Optical measuring device and optical measuring method

Abstract

In an optical measuring device, in a first process, excitation light is irradiated onto a reference member, and a light for calibration processing, including scattered light of the excitation light from the reference member, is detected as a detection light. A calibration signal corresponding to the light for calibration processing is used as a detection signal, and a calibration process is performed to remove a signal component due to scattered light from the detection signal in a second process. Additionally, in an optical measuring device, in a second process, excitation light is irradiated onto a sample, and a light to be measured, including fluorescence generated from the sample and scattered light from the sample irradiated with the excitation light, is detected as a detection light. A signal component due to scattered light in the result of the calibration process in the first process is removed from the measurement signal.

Description

Technology Field

[0001] One aspect of the present invention relates to an optical measuring device and an optical measuring method for measuring the optical properties of a sample. Background Technology

[0002] Patent document 1 discloses a technique for generating and removing a fluorescence noise component (fluorescence component from the substrate) included when irradiating an excitation light onto a fluorescent sample placed on a substrate and measuring the fluorescence. Specifically, in Patent document 1, a fluorescence noise component is removed by generating a fluorescence noise signal having the same phase as the fluorescence of the substrate and subtracting the fluorescence noise signal from the measured fluorescence signal. Prior art literature

[0003] Patent Document 1: Japanese Patent Application No. 2010-518394 The problem to be solved

[0004] As described above, in Patent Document 1, fluorescence noise components generated from sources other than the measurement target part of the sample are removed from the fluorescence generated by irradiating the sample with excitation light. Here, regarding the noise components included when light is irradiated onto a sample and light from the sample is detected, it is conceivable that the irradiated light (e.g., excitation light) itself (e.g., scattered light) may become a noise component, in addition to the light generated from the sample (the one irradiated). The technology of Patent Document 1 does not take into account the case where the irradiated light itself becomes a noise component, and therefore cannot remove such noise components.

[0005] Meanwhile, the inventors, etc., noticed the difference in phase between fluorescence and scattered light (light originating from the irradiation light itself) included in the detection light, and discovered an optical measuring device that eliminates noise components originating from the irradiation light itself by removing the signal component of scattered light, which is a signal component having the same phase as the irradiation light, from the detection light. In such an optical measuring device, for example, a calibration process is performed in advance. In the calibration process, a light for calibration processing (ideally, light containing only scattered light) is detected by irradiating a part of the sample where fluorescence is unlikely to occur, and based on the calibration signal corresponding to the light for calibration processing, only the signal component corresponding to approximately the scattered light component can be removed from the detection signal.

[0006] Here, in the calibration process, it is conceivable to detect calibration processing light containing generally only scattered light by irradiating a portion of the sample (e.g., a chip made to facilitate measurement, hereinafter referred to as the actual sample) that is difficult to generate fluorescence (e.g., an area where the amount of fluorescent material in the actual sample is judged to be low) with an irradiating light. However, in the method of detecting calibration processing light by irradiating the actual sample with an irradiating light, it is difficult to completely remove fluorescence from the calibration processing light detected by the photodetector, and thus trace amounts of fluorescent components are included in the calibration signal processing light. Consequently, it was found that calibration processing is performed based on trace amounts of fluorescent components in addition to scattered light components, and as a result of the calibration processing being affected by fluorescent components, there is a concern that subsequent fluorescence measurements cannot be performed with high precision.

[0007] One aspect of the present invention is made in consideration of the above circumstances and aims to provide an optical measuring device and an optical measuring method capable of performing fluorescence measurement with high precision by removing noise components caused by the irradiation light itself. means of solving the problem

[0008] An optical measuring device according to one aspect of the present invention is an optical measuring device for measuring the optical characteristics of a measurement target, comprising: an irradiation optical system for irradiating an irradiation target with irradiation light; a light detection unit for detecting a detection light originating from the irradiation light; and a signal processing unit for processing a detection signal according to the detection light. In a first processing step, the irradiation optical system irradiates a reference member for calibration processing that is different from the measurement target as the irradiation target and irradiates the irradiation light onto the reference member; the light detection unit detects a calibration processing light, which includes scattered light of the irradiation light from the reference member irradiated with the irradiation light, as the detection light; the signal processing unit takes a calibration signal according to the calibration processing light as the detection signal and performs a calibration processing step to remove a signal component according to the scattered light from the detection signal in a second processing step based on the calibration signal. In a second processing step, the irradiation optical system irradiates a measurement target as the irradiation target and irradiates the measurement target with irradiation light, and the light detection unit detects a measurement including fluorescence generated from the measurement target irradiated with the irradiation light and scattered light from the measurement target irradiated with the irradiation light. The target light is detected as the detection light, the signal processing unit takes the measurement signal corresponding to the target light as the detection signal, and removes the signal component corresponding to the scattered light in the calibration process of the first process from the measurement signal.

[0009] In an optical measuring device according to one embodiment of the present invention, in a first process related to calibration processing, an irradiating light is irradiated onto a reference member different from the object to be measured, and a calibration processing light containing scattered light is detected. Then, in a second process, based on the results of the calibration processing, a signal component due to scattered light is appropriately removed from the measurement signal, thereby removing a noise component caused by the irradiating light itself, and thus fluorescence measurement can be performed with high precision. Furthermore, since a reference member for calibration processing that is different from the object to be measured is used, deviations in the results of the calibration processing due to the characteristics of the object to be measured, which would be problematic when, for example, an irradiating light is irradiated onto the object to be measured to detect the calibration processing light, do not occur. Therefore, fluorescence measurement can be performed with higher precision. Thus, according to an optical measuring device according to one embodiment of the present invention, calibration processing can be performed more appropriately, and a noise component caused by the irradiating light itself can be removed more appropriately, and fluorescence measurement can be performed with higher precision.

[0010] The reference member may include a reflective member that reflects the illumination light. With this configuration, the amount of the detection light can be easily increased.

[0011] It is desirable that the reflective member does not generate fluorescence upon irradiation with illumination light. With this configuration, since fluorescence attributable to the reflective member is not generated even upon irradiation with illumination light (or is generated to a negligible degree), only the signal component corresponding to the scattered light component can be reliably removed from the measurement signal in the second processing.

[0012] The reflective member may include a reflective diffuser that diffuses the illumination light. With this configuration, scattered light at various angles can be easily generated by the reflective diffuser, thereby making it easier to increase the amount of detected light.

[0013] The reflective member may include a reflective substrate that reflects the irradiated light and a diffuser that is supported by the reflective substrate and diffuses the irradiated light. With this configuration, the amount of detected light can be more easily increased through the cooperation between the reflective substrate and the diffuser.

[0014] The reflective member may include a mirror. With this configuration, the amount of detected light can be more easily increased by adjusting the reflection angle of the mirror relative to the light detector.

[0015] An optical measuring device according to one aspect of the present invention is an optical measuring device for measuring the optical characteristics of a measurement target, comprising: an illumination optical system for irradiating illumination light; a light detection unit having a first detection optical system and a second detection optical system for detecting detection light originating from the illumination light; and a signal processing unit for processing a detection signal according to the detection light. In the first processing, the illumination optical system irradiates illumination light to the first detection optical system, and the first detection optical system of the light detection unit detects the illumination light, which is a light for calibration processing, as a detection light. The signal processing unit takes the calibration signal according to the light for calibration processing as a detection signal and, based on the calibration signal, performs a calibration processing to remove a signal component according to scattered light from the detection signal in the second processing. In the second processing, the illumination optical system irradiates illumination light to a measurement target, and the second detection optical system of the light detection unit detects a measurement target light, which includes fluorescence generated from the measurement target irradiated with the illumination light and scattered light from the measurement target irradiated with the illumination light, as a detection light. The signal processing unit then takes the measurement signal according to the measurement target light. The detection signal is used, and the signal component due to scattered light in the calibration process of the first process is removed from the measurement signal.

[0016] In an optical measuring device according to one embodiment of the present invention, in a first process related to calibration processing, direct illumination light is irradiated onto a first detection optical system, and the illumination light, which is the illumination light, is detected. The illumination light is light having the same phase as scattered light. Therefore, by the first detection optical system detecting the illumination light as the light for calibration processing, it becomes possible to appropriately detect light that does not contain fluorescence and has the same phase as scattered light as the light for calibration processing. With this, in a second process, based on the results of the calibration processing, the signal component due to scattered light is appropriately removed from the measurement signal, thereby removing noise components caused by the illumination light itself and enabling high-precision fluorescence measurement. Furthermore, since the light for calibration processing is detected without irradiating the object to be measured with illumination light, deviations in the results of the calibration processing due to the characteristics of the object to be measured, which would be problematic when, for example, the light for calibration processing is detected by irradiating the object to be measured with illumination light, do not occur. Therefore, fluorescence measurement can be performed with higher precision. Thus, according to one embodiment of the present invention, the optical measuring device can perform calibration processing more appropriately, thereby more appropriately removing noise components caused by the irradiation light itself, and thus fluorescence measurement can be performed with higher precision.

[0017] An optical measuring device according to one embodiment of the present invention is an optical measuring device for measuring the optical characteristics of a measurement target, comprising an illumination optical system that irradiates illumination light according to a modulation signal, a light detection unit that detects detection light caused by the illumination light, and a signal processing unit. In a first processing step, the signal processing unit generates a calibration signal in which the phase of the modulation signal is changed according to a delay corresponding to the time until scattered light of the illumination light irradiated from the illumination optical system is detected by the light detection unit as detection light, and based on the calibration signal, performs a calibration processing step to remove a signal component according to scattered light from the detection signal in a second processing step. In a second processing step, the illumination optical system irradiates illumination light onto a measurement target, and the light detection unit detects a measurement target light, which includes fluorescence generated from the measurement target irradiated by the illumination light and scattered light from the measurement target irradiated by the illumination light, as a detection light. The signal processing unit sets a measurement signal according to the measurement target light as a detection signal and removes a signal component according to scattered light in the calibration processing step in the first processing step from the measurement signal.

[0018] In an optical measuring device according to one embodiment of the present invention, in a first process related to calibration processing, a calibration signal is generated in which the phase of a modulation signal input to an illumination optical system is changed according to the delay of the illumination optical system. In this way, by generating a calibration signal according to the delay of the illumination optical system with respect to a modulation signal related to the illumination light, it is possible to obtain a calibration signal (a calibration signal having the same phase as the scattered light) that is equivalent to when scattered light is actually detected as the light for calibration processing, without detecting the light for calibration processing. That is, according to this configuration, a calibration signal consisting only of the signal component of scattered light, which does not include the signal component of fluorescence, can be obtained. With this, in a second process, by appropriately removing the signal component due to scattered light from the measurement signal based on the result of the calibration processing, noise components caused by the illumination light itself are removed, thereby enabling high-precision fluorescence measurement. In addition, since the light for calibration processing is detected without irradiating the object to be measured with illumination light, deviations in the results of the calibration processing due to the characteristics of the object to be measured, which are problematic when, for example, the light for calibration processing is detected by irradiating the object to be measured with illumination light, do not occur. Therefore, fluorescence measurement can be performed with higher precision. Thus, according to one embodiment of the present invention, the optical measuring device allows for more appropriate calibration processing, thereby more appropriately removing noise components caused by the illumination light itself, and enabling fluorescence measurement to be performed with higher precision.

[0019] In the first processing step, the signal processing unit may set the amplitude of the calibration signal according to the amplitude when the scattered light of the illumination light irradiated from the illumination optical system is detected by the light detector as the detection light. By doing so, a calibration signal that is closer to the case where the scattered light is actually detected as the light for calibration processing can be obtained.

[0020] An optical measurement method according to one embodiment of the present invention is an optical measurement method for measuring the optical characteristics of a measurement target, comprising: irradiating an irradiating light onto a reference member for calibration processing that is different from the measurement target; detecting a calibration processing light including scattered light of the irradiating light from the reference member irradiated with the irradiating light; and performing a calibration processing to remove a signal component according to scattered light from a measurement signal based on a calibration signal according to the calibration processing light; and performing a second processing including irradiating an irradiating light onto a measurement target; detecting a measurement target light including fluorescence generated from the measurement target irradiated with the irradiating light and scattered light from the measurement target irradiated with the irradiating light; and removing a signal component according to scattered light in the calibration processing of the first processing from a measurement signal according to the measurement target light.

[0021] An optical measurement method according to one embodiment of the present invention is an optical measurement method for measuring the optical characteristics of a measurement target, comprising: a first process including irradiating an irradiating light, detecting a light for calibration processing which is the irradiating light, and performing a calibration process to remove a signal component according to scattered light from a measurement signal based on a calibration signal according to the light for calibration processing; and a second process including irradiating an irradiating light onto a measurement target, detecting a measurement target light including fluorescence generated from the measurement target irradiated by the irradiating light and scattered light from the measurement target irradiated by the irradiating light, and removing a signal component according to the calibration process in the first process from a measurement signal according to the measurement target light.

[0022] An optical measurement method according to one embodiment of the present invention is an optical measurement method for measuring the optical characteristics of a measurement target, comprising: a first process including generating a calibration signal that changes the phase of a modulation signal input to an irradiation optical system according to a delay corresponding to the time until scattered light of an irradiation light irradiated from an irradiation optical system is detected by a light detector, and performing a calibration process to remove a signal component according to scattered light from a measurement signal based on the calibration signal; and a second process including irradiating an irradiation light onto a measurement target, detecting a measurement target light including fluorescence generated from the measurement target irradiated by the irradiation light and scattered light from the measurement target irradiated by the irradiation light, and removing a signal component according to scattered light in the calibration process of the first process from a measurement signal according to the measurement target light. Effects of the invention

[0023] According to one embodiment of the present invention, noise components caused by the irradiation light itself are removed, allowing for high-precision fluorescence measurement. Brief explanation of the drawing

[0024] FIG. 1 is a schematic diagram of an optical measuring device according to an embodiment of the present invention. Figure 2 is a diagram illustrating the phase difference between fluorescence and scattered light. Figure 3 is a diagram illustrating a method for canceling scattered light. Figure 4 is a diagram illustrating a fluorescence measurement method. Figure 5 is a flowchart showing the fluorescence measurement process by an optical measuring device. Figure 6 is a schematic diagram of an optical measuring device. Figure 7 is a diagram illustrating the removal of noise components. Figure 8 is a schematic diagram showing a sample. Figure 9 is a diagram explaining the definition of S / N. Figure 10 is a diagram illustrating the signal used in the calibration process. Figure 11 is a diagram illustrating the calibration process. Figure 12 is a table showing the ratio of the fluorescence component for each modulation frequency. Figure 13 is a flowchart showing the calibration process. Figure 14 is a diagram illustrating the effect of calibration processing. Figure 15 is a diagram illustrating the fluorescence measurement procedure. Figure 16 is a diagram illustrating the effect of calibration when using a reference member that generates fluorescence. Figure 17 is a diagram illustrating the effect of calibration when using a reference member that does not emit fluorescence. FIG. 18 is a diagram illustrating an overview of fluorescence measurement in the first embodiment. FIG. 19 is a diagram illustrating the acquisition of scattered light when a mirror is used as a reference element. FIG. 20 is a diagram illustrating the acquisition of scattered light when a reflective diffuser is used as a reference member. FIG. 21 is a diagram illustrating the acquisition of scattered light when opaque glass is used as a reference member. FIG. 22 is a flowchart illustrating the fluorescence measurement process of the first embodiment. FIG. 23 is a schematic diagram of an optical measuring device of the second embodiment. FIG. 24 is a diagram illustrating an overview of fluorescence measurement in the second embodiment. FIG. 25 is a diagram illustrating the acquisition of irradiation light of the second embodiment. FIG. 26 is a flowchart illustrating the fluorescence measurement process of the second embodiment. FIG. 27 is a schematic diagram of an optical measuring device of the third embodiment. FIG. 28 is a diagram illustrating an overview of fluorescence measurement in the third embodiment. FIG. 29 is a flowchart illustrating the fluorescence measurement process of the third embodiment. Specific details for implementing the invention

[0025] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In addition, in each drawing, the same or substantial parts are given the same reference numerals, and redundant descriptions are omitted.

[0026] FIG. 1 is a schematic diagram of an optical measuring device (1) according to the present embodiment. The optical measuring device (1) is a device that detects light generated from a sample according to light irradiated onto the sample. In the present embodiment, the optical measuring device (1) is described as a fluorescence measuring device that detects fluorescence generated from a sample according to excitation light (irradiation light) irradiated onto the sample. Excitation light is light that excites the sample, and fluorescence is light emitted by the sample according to the excitation light, and is light with a wavelength different from that of the excitation light. Also, in the present embodiment, the optical measuring device (1) is described as a device that detects fluorescence related to measurement using the immunochromatography method. The immunochromatography method is an immunoassay method using an antigen-antibody reaction, and is used, for example, for the detection of influenza viruses.

[0027] As shown in FIG. 1, in a measurement using the immunochromatography method, an immunochromatography test piece (100) is prepared as a sample. The immunochromatography test piece (100) contains an immunochromatography membrane to be measured within a reagent holder (101). At a specific location (measurement target) of the immunochromatography membrane of the immunochromatography test piece (100), a capture antibody against a predetermined antigen (e.g., an antibody against an influenza virus antigen) is fixed. The reagent holder (101) is provided with a sample adhesion window, which is an opening for dropping the sample onto the immunochromatography membrane, and a measurement window, which is an opening for measuring the measurement target where the capture antibody is fixed. When a sample is applied to the sample adhesion window of the reagent holder (101), the antigen in the sample first binds to a detection antibody labeled with a fluorescent reagent, and then causes an antigen-antibody reaction with the capture antibody to become trapped. The optical measuring device (1) measures the fluorescence intensity by irradiating excitation light onto the immunochromato membrane exposed through the measurement window of the immunochromato test specimen (100) and detecting fluorescence from the antigen-antibody complex (specifically, the fluorescent reagent of the antibody) in the measurement target area. In addition, as the fluorescent reagent, for example, europium, Q-dot (registered trademark), organic dye, etc., may be used.

[0028] Here, in the optical measuring device (1), the detection light that is incident on the detection optical system (20) described later and detected may include not only fluorescence but also light originating from the excitation light itself. Such light may include, for example, scattered light of the excitation light. Such scattered light is, for example, a part of the excitation light generated when the excitation light is irradiated onto the immunochromatographic test specimen (100) and scattered, and is light that has the same phase as the excitation light (no phase difference). Since the immunochromatographic membrane of the immunochromatographic test specimen (100) or the reagent holder (101) is generally white, the scattered light described above is likely to occur. Also, depending on the arrangement of the sample being measured or the detection optical system, the excitation light itself may be detected. Below, it will be explained that the detection light detected by the optical measuring device (1) includes fluorescence and scattered light.

[0029] As shown in FIG. 1, the optical measuring device (1) comprises an irradiation optical system (10), a detection optical system (20) (light detection unit), a light source driving circuit (30), an IV conversion amplifier (40), a waveform generation circuit (50), a cancellation circuit (60) (signal processing unit), a timing generator (70), an AD converter (80), and a CPU (90).

[0030] The irradiation optical system (10) irradiates excitation light (irradiation light) toward the immunochromatographic test specimen (100) (sample) which is the object to be measured. The irradiation optical system (10) has a light source (11), an aperture (12), an excitation light filter (13), and a collimating lens (14). The light source (11) irradiates excitation light onto the immunochromatographic test specimen (100) (sample). The light source (11) is, for example, a semiconductor light-emitting element. In this embodiment, the light source (11) is described as being a light-emitting diode (LED), but it is not limited to this, and, for example, an LD may be used to ensure a sufficient amount of light. The aperture (12) is a light beam shaping member for shaping the light emitted from the light source (11) into light having a desired cross-section of the light beam. The excitation light filter (13) is a wavelength selection filter that filters the wavelength required for the excitation light that has reached through the aperture (12). The excitation light filter (13) is an optical filter, such as a dielectric multilayer filter or a colored glass filter, and more specifically, a bandpass filter made of a dielectric multilayer filter that transmits only a specific wavelength range (the excitation wavelength of the fluorescent reagent). The collimating lens (14) is a lens that forms an image of the excitation light after filtering by the excitation light filter (13) onto an immunochromatographic test piece (100) (more specifically, a measurement target part of an immunochromatographic membrane).

[0031] The detection optical system (20) detects detection light originating from the excitation light. Specifically, the detection optical system (20) detects fluorescence from the immunochromatographic test specimen (100). However, in reality, detection light is incident on the detection optical system (20), which includes scattered light originating from the excitation light itself in addition to the fluorescence from the immunochromatographic test specimen (100) (fluorescence from the measurement target part of the immunochromatographic membrane), and detects the detection light. The detection optical system (20) has a light detection element (21), a fluorescence filter (22), and a condensing lens (23). The detection light is condensed by the condensing lens (23) and incident on the light detection element (21) through the fluorescence filter (22). The fluorescence filter (22) is a wavelength-selective filter provided to suppress light other than fluorescence from the detection light from the immunochromatographic test specimen (100) from reaching the light detection element (21). The fluorescence filter (22) is an optical filter, such as a dielectric multilayer filter or a colored glass filter, and more specifically, a band-pass filter that combines a dielectric multilayer filter and a colored glass filter that transmits only a specific wavelength range. However, for example, when the excitation light wavelength and the fluorescence wavelength are close, it is difficult to efficiently block only scattered light having the excitation light wavelength while appropriately transmitting fluorescence having the fluorescence wavelength using the fluorescence filter (22). Also, the dielectric multilayer filter, which is generally used as an efficient wavelength-selective filter, has its characteristics changed depending on the angle of incidence of the light. Therefore, in this embodiment, the fluorescence filter (22) is configured by combining a dielectric multilayer filter and a colored glass filter, so that scattered light from the oblique direction is effectively blocked by the colored glass filter. However, since it is difficult to achieve a sufficient effect with wavelength selection alone, it is difficult to efficiently block the entry of scattered light with various conditions.In the following description, by providing a fluorescent filter (22), the detection light reaching the light detection element (21) contains scattered light.

[0032] The light detection element (21) is a light sensor that detects detection light after filtering by a fluorescent filter (22). The light detection element (21) is, for example, a semiconductor light receiving element. In this embodiment, the light detection element (21) is described as a photodiode (PD), but is not limited thereto; as long as it can respond at high speed in response to the modulation frequency of the excitation light from the light source (11) described later, it may be an avalanche photodiode (APD) or a photomultiplier tube (PMT), etc. The light detection element (21) specifically detects fluorescence generated from an immunochromatographic test specimen (100) (specifically, a fluorescent reagent of an antigen-antibody complex in the measurement target part of an immunochromatographic membrane) irradiated with excitation light, and detection light containing the scattered light described above, which has no phase difference with the excitation light as light caused by the excitation light. The light detection element (21) outputs a detection signal corresponding to the detection light to an IV conversion amplifier (40).

[0033] The light source driving circuit (30) is a circuit that drives the light source (11), which is an LED, by outputting a driving current to the light source (11). The light source driving circuit (30) receives input of a reference sinusoidal frequency signal from the timing generator (70). The light source driving circuit (30) modulates the frequency of the driving current based on the input reference frequency signal. That is, the light source driving circuit (30) sets the modulation frequency of the light source (11) that outputs excitation light. Accordingly, the frequency of the excitation light output from the light source (11) is modulated, and the amount of light (excitation light amount) from the light source (11) changes in a sinusoidal shape. In addition, the modulation frequency may be determined based on the fluorescence lifetime of the fluorescent reagent used. For example, when europium with a fluorescence lifetime of several milliseconds is used as a fluorescent reagent, the modulation frequency may be about 1 kHz, when Q-dot with a fluorescence lifetime of several tens of nanoseconds is used, the modulation frequency may be about 100 MHz, and when an organic dye with a fluorescence lifetime of several nanoseconds to tens of nanoseconds is used, the modulation frequency may be about 1 GHz.

[0034] Generally, fluorescence lifetime is the time until the fluorescence intensity drops from its peak value to 1 / e (approximately 37%). By working backward from this definition of fluorescence lifetime, it can be assumed that, for example, when using europium with a fluorescence lifetime of several milliseconds, the optimal modulation frequency is 1 kHz, and when using organic dyes with a fluorescence lifetime of several nanoseconds to tens of nanoseconds, the optimal modulation frequency is 100 MHz to 1 GHz. However, when the fluorescence-derived signal output with respect to the modulation frequency was actually measured using a europium reagent, it was found that modulating at a frequency lower than the frequency determined by the fluorescence lifetime resulted in higher fluorescence intensity and a higher ratio of the fluorescence signal to the excitation light (see Fig. 12). As shown in Fig. 12, the fluorescence intensity is higher at frequencies lower than 1 kHz, which is the frequency determined by the fluorescence lifetime. Specifically, the fluorescence lifetime was defined not as 1 / e, but as "the time until the peak value of the fluorescence intensity drops to 1%," and by calculating the modulation frequency from that time, the fluorescence intensity could be increased. In this case, if it is europium, the fluorescence lifetime is about 10ms, and the modulation frequency of the light source (11) determined from this is about 100Hz.

[0035] As described above, the light source driving circuit (30) may determine the modulation frequency of the light source (11) by taking into account the fluorescence intensity. Specifically, the light source driving circuit (30) sets the modulation frequency of the light source (11) lower than a value corresponding to the fluorescence lifetime (specifically, 1 / fluorescence lifetime), which is the time until the fluorescence intensity drops from the peak value to 1 / e. The light source driving circuit (30) sets the modulation frequency of the light source (11) lower than the value corresponding to the fluorescence lifetime and also higher than the commercial frequency (50Hz, 60Hz), for example, to around 100Hz, and sets it to about 110Hz, which reduces the effect of noise by preventing the multiplication of the commercial frequency. The light source driving circuit (30) may set the modulation frequency of the light source (11) to other values ​​around 100Hz, for example, 90Hz, 80Hz, 70Hz, or 130Hz.

[0036] The IV conversion amplifier (40) converts the current signal (detection signal) output from the photodetection element (21) into a voltage signal. The IV conversion amplifier (40) outputs the detection signal converted into a voltage signal to the waveform generation circuit (50).

[0037] The waveform generation circuit (50) is a circuit that generates a waveform of a detection signal based on a detection signal output from an IV conversion amplifier (40). The waveform generation circuit (50) receives input of a reference frequency signal from a timing generator (70). The timing generator (70) inputs the reference frequency signal at the same timing to the light source driving circuit (30) and the waveform generation circuit (50). The waveform generation circuit (50) outputs information of the generated waveform (detection signal) to the cancel circuit (60).

[0038] The cancel circuit (60) is a signal processing unit that processes the waveform (detection signal) generated by the waveform generation circuit (50). The cancel circuit (60) removes the signal component corresponding to scattered light from the detection signal based on the phase difference (phase difference) between the fluorescence and scattered light. Additionally, the cancel circuit (60) acquires information on the phase of the excitation light (i.e., scattered light) by receiving the input of a reference frequency signal from the timing generator (70) at the same timing as the light source driving circuit (30) and the waveform generation circuit (50). By doing so, the cancel circuit (60) can remove the signal component of scattered light based on the phase difference between the fluorescence and scattered light. Details of the processing of the cancel circuit (60) will be explained with reference to FIGS. 2 to 4.

[0039] FIG. 2 is a conceptual diagram illustrating the phase difference between fluorescence and scattered light. As shown in FIG. 2, the sinusoidal detection light (detection light detected by the light detector (D)) from a sample (S) irradiated with sinusoidal excitation light from a light source (L) contains sinusoidal scattered light and fluorescence. Furthermore, the excitation light from the light source (L) is not limited to a sinusoidal shape and may be a periodic modulated waveform such as a square wave, in which case the detection light (scattered light and fluorescence) also has a periodic modulated waveform similar to the excitation light. While scattered light is light that has no phase difference with the excitation light, fluorescence is light generated from the sample (S) according to the excitation light, and is detected with a phase delay of several tens of milliseconds to nanoseconds relative to the scattered light. The inventors have focused on this phase difference and discovered a method to extract only fluorescence by removing only scattered light from the detection light. In addition, in FIG. 2, since the sample (S) and the light detector (D) are positioned on the optical axis of the light source (L), unlike FIG. 1, which detects fluorescence emitted in a direction intersecting the optical axis of the excitation light, fluorescence emitted in a direction coaxial with the optical axis of the excitation light is detected. In this case, in addition to fluorescence and scattered light, the excitation light itself may be included in the detected light. Also, the amount of light attributable to the excitation light incident on the light detector (D) is likely to increase. Therefore, the extraction of fluorescence by this method becomes effective.

[0040] FIG. 3 is a diagram illustrating a method for removing (canceling) scattered light. FIG. 3 shows only the waveform of the scattered light among the detected light. In addition, this waveform is identical to the waveform of the excitation light. In FIG. 3, the horizontal axis represents time and the vertical axis represents amplitude. For the waveform according to the phase of the scattered light shown in FIG. 3, if the time domain is separated (e.g., 1 / 4 of a period) and integrated for each time domain 1 to 4, the output of the scattered light in each time domain 1 to 4 can be obtained. Here, if a certain multiplier is applied to each of the integral values ​​of each time domain 1 to 4 and all are added together, the sum of the outputs can be made zero. That is, the absolute values ​​of the outputs of each time domain 1 to 4 are the same, and the amplitude ranges of time domains 1 and 2 are positive, while the amplitude ranges of time domains 3 and 4 are negative. As shown in FIG. 3, when time domain 1 is amplified by multiplying it by a multiplier "-1", the output of time domain 1 becomes a negative value ("positive × negative"); when time domain 2 is amplified by multiplying it by a multiplier "+1", the output of time domain 2 becomes a positive value ("positive × positive"); when time domain 3 is amplified by multiplying it by a multiplier "+1", the output of time domain 3 becomes a negative value ("negative × positive"); and when time domain 4 is amplified by multiplying it by a multiplier "-1", the output of time domain 4 becomes a positive value ("negative × negative"). Therefore, when the integral values ​​of each time domain 1 to 4, which are amplified by multiplying them by a predetermined multiplier, are all added together, the values ​​cancel each other out, and the sum of the outputs becomes zero. In this way, signal components due to scattered light can be separated into predetermined time units according to the phase of the scattered light, each separated component can be amplified, and each amplified component can be combined to eliminate (make the output zero).

[0041] Figure 4 is a diagram illustrating a fluorescence measurement method. Figure 4 shows the waveforms of scattered light and fluorescence included in the detection signal. In Figure 4, the horizontal axis represents time and the vertical axis represents amplitude. As described above, regarding the signal component due to scattered light, it can be removed (the output can be made zero) by separating it into predetermined time units according to the phase of the scattered light, amplifying each separated component, and synthesizing each amplified component. Here, as shown in Figure 4, since fluorescence has a phase difference with respect to scattered light, when separated into predetermined time units according to the phase of the scattered light, the integral values ​​of each time domain 1 to 4 do not become the same value. Therefore, the value obtained by multiplying each by the same multiplier as the scattered light, amplifying each, and adding them all together results in a non-zero output value. In this way, by separating the scattered light and fluorescence into the same time domain, amplifying, and synthesizing, the signal component of the scattered light can be removed while the output intensity of the fluorescence can be detected and output.

[0042] In this way, the cancel circuit (60) separates the detection signal into predetermined time units according to the phase of the scattered light, amplifies each component of the separated detection signal, and synthesizes each amplified component to remove the signal component due to scattered light from the detection signal and obtain the fluorescence signal component. The cancel circuit (60) outputs the fluorescence signal, which is a signal from which the signal component due to scattered light has been removed (i.e., a signal consisting only of the fluorescence signal component), to the AD converter (80). In addition, although 1 / 4 of a cycle time was exemplified as a predetermined time unit, it is not limited to this, and any time unit may be used as long as it is a time unit capable of removing the signal component due to scattered light after synthesis. Also, although "+1" and "-1" were exemplified as multipliers for amplification, they are not limited to this, and any multiplier may be used as long as it is a multiplier capable of removing the signal component due to scattered light after synthesis.

[0043] The AD converter (80) performs AD conversion on the fluorescent signal output from the cancel circuit (60) to convert it into a digital value and outputs it to the CPU (90). The CPU (90) performs a predetermined control and signal processing on the digital signal (fluorescent signal) output from the AD converter (80). The CPU (90) may transmit the signal processing result to an external computer, for example, via serial communication. In addition, the CPU (90) may generate a signal output from the timing generator (70), that is, a signal determining various operation timings in the optical measuring device (1), and output it to the timing generator (70). In addition, an FPGA may be used instead of the CPU (90). Through the above processing, the optical measuring device (1) can remove the influence of scattered light from the detection light and obtain only the signal regarding the fluorescence of the fluorescent reagent.

[0044] Next, the fluorescence measurement process (optical measurement method) performed by the optical measuring device (1) will be explained with reference to FIG. 5.

[0045] FIG. 5 is a flowchart illustrating a fluorescence measurement process by an optical measuring device (1). As shown in FIG. 5, in the fluorescence measurement process, first, a light source (11) of an irradiation optical system (10) (light source part) irradiates excitation light toward an immunochromatographic test piece (100) (sample) (Step S1). As the excitation light is irradiated toward the immunochromatographic test piece (100) (specifically, an antigen-antibody complex in the measurement target part of the immunochromatographic membrane), fluorescence is emitted from the fluorescent reagent of the antigen-antibody complex. Meanwhile, the excitation light is scattered from the immunochromatographic test piece (100), and scattered light is generated.

[0046] Next, the light detection element (21) of the detection optical system (20) (light detection unit) detects the detection light including the fluorescence and scattered light described above (Step S2). The light detection element (21) outputs the detection light to the IV conversion amplifier (40). Then, the current signal (detection signal) output from the light detection element (21) in the IV conversion amplifier (40) is converted into a voltage signal, and after the waveform of the detection signal is generated in the waveform generation circuit (50), the cancel circuit (60) (signal processing unit) removes the signal component corresponding to the scattered light from the detection signal based on the phase difference between the fluorescence and the scattered light (Step S3). Specifically, the cancel circuit (60) separates the detection signal into a predetermined time unit according to the phase of the scattered light, amplifies each component of the separated detection signal, and synthesizes each amplified component to remove the signal component corresponding to the scattered light from the detection signal and obtain the signal component of the fluorescence. After that, the fluorescent signal is converted into a digital value in the AD converter (80), and a predetermined control and signal processing is performed in the CPU (90) so that a signal regarding the fluorescent signal can be obtained.

[0047] In addition, although it has been described that the optical measuring device (1) removes the signal component due to scattered light from the detection signal in the cancel circuit (60), it is not limited to this. That is, as with the optical measuring device (1A) shown in FIG. 6, the cancel circuit (60) may not be provided, and after A / D conversion in the AD converter (80), the CPU (90A) (signal processing unit) may perform a process to remove the signal component due to scattered light from the detection signal. In this case, since there is no need to provide the cancel circuit (60), it can contribute to the miniaturization of the device.

[0048] Next, the removal of specific signal components (noise) described in the above-described embodiment will be explained in more detail with reference to FIGS. 7 to 14.

[0049] FIG. 7 is a diagram illustrating the removal of noise components. FIG. 7(a) shows the intensity of the detection light when no removal of the signal component (noise) caused by scattered light due to the excitation light is performed, and FIG. 7(b) shows the intensity of the detection light when the removal of the signal component (noise) caused by scattered light due to the excitation light is performed. In FIG. 7(a) and FIG. 7(b), the vertical axis represents the intensity of the detection light, and the horizontal axis represents a channel indicating the position in the measurement section (501), which is the measurement area of ​​the sample (500). One channel is, for example, 0.02 mm. FIG. 7(c) is a diagram showing the area of ​​the sample (500) corresponding to the channel positions in FIG. 7(a) and FIG. 7(b). FIG. 8 is an enlarged schematic diagram of the sample (500) shown in FIG. 7(c). As shown in FIG. 8, the sample (500) is arranged from upstream to downstream, comprising a dropping section (502) into which the sample is dropped, a holding section (503) that holds a detection antibody labeled with a fluorescent reagent, and a measuring section (501) that fixes the capture antibody to a measurement target section (504). The fluorescent reagent is, for example, DTBTA-Eu3+. Since the measuring section (501) is, for example, part of a white immunochromato membrane, it is easy to scatter the excitation light.

[0050] For such a sample (500), when the sample is dropped onto the dropping section (502), the sample moves downstream by capillary action. If there is a substance to be detected in the sample, the detection antibody of the maintenance section (503) reacts with the substance to be detected to form a complex, and this complex moves downstream to the measuring section (501). Then, when the complex reaches the measurement target section (504) on the measuring section (501), the complex is captured by the capture antibody of the measurement target section (504), and a complex is formed by the three substances to be detected, the detection antibody, and the capture antibody. In this state, by irradiating the measurement section (501), which is the measurement area, with the light-gathering position (channel) being changed, the detection light intensity for each channel can be derived as shown in FIG. 7 (a) and FIG. 7 (b). In FIG. 7(a) and FIG. 7(b), the channel in which the detection light intensity is greater than the others is the channel corresponding to the location of the measurement target part (504) in which the composite is captured.

[0051] As shown in Fig. 7(a), when the signal component (noise) due to scattered light is not removed, the detection light contains scattered light in addition to fluorescence, so the intensity of the detection light increases. Furthermore, since this noise increases as the amount of excitation light increases, as shown in Fig. 7(a), if the amount of excitation light is doubled, the noise also doubles. Generally, as a method to improve the S / N ratio, one can consider increasing the amount of fluorescence signal by increasing the amount of excitation light; however, as described above, in a manner where noise increases with the amount of excitation light as shown in Fig. 7(a), it is difficult to improve the S / N ratio. In addition, there is also the problem that the dynamic range narrows when the amount of excitation light is increased.

[0052] On the other hand, as shown in FIG. 7(b), when signal components (noise) due to scattered light are removed, the detection light generally contains only fluorescence, allowing only the signal to be detected (a signal based on fluorescence) to be detected. In this case, since the noise is almost zero, as shown in FIG. 7(b), even if the amount of excitation light is increased (e.g., doubled), as long as the photodetector does not become saturated, the influence of the excitation light (scattered light) can be canceled to almost zero, and the noise does not become extremely large. As described above, in a configuration that removes noise as shown in FIG. 7(b), when the excitation light is increased, only the signal component can be increased while canceling the noise component to almost zero, which leads to an improvement in S / N. Since this configuration is very robust against noise components, it becomes possible to increase the amount of excitation light or increase the multiplication factor of the IV conversion amplifier.

[0053] FIG. 9 is a diagram explaining the definition of S / N. FIG. 9 shows an example of the intensity of the detection light for each channel (the intensity of the detection light at each position in the measurement area). As shown in FIG. 9, there is a fluctuation component of ±4 around the intensity of the detection light of 10 count. This fluctuation (standard deviation) of the base light quantity is a value obtained by scanning the excitation light on the measurement part (501) on which nothing, such as fluorescent material, is coated (or the measurement part (501) which is in a wet state similar to the measurement state). Hereinafter, the fluctuation of the base light quantity is defined as noise (N). Also, the signal (S) is defined as "the value obtained by subtracting the average value of the noise component, excluding the position of the measurement target part (504) in all channels, from the peak fluorescence intensity of the measurement target part (504)." S / N is defined as the value obtained by dividing the signal defined above by the noise.

[0054] In addition, in the example shown in FIG. 9, the noise value is offset by about 10 counts. In principle, the noise value is canceled out to almost 0 by performing the calibration process described later. However, since there is a deviation in the background based on the noise value, and from the perspective of analysis in software, it is desirable for the signal to always be a positive value, background offset processing is performed. In addition, the offset amount is set so that the signal falls within the dynamic range (0 to 4096 counts). The offset amount is set so that the background signal obtained by scanning the excitation light on the measurement unit (501) is always (almost certainly) a positive value while minimizing it as much as possible from the perspective of the dynamic range. Specifically, the offset amount may be, for example, the average intensity value of the detection light obtained by scanning the excitation light on the measurement unit (501) on which nothing such as fluorescent material is coated (or the measurement unit (501) which is in a wet state similar to the measurement state) + 6σ of that average intensity value. In addition, to prepare for the possibility of sudden noise entering the circuit system, an appropriate margin may be added to the offset amount calculated above to obtain the final offset amount. The offset amount is selected so as not to sacrifice dynamic range and so that the signal is not output as a negative value, and for example, it may be about +20 count.

[0055] Next, a method for removing signal components (noise) due to scattered light will be explained in detail. In the optical measuring device (1), calibration processing is performed in the lock-in circuit, which is a cancel circuit (60), and the result of the calibration processing is taken into account to remove signal components (noise) due to scattered light from the detection signal.

[0056] Specifically, in an optical measurement method using an optical measuring device (1), an optical head of an irradiation optical system (10) is initially positioned so that excitation light is irradiated onto a reference member (600) for calibration processing (see FIG. 15 (a)) which is different from the sample (500). Subsequently, as the excitation light from the irradiation optical system (10) is irradiated onto the reference member (600), scattered light (a component of the excitation light scattered from the reference member (600)) is detected by a light detection element (21) of a detection optical system (20). The light detected by the detection optical system (20) is basically light consisting only of scattered light that does not contain fluorescence in the reference member (600), and is a light for calibration processing used for calibration processing.

[0057] Next, a calibration process is performed. Specifically, the cancel circuit (60) of the optical measuring device (1) performs a calibration process to remove a signal component due to scattered light from the detection signal based on the calibration signal according to the light for the calibration process described above. Details of the calibration process will be described later. Then, after the calibration process is completed, the fluorescence information of the measurement unit (501) is obtained by scanning the measurement area (measurement unit (501)) of the sample (500) with the optical head of the irradiation optical system (10). Specifically, the cancel circuit (60) obtains the fluorescence information by removing a signal component due to scattered light from the detection signal, taking into account the result of the calibration process described above.

[0058] Next, details of the calibration process will be explained. The cancel circuit (60) of the optical measuring device (1) is a lock-in circuit using, for example, a Field Programmable Gate Array (FPGA). In the calibration process, the cancel circuit (60) generates a lock-in switch signal with a phase shifted for a periodic signal that has a period at the operating frequency of the cancel circuit (60), which is set by the modulation frequency of the light source (11) (for example, the frequency of the Direct Digital Synthesizer (DDS)) set by the light source driving circuit (30). Then, the cancel circuit (60), which functions as a lock-in circuit, takes the calibration signal, which is a measurement signal, and the switch signal, which is a reference signal, as inputs, outputs a signal component according to scattered light, and adjusts the phase of the switch signal so that the voltage value of the signal component according to scattered light is within a predetermined range (slash level) that approximates 0.

[0059] FIG. 10 shows a signal used for calibration processing inside the FPGA of the cancel circuit (60). The periodic signal shown in FIG. 10 is a clock signal with a period divided into small segments to match the frequency of the DDS as described above. The reference signal is a signal of the same frequency as the periodic signal, which is at an arbitrary phase from the periodic signal (phase shifted relative to the periodic signal), and is a trigger for the XY signal described later. The XY signal is a switch signal for lock-in described above, and is a signal created using the reference signal as a trigger. The X signal (first signal) is a signal with no phase difference from the reference signal. The Y signal (second signal) is a signal with a 90-degree phase shift relative to the reference signal. In reality, the cancel circuit (60) generates, in addition to the X signal and the Y signal, an X′ signal (third signal) which is the inverted X signal and a Y′ signal (fourth signal) which is the inverted Y signal. The X signal, Y signal, X′ signal, and Y′ signal are each generated by independent dedicated circuits. Adjusting the phase of the switch signal so that the voltage value of the signal component according to the scattered light becomes a slash level means that the phase of the reference signal is continuously shifted with respect to the periodic signal until the output from the cancel circuit (60) becomes 0V (or a value close to it).

[0060] FIG. 11 is a diagram illustrating a process of adjusting the output to 0V by shifting the phase of the reference signal with respect to the periodic signal. Here, the phase relationship of the periodic signal, the reference signal, and the switch signal in the initial state is assumed to be the state shown in FIG. 11 (a). Then, based on the switch signal, integration processing is performed in the shaded section of FIG. 11, and the output (voltage value of the signal component due to scattered light) is not a slash level and is a positive value. In this case, as shown in FIG. 11 (b), the phase of the reference signal is adjusted to delay the phase of the switch signal. That is, in the calibration process, the cancel circuit (60) adjusts the phase of the switch signal to delay when the voltage value of the signal component due to scattered light is not a slash level and is a positive value.

[0061] Now, even in the state of FIG. 11 (b) where the phase of the switch signal is adjusted, as a result of the integration processing of the shaded section, the output (voltage value of the signal component due to scattered light) is not a slash level and is also a positive value. In this case, as shown in FIG. 11 (c), the phase of the reference signal is adjusted to further delay the phase of the switch signal.

[0062] Now, even in the state of (c) of FIG. 11 where the phase of the switch signal is adjusted, as a result of the integration processing of the shaded section, the output (voltage value of the signal component due to scattered light) is not a slash level and is also a positive value. In this case, as shown in (d) of FIG. 11, the phase of the reference signal is adjusted to further delay the phase of the switch signal.

[0063] Now, in the state of (d) of FIG. 11 where the phase of the switch signal is adjusted, as a result of the integration processing of the shaded section, the output (voltage value of the signal component due to scattered light) is not a slash level and is also a negative value. In this case, as shown in (e) of FIG. 11, the phase of the reference signal is adjusted to advance the phase of the switch signal. That is, in the calibration processing, the cancel circuit (60) adjusts the phase of the switch signal to advance when the voltage value of the signal component due to scattered light is not a slash level and is a negative value.

[0064] And, as a result of adjusting the phase of the switch signal to advance, as shown in (e) of FIG. 11, when the output (voltage value of the signal component according to scattered light) becomes a slash level (a value within a predetermined range approximating 0) as a result of the integration processing of the shaded section, the calibration processing is completed.

[0065] When the calibration process is completed, the cancel circuit (60) takes as input a detection signal according to the detection light including a fluorescent component and a scattered light component (excitation light component) and a switch signal whose phase is adjusted in the calibration process, and removes the signal component according to the scattered light component from the detection signal.

[0066] FIG. 13 is a flowchart illustrating a calibration process. As shown in FIG. 13, in the calibration process, the input to the AD converter is initially switched to a predetermined offset voltage and a 0 level is stored (Step S11). Then, by switching the switch, the signal of the cancel circuit (60) (lock-in circuit) is input to the AD converter (Step S12). In this state, the phase of the reference signal is forcibly shifted once (Step S13). The output of the cancel circuit (60) (lock-in circuit) becomes 0V when the phase of the reference signal relative to the periodic signal is 0 degrees and 180 degrees. However, if the phase happens to be 180 degrees in the initial state, the calibration process is completed incorrectly, and the positive and negative of the output signal are inverted, so the output signal cannot be detected by the configuration of the circuit in the subsequent stage. To prevent this, by forcibly shifting the phase of the reference signal at the start, it is possible to prevent the calibration process from being completed incorrectly. In addition, by aligning the phase at the start in this way, the positive and negative of the output signal are fixed. As a result, the sign bit is not needed when converting the output signal into a digital value, allowing the dynamic range of the AD converter to be used effectively. Also, if you want to measure with a negative output, the phase at the completion of calibration may be set to 180 degrees instead of 0 degrees.

[0067] When step S13 is completed, the input value of the current AD converter is recorded (step S14), and the calibration loop processing is executed. First, the input value of the current AD converter is compared with the 0 level, and it is determined whether the input value of the AD converter is smaller than the 0 level (negative value) (step S15). In step S15, if it is determined that the input value of the AD converter is a negative value, the phase of the switch signal (i.e., reference signal) of the cancel circuit (60) for the periodic signal according to the frequency of the DDS is advanced (step S16). On the other hand, if it is determined that the input value of the AD converter is a positive value in step S15, the phase of the switch signal (i.e., reference signal) of the cancel circuit (60) for the periodic signal is delayed (step S17).

[0068] Then, regarding the input value of the AD converter, it is determined whether the sign has not changed and has become a slash level (Step S18). If it is determined in Step S18 that the sign has not changed and has become a slash level, the calibration process is terminated. On the other hand, if it is determined in Step S18 that the condition is not satisfied, it is determined whether the sign of the input to the AD converter has changed by shifting the phase (Step S19). If it is determined in Step S19 that it has not changed, the processing of Step S14 is performed again; if it is determined that it has changed, the phase change range by control is changed to half of the current shape (Step S20), and the processing of Step S14 is performed again. The above is the calibration process.

[0069] In the present embodiment, light for calibration processing that includes scattered light and does not contain fluorescence is detected, and based on a calibration signal according to the light for calibration processing, a calibration process is performed to remove a signal component due to scattered light from a detected signal, and a signal component due to scattered light is removed from a detected signal by considering the result of the calibration process. By performing the calibration process to remove a signal component due to scattered light from a detected signal in advance based on the light for calibration processing that includes scattered light, the signal component due to scattered light can be appropriately removed from the detected signal.

[0070] The effect of appropriately removing scattered light (noise) in this manner is explained with reference to Fig. 14. Fig. 14(a) shows the intensity of the detected light when the signal component (noise) due to scattered light is not removed, and Fig. 14(b) shows the intensity of the detected light when the signal component (noise) due to scattered light is removed. Fig. 14 shows the results when measuring a membrane coated with DTBTA-Eu3+ as a fluorescent reagent. As shown in Fig. 14(a), in the case where noise is not removed, an offset of approximately 330 counts is required for the background (BKG) of the excitation light (scattered light). The noise (standard deviation) was 2.16, and the signal intensity was 404 counts. Regarding this, as shown in Fig. 14(b), when noise removal is performed, an offset is not required to account for the scattering of excitation light on the membrane, and only a minimal offset is applied for software processing (processing to make all signal values ​​positive). And, the noise (standard deviation) was set to 0.69 and the signal strength to 1475 counts. As such, when noise removal is performed, the amount of offset is small, so the amount of excitation light from the light source and the amplification rate of the IV conversion amplifier can be increased, and the signal strength can be appropriately increased. As a result, while the S / N when noise removal is not performed is 187, the S / N when noise removal is performed can be set to 2140, and the S / N can be improved by more than 10 times.

[0071] In the calibration process, a switch signal for locking in is generated by shifting the phase of a period signal that has a period set to the operating frequency of a cancel circuit (60) that matches the modulation frequency of the light source (11), and the calibration signal and the switch signal are inputs to output a signal component due to scattered light, and the phase of the switch signal is adjusted so that the voltage value of the signal component due to scattered light is within a predetermined range that approximates 0, and the signal component due to scattered light can be removed from the detection signal by inputting the detection signal and the switch signal whose phase has been adjusted in the calibration process. In this way, by using a lock-in circuit, the phase of the switch signal is adjusted so that the voltage value of the signal component due to scattered light in the calibration process approximates 0, and the signal component due to scattered light can be appropriately removed from the detection signal by inputting the switch signal after phase adjustment.

[0072] In the calibration process, if the voltage value of the signal component due to scattered light is not within a predetermined range and is greater than the value of the predetermined range, the phase of the switch signal may be adjusted to delay, and if the voltage value of the signal component due to scattered light is not within a predetermined range and is less than the value of the predetermined range, the phase of the switch signal may be adjusted to advance. By doing so, the voltage value of the signal component due to scattered light can be adjusted to an appropriately close value to zero during the calibration process.

[0073] The modulation frequency of the light source (11) may be lower than the value corresponding to the fluorescence lifetime, which is the time until the fluorescence intensity drops from the peak value to 1 / e. If the modulation frequency is high enough to correspond to the fluorescence lifetime, continuous signals may overlap, and thus the fluorescence intensity cannot be maximized. In this regard, by making the modulation frequency lower than the value corresponding to the fluorescence lifetime, the fluorescence intensity can be appropriately increased.

[0074] The modulation frequency of the light source (11) may be lower than the value corresponding to the fluorescence lifetime and higher than the commercial frequency. By doing so, it is possible to avoid increasing noise while avoiding the fluorescence intensity weakening as the modulation frequency becomes higher than the value corresponding to the fluorescence lifetime.

[0075] As switch signals for locking, an X signal, a Y signal with a phase shifted by 90 degrees relative to the X signal, an X′ signal with the X signal inverted, and a Y′ signal with the Y signal inverted may each be generated using independent dedicated circuits. By generating the inverted signal using independent dedicated circuits, it is possible to prevent the occurrence of minute delays (minute delays due to passing through the not circuit) that become a problem when, for example, the inverted signal is generated by a not circuit.

[0076] The cancel circuit (60) (lock-in circuit) may be set with two types of operating frequencies switched at a predetermined ratio. By doing so, it becomes easier to match the operating frequency of the lock-in circuit to the modulation frequency of the light source (11) than when the operating frequency is one type, thereby improving the synchronization system.

[0077] The light for calibration treatment may be detected by irradiating an excitation light to a downstream area of ​​the capture antibody fixed to the measurement target part (504) in the sample (500). Since the fluorescent component tends to remain upstream of the capture antibody, the light for calibration treatment can be detected by irradiating an excitation light to a downstream area of ​​the supplementary antibody and detecting the light for calibration treatment, thereby appropriately reducing the influence of the fluorescent component.

[0078] Next, specific embodiments of the calibration process in the fluorescence measurement described above (first to third embodiments) will be explained with reference to FIGS. 15 to 29. In each embodiment, the calibration process method differs from one another. Specifically, in the first embodiment, the calibration process is performed by irradiating a reference member with irradiation light and detecting the light for the calibration process. In the second embodiment, the calibration process is performed by the detection optical system directly detecting the irradiation light as the light for the calibration process. In the third embodiment, the calibration process is performed by generating a calibration signal, which is a pseudo-signal. Furthermore, the optical measuring device (1) of the first embodiment (see FIG. 1, configuration already described), the optical measuring device (701) of the second embodiment (see FIG. 23), and the optical measuring device (801) of the third embodiment (see FIG. 27) differ in some of their configurations. Each embodiment will be explained in detail below.

[0079] [First Mode]

[0080] FIG. 15 is a diagram illustrating a fluorescence measurement procedure of a first embodiment. As shown in FIG. 15 (a), in the fluorescence measurement of the first embodiment, initially, a light source (11) of an irradiation optical system (10) irradiates irradiation light onto a reference member (600) for calibration processing that is different from the sample (500) to be measured, and a light detection element (21) of a detection optical system (20) detects irradiation light for calibration processing that includes scattered light of the irradiation light from the reference member (600) to which the irradiation light was irradiated. Then, a cancel circuit (60) (see FIG. 1), which is a signal processing unit, performs calibration processing based on the calibration signal according to the irradiation light for calibration processing. The above is the first processing in the first embodiment.

[0081] After that, as shown in FIG. 15(b), a light source (11) irradiates an excitation light (irradiation light) onto a sample (500) which is the object to be measured, and a light detection element (21) detects a target light for measurement that includes fluorescence generated from the sample (500) irradiated with the excitation light, and scattered light of the excitation light from the sample (500) irradiated with the excitation light. Then, a cancel circuit (60) (see FIG. 1), which is a signal processing unit, removes the signal component corresponding to the scattered light from the measurement signal corresponding to the target light for measurement, taking into account the result of performing the calibration processing. The above is the second processing in the first embodiment. FIG. 15(a) and FIG. 15(b) are referred to as front views, FIG. 15(c) is a right side view. As shown in Fig. 15 (c), when acquiring fluorescence from a sample (500), the fluorescence information of the measurement target part (504) of the sample (500) is acquired by scanning the measurement area of ​​the sample (500) with the optical head of the irradiation optical system (10).

[0082] This fluorescence measurement is realized, more specifically, by the configuration shown in FIG. 18. That is, when the calibration process is first performed, the optical head of the irradiation optical system (10) is positioned so that the irradiation light is irradiated onto a reference member (600) fixed at a position different from the sample (500) placed on the sample stage, specifically, at a fixed part (601) that extends approximately vertically from the fixed part (602). Then, when the calibration process is completed, the optical head of the irradiation optical system (10) is moved so that the excitation light (irradiation light) is irradiated onto the sample (500), and the optical head of the irradiation optical system (10) performs a scanning operation so that the fluorescence information of the measurement target part (504) of the sample (500) is acquired.

[0083] Here, in the first embodiment, the reference member (600) is preferably a member that reflects the irradiation light and does not itself generate fluorescence upon irradiation of the irradiation light. Furthermore, in the present invention, not generating fluorescence upon irradiation of the irradiation light indicates that fluorescence attributable to the reference member (600) does not occur even upon irradiation of the irradiation light, or that fluorescence is generated only to a negligible degree. Meanwhile, FIG. 16 is a diagram explaining the effect of calibration when using a reference member that generates fluorescence, FIG. 16 (a) shows a calibration processing light containing fluorescence detected when irradiation light is irradiated onto a reference member, and FIG. 16 (b) shows the result of fluorescence measurement of a sample (500) based on the calibration result related to the calibration processing light shown in FIG. 16 (a). In FIG. 16(a), wavelength is shown on the horizontal axis and light intensity on the vertical axis, and the lowest wavelength detectable by the photodetector (photodetector element (21)) is shown as a dashed line. In FIG. 16(b), channel is shown on the horizontal axis and light intensity on the vertical axis. FIG. 16(a) shows that when a reference member that generates a certain fluorescence is excited by irradiation light of 380 nm, fluorescence of a wavelength detectable by the photodetector element (21), specifically fluorescence of about 600 to 850 nm, is detected. In this way, when irradiation light is irradiated onto a reference member that generates fluorescence, the photodetector element (21) detects not only scattered light (irradiation light) but also fluorescence from the reference member. That is, the photodetector element (21) detects light for calibration processing containing fluorescence.When calibration processing is performed based on light for calibration processing containing such fluorescence, when the phase of the switch signal is adjusted so that the voltage value of the device output becomes 0 using the calibration signal, the phase of the switch signal is adjusted to a phase affected by the fluorescence component (it becomes locked into the phase of the fluorescence component). As a result, as shown in FIG. 16 (b), if fluorescence measurement of the sample (500) is performed considering the calibration result, there is a risk that the waveform of the fluorescence from the sample (500) cannot be acquired due to the influence that the switch signal is adjusted so that the fluorescence component from the reference member is removed (canceled) during the calibration processing. FIG. 16 (b) shows an example in which the waveform of the fluorescence from the sample (500) is not acquired in any channel.

[0084] FIG. 17 is a diagram illustrating the effect of calibration when using a reference member that does not generate fluorescence, in contrast to the above. FIG. 17 (a) shows the light for calibration processing detected when irradiating a reference member with irradiation light, and FIG. 17 (b) shows the result of fluorescence measurement of a sample (500) based on the calibration result related to the light for calibration processing shown in FIG. 17 (a). In FIG. 17 (a), wavelength is shown on the horizontal axis and light intensity on the vertical axis, and the lowest wavelength detectable by the photodetector (photodetector element (21)) is shown as a dashed line. In FIG. 17 (b), channel is shown on the horizontal axis and light intensity on the vertical axis. FIG. 17(a) shows that when irradiating a reference member that does not emit fluorescence with 380 nm irradiation light, no fluorescence is detected (or only a negligible amount is detected). In this way, when the light for calibration processing does not contain fluorescence and generally contains only scattered light, as shown in FIG. 17(b), if fluorescence measurement of the sample (500) is performed considering the calibration result, only the signal component due to scattered light from the sample (500) is removed (canceled), and the waveform of the fluorescence from the sample (500) can be properly acquired. From the above, it can be said that it is preferable for the reference member (600) itself to be a member that does not emit fluorescence.

[0085] Next, as an example of a reference member (600), each example shown in FIGS. 19 to 21 will be explained. FIG. 19 is a drawing explaining the acquisition of scattered light when a mirror (600A) is used as a reference member. As shown in FIG. 19, the reference member (600) may include a mirror (600A). The mirror (600A) may be any mirror capable of reflecting irradiated light, but it is more preferable that the mirror (600A) be formed of a material that does not generate fluorescence upon irradiation with irradiated light (fluorescence caused by the mirror (600A) does not occur, or fluorescence is generated to a negligible degree). For example, the mirror (600A) may be a mirror whose surface is resistant to dirt. The mirror (600A) reflects irradiated light irradiated from a light source (11). The mirror (600A) is tilted at an angle such that the reflected illumination (scattered light) is detected by the light detection element (21). The mirror (600A) is fixed at a predetermined angle of inclination by an adjustment mechanism (not shown). By appropriately adjusting the angle of inclination of the mirror (600A) relative to the light detection element (21), the amount of light detected by the light detection element (21) can be increased.

[0086] FIG. 20 is a diagram illustrating the acquisition of scattered light when a reflective diffuser (600B) is used as a reference member (600). As shown in FIG. 20, the reference member (600) may include a reflective diffuser (600B). The reflective diffuser (600B) is, for example, a reflective diffuser plate formed of a white material that does not generate fluorescence upon irradiation with irradiation light (fluorescence caused by the reflective diffuser (600B) does not occur, or fluorescence is generated only to a negligible degree), and the amount of incident light to the light detection element (21) is relatively stable. The reflective diffuser (600B) reflects the irradiation light irradiated from the light source (11) to diffuse it at various angles. The reflective diffuser (600B) is positioned so that the reflected irradiation light (scattered light) is detected by the light detection element (21) with a large amount of light. The reflective diffuser (600B) is preferably an integrally molded member made of, for example, resin or ceramic, but may be formed by cutting or a combination of multiple members, and may be configured, for example, by providing a film capable of scattering irradiated light at a specific angle on the surface of a plate-shaped member.

[0087] FIG. 21 is a drawing illustrating the acquisition of scattered light when using a mirror (611) (reflective material) and opaque glass (610) (diffuser) as a reference member (600). As shown in FIG. 21, the reference member (600) may include a reference member (600C) comprising an opaque glass (610), which is a glass member that transmits irradiation light, and a mirror (611), which reflects irradiation light. In the reference member (600C), the opaque glass (610) is provided to be supported (laminated) on the mirror (611) and is provided so that irradiation light is incident on it before the mirror (611). The opaque glass (610) is formed of a material that transmits irradiation light of, for example, 380 nm or more, and has an uneven surface (610a) formed on its surface (the surface of incidence of irradiation light). Alternatively, a film capable of scattering the irradiated light at a specific angle may be provided on the surface of the opaque glass (610). The mirror (611) only needs to reflect the irradiated light and, for example, may be a plate-shaped metal member made of aluminum, or a plate-shaped member with its surface covered by a metal film such as aluminum, or may be a box-shaped member to accommodate the opaque glass (610). Furthermore, it is more preferable that both the mirror (611) and the opaque glass (610) be formed from a material that does not generate fluorescence upon irradiation with the irradiated light (fluorescence caused by the mirror (611) and the opaque glass (610) does not occur, or generates fluorescence to a negligible degree). As shown in FIG. 21, in the reference member (600C), the irradiated light incident on the opaque glass (610) diffuses through the uneven portion (610a), passes through the interior of the opaque glass (610), reaches the mirror (611), is reflected from the mirror (611), passes through the interior of the opaque glass (610) again, reaches the surface of the opaque glass (610), and diffuses in various directions from the uneven portion (610a) and is emitted (scattered light at various angles is generated).As scattered light is generated at various angles, a sufficient amount of scattered light is detected by the light detection element (21). Additionally, for the reference member (600C), a reflective material with a roughened surface may be used, and in that case, the roughened portion of the surface of the reflective material corresponds to a diffuser that diffuses the irradiated light.

[0088] Next, with reference to FIG. 22, a fluorescence measurement process of the first embodiment is described. FIG. 22 is a flowchart illustrating a fluorescence measurement process of the first embodiment. As shown in FIG. 22, in the fluorescence measurement process of the first embodiment, an optical head of an irradiation optical system (10) is initially positioned so that irradiation light is irradiated onto a reference member (600) for calibration processing that is different from the sample (500) (Step S31; see FIG. 15(a)). Then, a calibration processing light containing scattered light of the irradiation light from the reference member (600) to which the irradiation light was irradiated is detected by a light detection element (21) (Step S32; see FIG. 15(a)). Then, in the cancel circuit (60), a calibration process is performed to remove a signal component corresponding to scattered light from the detection signal based on the calibration signal corresponding to the calibration processing light (Step S33). As the details of the calibration process are as described above with reference to FIGS. 10 to 13, etc., the explanation is omitted.

[0089] Next, the optical head of the irradiation optical system (10) operates to scan the measurement area of ​​the sample (500), thereby irradiating the measurement target part (504) of the sample (500) with excitation light (irradiation light) (step S34. See (c) of FIG. 15), and the measurement target light, including fluorescence generated from the sample (500) irradiated with excitation light and scattered light of the excitation light from the sample (500) irradiated with excitation light, is detected by the light detection element (21) (step S35. See (b) of FIG. 15). Then, in the cancel circuit (60), based on the result of the calibration process of step S33, the signal component according to the scattered light is removed from the measurement signal according to the above-described measurement target light (step S36). Specifically, as described above, the cancel circuit (60) takes the measurement signal and the switch signal whose phase is adjusted in the calibration process as inputs, and removes the signal component according to the scattered light from the measurement signal.

[0090] Next, the effects of the action according to the first embodiment will be explained.

[0091] An optical measuring device (1) according to the first embodiment comprises an irradiation optical system (10) that irradiates an irradiation light (excitation light) onto a target for irradiation, a detection optical system (20) that detects a detection light originating from the irradiation light (excitation light), and a cancellation circuit (60) that processes a detection signal according to the detection light. And, in the optical measuring device (1), in the first process, the irradiation optical system (10) irradiates a reference member (600) for calibration processing that is different from the sample (500) as the target of irradiation and irradiates the reference member (600) with irradiation light (see FIG. 15 (a)), the detection optical system (20) detects the light for calibration processing, which includes scattered light of the irradiation light from the reference member (600) to which the irradiation light was irradiated, as the detection light (see FIG. 15 (a)), and the cancel circuit (60) takes the calibration signal according to the light for calibration processing as the detection signal and performs a calibration process to remove the signal component according to the scattered light from the detection signal in the second process described later based on the calibration signal. Also, in the optical measuring device (1), in the second processing, the irradiation optical system (10) targets the sample (500) for irradiation and irradiates the sample (500) with excitation light (irradiation light) (see FIG. 15 (b)), the detection optical system (20) detects the target light for measurement, which includes fluorescence generated from the sample (500) irradiated with excitation light and scattered light from the sample (500) irradiated with excitation light, as the detection light (see FIG. 15 (b)), and the cancel circuit (60) takes the measurement signal according to the target light for measurement as the detection signal and removes the signal component according to the scattered light in the calibration processing in the first processing from the measurement signal.

[0092] In this way, in the optical measuring device (1), during the first process related to calibration processing, an irradiating light is irradiated onto a reference member (600) that is different from the sample (500), and a calibration processing light containing scattered light is detected. By this, for example, by preparing a reference member (600) that does not generate fluorescence (or generates fluorescence to a negligible degree) upon irradiation with the irradiating light, it becomes possible to detect a calibration processing light that does not contain fluorescence (or contains fluorescence to a negligible degree). With this, in the second process, based on the results of the calibration processing, only the signal component corresponding to scattered light is appropriately removed from the measurement signal, thereby removing noise components caused by the irradiating light itself and enabling high-precision fluorescence measurement. In addition, by using a reference member (600) for calibration processing that is different from the sample (500) (actual sample), deviations in the results of the calibration processing due to the characteristics of the actual sample (characteristics determined by differences such as the moving speed of the fluorescent material), which are problematic when, for example, the actual sample is irradiated with irradiation light to detect the light for calibration processing, do not occur. More specifically, the actual sample may have deviations in characteristics due to differences in manufacturing lots, for example, and may have differences such as the moving speed of the fluorescent material. In that case, there is a possibility that the amount of fluorescent material (i.e., the amount of fluorescent emission) in the area where the irradiation light is irradiated during the calibration processing will differ from one another for each actual sample. Due to this, there is a concern that the results of the calibration processing will not be stable and that sufficient reliability will not be obtained, for example, in determining the results of optical measurement, but by using a reference member (600), the fluorescence measurement can be performed with higher precision.In addition, when the output of the device differs depending on the difference in characteristics of each actual sample, for example, it becomes difficult to set a threshold value for determining positive or negative in measurement using immunochromatography, but this problem can be suppressed when calibration is performed using a reference member (600). Also, for example, when measuring multiple samples (500) with different shapes (for example, multiple immunochromatography test kits corresponding individually to each antigen) with one optical measuring device (1), there is no need to set an appropriate calibration area for each sample (500), and since calibration processing can be performed using one reference member (600), control in terms of hardware or software can be simplified. Furthermore, compared to the case where light for calibration processing is detected by irradiating an actual sample with an irradiation light, for example, there is no need to significantly change the configuration of the optical measuring device (1), so this embodiment has an advantage in terms of cost. In addition, since calibration processing is performed from actual measured values, the present embodiment can guarantee the precision of the calibration processing (accurate phase alignment can be performed in the calibration processing). Thus, according to the optical measuring device (1) according to the present embodiment, calibration processing can be performed more appropriately, and by removing noise components caused by the irradiation light itself more appropriately, fluorescence measurement can be performed with high precision.

[0093] The reference member (600) may include a reflective member (mirror (600A), mirror (611), reflective diffuser (600B)) that reflects the irradiation light as shown in FIGS. 19 to 21. With this configuration, the amount of light of the detection light can be easily increased. In addition, it is preferable that the reflective member does not generate fluorescence when irradiated with the irradiation light. With this configuration, even when irradiated with the irradiation light, fluorescence caused by the reflective member is not generated (or only generates fluorescence to a negligible degree), so in the second processing, only the signal component corresponding to the scattered light component can be reliably removed from the measurement signal.

[0094] The reference member (600) may include a reflective diffuser (600B) that diffuses the irradiated light, as shown in FIG. 20. With this configuration, scattered light at various angles can be easily generated by the reflective diffuser (600B), making it easier to increase the amount of light of the detection light. In addition, with this configuration, the reference member can be made simple, so it is superior in terms of processability and cost.

[0095] As shown in FIG. 21, the reference member (600) may include a mirror (611) that reflects the illumination light and an opaque glass (610) that is supported by the mirror (611) and diffuses the illumination light. With this configuration, the amount of light of the detection light can be more easily increased through the cooperation of the mirror (611) and the glass (610). In addition, since an uneven portion (610a) is formed on the surface of the opaque glass (610), scattered light at various angles can be easily generated, thereby making it easier to increase the amount of light of the detection light.

[0096] The reference member (600) may include a mirror, as shown in FIG. 19. With this configuration, the amount of light from the detection light can be more easily increased by adjusting the reflection angle of the mirror relative to the light detection element (21).

[0097] [Second Mode]

[0098] FIG. 23 is a schematic diagram of an optical measuring device (701) of a second embodiment. The optical measuring device (701) of the second embodiment has a configuration generally similar to the optical measuring device (1) described above (see FIG. 1), but differs from the optical measuring device (1) in that it is equipped with a light detection element (721) of a detection optical system (720) (first detection optical system) and an IV conversion amplifier (740), as shown in FIG. 23. The light detection element (721) is a separate configuration from the light detection element (21) of the detection optical system (20) (second detection optical system). Also, the IV conversion amplifier (740) is a separate configuration from the IV conversion amplifier (40). The light detection element (721) directly detects the irradiated light (light for calibration processing) irradiated from the irradiated optical system (10) as a detection light, as a process related to calibration processing. The light detection element (721) outputs a detection signal corresponding to the detection light to the IV conversion amplifier (740). The IV conversion amplifier (740) converts the power signal (detection signal) input from the light detection element (721) into a voltage signal as a process related to calibration processing. The IV conversion amplifier (740) outputs the detection signal converted into a voltage signal to the waveform generation circuit (50). In this case, the waveform generation circuit (50) generates a waveform of the detection signal based on the detection signal input from the IV conversion amplifier (740) and outputs information of the generated waveform (detection signal) to the cancellation circuit (60).

[0099] FIG. 24 is a diagram illustrating an overview of fluorescence measurement in a second embodiment. As shown in FIG. 24, in the fluorescence measurement of the second embodiment, when calibration processing is first performed, the optical head of the irradiation optical system (10) is positioned so that irradiation light is irradiated onto a light detection element (721) fixed at a position different from the sample (500) placed on the sample stage, specifically, a fixed part (601) that extends approximately vertically from a fixed part (602). By acquiring irradiation light in this way, the irradiation light, which is the light for calibration processing, is detected. Then, when calibration processing is completed, the optical head of the irradiation optical system (10) moves so that excitation light (irradiation light) is irradiated onto the sample (500), and the optical head of the irradiation optical system (10) performs a scanning operation so that fluorescence information of the measurement target part (504) of the sample (500) is acquired.

[0100] FIG. 25 is a diagram illustrating the acquisition of excitation light in a second embodiment. As shown in FIG. 25 (a), in the acquisition of irradiation light in the second embodiment, the light source (11) of the irradiation optical system (10) irradiates irradiation light to the light detection element (721) of the detection optical system (720). By doing so, the light detection element (721) directly detects the irradiation light, which is the light for calibration processing. Then, a cancel circuit (60), which is a signal processing unit, performs calibration processing based on the calibration signal corresponding to the light for calibration processing. The irradiation light is light that has the same phase as scattered light. Therefore, by the light detection element (721) detecting the irradiation light as the light for calibration processing, calibration processing can be appropriately performed to remove the signal component corresponding to scattered light from the detection signal. Furthermore, the processing after calibration processing (second processing) is the same as in the first embodiment, so the explanation is omitted.

[0101] Next, with reference to FIG. 26, a fluorescence measurement process of the second embodiment is described. FIG. 26 is a flowchart illustrating a fluorescence measurement process of the second embodiment. As shown in FIG. 26, in the fluorescence measurement process of the second embodiment, an optical head of an irradiation optical system (10) is initially positioned so that irradiation light is irradiated onto a light detection element (721) fixed at a position different from that of the sample (500) (Step S41. See FIG. 25 (a)). Then, the irradiation light, which is a light for calibration processing, is detected by the light detection element (721) (Step S42. See FIG. 25 (a)). The irradiation light is light having the same phase as scattered light. Then, in the cancel circuit (60), a calibration process is performed to remove the signal component corresponding to scattered light from the detection signal based on the calibration signal corresponding to the light for calibration processing (Step S43). As the details of the calibration process are as described above with reference to FIGS. 10 to 13, etc., the explanation is omitted.

[0102] Next, the optical head of the irradiation optical system (10) operates to scan the measurement area of ​​the sample (500) so that the excitation light (irradiation light) is irradiated onto the measurement target part (504) of the sample (500) (step S44), and the measurement target light, including the fluorescence generated from the sample (500) irradiated with the excitation light and the scattered light of the excitation light from the sample (500) irradiated with the excitation light, is detected by the light detection element (21) (step S45). Then, in the cancel circuit (60), the signal component due to the scattered light in the calibration process of step S43 is removed from the measurement signal according to the measurement target light described above (step S46). Specifically, as described above, the cancel circuit (60) takes the measurement signal and the switch signal with phase adjusted in the calibration process as inputs and removes the signal component due to the scattered light from the measurement signal.

[0103] Next, the effects of the action according to the second mode will be explained.

[0104] An optical measuring device (701) according to a second embodiment (see FIG. 23) comprises an irradiating optical system (10) that irradiates irradiating light (excitation light), a detection optical system (20) that detects detection light caused by irradiating light (excitation light), and a light detection unit having a detection optical system (720), and a cancellation circuit (60) that processes a detection signal according to the detection light. In the optical measuring device (701), in the first processing, the irradiating optical system (10) irradiates irradiating light to a light detection element (721) of the detection optical system (720), and the light detection element (721) detects the light for calibration processing, which is the irradiating light, as the detection light (see FIG. 25 (a)), and the cancellation circuit (60) takes the calibration signal according to the light for calibration processing as the detection signal, and based on the calibration signal, performs a calibration processing to remove a signal component according to scattered light from the detection signal in the second processing described later. Additionally, in the optical measuring device (701), in the second processing, the irradiation optical system (10) irradiates the sample (500) with excitation light (irradiation light), and the detection optical system (20) detects the light to be measured, which includes fluorescence generated from the sample (500) irradiated with the excitation light and scattered light from the sample (500) irradiated with the excitation light, as the detection light, and the cancel circuit (60) takes the measurement signal according to the light to be measured as the detection signal and removes the signal component according to the scattered light in the calibration processing in the first processing from the measurement signal.

[0105] In this way, in the optical measuring device (701), during the first process related to calibration processing, direct irradiation light is irradiated onto the light detection element (721) of the detection optical system (720), and the irradiation light, which is the light for calibration processing, is detected. The irradiation light is light that has the same phase as scattered light. Therefore, by the detection optical system (720) detecting the irradiation light as the light for calibration processing, it becomes possible to appropriately detect light that does not contain fluorescence and has the same phase as scattered light as the light for calibration processing. With this, in the second process, based on the results of the calibration processing, the signal component due to scattered light is appropriately removed from the measurement signal, thereby removing the noise component caused by the irradiation light itself, and thus fluorescence measurement can be performed with high precision. In addition, since the light for calibration treatment is detected without irradiating the sample (500) with irradiating light, there is no deviation in the results of the calibration treatment due to the characteristics of the sample (500), which would be a problem if the light for calibration treatment were detected by irradiating the sample (500) with irradiating light. More specifically, actual samples may have deviations in characteristics due to differences in manufacturing lots, for example, and differences such as the migration speed of fluorescent material may occur. In that case, there is a possibility that the amount of fluorescent material (i.e., the amount of fluorescent emission) in the area where the irradiating light is irradiated during the calibration treatment may differ from one another for each actual sample. Due to this, there is a concern that the results of the calibration treatment will not be stable and that sufficient reliability may not be obtained, for example, in determining the results of optical measurement, but since the light for calibration treatment is detected without irradiating the sample (500) with irradiating light, the fluorescence measurement can be performed with higher precision.In addition, if the results output by the device differ depending on the differences in characteristics of each actual sample, for example, it becomes difficult to set a threshold value for determining positive or negative in measurement using an immunochromatography method, but if light for calibration processing is detected without irradiating the sample (500), this problem can also be suppressed. Also, for example, when measuring multiple samples (500) with different shapes (for example, multiple immunochromatography test kits corresponding individually to each antigen) with one optical measuring device (1), there is no need to set an appropriate calibration area for each sample (500), and regarding the calibration processing, since light for calibration processing can be detected without irradiating the sample (500), control in terms of hardware or software can be simplified. Also, since the irradiated light is directly detected and becomes light for calibration processing, sufficient light amount for calibration processing can be guaranteed. In this way, according to the optical measuring device (701) of the present embodiment, calibration processing can be performed more appropriately, and noise components caused by the irradiation light itself can be removed more appropriately, thereby allowing fluorescence measurement to be performed with high precision.

[0106] Additionally, although the above description describes the optical measuring device (701) having a first detection optical system (720) and a second detection optical system (20), for example, as shown in FIG. 25 (b), the optical measuring device may have one detection optical system (820) that functions as the first detection optical system and the second detection optical system. The detection optical system (820) has a light detection element (821). In this case, the irradiation optical system includes, for example, an irradiation optical system (910) that irradiates irradiation light to the detection optical system (820) when the detection optical system (820) functions as a first detection optical system (i.e., a detection optical system that detects irradiation light as light for calibration processing), and an irradiation optical system (10) that irradiates excitation light (irradiation light) to the sample (500) when the detection optical system (820) functions as a second detection optical system (i.e., a detection optical system that detects light to be measured including fluorescence and scattered light from the sample (500). In addition, the light source (911) of the irradiation optical system (910) and the light source (11) of the irradiation optical system (10) in this case need to be modulated with respect to a reference signal.

[0107] [Third Mode]

[0108] FIG. 27 is a schematic diagram of the optical measuring device (801) of the third embodiment. The optical measuring device (801) of the fifth embodiment has a configuration generally similar to the optical measuring device (1) described above (see FIG. 1), but differs from the optical measuring device (1) in that it is equipped with a delay / amplification circuit (802) as shown in FIG. 27. The delay / amplification circuit (802) is configured to function as a signal processing unit together with the cancel circuit (60). Here, as described above, the light source driving circuit (30) sets the modulation frequency of the light source (11) based on a reference frequency signal input from the timing generator (70). Based on the frequency signal input from the timing generator (70), the delay / amplification circuit (802) generates a pseudo-signal (calibration signal) that changes the phase of the signal (modulation signal) of the modulation frequency set to the light source (11) from the light source driving circuit (30).

[0109] Specifically, the delay / amplification circuit (802) generates a pseudo-signal (calibration signal) by changing the phase of the aforementioned modulation signal based on a delay corresponding to the time until the scattered light of the illumination light irradiated from the light source (11) of the illumination optical system (10) is detected as a detection light by the detection optical system (20). Thus, the pseudo-signal is a signal that simulates the scattered light (illumination light) detected by the detection optical system (20) in the calibration process. In addition, the delay / amplification circuit (802) sets the amplitude of the pseudo-signal (calibration signal) based on the amplitude when the scattered light of the illumination light irradiated from the light source (11) of the illumination optical system (10) is detected as a detection light by the detection optical system (20). In this way, the delay and amplification circuit (802) generates a pseudo-signal that processes the phase and amplitude of the modulation signal set in the light source (11), thereby obtaining a calibration signal without detecting light for calibration processing (without performing irradiation and detection of light for calibration processing). Specifically, the delay and amplification circuit (802) has a delay circuit for changing the phase and an amplification circuit for changing the amplitude. As shown in FIG. 28, since irradiation of light for calibration processing is not performed, the position of the optical head of the irradiation optical system (10) when performing calibration processing is not particularly limited. Also, regarding the processing after calibration processing (second processing), the description is omitted as it is the same as the first embodiment.

[0110] Next, with reference to FIG. 29, the fluorescence measurement process of the third embodiment is described. FIG. 29 is a flowchart illustrating the fluorescence measurement process of the third embodiment. As shown in FIG. 29, in the fluorescence measurement process of the third embodiment, first, in the delay / amplification circuit (802), a pseudo-signal (calibration signal) is generated by changing the phase of the modulation signal by an amount corresponding to the delay until the scattered light of the irradiation light irradiated from the light source (11) of the irradiation optical system (10) is detected as a detection light by the detection optical system (20) (step S51). Subsequently, in the cancel circuit (60), a calibration process is performed to remove the signal component corresponding to the scattered light from the detection signal based on the pseudo-signal (calibration signal) (step S52). The details of the calibration process are as described above with reference to FIG. 10 to FIG. 13, etc., so the description is omitted.

[0111] Next, the optical head of the irradiation optical system (10) operates to scan the measurement area of ​​the sample (500) so that the excitation light (irradiation light) is irradiated onto the measurement target part (504) of the sample (500) (step S53), and the measurement target light, including the fluorescence generated from the sample (500) irradiated with the excitation light and the scattered light of the excitation light from the sample (500) irradiated with the excitation light, is detected by the light detection element (21) (step S54). Then, in the cancel circuit (60), the result of the calibration processing of step S52 is taken into account, and the signal component due to the scattered light is removed from the measurement signal due to the measurement target light described above (step S55). Specifically, as described above, the cancel circuit (60) takes the measurement signal and the switch signal with phase adjusted in the calibration processing as inputs and removes the signal component due to the scattered light from the measurement signal.

[0112] Next, the effects of action according to the third mode will be explained.

[0113] An optical measuring device (801) according to a third embodiment (see FIG. 27) comprises an irradiation optical system (10) that irradiates an irradiation light (excitation light) according to a modulation signal, a detection optical system (20) that detects a detection light caused by the irradiation light (excitation light), and a delay / amplification circuit (802) and a cancellation circuit (60) that function as a signal processing unit. In the optical measuring device (801), in the first processing, the delay / amplification circuit (802) generates a pseudo-signal (calibration signal) that changes the phase of the modulation signal by considering a delay corresponding to the time until the scattered light of the irradiation light irradiated from the irradiation optical system (10) is detected as a detection light by the detection optical system (20), and the cancellation circuit (60) performs a calibration processing to remove a signal component due to scattered light from the detection signal in the second processing based on the pseudo-signal. Additionally, in the optical measuring device (801), in the second processing, the irradiation optical system (10) irradiates the sample (500) with excitation light (irradiation light), and the detection optical system (20) detects the measurement target light, which includes fluorescence generated from the sample (500) irradiated with the excitation light and scattered light from the sample (500) irradiated with the excitation light, as the detection light, and the cancel circuit (60) takes the measurement signal according to the measurement target light as the detection signal and removes the signal component according to the scattered light in the calibration processing in the first processing from the measurement signal.

[0114] In this way, in the optical measuring device (801), in the first processing related to calibration processing, a pseudo-signal (calibration signal) is generated in which the phase of the modulation signal input to the irradiation optical system (10) is changed according to the delay of the irradiation optical system (10). In this way, by generating a calibration signal according to the delay of the irradiation optical system (10) for the modulation signal related to the irradiation light, it is possible to obtain a calibration signal (a calibration signal having the same phase as the scattered light) that is the same as when the scattered light is actually detected as the light for calibration processing, without detecting the light for calibration processing. That is, according to this configuration, a calibration signal containing only the signal component of the scattered light, which does not include the signal component of the fluorescence, can be obtained. With this, in the second processing, by appropriately removing the signal component due to the scattered light from the measurement signal based on the result of the calibration processing, the noise component caused by the irradiation light itself is removed, and fluorescence measurement can be performed with high precision. In addition, since the light for calibration processing is detected without irradiating the sample (500) with irradiating light, deviations in the results of the calibration processing due to the characteristics of the sample (500), which would be problematic when irradiating the sample (500) with irradiating light to detect the light for calibration processing, are suppressed. More specifically, actual samples may have deviations in characteristics due to differences in manufacturing lots, for example, and differences such as the movement speed of fluorescent materials may occur. In that case, there is a possibility that the amount of fluorescent material (i.e., the amount of fluorescent emission) in the area where irradiating light is irradiated during the calibration processing may differ from one another for each actual sample. Due to this, there is a concern that the results of the calibration processing will not be stable and, for example, sufficient reliability may not be obtained in determining the results of optical measurement, but by generating a calibration signal according to the delay of the irradiating optical system (10) with respect to the modulation signal related to the irradiating light, the fluorescence measurement can be performed with higher precision.In addition, if the results output by the device differ depending on the differences in characteristics of each actual sample, for example, it becomes difficult to set a threshold value for determining positive or negative in measurement using an immunochromatography method, but if a calibration signal is generated according to the delay of the irradiation optical system (10) with respect to the modulation signal related to the irradiation light, this problem can also be suppressed. Also, for example, when measuring multiple samples (500) with different shapes (for example, multiple immunochromatography test kits corresponding individually to each antigen) with one optical measuring device (1), there is no need to set an appropriate calibration area for each sample (500), and since a calibration signal is generated according to the delay of the irradiation optical system (10) with respect to the modulation signal related to the irradiation light, control in terms of hardware or software can be simplified. Also, since a calibration signal is generated electrically without actually detecting the light for calibration processing, a detection optical system for calibration processing becomes unnecessary, and a simple and inexpensive device configuration can be achieved. In this way, according to the optical measuring device (801) of the present embodiment, calibration processing can be performed more appropriately, thereby removing noise components caused by the irradiation light itself more appropriately, and fluorescence measurement can be performed with higher precision.

[0115] Additionally, in the first processing described above, the delay / amplification circuit (802) may set the amplitude of the calibration signal by considering the amplitude when the scattered light of the irradiated light from the irradiated optical system (10) is detected by the detection optical system (20) as the detection light. By doing so, a calibration signal that is closer to the case where the scattered light is actually detected as the light for calibration processing can be obtained.

[0116] Regarding the calibration process described above, in addition to ensuring high reproducibility in actual measurements, it is desirable to perform the calibration process with the same amount of irradiated light each time. To this end, when using the member (reference member) described in the first embodiment, it is desirable that the member can be mechanically stabilized and fixed. Furthermore, it is desirable that the member is resistant to change over time. Additionally, regarding the calibration process described in the second embodiment, when irradiating light onto a light detection element, it is desirable to provide circuit-based feedback to maintain the amount of light from the light source constant. Moreover, in an embodiment where the calibration process is performed using a pseudo-generated signal as in the third embodiment, it can be assumed that calibration can be performed with the same amount of light each time. Explanation of the symbols

[0117] 1, 701, 801… Optical measuring device 10… Illumination optical system 20… detection optical system (light detector, second detection optical system) 60… Cancel circuit (signal processing unit) 600… Absence of reference 600A… Mirror 600B… Reflective diffuser 610… Opaque glass 611… Mirror 720… detection optical system (light detector, first detection optical system) 802… Delay / amplification circuit (signal processing unit) 820… detection optical system (first detection optical system and second detection optical system) 910… Survey optical system

Claims

Claim 1 An optical measuring device for measuring the optical characteristics of a measurement target comprises an illumination optical system that irradiates an illumination target with illumination light, a light detection unit that detects a detection light originating from the illumination light, and a signal processing unit that processes a detection signal according to the detection light. In a first processing step, the illumination optical system irradiates the illumination light onto a reference member for calibration processing that is different from the measurement target, and the light detection unit detects the illumination light for calibration processing, which includes scattered light of the illumination light from the reference member irradiated with the illumination light, as the detection light. The signal processing unit sets the calibration signal according to the calibration processing light as the detection signal, and performs a calibration processing step to remove a signal component according to the scattered light from the detection signal in a second processing step based on the calibration signal. In a second processing step, the illumination optical system irradiates the illumination light onto the measurement target, and the light detection unit detects the fluorescence generated from the measurement target irradiated with the illumination light, and the An optical measuring device that detects a measurement target light containing scattered light from a measurement target object irradiated with an irradiating light as the detection light, and the signal processing unit takes a measurement signal according to the measurement target light as the detection signal, and removes a signal component according to the scattered light in the calibration process in the first process from the measurement signal based on the phase difference between the fluorescence and the scattered light. Claim 2 An optical measuring device according to claim 1, wherein the reference member comprises a reflective member that reflects the illumination light. Claim 3 An optical measuring device according to claim 2, wherein the reflective member does not generate fluorescence upon irradiation with the irradiation light. Claim 4 An optical measuring device according to claim 2 or claim 3, wherein the reflective member comprises a reflective diffuser that diffuses the irradiated light. Claim 5 An optical measuring device according to claim 2 or claim 3, wherein the reflective member comprises a reflective substrate that reflects the irradiated light and a diffuser supported on the reflective substrate and diffusing the irradiated light. Claim 6 An optical measuring device according to claim 2 or claim 3, wherein the reflective member comprises a mirror. Claim 7 An optical measuring device for measuring the optical characteristics of a measurement target comprises: an illumination optical system for irradiating illumination light; a light detection unit having a first detection optical system and a second detection optical system for detecting detection light originating from the illumination light; and a signal processing unit for processing a detection signal according to the detection light. In a first processing step, the illumination optical system irradiates the illumination light to the first detection optical system, and the first detection optical system of the light detection unit detects the illumination light, which is a light for calibration processing, as the detection light. The signal processing unit sets the calibration signal according to the light for calibration processing as the detection signal. Based on the calibration signal, a calibration processing step is performed to remove a signal component according to scattered light from the detection signal in a second processing step. In a second processing step, the illumination optical system irradiates the illumination light to the measurement target, and the second detection optical system of the light detection unit sets the measurement target light, which includes fluorescence generated from the measurement target irradiated with the illumination light, and scattered light from the measurement target irradiated with the illumination light, as the detection light. An optical measuring device that detects, wherein the signal processing unit uses a measurement signal according to the light to be measured as the detection signal, and removes a signal component according to the scattered light in the calibration process in the first process from the measurement signal based on the phase difference between the fluorescence and the scattered light. Claim 8 An optical measuring device for measuring the optical characteristics of a measurement target comprises an illumination optical system that irradiates illumination light according to a modulation signal, a light detector that detects detection light originating from the illumination light, and a signal processing unit. In a first processing step, the signal processing unit generates a calibration signal that changes the phase of the modulation signal according to a delay corresponding to the time until scattered light of the illumination light irradiated from the illumination optical system is detected by the light detector as the detection light. Based on the calibration signal, a calibration processing step is performed to remove a signal component corresponding to scattered light from a detection signal in a second processing step. In a second processing step, the illumination optical system irradiates the illumination light onto the measurement target, and the light detector detects a measurement target light as the detection light, which includes fluorescence generated from the measurement target irradiated by the illumination light and scattered light from the measurement target irradiated by the illumination light. The signal processing unit sets a measurement signal corresponding to the measurement target light as the detection signal, and based on the phase difference between the fluorescence and the scattered light, from the measurement signal, the first processing step An optical measuring device that removes signal components due to scattered light in the calibration process above. Claim 9 An optical measuring device according to claim 8, wherein in the first processing, the signal processing unit sets the amplitude of the calibration signal according to the amplitude when the scattered light of the irradiated light irradiated from the irradiated optical system is detected by the light detection unit as the detection light. Claim 10 An optical measurement method for measuring optical characteristics of a measurement target, comprising: irradiating an irradiating light onto a reference member for calibration processing that is different from the measurement target; detecting a calibration processing light including scattered light of the irradiating light from the reference member on which the irradiating light is irradiated; and performing a calibration processing to remove a signal component according to the scattered light from a measurement signal based on a calibration signal according to the calibration processing light; and performing a second processing including irradiating the irradiating light onto the measurement target; detecting a measurement target light including fluorescence generated from the measurement target on which the irradiating light is irradiated and scattered light from the measurement target on which the irradiating light is irradiated; and removing a signal component according to the scattered light in the calibration processing of the first processing from the measurement signal according to the measurement target light based on the phase difference between the fluorescence and the scattered light. Claim 11 An optical measurement method for measuring the optical characteristics of a measurement target, comprising: a first process including irradiating an irradiating light, detecting a light for calibration processing which is the irradiating light, and performing a calibration process to remove a signal component according to scattered light from a measurement signal based on a calibration signal according to the light for calibration processing; and a second process including irradiating the irradiating light onto the measurement target, detecting a measurement target light including fluorescence generated from the measurement target irradiated by the irradiating light and scattered light from the measurement target irradiated by the irradiating light, and removing a signal component according to the calibration process in the first process from the measurement signal according to the measurement target light based on the phase difference between the fluorescence and the scattered light. Claim 12 An optical measurement method for measuring the optical characteristics of a measurement target, comprising: generating a calibration signal that changes the phase of a modulation signal input to an illumination optical system according to a delay corresponding to the time until scattered light of illumination light irradiated from an illumination optical system is detected by a light detector, and performing a calibration process to remove a signal component according to scattered light from a measurement signal based on the calibration signal; and irradiating the illumination light onto the measurement target, detecting a measurement target light including fluorescence generated from the measurement target irradiated by the illumination light and scattered light from the measurement target irradiated by the illumination light, and performing a second process to remove a signal component according to the calibration process in the first process from the measurement signal according to the measurement target light based on the phase difference between the fluorescence and the scattered light.

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