P with high measurement sensitivity while suppressing noise. Furthermore, the fluorescence polarization measurement apparatus 100 of this embodiment can detect the degree of polarization P with high measurement sensitivity while suppressing the influence of the intensity distribution of the excitation light EL. Because the measurement sensitivity of the degree of polarization P is high, the fluorescence polarization measurement apparatus 100 can accurately detect the substance to be measured even if the concentration of the substance to be measured is low.

[0075] Furthermore, the fluorescence polarization measurement device 100 of this embodiment can easily find noise due to foreign matter in the microflow channel 220, bright spot noise in the first light receiving section 58A, and the like.

[0076] <Embodiment 3> In the first embodiment, the light source unit 10 emits excitation light EL having a polarization direction in the X direction to the polarization direction modulation element 30. The light source unit 10 may switch the polarization direction of the excitation light EL between two orthogonal directions (X direction and Y direction).

[0077] Similar to the fluorescence polarization measurement device 100 of embodiment 1, the fluorescence polarization measurement device 100 of this embodiment includes a light source section 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detection section 50, and a control section 70. Since the configurations from the polarization direction modulation element 30 to the objective lens 42 of this embodiment are similar to those of embodiment 1, the light source section 10, the detection section 50, and the control section 70 of this embodiment will be described below.

[0078] The light source section 10 of this embodiment, like the light source section 10 of embodiment 1, has a light source 12, a condenser lens 14, an iris 16, a collimator 18, a first polarization adjustment element 20, and an excitation light filter 22. Since the configurations of the light source 12 to the collimator 18 and the excitation light filter 22 of this embodiment are similar to those of embodiment 1, only the first polarization adjustment element 20 of this embodiment will be described.

[0079] The first polarization adjustment element 20 of this embodiment switches the polarization direction of light including the excitation light EL emitted from the first polarization adjustment element 20 between the X direction and the Y direction. The first polarization adjustment element 20 of this embodiment is a TN liquid crystal cell, a rotatably held polarizing plate, or the like. The first polarization adjustment element 20 of this embodiment is controlled by the light source control unit 82 of the control unit 70.

[0080] The light emitted from the first polarization adjustment element 20 of this embodiment is output from the light source unit 10 via the excitation light filter 22. Therefore, the light source unit 10 of this embodiment switches the polarization direction of the excitation light EL between the X direction and the Y direction and outputs the excitation light EL.

[0081] In this 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, the intensity of the polarization component of which increases and decreases periodically at the spatial frequency of the polarization direction modulation element 30, is irradiated onto the measurement target solution (microdevice 200) via 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, the intensity 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 increases and decreases periodically at the spatial frequency of the polarization direction modulation element 30, and are in opposite phase to each other, as in the first embodiment (FIG. 4). Hereinafter, the excitation light EL irradiated 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 the first excitation light EL.

[0082] On the other hand, when excitation light EL having a polarization direction in the Y direction is emitted from the light source unit 10, the solution to be measured is irradiated with excitation light EL (FIG. 17) in which the component of the first excitation light EL having a polarization direction in the X direction is replaced with a component having a polarization direction in the Y direction, and the component of the first excitation light EL having a polarization direction in the Y direction is replaced with a component having a polarization direction in the X direction. Hereinafter, the excitation light EL irradiated to the solution to be measured when excitation light EL having a polarization direction in the Y direction is emitted from the light source unit 10 is referred to as second excitation light EL.

[0083] The detection unit 50 of this embodiment has an absorption filter 52, a second polarization adjustment element 54, an imaging lens 56, and a first light receiving unit 58A, similar to the detection unit 50 of embodiment 1. The configurations from the absorption filter 52 to the imaging lens 56 of this embodiment are similar to those of embodiment 1.

[0084] The first light receiving unit 58A of this embodiment detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction, similarly to the first light receiving unit 58A of the first embodiment. In this embodiment, the first light receiving unit 58A detects the spatial distribution of the fluorescence intensity (hereinafter referred to as the spatial distribution of the first fluorescence intensity) of the fluorescence FL having the polarization direction in the X direction among the fluorescence FL light emitted from the solution to be measured when the first excitation light is irradiated to the solution to be measured (i.e., when the light source unit 10 emits the excitation light EL having the polarization direction in the X direction). In addition, the first light receiving unit 58A detects the spatial distribution of the fluorescence intensity (hereinafter referred to as the spatial distribution of the second fluorescence intensity) of the fluorescence FL having the polarization direction in the X direction among the fluorescence FL light emitted from the solution to be measured when the second excitation light is irradiated to the solution to be measured (i.e., when the light source unit 10 emits the excitation light EL having the polarization direction in the Y direction). The first light receiving section 58A of the present embodiment transmits to the control section 70 image data corresponding to the spatial distribution of the first fluorescent light intensity and image data corresponding to the spatial distribution of the second fluorescent light intensity.

[0085] The control unit 70 of this embodiment extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the spatial distribution of the first fluorescence intensity. The control unit 70 of this embodiment extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the spatial distribution of the second fluorescence intensity. The control unit 70 of this embodiment calculates the degree of polarization P of the measurement target solution based on the extracted component and the DC component, and calculates the concentration of the measurement target substance from the degree of polarization P and the calibration curve. The control unit 70 of this embodiment has an input / output unit 72 to a detection control unit 84, similar to the control unit 70 of the first embodiment. The configurations of the input / output unit 72, the storage unit 74, the concentration calculation unit 80, the light source control unit 82, and the detection control unit 84 of this embodiment are similar to those of the first embodiment.

[0086] The extraction unit 76 of the present embodiment obtains the spatial distribution of the fluorescence intensity of the fluorescence FL from image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL. In the present embodiment, the extraction unit 76 obtains the spatial distribution of the first fluorescence intensity and the spatial distribution of the second fluorescence intensity. The obtained spatial distribution of the first fluorescence intensity and the spatial distribution of the second fluorescence intensity contain noise, as in the first embodiment.

[0087] The extraction section 76 of this embodiment, like the extraction section 76 of the first embodiment, extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component from the spatial distribution of the obtained fluorescence intensity of the fluorescence FL. In this embodiment, the extraction section 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as the first component) and a DC component (hereinafter referred to as the first DC component) from the spatial distribution of the first fluorescence intensity. In addition, the extraction section 76 extracts a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 (hereinafter referred to as the second component) and a DC component (hereinafter referred to as the second DC component) from the spatial distribution of the second fluorescence intensity. This makes it possible to improve the SN ratio of the fluorescence intensity of the fluorescence FL, like the first embodiment.

[0088] Specifically, the extraction unit 76 of this embodiment performs a Fourier transform and an inverse Fourier transform on the spatial distribution of the first fluorescence intensity, using the spatial frequency of the polarization direction modulation element 30, as in the first embodiment. This results in a first component and a first DC component as shown in Fig. 18. Moreover, the extraction unit 76 of this embodiment performs a Fourier transform and an inverse Fourier transform on the spatial distribution of the second fluorescence intensity, using the spatial frequency of the polarization direction modulation element 30. This results in a second component and a second DC component as shown in Fig. 19.

[0089] The polarization degree calculation unit 78 of this embodiment determines the polarization degree P from the first component and the first DC component or the second component and the second DC component. In this embodiment, as in the first embodiment, 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 Figs. 18 and 19. Moreover, 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 parallel to the polarization direction of the excitation light EL. Therefore, in this embodiment, the polarization degree calculation unit 78 determines the polarization degree P from the maximum and minimum values ​​of the first component or the maximum and minimum values ​​of the second component.

[0090] 20, the first and second components are in an inverse 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 micro-channel 220. Therefore, by comparing the fluorescence intensity Ih of the fluorescence FL of the first and second components with the fluorescence intensity Iv of the fluorescence FL, it is possible to easily find noise due to foreign matter in the micro-channel 220, bright spot noise of the first light receiving unit 58A, and the like.

[0091] Next, the detection process of this embodiment will be described. As in the first embodiment, the detection process of this embodiment is performed in the order of a degree of polarization measurement process (step S100) and a concentration calculation process (step S200) (FIG. 12). Since the concentration calculation process (step S200) of this embodiment is the same as in the first embodiment, the degree of polarization measurement process (step S100) of this embodiment will be described with reference to FIG. 21.

[0092] In the polarization degree measurement process (step S100) of this embodiment, the control unit 70 first controls the polarization direction of the light emitted from the first polarization adjustment element 20 (excitation light EL emitted from the light source unit 10) in the X direction to irradiate the measurement target solution with the first excitation light EL (step S116a). Next, the control unit 70 acquires image data corresponding to the spatial distribution of the first fluorescence intensity from the first light receiving unit 58A (step S118a). Furthermore, the control unit 70 controls the light emitted from the first polarization adjustment element 20 in the Y direction to irradiate the measurement target solution with the second excitation light EL (step S116b). Then, the control unit 70 acquires image data corresponding to the spatial distribution of the second fluorescence intensity from the first light receiving unit 58A (step S118b).

[0093] The control unit 70 detects the spatial distribution of the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction (X direction) among the fluorescence FL emitted from the measurement target solution (step S120). Specifically, the extraction unit 76 of the control unit 70 obtains the spatial distribution of the first fluorescence intensity from image data corresponding to the spatial distribution of the first fluorescence intensity. The extraction unit 76 of the control unit 70 also obtains the spatial distribution of the second fluorescence intensity from image data corresponding to the spatial distribution of the second fluorescence intensity.

[0094] Furthermore, the control unit 70 extracts a component having the same frequency as a predetermined frequency (spatial frequency of the polarization direction modulation element 30) and a DC component from the spatial distribution of the obtained fluorescence intensity (step S130). Specifically, the extraction unit 76 extracts a first component and a first DC component from the spatial distribution of the first fluorescence intensity. The extraction unit 76 also extracts a second component and a second DC component from the spatial distribution of the second fluorescence intensity. The first component and the first DC component, and the second component and the second DC component are extracted by performing a Fourier transform and an inverse Fourier transform on the spatial distribution of the fluorescence intensity using the spatial frequency of the polarization direction modulation element 30, as in the first embodiment.

[0095] The control unit 70 determines the degree of polarization 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 calculation unit 78 of the control unit 70 determines the degree of polarization P from 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).

[0096] As described above, like the fluorescence polarization measurement device 100 of embodiment 1, the fluorescence polarization measurement device 100 of this embodiment can detect the degree of polarization P with high measurement sensitivity while suppressing noise. Furthermore, the fluorescence polarization measurement device 100 of this embodiment can detect the degree of polarization P with high measurement sensitivity while suppressing the influence of the intensity distribution of the excitation light EL. Since the measurement sensitivity of the degree of polarization P is high, the fluorescence polarization measurement device 100 can accurately detect the substance to be measured even if the concentration of the substance to be measured is low. Furthermore, the fluorescence polarization measurement device 100 of this embodiment can easily find noise due to foreign matter in the microchannel 220, bright spot noise of the first light receiving unit 58A, etc.

[0097] <Embodiment 4> In the second embodiment, the detection unit 50 detects the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction by switching the polarization direction of the fluorescence FL output from the second polarization adjustment element 54 between the X direction and the Z direction. The detection unit 50 may detect the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction and the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Y direction by separating the fluorescence FL into its polarization components.

[0098] Similar to the fluorescence polarization measurement device 100 of embodiment 1, the fluorescence polarization measurement device 100 of this embodiment includes a light source section 10, a polarization direction modulation element 30, a dichroic mirror 40, an objective lens 42, a detection section 50, and a control section 70. Since the configurations from the light source section 10 to the objective lens 42 of this embodiment are similar to those of embodiment 1, only the detection section 50 and control section 70 of this embodiment will be described.

[0099] 22, the detection unit 50 of this embodiment has an absorption filter 52, a second polarization adjustment element 54, two imaging lenses 56, a first light receiving unit 58A, and a second light receiving unit 58B. The configuration of the absorption filter 52 of this embodiment is the same as that of the first embodiment.

[0100] The second polarization adjusting element 54 in this embodiment is a polarizing beam splitter. In this embodiment, the second polarization adjusting element 54 transmits the fluorescence FL polarized in the X direction among the fluorescence FL incident from the -Y direction, and reflects the fluorescence FL polarized in the Z direction in the +Z direction as fluorescence FL polarized in the Y direction.

[0101] The fluorescence FL having the polarization direction in the X direction is incident on the first light receiving part 58A via the imaging lens 56. The fluorescence FL having the polarization direction in the Y direction is incident on the second light receiving part 58B via the imaging lens 56.

[0102] The first light receiving unit 58A of the present embodiment detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction, similarly to the first light receiving unit 58A of embodiment 1. The first light receiving unit 58A of the present embodiment transmits image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction to the control unit 70.

[0103] The second light receiving unit 58B is, for example, a CCD (Charge Coupled Device) image sensor. It detects the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Y direction as an image. The second light receiving unit 58B transmits image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Y direction to the control unit 70.

[0104] The control unit 70 of the fluorescence polarization measurement device 100 extracts a component (X-direction component) having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component (X-direction DC component) from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction detected by the first light receiving unit 58A. The control unit 70 of this embodiment also extracts a component (hereinafter, Y-direction component) having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a DC component (hereinafter, Y-direction DC component) from the spatial distribution of the fluorescence intensity of the fluorescence FL having the polarization direction in the Y direction detected by the second light receiving unit 58B. The control unit 70 of this embodiment determines the degree of polarization P of the measurement target solution based on the extracted component and the DC component, and determines the concentration of the measurement target substance from the degree of polarization P and the calibration curve.

[0105] The control unit 70 of this embodiment has an input / output unit 72 to a detection control unit 84, similar to the control unit 70 of embodiments 1 and 2. The configuration of each part of the control unit 70 of this embodiment is similar to the configuration of each part of the control unit 70 of embodiment 2, except that the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Y direction is used instead of the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction in embodiment 2.

[0106] In this embodiment, as shown in Fig. 23, the maximum value of the X-direction component (including a DC component) or the Y-direction component (including a 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. The minimum value of the X-direction component (including a DC component) or the Y-direction component (including a 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 degree of polarization P is determined from 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. Furthermore, the concentration of the substance to be measured is determined from a calibration curve of the degree of polarization P and the concentration of the substance to be measured.

[0107] The fluorescence polarization measurement device 100 of this embodiment, like the fluorescence polarization measurement device 100 of embodiment 2, can detect the degree of polarization P with high measurement sensitivity while suppressing noise. Furthermore, since 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 microflow channel 220, the fluorescence polarization measurement device 100 of this embodiment can easily find noise due to foreign matter in the microflow channel 220, bright spot noise of the first light receiving unit 58A, and the like. Furthermore, the fluorescence polarization measurement device 100 of this embodiment can measure the degree of polarization P without switching either the polarization direction of the excitation light EL irradiated to the measurement target solution or the polarization direction of the fluorescence FL to be detected, thereby shortening the measurement time.

[0108] <Modification> Although the embodiments have been described above, the present disclosure can be modified in various ways without departing from the gist of the present disclosure.

[0109] For example, the microdevice 200 of the embodiment includes three microchannels 220. The microdevice 200 may include at least one microchannel 220. The microdevice 200 may include a plurality of microchannels 220.

[0110] The first light receiving section 58A and the second light receiving section 58B may be a complementary metal oxide semiconductor (CMOS) image sensor. Moreover, the first light receiving section 58A and the second light receiving section 58B are not limited to imaging elements.

[0111] In the first to fourth embodiments, the polarization direction modulation element 30 continuously modulates the polarization direction of the excitation light EL along the X direction at a predetermined frequency. The polarization direction modulation element 30 only needs to modulate the polarization direction of the excitation light EL at a predetermined frequency, and does not need to modulate continuously. The polarization direction modulation element 30 only needs to modulate the polarization direction of the excitation light EL so that the control unit 70 can extract a component that is the same as the spatial frequency of the polarization direction modulation element 30 from the fluorescence intensity.

[0112] In the detection process of the second embodiment, after acquiring image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction is acquired (steps S112a, S114a → steps S112b, S114b). In the second embodiment, after acquiring image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the Z direction, image data corresponding to the spatial distribution of the fluorescence intensity of the fluorescence FL polarized in the X direction may be acquired (steps S112b, S114b → steps S112a, S114a).

[0113] In the detection process of the third embodiment, after acquiring image data corresponding to the spatial distribution of the first fluorescence intensity, image data corresponding to the spatial distribution of the second fluorescence intensity is acquired (steps S116a, S118a → steps S116b, S118b). In the third embodiment, after acquiring image data corresponding to the spatial distribution of the second fluorescence intensity, image data corresponding to the spatial distribution of the first fluorescence intensity may be acquired (steps S116b, S118b → steps S116a, S118a).

[0114] In the degree of polarization measurement process, the spatial distribution of the fluorescence intensity may be detected from a plurality of image data.

[0115] Furthermore, in the polarization degree measurement process, the background fluorescence intensity may be detected without irradiating the excitation light EL. After subtracting the background fluorescence intensity from the spatial distribution of the detected fluorescence intensity, a component having the same frequency as the spatial frequency of the polarization direction modulation element 30 and a direct current component may be extracted. This makes it possible to suppress dark noise of the first light receiving section 58A and the second light receiving section 58B, noise from the circuits of the first light receiving section 58A and the second light receiving section 58B, and the like.

[0116] In the fluorescence polarization measuring device 100, the polarization direction modulating element 30 and the first light receiving section 58A (the first light receiving section 58A and the second light receiving section 58B) may be aligned by irradiating a fluorescent standard sample with excitation light EL so that the polarization direction of the excitation light EL and the positions of the maximum and minimum values ​​of the fluorescence intensity are aligned (Figure 24).

[0117] Although the preferred embodiments have been described above, the present disclosure is not limited to such specific embodiments, and the present disclosure includes the invention described in the claims and their equivalents. [Explanation of symbols]

[0118] 10 light source unit, 12 light source, 14 condenser lens, 16 iris, 18 collimator, 20 first polarization adjustment element, 22 excitation light filter, 30 polarization direction modulation element, 40 dichroic mirror, 42 objective lens, 50 detection unit, 52 absorption filter, 54 second polarization adjustment element, 56 imaging lens, 58A first light receiving unit, 58B second light receiving unit, 70 control unit, 72 input / output unit, 74 memory unit, 76 extraction unit, 78 polarization degree calculation unit, 80 concentration calculation unit, 82 light source control unit, 84 detection control unit, 92 CPU, 93 ROM, 94 RAM, 96 input / output interface, 100 fluorescence polarization measurement device, 200 microdevice, 202 first substrate, 202a main surface, 204 second substrate, 206 partition wall, 220 microchannel, 222 opening, P Degree of polarization, Ih Fluorescence intensity of fluorescence with a polarization direction parallel to the polarization direction of the excitation light, Iv Fluorescence intensity of fluorescence with a polarization direction perpendicular to the polarization direction of the excitation light, EL Excitation light, 1st excitation light, 2nd excitation light, FL Fluorescence, ST Stage

Claims

1. 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, the polarization direction of which has been spatially modulated at the predetermined frequency, to a measurement target solution; a detection unit that detects a spatial distribution of fluorescence intensity of the fluorescence having a polarization direction in a predetermined direction among the fluorescence emitted from the measurement target solution by the excitation light whose polarization direction has been spatially modulated at the predetermined frequency; and a control unit that extracts a component having the same frequency as the predetermined frequency and a direct current component from the spatial distribution of the detected fluorescence intensity, and determines the degree of polarization of the measurement target solution based on the extracted component and the direct current component. Fluorescence polarization measurement device.

2. The light source unit switches the polarization direction of the excitation light between two orthogonal directions and emits the excitation light. The fluorescence polarization measurement device according to claim 1 .

3. the detection unit includes a polarization adjustment element that selects the fluorescence having the predetermined polarization direction from the fluorescence emitted from the measurement target solution, and a light receiving unit that detects a spatial distribution of the fluorescence intensity of the fluorescence having the predetermined polarization direction. The fluorescence polarization measurement device according to claim 1 .

4. the polarization adjustment element switches the fluorescence having the polarization direction in the predetermined direction between the fluorescence having a polarization direction horizontal 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 measurement device according to claim 3 .

5. The polarization adjusting element is a polarizing beam splitter. The fluorescence polarization measurement device according to claim 3 .

6. The measurement target solution contains a measurement target substance, a fluorescently labeled derivative of the measurement target substance, and an antibody that specifically binds to the measurement target substance. The fluorescence polarization measurement device according to claim 1 .

7. Irradiating a solution to be measured with excitation light whose polarization direction has been spatially modulated at a predetermined frequency; detecting a spatial distribution of the fluorescence intensity of the fluorescence having a polarization direction in a predetermined direction among the fluorescence emitted from the solution to be measured; extracting a component having the same frequency as the predetermined frequency and a direct current component from the spatial distribution of the detected fluorescence intensity; determining a degree of polarization of the measurement target solution based on the extracted component and the DC component; How to measure the degree of polarization.

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