Fluorescence intensity measuring equipment and method for measuring fluorescence intensity

Abstract

The invention discloses fluorescence intensity measuring equipment and a method for measuring fluorescence intensity. The fluorescence intensity measuring apparatus includes: a light source configured to emit a plurality of pulse-shaped excitation lights having modulated pulse widths to a measurement target; a CMOS image sensor for detecting fluorescence emitted from the measurement target by one of the plurality of pulse-shaped excitation lights as an image of a pulse of one of the plurality of pulse-shaped excitation lights; and a controller for calculating the fluorescence intensity of the fluorescence. The controller calculates the fluorescence intensity of the fluorescence on the basis of two duty cycles of two pieces of pulsed excitation light having different pulse widths and two light intensities obtained from an image detected by the two pieces of pulsed excitation light.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of Japanese Patent Application No. 2023 - 220652, filed on December 27, 2023, the entire disclosure of which is incorporated herein by reference. Technical field

[0003] The present invention generally relates to a fluorescence intensity measuring device and a method for measuring fluorescence intensity. Background art

[0004] In the related art, analysis methods and observation methods using fluorescence are known. For example, a fluorescence polarization immunoassay (FPIA) method for detecting a detection substance using an antigen - antibody reaction is known. Unexamined Japanese Patent Application Publication No. H03 - 103765 describes a method for calculating the concentration of a measured antigen (detection substance) based on the measured fluorescence polarization degree.

[0005] In addition, in the related art, a fluorescence observation device is known, which irradiates a measurement target with excitation light and captures an image with the fluorescence emitted from the measurement target. For example, Unexamined Japanese Patent Application Publication No. 2000 - 210246 describes a fluorescence observation device including an excitation light emission device for emitting excitation light and a fluorescence image capture device for detecting fluorescence to capture a fluorescence image of a living body.

[0006] The fluorescence observation device of Unexamined Japanese Patent Application Publication No. 2000 - 210246 further includes: a modulation device for modulating the fluorescence intensity entering the capture device based on a predetermined periodic function; and an analysis device for obtaining a characteristic value corresponding to the amplitude of the periodic change of the fluorescence intensity of each pixel from the time - series image data output by the fluorescence image capture device. In the fluorescence observation device of Unexamined Japanese Patent Application Publication No. 2000 - 210246, by modulating the intensity of the excitation light in a sine - wave form, performing a Fourier transform on the time - series image data of each pixel, and then removing the influence of external disturbances such as background light or noise on the characteristic value, a fluorescence image with a high signal - to - noise ratio (high SN ratio) is obtained.

[0007] If a CMOS image sensor is used in fluorescence detection, the sensitivity can be improved by, for example, cooling the CMOS image sensor and extending the exposure time of the CMOS image sensor. However, there are limitations to cooling the CMOS image sensor. In addition, if the exposure time is extended, noise (also known as amplifier light, amplifier growth, etc.) caused by heat or infrared light, etc. from the peripheral circuits (amplifier circuits, AD converters, etc.) of the CMOS image sensor will significantly appear. The noise caused by the peripheral circuits can be removed by measuring the background noise, but the background noise should be measured each time to accurately measure the fluorescence.

[0008] In addition, as described in Japanese Patent Application Laid-Open No. 2000-210246, if intensity modulation of the excitation light and Fourier transform are used to remove noise, the intensity of the excitation light should be accurately modulated. For example, if the intensity-modulated excitation light includes a DC component, it is difficult to distinguish the fluorescence component from the noise caused by peripheral circuits, background light, etc. In the case where the excitation light source includes a light-emitting diode (LED), since the voltage-current characteristic of the LED is in a non-linear manner, it is difficult to accurately modulate the intensity of the excitation light.

[0009] The present disclosure has been made in view of the above circumstances, and an object of the present disclosure is to provide a fluorescence intensity measurement device and a method for measuring fluorescence intensity with high measurement sensitivity. Summary of the Invention

[0010] To achieve the above object, a fluorescence intensity measurement device according to a first aspect of the present disclosure includes:

[0011] A light source for emitting a plurality of pulsed excitation lights with modulated pulse widths to a measurement target;

[0012] A CMOS image sensor for detecting fluorescence emitted from the measurement target by one of the plurality of pulsed excitation lights as an image for each pulse of one pulsed excitation light among the plurality of pulsed excitation lights; and

[0013] A controller for calculating the fluorescence intensity of the fluorescence, where

[0014] The controller calculates the fluorescence intensity of the fluorescence based on two duty ratios of two pulsed excitation lights having different pulse widths among the plurality of pulsed excitation lights and two light intensities obtained from the images detected by the two pulsed excitation lights.

[0015] A method for measuring fluorescence intensity according to a second aspect of the present disclosure includes:

[0016] Emitting a plurality of pulsed excitation lights with modulated pulse widths to a measurement target;

[0017] A CMOS image sensor detects fluorescence emitted from a measurement target by one of a plurality of pulsed excitation lights as an image for each pulse of one of the plurality of pulsed excitation lights; and

[0018] calculate the fluorescence intensity of the fluorescence, where

[0019] in the calculation,

[0020] calculate the fluorescence intensity of the fluorescence based on two duty cycles of two pulsed excitation lights having different pulse widths among the plurality of pulsed excitation lights and two light intensities obtained from images detected by the two pulsed excitation lights.

[0021] It should be understood that the above general description and the following detailed description are both exemplary and explanatory, and do not limit the present disclosure.

[0022] The present disclosure can improve the measurement sensitivity by removing noise caused by the peripheral circuit of the CMOS image sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present application can be more fully understood when the following detailed description is considered in conjunction with the following drawings, in which:

[0024] Figure 1 is a schematic diagram showing a fluorescence intensity measurement device according to Embodiment 1;

[0025] Figure 2 is a plan view showing a micro device according to Embodiment 1;

[0026] Figure 3 is Figure 2 a cross-sectional view taken along line C-C of the shown micro device;

[0027] Figure 4 is a block diagram showing the configuration of a controller according to Embodiment 1;

[0028] Figure 5 is a schematic diagram showing an excitation light according to Embodiment 1;

[0029] Figure 6 is a graph showing the light intensity according to Embodiment 1;

[0030] Figure 7 is another graph showing the light intensity according to Embodiment 1;

[0031] Figure 8 is a graph showing the fluorescence intensity according to Embodiment 1;

[0032] Figure 9 is a graph showing the hardware configuration of a controller according to Embodiment 1;

[0033] Figure 10 is a flowchart showing the detection process according to Embodiment 1;

[0034] Figure 11 is a flowchart showing the fluorescence intensity measurement process according to Embodiment 1;

[0035] Figure 12 is a schematic diagram showing an example of the excitation light according to Embodiment 2;

[0036] Figure 13 is a graph showing the light intensity according to Embodiment 2; and

[0037] Figure 14 is a graph showing the fluorescence intensity according to Embodiment 2. DETAILED DESCRIPTION

[0038] Hereinafter, a fluorescence intensity measurement device according to various embodiments will be described with reference to the accompanying drawings.

[0039] Embodiment 1

[0040] First, with reference to Figures 1-11 a fluorescence intensity measurement device 100 according to the present embodiment is described. In one example, the fluorescence intensity measurement device 100 is used to detect a detection substance contained in a measurement target solution to be measured using a fluorescence polarization immunoassay method. The measurement target solution corresponds to the measurement target to be measured.

[0041] As Figure 1 shown, the fluorescence intensity measurement device 100 includes a light source unit 10, a dichroic mirror 30, an objective lens 40, a detector 50, and a controller 70. The light source unit 10 emits excitation light EL whose pulse width is modulated and linearly polarized. The excitation light EL is a plurality of pulsed excitation lights. The excitation light EL emitted from the light source unit 10 irradiates the measurement target solution introduced into the microchannel 220 of the microdevice 200 to be described later through the dichroic mirror 30 and the objective lens 40. The detector 50 detects, as an image, one fluorescence FL whose polarization direction is in a predetermined direction among the plurality of fluorescence FLs emitted from the measurement target solution. The controller 70 controls various components of the fluorescence intensity measurement device 100. In addition, the controller 70 calculates the fluorescence intensity of one fluorescence FL whose polarization direction is in a predetermined direction based on the duty ratio of the excitation light EL and the light intensity obtained from the detected image. The controller 70 also calculates the concentration of the detection substance by calculating the degree of polarization P of the measurement target solution.

[0042] Note that, in the present disclosure, for ease of understanding, in Figure 1In the fluorescence intensity measurement device 100, the left direction (the left direction on the paper) is referred to as the “+Z direction”, the upward direction (the upward direction on the paper) is referred to as the “+Y direction”, and the direction perpendicular to the +Y direction and the +Z direction (the front direction on the paper) is referred to as the “+X direction”. The excitation light EL is light that excites the fluorescently labeled derivative described later and causes the fluorescently labeled 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 detector 50 form an observation optical system.

[0043] First, the measurement target solution and the microdevice 200 are described. The measurement target solution includes a detection substance, a fluorescently labeled derivative, and an antibody. The detection substance is the detection target of the fluorescence intensity measurement device 100. The detection substance can be any compound detectable in a fluorescence immunoassay. Examples of the detection substance include antibiotics, bioactive substances, and mycotoxins. 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 (wavelength of the excitation light EL: 494 nm, wavelength of the fluorescence FL: 521 nm). Due to the antigen-antibody reaction, the antibody specifically binds to the detection substance. In one example, the antibody is obtained by inoculating a host animal (e.g., a mouse or a cow) with the detection substance and then collecting the antibody in the blood produced by the host animal and purifying the antibody collected in the blood. Alternatively, a commercially available antibody can be used as the antibody.

[0044] Due to the antigen-antibody reaction, the detection substance and the fluorescently labeled derivative specifically bind to the antibody in a competitive manner. In fluorescence polarization immunoassay, the degree of polarization P of the fluorescence FL emitted by the fluorescently labeled derivative included in the measurement target solution is obtained. The concentration of the detection substance can be calculated based on the obtained degree of polarization P and a calibration curve created in advance.

[0045] The fluorescently labeled derivatives not bound to the antibody move vigorously in the measurement target solution. Therefore, when the excitation light EL, which is polarized light, irradiates the fluorescently labeled derivatives not bound to the antibody, fluorescence FL is emitted randomly. At the same time, the movement of the fluorescently labeled derivatives bound to the antibody in the measurement target solution is restricted. Therefore, when the excitation light EL, which is polarized light, irradiates the fluorescently labeled derivatives bound to the antibody, fluorescence FL biased in the polarization direction of the excitation light EL is emitted. The fluorescence intensity Ih of fluorescence FL with a polarization direction parallel to the polarization direction of the excitation light EL and the fluorescence intensity Iv of fluorescence FL with a polarization direction perpendicular to the polarization direction of the excitation light EL are measured, and the bias of the fluorescence intensity is calculated as the degree of polarization P. The degree of polarization P depends on the amount of the fluorescently labeled derivatives bound to the antibody. Therefore, the concentration of the analyte can be calculated based on the obtained degree of polarization P and the calibration curve created in advance. Note that the degree of polarization P is expressed as P = (Ih - Iv) / (Ih + Iv).

[0046] As Figure 2 and Figure 3 shown, the microdevice 200 includes a first substrate 202, a second substrate 204, a partition wall 206, and three microchannels 220. The measurement target solution is introduced into each of the microchannels 220. The microdevice 200 is placed on the stage ST of the fluorescence intensity measurement device 100.

[0047] The first substrate 202 of the microdevice 200 is implemented as a flat quartz glass substrate. The excitation light EL emitted from the light source unit 10 of the fluorescence intensity measurement device 100 enters the microdevice 200 from the first substrate 202. The excitation light EL falls on Figure 2 the measurement region R shown, and perpendicularly enters the main surface 202a of the first substrate 202.

[0048] The second substrate 204 of the microdevice 200 is implemented as a flat substrate. The second substrate 204 is formed of a material with low autofluorescence. In one example, the second substrate 204 is formed of carbon black polydimethylsiloxane (PDMS). The second substrate 204 faces the first substrate 202. The second substrate 204 and the first substrate 202 sandwich the partition wall 206.

[0049] The partition wall 206 of the microdevice 200 is sandwiched by the first substrate 202 and the second substrate 204 to form the microchannels 220. The partition wall 206 is formed of a material with low autofluorescence. In addition, preferably, the partition wall 206 is formed of a material that absorbs light such as the excitation light EL, fluorescence FL, etc. In the present embodiment, the partition wall 206 is integrally formed with the second substrate 204.

[0050] The microchannels 220 of the microdevice 200 extend parallel to the X direction in the measurement region R. In one example, the width of each of the microchannels 220 in the measurement region R is 200 μm. Each of the microchannels 220 includes two openings 222 that penetrate the second substrate 204 and the partition wall 206. The measurement target solution is introduced or discharged through the openings 222.

[0051] Next, various components of the fluorescence intensity measurement device 100 are described. As Figure 1 shown, the light source unit 10 of the fluorescence intensity measurement device 100 includes a light source 12, a condenser lens 14, an aperture 16, a collimator 18, a polarization filter 22, and an excitation light filter 24.

[0052] The light source 12 emits light including the excitation light EL in the +Z direction, that is, the excitation light EL. In one example, the light source 12 includes an LED element. The light source 12 is controlled by the controller 70 and emits pulsed light, which includes pulsed excitation light EL, the pulse width of which is modulated based on a pulse width modulation (PWM) signal from the controller 70. The excitation light EL with the modulated pulse width will be described later.

[0053] The light emitted from the light source 12 is focused by the condenser lens 14 and then passes through the aperture 16. The aperture 16 reduces the influence of external light. External light is light other than the light emitted from the light source 12. The light that has passed through the aperture 16 enters the collimator 18.

[0054] The collimator 18 converts the incident light into parallel light. The light converted into parallel light enters the polarization filter 22.

[0055] The polarization filter 22 emits light with a polarization direction in a predetermined direction from 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 with a polarization direction in the X direction. Specifically, the light including the excitation light EL that enters the polarization filter 22 is emitted as linearly polarized light with a polarization direction in the X direction and enters the excitation light filter 24. In one example, the polarization filter 22 is implemented as a polarizing plate.

[0056] The excitation light filter 24 removes light other than the excitation light EL from the light emitted from the light source 12. In one example, the excitation light filter 24 is implemented as a band-pass filter.

[0057] Therefore, multiple pulsed excitation lights EL with the modulated pulse width and a polarization direction in the X direction are emitted from the light source unit 10 in the +Z direction. The pulsed excitation light EL with the modulated pulse width and a polarization direction in the X direction enters the dichroic mirror 30.

[0058] The dichroic mirror 30 of the fluorescence intensity measurement device 100 transmits pulsed excitation light EL whose pulse width is modulated and whose polarization direction is in the X direction in the +Z direction, and reflects fluorescence FL emitted from the microdevice 200 to the detector 50 (+Y direction). The objective lens 40 of the fluorescence intensity measurement device 100 focuses the fluorescence FL and the excitation light EL that have passed through the dichroic mirror 30.

[0059] The pulsed excitation light EL whose pulse width is modulated and whose polarization direction is in the X direction is emitted onto the measurement region R of the microdevice 200 through the dichroic mirror 30 and the objective lens 40. As a result, 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, passes through the objective lens 40 and the dichroic mirror 30, and enters the detector 50 (absorption filter 52 described later).

[0060] The detector 50 of the fluorescence intensity measurement device 100 is provided on the +Y side of the dichroic mirror 30. The detector 50 includes an absorption filter 52, a polarization adjustment element 54, an imaging lens 56, and a CMOS image sensor 58.

[0061] The absorption filter 52 separates the fluorescence FL emitted from the microdevice 200 from light such as scattered light and leakage light, and transmits the fluorescence FL. In one example, the absorption filter 52 is implemented as a band-pass filter. The fluorescence FL emitted from the absorption filter 52 enters the polarization adjustment element 54.

[0062] The polarization adjustment element 54 adjusts the fluorescence FL that has passed through the absorption filter 52 to linearly polarized light. In addition, the polarization adjustment element 54 switches the polarization direction of the linearly polarized fluorescence FL that enters the CMOS image sensor 58 to a direction parallel to the polarization direction of the excitation light EL emitted from the light source unit 10 (X direction) 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 through the imaging lens 56. In one example, the polarization adjustment element 54 is implemented as a twisted nematic (TN) liquid crystal element.

[0063] Based on the trigger signal from the controller 70, the CMOS image sensor 58 detects, for each pulse of one of the plurality of pulsed excitation lights EL, 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 of polarized light in the Z direction) as an image. That is, the CMOS image sensor 58 synchronously detects the spatial distribution of the fluorescence FL having a predetermined polarization direction as an image with the emission of the excitation light EL. The CMOS image sensor 58 generates image data representing the captured image to send the image data to the controller 70. Note that the main surface 202a of the first substrate 202 of the microdevice 200 and the imaging surface of the CMOS image sensor 58 have an imaging relationship.

[0064] The controller 70 of the fluorescence intensity measuring device 100 controls various components of the fluorescence intensity measuring device 100. In addition, the controller 70 calculates the fluorescence intensity of the fluorescence FL having a predetermined polarization direction (i.e., the predetermined polarization direction in the X direction or the polarization direction in the Z direction) based on the duty ratio and light intensity of the pulsed excitation light EL. The light intensity is obtained from the image detected by the CMOS image sensor 58, that is, the image data sent from the CMOS image detector 58. In addition, the controller 70 calculates the degree of polarization P based on the calculated fluorescence intensity of the fluorescence FL, and calculates the concentration of the detected substance based on the degree of polarization P. As Figure 4 shown, the controller 70 includes an input / output device 72, a memory 74, a polarization controller 76, a synchronization signal generator 78, a light source controller 80, a detection controller 82, and a calculator 85. The calculator 85 includes a light intensity calculator 86, a fluorescence intensity calculator 87, and a concentration calculator 88.

[0065] The input / output device 72 inputs / outputs signals, data, etc. between the controller 70 and various components.

[0066] The memory 74 stores programs, image data sent from the CMOS image sensor 58, data of a calibration curve representing the degree of polarization P and the concentration of the detected substance, etc.

[0067] The polarization controller 76 controls the polarization adjustment element 54 of the fluorescence intensity measuring device 100, thereby controlling the polarization direction of the fluorescence FL incident on the CMOS image sensor 58. For example, the polarization controller 76 first adjusts the polarization direction of the fluorescence FL entering the CMOS image sensor 58 to the X direction. After the CMOS image sensor 58 detects the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction as an image, the polarization controller 76 switches the polarization direction of the fluorescence FL entering the CMOS image sensor 58 to the Z direction.

[0068] The synchronization signal generator 78 generates a synchronization signal. The synchronization signal synchronizes the emission (irradiation) of the excitation light EL from the light source 12 (light source unit 10) with the detection (imaging) of the CMOS image sensor 58. The synchronization signal generator 78 sends the synchronization signal to the light source controller 80 and the detection controller 82.

[0069] Based on the synchronization signal sent from the synchronization signal generator 78, the light source controller 80 sends a PWM signal to the light source 12. The light source 12 emits a pulsed light including a pulsed excitation light EL whose pulse width is modulated based on the PWM signal. The light with the modulated pulse width emitted from the light source 12 passes through the polarization filter 22, the excitation light filter 24, etc., and is emitted onto the measurement target solution (microdevice 200) as a pulsed excitation light EL whose pulse width is modulated and whose polarization direction is in the X direction.

[0070] In this embodiment, a plurality of pulsed excitation lights E1 - E4 whose pulse widths t1 - t4 are sequentially widened are sequentially emitted onto the measurement target solution at intervals of T. The plurality of pulsed excitation lights E1 - E4 are as Figure 5 shown. Then, the pulse trains of the plurality of excitation lights E1 - E4 are repeatedly emitted onto the measurement target solution. Here, the polarization direction of each of the plurality of excitation lights E1 - E4 is in the X direction and has an equal excitation light intensity.

[0071] Based on the synchronization signal sent from the synchronization signal generator 78, the detection controller 82 sends a trigger signal to the CMOS image sensor 58. The CMOS image sensor 58 detects each of the plurality of fluorescence FLs as an image at intervals of T based on the trigger signal. Each of the plurality of fluorescence FLs has a predetermined polarization direction (polarization direction in the X direction or polarization direction in the Z direction) emitted from the measurement target solution by the corresponding plurality of excitation lights E1 - E4. Hereinafter, an example of fluorescence FL with a polarization direction in the X direction is used to describe the light intensity calculator 86 and the fluorescence intensity calculator 87. Here, the image detected by the excitation light E1 is defined as Pc1, the image detected by the excitation light E2 is defined as Pc2, the image detected by the excitation light E3 is defined as Pc3, and the image detected by the excitation light E4 is defined as Pc4.

[0072] The light intensity calculator 86 of the calculator 85 calculates the light intensity (brightness) of the microchannel 220 at a predetermined position for each of the images Pc1 - Pc4 detected by the CMOS image sensor 58. For example, at the position A of the microchannel 220 as Figure 2 shown, the light intensities A1 - A4 as Figure 6 shown are calculated. In addition, at the position B of the microchannel 220 as Figure 2 shown, the light intensities B1 - B4 as Figure 7 shown are calculated. AsFigure 6 and Figure 7 As shown in Figure 7 , the light intensities calculated from images Pc1 - Pc4 include the fluorescence intensities ΔF1 - ΔF4 of fluorescence FL with the polarization direction in the X direction and noise components. The fluorescence intensities ΔF1 - ΔF4 are fluorescence components according to the pulse widths t1 - t4 of multiple excitation lights E1 - E4. The noise components are caused by heat, infrared light, etc. from the peripheral circuit of the CMOS image sensor 58, and are hereinafter referred to as noise components caused by the peripheral circuit. The fluorescence intensities ΔF1 - ΔF4 of fluorescence FL are independent of the position of the microchannel 220, but the magnitude of the noise components caused by the peripheral circuit depends on the position of the microchannel 220.

[0073] The fluorescence intensity calculator 87 of the calculator 85 calculates the fluorescence intensities ΔF1 - ΔF4 of fluorescence FL with the polarization direction in the X direction emitted from the measurement target solution by the corresponding multiple excitation lights E1 - E4 according to the duty cycles and light intensities A1 - A4 and B1 - B4 of the multiple excitation lights E1 - E4 calculated by the light intensity calculator 86 from images Pc1 - Pc4. Here, an example of the fluorescence intensity ΔF1 - ΔF4 of fluorescence FL with the polarization direction in the X direction at the position A of the microchannel 220 is used to describe how to calculate the fluorescence intensity of fluorescence FL.

[0074] The fluorescence intensities ΔF1 - ΔF4 of fluorescence FL depend on the pulse widths t1 - t4 of the multiple excitation lights E1 - E4, and the magnitude of the noise components caused by the peripheral circuit is independent of the pulse widths t1 - t4 of the multiple excitation lights E1 - E4. Therefore, when defining two excitation lights among the multiple excitation lights E1 - E4, one excitation light En has a pulse width tn and a duty cycle dutyn, and the other excitation light Em has a pulse width tm and a duty cycle dutym, the light intensity obtained from the image Pcn detected by the excitation light En is An, the light intensity obtained from the image Pcm detected by the excitation light Em is Am, and the interval between each of the multiple excitation lights E1 - E4 being emitted is T, the fluorescence intensity ΔFn of fluorescence FL emitted from the measurement target solution by one excitation light En is represented by the following formulas (1) - (3): where n = 1, 2, 3, or 4, m = 1, 2, 3, or 4 and n ≠ m. That is, the fluorescence intensities ΔF1 - ΔF4 of fluorescence FL are calculated according to the duty cycles dutyn and dutym of two excitation lights En and Em with different pulse widths, and the light intensities An and Am obtained from the images Pcn and Pcm detected by these two excitation lights En and Em respectively.

[0075]

[0076] The fluorescence intensity calculator 87 calculates the fluorescence intensity ΔFn of fluorescence FL emitted from the measurement target solution by one excitation light En according to formulas (1) - (3). AsFigure 8 As shown, according to formulas (1)-(3), the fluorescence intensities ΔF1-ΔF4 of the fluorescence FL with the polarization direction in the X direction can be obtained, where the light intensities A1-A4 obtained from the images Pc1-Pc4 ( Figure 6 ) have had the noise components caused by the peripheral circuit removed.

[0077] In this embodiment, based on the duty cycles dutyn and dutym of the two excitation lights En and Em and the light intensities An and Am obtained from the detected images Pcn and Pcm, the fluorescence intensities ΔF1-ΔF4 of the fluorescence FL are calculated. Therefore, the noise components caused by the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. In addition, even if the excitation light EL includes a DC component, the noise components caused by the peripheral circuit can be removed.

[0078] In addition, the excitation light intensities of the multiple excitation lights E1-E4 are constant, and the pulse widths (pulse widths t1-t4) of the multiple excitation lights E1-E4 are modulated. Therefore, the excitation light EL can be easily generated, and waveform distortion 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.

[0079] Note that the fluorescence intensity of the fluorescence FL with the polarization direction in the Z direction can be calculated similarly to the fluorescence intensities ΔF1-ΔF4 of the fluorescence FL with the polarization direction in the X direction. The above light intensity calculator 86 calculates the light intensity based on one image, but the light intensity can be calculated from the accumulated images by accumulating multiple images detected by multiple excitation lights EL having equal pulse widths.

[0080] The concentration calculator 88 of the calculator 85 calculates the degree of polarization P based on the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction and the fluorescence intensity of the fluorescence FL with the polarization direction in the Z direction. In this embodiment, the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction corresponds to the fluorescence intensity Ih of the fluorescence FL with the polarization direction parallel to the polarization direction of the excitation light EL. In addition, the fluorescence intensity of the fluorescence FL with the polarization direction in the Z direction corresponds to the fluorescence intensity Iv of the fluorescence FL with the polarization direction perpendicular to the polarization direction of the excitation light EL. In this case, the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction and the fluorescence intensity of the fluorescence FL with the 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 multiple excitation lights EL having the same pulse width.

[0081] The concentration calculator 88 also calculates the concentration of the detected substance based on the calculated degree of polarization P and the calibration curve of the degree of polarization P and the concentration of the detected substance.

[0082] Figure 9 shows the hardware configuration of the controller 70. The controller 70 includes a central processing unit (CPU) 92, a read-only memory (ROM) 93, a random access memory (RAM) 94, and an input / output interface 96. The CPU 92 executes programs 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 various components. The functions of the controller 70 are implemented by the CPU 92 executing programs.

[0083] As described above, the fluorescence intensity of the fluorescence FL is calculated by irradiating the measurement target with the excitation light EL whose pulse width is modulated. Therefore, by removing the noise components caused by the peripheral circuit, the fluorescence intensity of the fluorescence FL emitted from the measurement target solution can be easily calculated, and the measurement sensitivity of the fluorescence intensity can be improved with a simple configuration. In addition, since the fluorescence intensity of the fluorescence FL is calculated based on the duty ratio and the light intensity obtained from the detected image, even if the excitation light EL includes a DC component, the noise components caused by the peripheral circuit can be removed.

[0084] Next, with reference to Figure 10 and Figure 11 the detection process (i.e., the detection substance detection method) of the fluorescence intensity measurement device 100 is described. As Figure 10 shown, the detection process sequentially executes a fluorescence intensity measurement process (step S100) for calculating the fluorescence intensity of the fluorescence FL and a concentration calculation process (step S200) for detecting the concentration of the detection substance. The fluorescence intensity measurement process (step S100) corresponds to the method of measuring the fluorescence intensity.

[0085] With reference to Figure 11 the fluorescence intensity measurement process (step S100) is described. In the fluorescence intensity measurement process (step S100), the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction and the fluorescence intensity of the fluorescence FL with the polarization direction in the Z direction emitted from the measurement target solution introduced into the microchannel 220 of the microdevice 200 are calculated.

[0086] First, the controller 70 controls the polarization adjustment element 54 to control the polarization direction of the fluorescence FL entering 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 placed on the stage ST with the excitation light EL whose pulse width is modulated at intervals T and whose polarization direction is in the X direction (step S112). The light with the modulated pulse width emitted from the light source 12 passes through the polarization filter 22, the excitation light filter 24, etc., and is emitted onto the measurement target solution as the excitation light EL with the modulated pulse width and the polarization direction in the X direction. In this embodiment, a plurality of pulsed excitation lights E1 - E4 with the pulse widths t1 - t4 sequentially widened are emitted onto the measurement target solution at intervals T( Figure 5 ). As a result, fluorescence FL is emitted from the measurement target solution.

[0087] Next, the controller 70 controls the CMOS image sensor 58 to detect, through the CMOS image sensor 58, the fluorescence FL with the polarization direction in the X direction among the plurality of fluorescence FL emitted from the measurement target solution as an image of each pulse of the excitation light EL (step S114). Specifically, the fluorescence FL emitted from the measurement target solution passes through the dichroic mirror 30, the polarization adjustment element 54, etc., and enters the CMOS image sensor 58 as the fluorescence FL with the polarization direction in the X direction. The CMOS image sensor 58 detects the spatial distribution of the fluorescence FL with the polarization direction in the X direction as an image at intervals T for each pulse of the excitation light EL. The controller 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. The image Pc3 is detected by the excitation light E3, and the image Pc4 is detected by the excitation light E4.

[0088] The controller 70 controls the polarization adjustment element 54 to control the polarization direction of the fluorescence FL entering 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 placed on the stage ST with the excitation light EL whose pulse width is modulated at intervals T and whose polarization direction is in the X direction (step S116). Thus, similar to step S112, the measurement target solution is irradiated with the excitation light EL with the modulated pulse width and the polarization direction in the X direction, and fluorescence FL is emitted from the measurement target solution.

[0089] Next, the controller 70 controls the CMOS image sensor 58 to detect, from among the multiple fluorescence FL emitted from the measurement target solution, the fluorescence FL with a polarization direction in the Z direction as an image of each pulse of the excitation light EL by the CMOS image sensor 58 (step S118). Similar to step S114, the CMOS image sensor 58 detects the spatial distribution of the fluorescence FL with a polarization direction in the Z direction as an image at intervals of T for each pulse of the excitation light EL. The controller 70 acquires image data representing the captured image.

[0090] Next, the controller 70 calculates the fluorescence intensity ΔFn of the fluorescence FL with a polarization direction in the X direction (step S120). The controller 70 calculates the fluorescence intensity ΔFn of the fluorescence FL with a polarization direction in the X direction according to the duty ratios dutyn and dutym of two excitation lights En and Em with different pulse widths, and the light intensities An and Am of the fluorescence FL with a polarization direction in the X direction obtained from the images Pcn and Pcm detected by the two excitation lights En and Em respectively, by the above formulas (1)-(3), where n = 1, 2, 3, or 4, m = 1, 2, 3, or 4, and n ≠ m.

[0091] Specifically, the controller 70 first calculates the light intensities A1 - A4 of the microchannel 220 at a predetermined position (position A) according to the images Pc1 - Pc4 ( Figure 6 ) that detect the fluorescence intensity of the fluorescence FL with a polarization direction in the X direction. Next, according to formulas (1)-(3), based on the duty ratios duty1 - duty4 of the multiple excitation lights E1 - E4 and the calculated light intensities A1 - A4, the controller 70 calculates the fluorescence intensities ΔF1 - ΔF4 of the fluorescence FL with a polarization direction in the X direction, where the noise components caused by the peripheral circuit are removed from the light intensities A1 - A4 ( Figure 8 ).

[0092] Next, the controller 70 calculates the fluorescence intensity of the fluorescence FL with a polarization direction in the Z direction (step S122). The controller 70 calculates the fluorescence intensity of the fluorescence FL with a polarization direction in the Z direction according to the duty ratios dutyn and dutym of two excitation lights En and Em with different pulse widths, and the light intensity of the fluorescence FL with a polarization direction in the Z direction obtained from the images detected by the two excitation lights En and Em respectively, by the above formulas (1)-(3), where n = 1, 2, 3, or 4, m = 1, 2, 3, or 4, and n ≠ m. The specific steps for calculating the fluorescence intensity of the fluorescence FL with a polarization direction in the Z direction are the same as those in step S120. When the processing in step S122 ends, the fluorescence intensity measurement processing (step S100) ends.

[0093] In this embodiment, the fluorescence intensity of the fluorescence FL is calculated based on the duty cycles of the two excitation lights EL and the light intensity obtained from the images detected by the two excitation lights EL. Therefore, the noise components caused by the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. In addition, even if the excitation light EL includes a DC component, the noise components caused by the peripheral circuit can be removed.

[0094] The excitation light EL with a modulated pulse width is used. Therefore, the excitation light EL can be easily emitted onto the measurement target solution. It can also suppress the waveform distortion of the excitation light EL. In addition, 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.

[0095] Return to Figure 10 , the concentration calculation process (step S200) is described. The concentration calculation process (step S200) calculates the concentration of the detected substance. First, the controller 70 calculates the degree of polarization P using the fluorescence intensity of the fluorescence FL with the polarization direction in the X direction as the fluorescence intensity Ih and the fluorescence intensity of the fluorescence FL with the polarization direction in the Z direction as the fluorescence intensity Iv. The controller 70 also calculates the concentration of the detected substance based on the degree of polarization P and the calibration curve of the degree of polarization P and the concentration of the detected substance. When the concentration calculation process (step S200) ends, the detection process ends.

[0096] As described above, the fluorescence intensity measuring device 100 calculates the fluorescence intensity of the fluorescence FL based on the duty cycles of the two excitation lights EL and the light intensity obtained from the images detected by the two excitation lights EL. Therefore, the noise components caused by the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. In addition, even if the excitation light EL includes a DC component, the noise components caused by the peripheral circuit can be removed.

[0097] In addition, in the fluorescence intensity measuring device 100, the excitation light intensity of the excitation light EL is constant, and the pulse widths of multiple excitation lights EL are modulated. Therefore, the excitation light EL can be easily generated. It can also suppress the waveform distortion of the excitation light EL. 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.

[0098] Embodiment 2

[0099] In Embodiment 1, multiple pulse-shaped excitation lights E1 - E4 with sequentially widened pulse widths t1 - t4 are emitted onto the measurement target solution as the excitation light EL. Any method can be used to modulate the pulse width of the excitation light EL. For example, the pulse width of the excitation light EL can be randomly modulated.

[0100] Except that the excitation light EL with a randomly modulated pulse width is emitted onto the measurement target solution, the configuration of the fluorescence intensity measurement device 100 in this embodiment is the same as that of the fluorescence intensity measurement device 100 in Embodiment 1. In this embodiment, the light source controller 80, the detection controller 82, the light intensity calculator 86, and the fluorescence intensity calculator 87 of the controller 70 and the light source 12 are described.

[0101] The light emitted by the light source 12 in this embodiment includes the excitation light EL, and the pulse width of the excitation light EL is modulated based on the PWM signal from the light source controller 80. The excitation light EL in this embodiment has a randomly modulated pulse width. In this embodiment, similar to Embodiment 1, the excitation light EL with a modulated pulse width and a polarization direction in the X direction is emitted onto the measurement region R of the microdevice 200 through the dichroic mirror 30 and the objective lens 40. The excitation light EL with a modulated pulse width will be described later.

[0102] Similar to the light source controller 80 in Embodiment 1, the light source controller 80 in this embodiment controls the light source 12. The light source 12 in this embodiment emits light including the excitation light EL, and the pulse width of the excitation light EL is randomly modulated based on the PWM signal from the light source controller 80. In this embodiment, Figure 12 The examples of the four pulsed excitation lights E1 - E4 emitted sequentially as shown are used to describe the detection controller 82, the light intensity calculator 86, and the fluorescence intensity calculator 87.

[0103] Figure 12 The multiple excitation lights E1 - E4 as shown respectively have pulse widths t1 - t4. The pulse widths become wider 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).

[0104] Similar to the detection controller 82 in Embodiment 1, the detection controller 82 in this embodiment controls the CMOS image sensor 58. The CMOS image sensor 58 detects, at intervals of T, the fluorescence FL emitted from the measurement target solution and having a predetermined polarization direction from the multiple excitation lights E1 - E4 as an image. Also in this embodiment, the image detected by the excitation light E1 is defined as Pc1, the image detected by the excitation light E2 is defined as Pc2, the image detected by the excitation light E3 is defined as Pc3, and the image detected by the excitation light E4 is defined as Pc4.

[0105] Similar to the light intensity calculator 86 in Embodiment 1, the light intensity calculator 86 in this embodiment calculates the light intensity at a predetermined position in the microchannel 220 for each of the images Pc1 - Pc4. For example, at Figure 2 the position A of the microchannel 220 as shown, it calculatesFigure 13 The light intensities A1 - A4 shown.

[0106] Similar to the fluorescence intensity calculator 87 of Embodiment 1, the fluorescence intensity calculator 87 of this embodiment calculates the fluorescence intensities ΔF1 - ΔF4 of the fluorescence FL according to the above formulas (1) - (3) Figure 14 ). As described above, the fluorescence intensity of the fluorescence FL is calculated 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 method of modulating the pulse width of the excitation light EL, the fluorescence intensity of the fluorescence FL can be calculated by removing the noise components caused by the peripheral circuit. The excitation light EL of this embodiment has a randomly modulated pulse width. Therefore, the influence of device - specific repetitive noise can be suppressed.

[0107] In this embodiment, the fluorescence intensity of the fluorescence FL is also calculated 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 caused by the peripheral circuit can be easily removed, and the measurement sensitivity can be improved. In addition, even if the excitation light EL includes a DC component, the noise components caused by the peripheral circuit can be removed.

[0108] In the fluorescence intensity measurement device 100 of this embodiment, the excitation light intensity of the excitation light EL is constant, and the pulse widths of multiple excitation lights EL are modulated. Therefore, the excitation light EL can be easily generated. It can also suppress the waveform distortion of the excitation light EL. 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.

[0109] Modification

[0110] Although the embodiments have been described above, various modifications can be made without departing from the scope of the present disclosure.

[0111] For example, in the above - described embodiment, the micro - device 200 includes three micro - channels 220. It is sufficient that the micro - device 200 includes at least one micro - channel 220. A configuration in which the micro - device 200 includes a plurality of micro - channels 220 is also possible.

[0112] As the excitation light EL, the embodiments describe four pulsed excitation lights E1 - E4 as an example. It is sufficient that the excitation light EL is formed by a plurality of pulsed excitation lights EL.

[0113] In the embodiment, the linearly polarized excitation light EL is emitted onto the measurement target solution (measurement target). The excitation light EL emitted onto the measurement target solution can be in an unpolarized state.

[0114] The fluorescence intensity measurement device of the present disclosure can measure the fluorescence intensity of fluorescence FL in an unpolarized state. For example, the fluorescence intensity measurement device of the present disclosure can measure the fluorescence intensity of fluorescence emitted from a deoxyribonucleic acid (DNA) array.

[0115] For purposes of explanation, some example embodiments have been described above. Although the foregoing discussion has presented specific embodiments, those skilled in the art will recognize that changes may be made in form and detail without departing from the broader spirit and scope of the invention. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive. Thus, this detailed description is not to be considered limiting, and the scope of the invention is defined only by the included claims and all equivalents to which those claims are entitled.

Claims

1. A fluorescence intensity measuring device, comprising: A light source, used for emitting a plurality of pulse-shaped excitation lights with modulated pulse widths to a measurement target; a CMOS image sensor for detecting fluorescence emitted from the measurement target by one of the plurality of pulse-shaped excitation lights as an image of each pulse of the one of the plurality of pulse-shaped excitation lights; and A controller for calculating the fluorescence intensity of the fluorescence, wherein The controller calculates the fluorescence intensity of the fluorescence based on two duty ratios of two pulse-shaped excitation lights having different pulse widths among the plurality of pulse-shaped excitation lights and two light intensities obtained from images detected by the two pulse-shaped excitation lights.

2. The fluorescence intensity measuring device according to claim 1, wherein The pulse width of the pulse-shaped excitation light emitted from the light source is randomly modulated.

3. The fluorescence intensity measuring device according to claim 1 or 2, wherein A polarization adjustment element is used to adjust the fluorescence incident on the CMOS image sensor into linearly polarized light and switch the polarization direction of the linearly polarized fluorescence, wherein The CMOS image sensor detects the fluorescence whose polarization direction is in a predetermined direction as an image of each pulse of one pulse-shaped excitation light among the plurality of pulse-shaped excitation lights, and The controller calculates the fluorescence intensity of the fluorescence having a polarization direction in the predetermined direction.

4. A method for measuring fluorescence intensity, comprising: emitting a plurality of pulse-shaped excitation lights with modulated pulse widths toward a measurement target; detecting, by the CMOS image sensor, fluorescence emitted from the measurement target by one of the plurality of pulse-shaped excitation lights as an image of each pulse of the one of the plurality of pulse-shaped excitation lights; and Calculate the fluorescence intensity of the fluorescence, where In the calculation, The fluorescence intensity of the fluorescence is calculated based on two duty ratios of two pulse-shaped excitation lights having different pulse widths among the plurality of pulse-shaped excitation lights and two light intensities obtained from images detected by the two pulse-shaped excitation lights.

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