Fluorescence intensity measurement device and method for measuring fluorescence intensity

Abstract

To provide a fluorescence intensity measurement device and a method for measuring a fluorescence intensity which have a high measurement sensitivity.SOLUTION: A fluorescence intensity measurement device 100 includes: a light source 12 for emitting a pulse-like excitation light EL with a modulated width to a measurement target; a CMOS image sensor 58 for detecting a fluorescence FL emitted from the measurement target by the excitation light EL as an image for each pulse of the excitation light EL and determining the fluorescence intensity of the fluorescence FL; and a control unit 70 for determining the fluorescence intensity of the fluorescence FL. The control unit 70 determines the fluorescence intensity of the fluorescence FL on the basis of the duty ratio of two different excitation lights EL with different pulse widths and the optical intensity obtained from each of images detected by the two excitation lights EL.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a fluorescence intensity measurement device and a method for measuring fluorescence intensity.

Background Art

[0002] Analysis methods, observation methods, etc. using fluorescence are known. For example, a fluorescence polarization immunoassay (FPIA: Fluorescence Polarization Immunoassay) for detecting a substance to be detected by utilizing an antigen-antibody reaction is known. Patent Document 1 discloses a method for obtaining the concentration of a measurement antigen (substance to be detected) from the measured fluorescence polarization degree.

[0003] In addition, a fluorescence observation device that irradiates an excitation light to a measurement target and captures an image of fluorescence emitted from the measurement target is known. For example, Patent Document 2 discloses a fluorescence observation device including an excitation light irradiation unit that irradiates an excitation light and a fluorescence image capturing unit that detects fluorescence and captures a fluorescence image of a living body.

[0004] The fluorescence observation device of Patent Document 2 further includes a modulation unit that intensity-modulates the fluorescence incident on the imaging unit according to a predetermined periodic function, and an analysis unit that obtains a characteristic value corresponding to the amplitude of the periodic change of the fluorescence intensity for each pixel from the time-series image data output by the fluorescence image capturing unit. In the fluorescence observation device of Patent Document 2, the excitation light is intensity-modulated in a sine wave shape, and the time-series image data for each pixel is subjected to Fourier transform, thereby eliminating the influence of disturbances such as background light and noise on the characteristic value and obtaining a fluorescence image with a high S / N ratio.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] When a CMOS image sensor is used for fluorescence detection, the sensitivity can be increased by cooling the CMOS image sensor, increasing the exposure time of the CMOS image sensor, etc. However, there is a limit to cooling the CMOS image sensor. Also, when the exposure time is increased, noise (also referred to as amplifier bright, amplifier glow, etc.) due to heat from the peripheral circuits (amplifier circuits, AD converters, etc.) of the CMOS image sensor, infrared light, etc. becomes prominent. Noise from the peripheral circuits can be removed by measuring the background, but for precise measurement of fluorescence, the background must also be measured for each fluorescence measurement.

[0007] Also, when removing noise using intensity modulation of the excitation light and Fourier transform as in Patent Document 2, it is necessary to accurately modulate the intensity of the excitation light. For example, if there is a DC component in the intensity-modulated excitation light, it becomes difficult to distinguish between the background light, noise from the peripheral circuits, etc. and the fluorescence component. Also, when the light source of the excitation light is composed of an LED (Light emitting diode) element, since the voltage-current characteristics of the LED element are non-linear, it is difficult to accurately modulate the intensity of the excitation light.

[0008] The present disclosure has been made in view of the above circumstances, and an object thereof is to provide a fluorescence intensity measurement device and a fluorescence intensity measurement method with high measurement sensitivity.

Means for Solving the Problems

[0009] To achieve the above object, a fluorescence intensity measurement device according to a first aspect of the present disclosure includes: a light source that emits pulsed excitation light with pulse width modulation to a measurement target; a CMOS image sensor that detects fluorescence emitted from the measurement target by the excitation light as an image for each pulse of the excitation light; a control unit that obtains the fluorescence intensity of the fluorescence, and is provided with The control unit obtains the fluorescence intensity of the fluorescence based on the duty ratios of the two excitation lights with different pulse widths and the light intensities obtained from the respective images detected by the respective two excitation lights.

[0010] The method for measuring fluorescence intensity according to the second aspect of the present disclosure is an irradiation step of irradiating a measurement target with pulsed excitation light whose pulse width is modulated; a detection step of detecting, as an image for each pulse of the excitation light, fluorescence emitted from the measurement target by the excitation light using a CMOS image sensor; a calculation step of obtaining the fluorescence intensity of the fluorescence, and includes in the calculation step, the fluorescence intensity of the fluorescence is obtained based on the duty ratios of the two excitation lights with different pulse widths and the light intensities obtained from the respective images detected by the respective two excitation lights.

Advantages of the Invention

[0011] According to the present disclosure, the measurement sensitivity can be improved by removing noise from the peripheral circuit of the CMOS image sensor.

Brief Description of the Drawings

[0012]

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Modes for Carrying Out the Invention

[0013] Hereinafter, the fluorescence intensity measurement apparatus according to the embodiment will be described with reference to the drawings.

[0014] <Embodiment 1> With reference to FIGS. 1 to 11, the fluorescence intensity measurement apparatus 100 according to the present embodiment will be described. The fluorescence intensity measurement apparatus 100 is used, for example, for detecting a detection substance contained in a measurement target solution using a fluorescence polarization immunoassay method. The measurement target solution corresponds to the measurement target.

[0015] As shown in Fig. 1, the fluorescence intensity measurement device 100 includes a light source unit 10, a dichroic mirror 30, an objective lens 40, a detection unit 50, and a control unit 70. The light source unit 10 emits linearly polarized excitation light EL with a pulse width modulation. The excitation light EL emitted from the light source unit 10 is irradiated onto a measurement target solution introduced into a microchannel 220 of a microdevice 200, which will be described later, via the dichroic mirror 30 and the objective lens 40. The detection unit 50 detects, as an image, 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 part of the fluorescence intensity measurement device 100. Further, the control unit 70 obtains the fluorescence intensity of the fluorescence FL having a polarization direction in a predetermined direction based on the duty ratio of the excitation light EL and the light intensity obtained from the detected image. Furthermore, the control unit 70 obtains the concentration of the detection substance by obtaining the degree of polarization P of the measurement target solution.

[0016] For easy understanding, in this specification, the left direction (the left direction on the paper surface) of the fluorescence intensity measurement device 100 in Fig. 1 is defined as the +Z direction, the upper direction (the upper direction on the paper surface) is defined as the +Y direction, and the direction perpendicular to the +Y direction and the +Z direction (the front direction on the paper surface) is defined as the +X direction for explanation. Further, the excitation light EL is light that excites a fluorescence labeling derivative, which will be described later, and causes the fluorescence labeling derivative to emit fluorescence. The light source unit 10, the dichroic mirror 30, and the objective lens 40 form an illumination optical system, and the objective lens 40, the dichroic mirror 30, and the detection unit 50 form an observation optical system.

[0017] First, the measurement target solution and the microdevice 200 will be described. The measurement target solution contains a detection substance, a fluorescently labeled derivative, and an antibody. The detection substance is the object to be detected by the fluorescence intensity measurement device 100. The detection substance may be any compound that can be detected by fluorescence immunoassay. Examples of the detection substance include antibiotics, physiologically active substances, and mold toxins. Specific examples of the detection substance include β-lactoglobulin, chloramphenicol, and deoxynivalenol. The fluorescently labeled derivative is a derivative obtained by fluorescently labeling the detection substance with a fluorescent substance. The fluorescently labeled derivative can be obtained by binding a fluorescent substance to the detection substance using a known method. The fluorescent substance is, for example, fluorescein (excitation light EL wavelength: 494 nm, fluorescence FL wavelength: 521 nm). The antibody specifically binds to the detection substance through an antigen-antibody reaction. The antibody can be obtained, for example, by inoculating a host animal (e.g., mouse, cow) with the detection substance and then collecting and purifying the antibody in the blood produced by the host animal. Also, commercially available antibodies can be used as the antibody.

[0018] The detection substance and the fluorescently labeled derivative competitively and specifically bind to the antibody through an antigen-antibody reaction. In the fluorescence polarization immunoassay, the polarization degree P of the fluorescence FL emitted by the fluorescently labeled derivative contained in the measurement target solution is obtained, and the concentration of the detection substance is determined from the obtained polarization degree P and a calibration curve prepared in advance.

[0019] Since the fluorescently labeled derivative not bound to the antibody moves vigorously in the solution to be measured, when the polarized excitation light EL irradiates the fluorescently labeled derivative not bound to the antibody, the fluorescence FL is emitted randomly. On the other hand, since the movement of the fluorescently labeled derivative bound to the antibody is restricted in the solution to be measured, when the excitation light EL irradiates the fluorescently labeled derivative bound to the antibody, fluorescence FL polarized in the polarization direction of the excitation light EL is emitted. 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 deviation of the fluorescence intensity is determined as the degree of polarization P. Since the degree of polarization P depends on the amount of the fluorescently labeled derivative bound to the antibody, the concentration of the analyte can be obtained from the obtained degree of polarization P and the calibration curve prepared in advance. Note that the degree of polarization P is expressed as P = (Ih - Iv) / (Ih + Iv).

[0020] As shown in FIGS. 2 and 3, the microdevice 200 includes a first substrate 202, a second substrate 204, a partition wall 206, and three microchannels 220. A solution to be measured is introduced into each of the microchannels 220. The microdevice 200 is placed on the stage ST of the fluorescence intensity measuring device 100.

[0021] The first substrate 202 of the microdevice 200 is a flat quartz glass substrate. The excitation light EL emitted from the light source unit 10 of the fluorescence intensity measuring device 100 enters the microdevice 200 from the first substrate 202. The excitation light EL irradiates the measurement region R shown in FIG. 2 from the -Z direction and enters perpendicularly to the main surface 202a of the first substrate 202.

[0022] The second substrate 204 of the microdevice 200 is a flat substrate. The second substrate 204 is formed of a material with low autofluorescence. The second substrate 204 is formed of, for example, polydimethylsiloxane (PDMS) containing carbon black. The second substrate 204 faces the first substrate 202. The second substrate 204 and the first substrate 202 sandwich the partition wall 206.

[0023] The partition wall 206 of the microdevice 200 is sandwiched between the first substrate 202 and the second substrate 204 to form a microchannel 220. The partition wall 206 is formed of a material with low autofluorescence. Further, the partition wall 206 is preferably formed of a material that absorbs light such as excitation light EL and fluorescence FL. In the present embodiment, the partition wall 206 is integrally formed with the second substrate 204.

[0024] The microchannel 220 of the microdevice 200 extends parallel to the X direction within the measurement region R. The width of the microchannel 220 within the measurement region R is, for example, 200 μm. Each of the microchannels 220 has two openings 222 that penetrate through the second substrate 204 and the partition wall 206. The solution to be measured is introduced or discharged through the openings 222.

[0025] Next, each part of the fluorescence intensity measuring device 100 will be described. As shown in FIG. 1, the light source unit 10 of the fluorescence intensity measuring device 100 includes a light source 12, a condenser lens 14, an iris 16, a collimator 18, a polarizing filter 22, and an excitation light filter 24.

[0026] The light source 12 emits light including excitation light EL (i.e., excitation light EL) in the +Z direction. The light source 12 is composed of, for example, an LED element. The light source 12 is controlled by the control unit 70 and emits light including pulsed excitation light EL that is pulse-width modulated based on a PWM (Pulse Width Modulation) signal from the control unit 70. The pulse-width modulated excitation light EL will be described later.

[0027] The light emitted from the light source 12 is condensed by the condenser lens 14 and then passes through the 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 has passed through the iris 16 enters the collimator 18.

[0028] The collimator 18 converts the incident light into parallel light. The light converted into parallel light enters the polarizing filter 22.

[0029] The polarization filter 22 emits light having a polarization direction in a predetermined direction among the incident light. The light emitted from the polarization filter 22 enters the excitation light filter 24. In the present embodiment, the polarization filter 22 emits light having a polarization direction in the X direction. That is, the light including the excitation light EL incident on the polarization filter 22 is emitted as linearly polarized light having a polarization direction in the X direction and enters the excitation light filter 24. The polarization filter 22 is, for example, a polarizing plate.

[0030] The excitation light filter 24 removes light other than the excitation light EL from the light emitted from the light source 12. The excitation light filter 24 is, for example, a band-pass filter.

[0031] Therefore, the excitation light EL that is pulse-width modulated and has a polarization direction in the X direction is emitted from the light source unit 10 in the +Z direction. The excitation light EL that is pulse-width modulated and has a polarization direction in the X direction enters the dichroic mirror 30.

[0032] The dichroic mirror 30 of the fluorescence intensity measuring device 100 transmits the excitation light EL that is pulse-width modulated and has a polarization direction in the X direction in the +Z direction and reflects the fluorescence FL emitted from the microdevice 200 to the detection unit 50 (+Y direction). The objective lens 40 of the fluorescence intensity measuring device 100 condenses the excitation light EL transmitted through the dichroic mirror 30 and the fluorescence FL.

[0033] The excitation light EL that is pulse-width modulated and has a polarization direction in the X direction is irradiated onto the measurement region R of the microdevice 200 via the dichroic mirror 30 and the objective lens 40. As a result, the fluorescence FL is emitted from the measurement target solution (fluorescently labeled derivative) introduced into the microchannel 220 of the microdevice 200. The fluorescence FL travels in the +Y direction via the objective lens 40 and the dichroic mirror 30 and enters the detection unit 50 (absorption filter 52 described later).

[0034] The detection unit 50 of the fluorescence intensity measurement device 100 is arranged on the +Y side of the dichroic mirror 30. The detection unit 50 includes an absorption filter 52, a polarization adjustment element 54, an imaging lens 56, and a CMOS image sensor 58.

[0035] The absorption filter 52 separates the fluorescence FL emitted from the microdevice 200 from scattered light, leakage light, etc., 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 enters the polarization adjustment element 54.

[0036] The polarization adjustment element 54 makes the fluorescence FL transmitted through the absorption filter 52 linearly polarized. Also, the polarization adjustment element 54 switches the polarization direction of the linearly polarized fluorescence FL incident on the CMOS image sensor 58 to a direction parallel to the polarization direction (X direction) of the excitation light EL emitted from the light source unit 10 and a direction perpendicular to the polarization direction of the excitation light EL emitted from the light source unit 10 (Z direction). The linearly polarized fluorescence FL enters the CMOS image sensor 58 via the imaging lens 56. The polarization adjustment element 54 is, for example, a TN (Twisted Nematic) liquid crystal element.

[0037] The CMOS image sensor 58 detects, as an image, the spatial distribution of the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (the polarization direction in the X direction or the polarization direction in the Z direction) for each pulse of the excitation light EL based on the trigger signal from the control unit 70. That is, the CMOS image sensor 58 detects, as an image, the spatial distribution of the fluorescence FL having a predetermined polarization direction in synchronization with the emission of the excitation light EL. The CMOS image sensor 58 generates image data representing the captured image 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 CMOS image sensor 58 are in an imaging relationship.

[0038] The control unit 70 of the fluorescence intensity measurement device 100 controls each part of the fluorescence intensity measurement device 100. Further, the control unit 70 obtains the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (the polarization direction in the X direction or the polarization direction in the Z direction) based on the duty ratio of the excitation light EL and the light intensity obtained from the image detected by the CMOS image sensor 58 (that is, the image data transmitted from the CMOS image sensor 58). Furthermore, the control unit 70 obtains the degree of polarization P from the obtained fluorescence intensity of the fluorescence FL, and obtains the concentration of the detection substance from the degree of polarization P. As shown in FIG. 4, the control unit 70 includes an input / output unit 72, a storage unit 74, a polarization control unit 76, a synchronization signal generation unit 78, a light source control unit 80, a detection control unit 82, and a calculation unit 85. The calculation unit 85 includes a light intensity calculation unit 86, a fluorescence intensity calculation unit 87, and a concentration calculation unit 88.

[0039] The input / output unit 72 inputs and outputs signals, data, etc. between the control unit 70 and each part.

[0040] The storage unit 74 stores programs, image data transmitted from the CMOS image sensor 58, data representing a calibration curve of the degree of polarization P and the concentration of the detection substance, and the like.

[0041] The polarization control unit 76 controls the polarization direction of the fluorescence FL incident on the CMOS image sensor 58 by controlling the polarization adjustment element 54 of the fluorescence intensity measurement device 100. For example, first, the polarization control unit 76 sets the polarization direction of the fluorescence FL incident on the CMOS image sensor 58 to the X direction, and after the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction is detected as an image by the CMOS image sensor 58, the polarization direction of the fluorescence FL incident on the CMOS image sensor 58 is switched to the Z direction.

[0042] The synchronization signal generation unit 78 generates a synchronization signal that synchronizes the emission (irradiation) of the excitation light EL from the light source 12 (light source unit 10) and the detection (imaging) by the CMOS image sensor 58. The synchronization signal generation unit 78 transmits the synchronization signal to the light source control unit 80 and the detection control unit 82.

[0043] The light source control unit 80 transmits a PWM signal to the light source 12 based on the synchronization signal transmitted from the synchronization signal generation unit 78. The light source 12 emits light including excitation light EL that is pulse-width modulated based on the PWM signal. The pulse-width modulated light from the light source 12 is irradiated onto the measurement target solution (microdevice 200) as excitation light EL having a polarization direction in the X direction that is pulse-width modulated through a polarization filter 22, an excitation light filter 24, and the like.

[0044] In this embodiment, pulsed excitation lights E1 to E4 shown in FIG. 5, whose pulse widths t1 to t4 gradually become wider in sequence, are sequentially irradiated onto the measurement target solution at a period T. Further, the pulse trains of the excitation lights E1 to E4 are repeatedly irradiated onto the measurement target solution. Note that the excitation lights E1 to E4 have a polarization direction in the X direction, and the excitation light intensities of the excitation lights E1 to E4 are equal.

[0045] The detection control unit 82 transmits a trigger signal to the CMOS image sensor 58 based on the synchronization signal transmitted from the synchronization signal generation unit 78. The CMOS image sensor 58 detects, as an image at a period T, each of the fluorescent lights FL having a predetermined polarization direction (polarization direction in the X direction or polarization direction in the Z direction) emitted from the measurement target solution by each of the excitation lights E1 to E4. Hereinafter, the light intensity calculation unit 86 and the fluorescence intensity calculation unit 87 will be described by taking the fluorescent light FL having a polarization direction in the X direction as an example. Also, let the image detected by the excitation light E1 be Pc1, the image detected by the excitation light E2 be Pc2, the image detected by the excitation light E3 be Pc3, and the image detected by the excitation light E4 be Pc4.

[0046] The light intensity calculation unit 86 of the calculation unit 85 obtains the light intensity (brightness) at a predetermined position in the microchannel 220 for each of the images Pc1 to Pc4 detected by the CMOS image sensor 58. For example, at position A of the microchannel 220 in FIG. 2, the light intensities A1 to A4 shown in FIG. 6 are obtained. Also, at position B of the microchannel 220 in FIG. 2, the light intensities B1 to B4 shown in FIG. 7 are obtained. The light intensities obtained from the images Pc1 to Pc4 are, as shown in FIGS. 6 and 7, the fluorescence intensities (fluorescence components) ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction corresponding to the pulse widths t1 to t4 of the excitation lights E1 to E4, and noise components (hereinafter referred to as noise components from the peripheral circuit) due to heat, infrared light, etc. from the peripheral circuit of the CMOS image sensor 58. The fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL do not depend on the position of the microchannel 220, and the magnitude of the noise component from the peripheral circuit depends on the position of the microchannel 220.

[0047] The fluorescence intensity calculation unit 87 of the calculation unit 85 obtains the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction emitted from the measurement target solution by each of the excitation lights E1 to E4 from the duty ratios of the excitation lights E1 to E4 and the light intensities A1 to A4, B1 to B4 obtained from the images Pc1 to Pc4 by the light intensity calculation unit 86. Here, taking the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction at position A of the microchannel 220 as an example, the calculation of the fluorescence intensity of the fluorescence FL will be described.

[0048] The fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL depend on the pulse widths t1 to t4 of the excitation lights E1 to E4, and the magnitude of the noise component from the peripheral circuit does not depend on the pulse widths t1 to t4 of the excitation lights E1 to E4. Therefore, for two of the excitation lights E1 to E4, the pulse width of one excitation light En is tn and the duty ratio is duty n and the pulse width of the other excitation light Em is tm and the duty ratio is duty m and the light intensity obtained from the image Pcn detected by the excitation light En is A n and the light intensity obtained from the image Pcm detected by the excitation light Em is A mWhen the period for irradiating each of the excitation lights E1 to E4 is T, the fluorescence intensity ΔF of the fluorescence FL emitted from the measurement target solution by one of the excitation lights En n is represented by the following formulas (1) to (3) (n = 1, 2, 3, 4, m = 1, 2, 3, 4, n ≠ m). That is, the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL are the duty ratios of the excitation lights En and Em with different pulse widths n , duty m and the light intensities A n , A m obtained from each of the images Pcm and Pcm detected by each of the two excitation lights En and Em.

[0049]

Equation

Equation

Equation

[0050] The fluorescence intensity calculation unit 87 obtains the fluorescence intensity ΔF n of the fluorescence FL emitted from the measurement target solution by one of the excitation lights En from formulas (1) to (3). From formulas (1) to (3), as shown in FIG. 8, the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction, obtained by removing the noise components from the peripheral circuit from the light intensities A1 to A4 (FIG. 6) obtained from the images Pc1 to Pc4, can be obtained.

[0051] In the present embodiment, since the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL are obtained based on the duty ratios n , duty m of the two excitation lights En and Em and the light intensities A n , A m obtained from the detected images Pcn and Pcm, the noise components from the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. Further, even if the excitation light EL contains a DC component, the noise components from the peripheral circuit can be removed.

[0052] Further, since the excitation light intensities of the excitation lights E1 to E4 are made constant and the pulse widths (pulse widths t1 to t4) of the excitation lights E1 to E4 are modulated, the excitation light EL can be easily generated and the distortion of the waveform of the excitation light EL can be suppressed. With a simple configuration, the emission (irradiation) of the excitation light EL from the light source unit 10 (light source 12) and the detection (imaging) by the CMOS image sensor 58 can be synchronized.

[0053] Note that the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction can also be obtained in the same manner as the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction. Further, in the above light intensity calculation unit 86, the light intensity is obtained from one image, but a plurality of images detected by a plurality of excitation lights EL having equal pulse widths may be integrated, and the light intensity may be obtained from the integrated image.

[0054] The concentration calculation unit 88 of the calculation unit 85 obtains the degree of polarization P from the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction and the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction. In the present embodiment, the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction corresponds to the fluorescence intensity Ih of the fluorescence FL having a polarization direction parallel to the polarization direction of the excitation light EL. Further, the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction corresponds to the fluorescence intensity Iv of the fluorescence FL having a polarization direction perpendicular to the polarization direction of the excitation light EL. In this case, the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction and the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction are the fluorescence intensities of the polarization components of the fluorescence FL emitted from the measurement target solution by the excitation light EL having the same pulse width.

[0055] Furthermore, the concentration calculation unit 88 obtains the concentration of the detection substance from the obtained degree of polarization P and the calibration curve between the degree of polarization P and the concentration of the detection substance.

[0056] FIG. 9 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 the 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 part. The functions of the control unit 70 are realized by the execution of the program of the CPU 92.

[0057] As described above, by irradiating the measurement target with the pulse-width modulated excitation light EL, the fluorescence intensity of the fluorescence FL is obtained. Therefore, with a simple configuration, it is possible to easily obtain the fluorescence intensity of the fluorescence FL emitted from the measurement target solution while removing the noise components from the peripheral circuit, and the measurement sensitivity of the fluorescence intensity can be improved. Further, since the fluorescence intensity of the fluorescence FL is obtained based on the duty ratio and the light intensity obtained from the detected image, even if the excitation light EL contains a DC component, the noise components from the peripheral circuit can be removed.

[0058] Next, with reference to FIGS. 10 and 11, the detection process (that is, the detection method of the detection substance) of the fluorescence intensity measurement device 100 will be described. As shown in FIG. 10, the detection process is performed in the order of the fluorescence intensity measurement process (step S100) for obtaining the fluorescence intensity of the fluorescence FL and the concentration calculation process (step S200) for detecting the concentration of the detection substance. The fluorescence intensity measurement process (step S100) corresponds to the measurement method of the fluorescence intensity.

[0059] With reference to FIG. 11, the fluorescence intensity measurement process (step S100) will be described. In the fluorescence intensity measurement process (step S100), the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction and the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction emitted from the measurement target solution introduced into the microchannel 220 of the microdevice 200 are obtained.

[0060] First, the control unit 70 controls the polarization adjustment element 54 to control the polarization direction of the fluorescence FL incident on the CMOS image sensor 58 in the X direction, and controls the light source 12 to irradiate the measurement target solution introduced into the microchannel 220 of the microdevice 200 installed on the stage ST with the excitation light EL having the polarization direction in the X direction and pulse-width modulated at a period T (step S112). The pulse-width modulated light emitted from the light source 12 is irradiated onto the measurement target solution as the excitation light EL having the polarization direction in the X direction and pulse-width modulated through the polarization filter 22, the excitation light filter 24, etc. In this embodiment, the pulsed excitation lights E1 to E4 whose pulse widths t1 to t4 gradually widen in sequence are irradiated onto the measurement target solution at a period T (FIG. 5). Thereby, the fluorescence FL is emitted from the measurement target solution.

[0061] Next, the control unit 70 controls the CMOS image sensor 58 to detect, as an image by the CMOS image sensor 58 for each pulse of the excitation light EL, the fluorescence FL having the polarization direction in the X direction among the fluorescence FL emitted from the measurement target solution (step S114). Specifically, the fluorescence FL emitted from the measurement target solution is incident on the CMOS image sensor 58 as the fluorescence FL having the polarization direction in the X direction through the dichroic mirror 30, the polarization adjustment element 54, etc. The CMOS image sensor 58 detects, as an image, the spatial distribution of the fluorescence FL having the polarization direction in the X direction for each pulse of the excitation light EL at a period T. The control unit 70 acquires the image data representing the captured image. In this embodiment, the image Pc1 is detected by the excitation light E1, and the image Pc2 is detected by the excitation light E2. Also, the image Pc3 is detected by the excitation light E3, and the image Pc4 is detected by the excitation light E4.

[0062] Furthermore, the control unit 70 controls the polarization adjustment element 54 to control the polarization direction of the fluorescence FL incident on the CMOS image sensor 58 in the Z direction, and controls the light source 12 to irradiate the measurement target solution introduced into the microchannel 220 of the microdevice 200 installed on the stage ST with excitation light EL having a polarization direction in the X direction and pulse-width modulated at a period T (step S116). As a result, similarly to step S112, the measurement target solution is irradiated with the excitation light EL having a polarization direction in the X direction and pulse-width modulated, and the fluorescence FL is emitted from the measurement target solution.

[0063] Next, the control unit 70 controls the CMOS image sensor 58 to detect, as an image, the fluorescence FL having a polarization direction in the Z direction among the fluorescence FL emitted from the measurement target solution by the CMOS image sensor 58 for each pulse of the excitation light EL (step S118). Similarly to step S114, the CMOS image sensor 58 detects, as an image, the spatial distribution of the fluorescence FL having a polarization direction in the Z direction for each pulse of the excitation light EL at a period T. The control unit 70 acquires image data representing the captured image.

[0064] Next, the control unit 70 determines the fluorescence intensity ΔF of the fluorescence FL having a polarization direction in the X direction n (step S120). The control unit 70 uses the above equations (1) to (3) to determine the duty ratios duty n , duty m of the two excitation lights En and Em with different pulse widths, and the light intensities A n , A m obtained from the images Pcm and Pcm of the fluorescence FL having a polarization direction in the X direction detected by the two excitation lights En and Em respectively, and determines the fluorescence intensity ΔF n of the fluorescence FL having a polarization direction in the X direction (n = 1, 2, 3, 4, m = 1, 2, 3, 4, n ≠ m).

[0065] Specifically, first, the control unit 70 obtains the light intensities A1 to A4 at a predetermined position (position A) of the microchannel 220 from the images Pc1 to Pc4 in which the fluorescence intensity of the fluorescence FL having a polarization direction in the X direction is detected (FIG. 6). Next, from the duty ratios duty1 to duty4 of the excitation lights E1 to E4 and the obtained light intensities A1 to A4, using Expressions (1) to (3), the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL having a polarization direction in the X direction, from which the noise components from the peripheral circuit are removed from the light intensities A1 to A4, are obtained (FIG. 8).

[0066] Next, the control unit 70 obtains the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction (step S122). The control unit 70 determines the duty ratios duty n , duty m of the two excitation lights En and Em having different pulse widths, and the light intensities obtained from the respective images of the fluorescence FL having a polarization direction in the Z direction detected by the two excitation lights En and Em, and obtains the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction (n = 1, 2, 3, 4, m = 1, 2, 3, 4, n ≠ m). The specific process for obtaining the fluorescence intensity of the fluorescence FL having a polarization direction in the Z direction is the same as that in step S120. When step S122 ends, the fluorescence intensity measurement process (step S100) ends.

[0067] In the present embodiment, since the fluorescence intensity of the fluorescence FL is obtained based on the duty ratios of the two excitation lights EL and the light intensities obtained from the images detected by the two excitation lights, the noise components from the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. Further, even if the excitation light EL contains a DC component, the noise components from the peripheral circuit can be removed.

[0068] Also, since the pulse width-modulated excitation light EL is used, the excitation light EL can be easily irradiated onto the measurement target solution. The distortion of the waveform of the excitation light EL can also be suppressed. Further, the emission of the excitation light EL from the light source 12 and the detection by the CMOS image sensor 58 can be easily synchronized.

[0069] Returning to FIG. 10, the concentration calculation process (step S200) will be described. In the concentration calculation process (step S200), the concentration of the detected substance is determined. First, the control unit 70 obtains the polarization degree P by setting the fluorescence intensity of the fluorescence FL having the polarization direction in the X direction as the fluorescence intensity Ih and the fluorescence intensity of the fluorescence FL having the polarization direction in the Z direction as the fluorescence intensity Iv. Further, the control unit 70 obtains the concentration of the detected substance from the calibration curve of the polarization degree P and the concentration of the detected substance. When the concentration calculation process (step S200) ends, the detection process ends.

[0070] As described above, the fluorescence intensity measuring device 100 obtains the fluorescence intensity of the fluorescence FL based on the duty ratios of the two excitation lights EL and the light intensity obtained from the images detected by the two excitation lights EL. Therefore, noise components from peripheral circuits can be easily removed, and the measurement sensitivity can be improved. Also, even if the excitation light EL contains a DC component, noise components from peripheral circuits can be removed.

[0071] Further, in the fluorescence intensity measuring device 100, since the excitation light intensity of the excitation light EL is constant and the pulse width of the excitation light EL is modulated, the excitation light EL can be easily generated. The distortion of the waveform of the excitation light EL can also be suppressed. With a simple configuration, the emission of the excitation light EL from the light source 12 and the detection by the CMOS image sensor 58 can be synchronized.

[0072] <Embodiment 2> In Embodiment 1, pulsed excitation lights E1 to E4 whose pulse widths t1 to t4 gradually widen are irradiated onto the measurement target solution as the excitation light EL. The method of modulating the pulse width of the excitation light EL is arbitrary. For example, the pulse width of the excitation light EL may be modulated randomly.

[0073] The configuration of the fluorescence intensity measuring device 100 of this embodiment is the same as that of the fluorescence intensity measuring device 100 of Embodiment 1, except that the measurement target solution is irradiated with the excitation light EL whose pulse width is modulated randomly. Here, the light source 12, the light source control unit 80, the detection control unit 82, the light intensity calculation unit 86, and the fluorescence intensity calculation unit 87 of the control unit 70 will be described.

[0074] The light source 12 of this embodiment emits light including excitation light EL that is pulse-width modulated based on the PWM signal from the light source control unit 80. In the excitation light EL of this embodiment, the pulse width of the excitation light EL is randomly modulated. Also in this embodiment, similar to Embodiment 1, the excitation light EL that is pulse-width modulated and has a polarization direction in the X direction is irradiated onto the measurement region R of the microdevice 200 via the dichroic mirror 30 and the objective lens 40. The pulse-width modulated excitation light EL will be described later.

[0075] The light source control unit 80 of this embodiment controls the light source 12 in the same manner as the light source control unit 80 of Embodiment 1. The light source 12 of this embodiment emits light including excitation light EL whose pulse width is randomly modulated based on the PWM signal from the light source control unit 80. In this embodiment, taking the four pulsed excitation lights E1 to E4 sequentially emitted shown in FIG. 12 as an example, the detection control unit 82, the light intensity calculation unit 86, and the fluorescence intensity calculation unit 87 will be described.

[0076] In the excitation lights E1 to E4 shown in FIG. 12, the pulse widths are wide in the order of the pulse width t4 of the excitation light E4, the pulse width t1 of the excitation light E1, the pulse width t3 of the excitation light E3, and the pulse width t2 of the excitation light E2 (t4 < t1 < t3 < t2).

[0077] The detection control unit 82 of this embodiment controls the CMOS image sensor 58 in the same manner as the detection control unit 82 of Embodiment 1. The CMOS image sensor 58 detects each of the fluorescence FLs having a predetermined polarization direction emitted from the measurement target solution by each of the excitation lights E1 to E4 as an image at a period T. Also in this embodiment, let the image detected by the excitation light E1 be Pc1, the image detected by the excitation light E2 be Pc2, the image detected by the excitation light E3 be Pc3, and the image detected by the excitation light E4 be Pc4.

[0078] The light intensity calculation unit 86 of the present embodiment obtains the light intensity at a predetermined position in the microchannel 220 for each of the images Pc1 to Pc4, in the same manner as the light intensity calculation unit 86 of the first embodiment. At position A of the microchannel 220 in FIG. 2, for example, the light intensities A1 to A4 shown in FIG. 13 are obtained.

[0079] The fluorescence intensity calculation unit 87 of the present embodiment obtains the fluorescence intensities ΔF1 to ΔF4 of the fluorescence FL from the above equations (1) to (3), in the same manner as the fluorescence intensity calculation unit 87 of the first embodiment (FIG. 14). In equations (1) to (3), the fluorescence intensity of the fluorescence FL is obtained based on the duty ratios of the two excitation lights EL and the light intensities obtained from the images detected by the two excitation lights EL. Therefore, regardless of the modulation method of the pulse width of the excitation light EL, the fluorescence intensity of the fluorescence FL can be obtained by removing the noise components from the peripheral circuit. In the present embodiment, since the pulse width of the excitation light EL is randomly modulated, the influence of the repetitive noise specific to the apparatus can be reduced.

[0080] Also in the present embodiment, the fluorescence intensity measuring apparatus 100 obtains the fluorescence intensity of the fluorescence FL based on the duty ratios of the two excitation lights EL and the light intensities obtained from the images detected by the two excitation lights EL. Therefore, the noise components from the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. Further, even if the excitation light EL includes a DC component, the noise components from the peripheral circuit can be removed.

[0081] Further, also in the fluorescence intensity measuring apparatus 100 of the present embodiment, since the excitation light intensity of the excitation light EL is constant and the pulse width of the excitation light EL is modulated, the excitation light EL can be easily generated. The distortion of the waveform of the excitation light EL can also be suppressed. With a simple configuration, the emission of the excitation light EL from the light source 12 and the detection by the CMOS image sensor 58 can be synchronized.

[0082] <Modification Example> Although the embodiments have been described above, the present disclosure can be variously modified without departing from the gist thereof.

[0083] 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.

[0084] In the embodiment, four pulsed excitation lights E1 to E4 are exemplified as the excitation light EL. The excitation light EL may be formed from a plurality of pulsed excitation lights.

[0085] Also, in the embodiment, the linearly polarized excitation light EL is irradiated onto the measurement target solution (measurement target). The excitation light EL irradiated onto the measurement target solution may be in an unpolarized state.

[0086] The fluorescence intensity measurement device of the present disclosure may measure the fluorescence intensity of the unpolarized fluorescence FL. For example, the fluorescence intensity measurement device of the present disclosure may measure the fluorescence intensity of the fluorescence emitted from a DNA (deoxyribonucleic acid) array.

[0087] As described above, the preferred embodiments have been explained, but the present disclosure is not limited to such specific embodiments, and the present disclosure includes the invention described in the claims and its equivalent scope.

Description of Reference Numerals

[0088] 10 Light source unit, 12 Light source, 14 Condensing lens, 16 Iris, 18 Collimator, 22 Polarizing filter, 24 Excitation light filter, 30 Dichroic mirror, 40 Objective lens, 50 Detection unit, 52 Absorption filter, 54 Polarization adjustment element, 56 Imaging lens, 58 CMOS image sensor, 70 Control unit, 72 Input / output unit, 74 Storage unit, 76 Polarization control unit, 78 Synchronization signal generation unit, 80 Light source control unit, 82 Detection control unit, 85 Calculation unit, 86 Light intensity calculation unit, 87 Fluorescence intensity calculation unit, 88 Concentration calculation unit, 92 CPU, 93 ROM, 94 RAM, 96 Input / output interface, 100 Fluorescence intensity measurement device, 200 Microdevice, 202 First substrate, 202a Main surface, 204 Second substrate, 206 Partition wall, 220 Microchannel, 222 Opening, A, B positions, P Degree of polarization, Ih Fluorescence intensity of fluorescence having a polarization direction parallel to the polarization direction of the excitation light, Iv Fluorescence intensity of fluorescence having a polarization direction perpendicular to the polarization direction of the excitation light, EL, E1~E4, En, Em Excitation light, t1~t4 Pulse widths, FL Fluorescence, Pc1~Pc4, Pcn, Pcm Images, A1~A4, A n , A m , B1~B4, Af, Ag Light intensities, ΔF, ΔF1~ΔF4, ΔF n Fluorescence intensity, duty n , duty m , duty1~duty4 Duty ratios, T Period, R Measurement region, ST Stage

Claims

1. A light source that emits pulsed excitation light with pulse-width modulation to a measurement target, A CMOS image sensor that detects fluorescence emitted from the measurement target by the excitation light as an image for each pulse of the excitation light, A control unit that obtains the fluorescence intensity of the fluorescence, and includes: The control unit obtains the fluorescence intensity of the fluorescence based on the duty ratios of two pieces of the excitation light with different pulse widths and the light intensities obtained from each of the images detected by each of the two pieces of excitation light. A fluorescence intensity measurement device.

2. The pulse width of the excitation light emitted from the light source is randomly modulated. The fluorescence intensity measurement device according to Claim 1.

3. The fluorescence incident on the CMOS image sensor is linearly polarized, and includes a polarization adjustment element that switches the polarization direction of the linearly polarized fluorescence. The CMOS image sensor detects the fluorescence having a polarization direction in a predetermined direction as the image for each pulse of the excitation light. The control unit obtains the fluorescence intensity of the fluorescence having a polarization direction in the predetermined direction. The fluorescence intensity measurement device according to Claim 1 or 2.

4. An irradiation step of irradiating a measurement target with pulsed excitation light with pulse-width modulation, A detection step of detecting fluorescence emitted from the measurement target by the excitation light as an image for each pulse of the excitation light by a CMOS image sensor, An arithmetic step of obtaining the fluorescence intensity of the fluorescence, and includes: In the arithmetic step, the fluorescence intensity of the fluorescence is obtained based on the duty ratios of two pieces of the excitation light with different pulse widths and the light intensities obtained from each of the images detected by each of the two pieces of excitation light. A method for measuring fluorescence intensity.

Images