Optical measuring device and optical measuring method

Abstract

In the optical measurement apparatus, in the first process, the reference member is irradiated with excitation light, and calibration-process light including scattered light from the reference member is detected as detection light, and a calibration signal corresponding to the calibration-process light is detected as a detection signal, and a calibration process for removing a signal component corresponding to the scattered light from the detection signal in the second process is performed. In the optical measurement apparatus, in the second process, the sample is irradiated with excitation light, and measurement target light including fluorescence generated from the sample and scattered light from the sample irradiated with the excitation light is detected as detection light, and a signal component corresponding to the scattered light in the result of the calibration process in the first process is removed from the measurement signal.

Description

TECHNICAL FIELD

[0001] One embodiment of the present application relates to an optical measurement device and an optical measurement method for measuring an optical characteristic of a sample. BACKGROUND

[0002] In Patent Literature 1, a technique of generating and subtracting an attenuation signal in order to remove a fluorescent noise component (a fluorescent component from a substrate) contained when exciting light is irradiated on a fluorescent sample placed on a substrate and fluorescence is measured. Specifically, in Patent Literature 1, by generating an attenuation signal of the same phase as the fluorescence of the substrate, the fluorescent noise component is removed by subtracting the attenuation signal from the measured fluorescent signal.

[0003] PRIOR ART DOCUMENTS

[0004] PATENT LITERATURE

[0005] Patent Literature 1: Japanese Patent Application Laid-Open No. 2010-518394 SUMMARY

[0006] PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] As described above, in Patent Literature 1, from the fluorescence generated by irradiating exciting light on a sample, a fluorescent noise component generated from a part other than the measurement target part of the sample is removed. Here, it is considered that a noise component contained when light is irradiated on a sample and light from the sample is detected is caused not only by light (e.g., fluorescence) generated from the sample (the sample to be irradiated) but also by the irradiated light (e.g., exciting light) itself (e.g., scattered light). In the technique of Patent Literature 1, the case where the irradiated light itself becomes a noise component is not considered, and the noise component cannot be removed.

[0008] On the other hand, the present inventors have found an optical measurement device that removes a noise component caused by the irradiated light itself by removing a signal component having the same phase as the irradiated light from a detection light, by focusing on the difference in phase of fluorescence and scattered light (light caused by the irradiated light itself) contained in the detection light. In such an optical measurement device, for example, a calibration process is performed in advance. In the calibration process, by irradiating the irradiated light on a part of the sample where fluorescence is not easily generated, a calibration process light (ideally, light containing only scattered light) is detected, and based on a calibration signal corresponding to the calibration process light, a signal component corresponding to the scattered light component is removed from the detection signal.

[0009] Here, in the calibration processing, the calibration processing light containing substantially only scattered light is detected by irradiating a portion of the sample (for example, a sample that is chipped in a manner that makes it easy to perform the measurement, hereinafter referred to as an actual sample) that is not likely to generate fluorescence (for example, a region in which it is judged that the fluorescent substance is less in the actual sample) with the irradiation light. However, in the method in which the actual sample is irradiated with the irradiation light and the calibration processing light is detected, it is difficult to completely remove the fluorescence from the calibration processing light detected in the light detector, and a trace amount of the fluorescent component is included in the calibration signal processing light. Therefore, it is known that the calibration processing is performed based on the trace amount of the fluorescent component in addition to the scattered light component, and as a result of the calibration processing being affected by the fluorescent component, there is a concern that the subsequent fluorescence measurement cannot be performed with high accuracy.

[0010] One embodiment of the present application was completed in view of the above-described actual situation, and has an object to provide an optical measurement apparatus and an optical measurement method that can remove a noise component caused by the irradiation light itself and perform fluorescence measurement with high accuracy.

[0011] Technical means for solving the problem

[0012] The optical measurement apparatus of one embodiment of the present application is an optical measurement apparatus that measures an optical property of a measurement target, and includes an irradiation optical system that irradiates a reference member for calibration processing with irradiation light, a light detection unit that detects calibration processing light containing scattered light from the reference member irradiated with the irradiation light as detection light, and a signal processing unit that processes a calibration signal corresponding to the calibration processing light as a detection signal. The signal processing unit removes a signal component corresponding to the scattered light from a measurement signal in second processing, based on the calibration signal. The irradiation optical system irradiates the measurement target with the irradiation light in the second processing. The light detection unit detects measurement target light containing fluorescence generated from the measurement target irradiated with the irradiation light and scattered light from the measurement target irradiated with the irradiation light as the detection light. The signal processing unit removes the signal component corresponding to the scattered light in the calibration processing in the first processing from the measurement signal.

[0013] In the optical measurement apparatus according to the aspect of the present application, in the first process relating to the calibration process, the reference member different from the measurement target is irradiated with the irradiation light to detect the calibration process light including the scattered light. Then, in the second process, by appropriately removing the signal component corresponding to the scattered light from the measurement signal based on the result of the calibration process, the noise component due to the irradiation light itself can be removed and the fluorescence measurement can be performed with high accuracy. Further, by using the reference member for the calibration process which is different from the measurement target, the deviation of the result of the calibration process corresponding to the characteristics of the measurement target, which becomes a problem in the case where, for example, the irradiation light is irradiated to the measurement target and the calibration process light is detected, does not occur. Therefore, the fluorescence measurement can be performed with higher accuracy. Thus, according to the optical measurement apparatus according to the aspect of the present application, the calibration process can be performed more appropriately, the noise component due to the irradiation light itself can be removed more appropriately, and the fluorescence measurement can be performed with higher accuracy.

[0014] The reference member can include a reflection member which reflects the irradiation light. According to such a configuration, the amount of light of the detection light can be easily increased.

[0015] The reflection member is preferably a member which does not generate fluorescence by irradiation of the irradiation light. According to such a configuration, since even if the irradiation of the irradiation light is performed, fluorescence due to the reflection member is not generated (or only fluorescence of a degree which can be ignored is generated), in the second process, the signal component corresponding to the scattered light component can be reliably removed from the measurement signal.

[0016] The reflection member can include a reflection diffuser which diffuses the irradiation light. According to such a configuration, by the reflection diffuser, scattered light of various angles can be easily generated, and the amount of light of the detection light can be more easily increased.

[0017] The reflection member can include a reflection base material which reflects the irradiation light, and a diffuser which is supported by the reflection base material and diffuses the irradiation light. According to such a configuration, by the cooperation of the reflection base material and the diffuser, the amount of light of the detection light can be more easily increased.

[0018] The reflection member can include a mirror. According to such a configuration, by adjusting the reflection angle of the mirror with respect to the light detection portion, the amount of light of the detection light can be more easily increased.

[0019] An optical measurement apparatus according to an embodiment of the present application is an optical measurement apparatus that measures an optical property of a measurement target, and includes: an irradiation optical system that irradiates an irradiation light; a light detection unit that includes a first detection optical system and a second detection optical system that detect a detection light caused by the irradiation light; and a signal processing unit that processes a detection signal corresponding to the detection light. In a first process, the irradiation optical system irradiates the first detection optical system with the irradiation light, the first detection optical system of the light detection unit detects the irradiation light as a calibration processing light, the signal processing unit uses a calibration signal corresponding to the calibration processing light as the detection signal, and based on the calibration signal, performs a calibration process for removing a signal component corresponding to scattered light from a detection signal in a second process. In the second process, the irradiation optical system irradiates the measurement target with the irradiation light, the second detection optical system of the light detection unit detects a measurement target light including a fluorescent light generated from the measurement target irradiated with the irradiation light and the scattered light from the measurement target irradiated with the irradiation light as the detection light, the signal processing unit uses a measurement signal corresponding to the measurement target light as the detection signal, and removes the signal component corresponding to the scattered light in the calibration process in the first process from the measurement signal.

[0020] In the optical measurement apparatus according to the embodiment of the present application, in the first process involved in the calibration process, the irradiation light is directly irradiated to the first detection optical system, and the calibration processing light that is the irradiation light is detected. The irradiation light is light having the same phase as the scattered light. Therefore, the irradiation light is detected as the calibration processing light by the first detection optical system, and light that does not include the fluorescent light and has the same phase as the scattered light can be appropriately detected as the calibration processing light. Thus, in the second process, by removing the signal component corresponding to the scattered light from the measurement signal based on the result of the calibration process, noise components caused by the irradiation light itself can be removed and the fluorescent measurement can be performed with high accuracy. In addition, since the calibration processing light is detected without irradiating the measurement target with the irradiation light, a deviation of a result of the calibration process corresponding to a property of the measurement target, which becomes a problem in a case where the irradiation light is irradiated to the measurement target and the calibration processing light is detected, for example, does not occur. Therefore, the fluorescent measurement can be performed with higher accuracy. Thus, according to the optical measurement apparatus according to the embodiment of the present application, the calibration process can be appropriately performed, noise components caused by the irradiation light itself can be appropriately removed, and the fluorescent measurement can be performed with higher accuracy.

[0021] The optical measurement apparatus according to one embodiment of the present application is an optical measurement apparatus that measures an optical property of a measurement target, and includes an irradiation optical system that irradiates an irradiation light corresponding to a modulation signal, a light detection unit that detects a detection light due to the irradiation light, and a signal processing unit. In a first process, the signal processing unit generates a calibration signal that changes a phase of the modulation signal in accordance with a delay from the irradiation light irradiated from the irradiation optical system until the detection light is detected by the light detection unit, and performs a calibration process for removing a signal component corresponding to the scattered light from a detection signal in a second process on the basis of the calibration signal. In the second process, the irradiation optical system irradiates the irradiation light to the measurement target, the light detection unit detects a measurement target light including a fluorescence generated from the measurement target irradiated with the irradiation light and the scattered light from the measurement target irradiated with the irradiation light as the detection light, and the signal processing unit removes the signal component corresponding to the scattered light in the calibration process in the first process from the detection signal as the detection signal.

[0022] In the optical measurement apparatus according to one embodiment of the present application, in the first process involved in the calibration process, the calibration signal that changes the phase of the modulation signal input to the irradiation optical system in accordance with the delay of the irradiation optical system is generated. Thus, by generating the calibration signal in accordance with the delay of the irradiation optical system with respect to the modulation signal involved in the irradiation light, the calibration signal can be obtained without detecting the light for the calibration process, and actually the same calibration signal as that obtained when the scattered light is detected as the light for the calibration process (the calibration signal having the same phase as that of the scattered light) can be obtained. That is, according to such a configuration, the calibration signal of only the signal component of the scattered light that does not include the signal component of the fluorescence can be obtained. Thus, in the second process, by appropriately removing the signal component corresponding to the scattered light from the detection signal on the basis of the result of the calibration process, the noise component due to the irradiation light itself can be removed and the fluorescence measurement can be performed with high accuracy. In addition, since the light for the calibration process is detected without irradiating the irradiation light to the measurement target, a deviation of the result of the calibration process corresponding to the property of the measurement target, which becomes a problem when the irradiation light is irradiated to the measurement target and the light for the calibration process is detected, for example, does not occur. Thus, the fluorescence measurement can be performed with higher accuracy. Thus, according to the optical measurement apparatus according to one embodiment of the present application, the calibration process can be performed more appropriately, the noise component due to the irradiation light itself can be removed more appropriately, and the fluorescence measurement can be performed with higher accuracy.

[0023] In the first process, the signal processing unit can set the amplitude of the calibration signal in accordance with the amplitude when the scattered light of the irradiation light irradiated from the irradiation optical system is detected by the light detection unit as the detection light. Thus, the calibration signal that is more similar to that obtained when the scattered light is actually detected as the light for the calibration process can be obtained.

[0024] An optical measurement method according to one embodiment of the present application is an optical measurement method of measuring an optical property of a measurement target, and performs a first process and a second process. The first process includes: irradiating a reference member for a calibration process with irradiation light; detecting calibration-process light including scattered light from the reference member irradiated with the irradiation light; and performing a calibration process for removing a signal component corresponding to the scattered light from a measurement signal based on a calibration signal corresponding to the calibration-process light. The second process includes: irradiating the measurement target with the irradiation light; detecting measurement-target light including fluorescence generated from the measurement target irradiated with the irradiation light and scattered light from the measurement target irradiated with the irradiation light; and removing a signal component corresponding to the scattered light of the calibration process in the first process from a measurement signal corresponding to the measurement-target light.

[0025] An optical measurement method according to one embodiment of the present application is an optical measurement method of measuring an optical property of a measurement target, and performs a first process and a second process. The first process includes: irradiating a reference member for a calibration process with irradiation light; detecting calibration-process light including scattered light from the reference member irradiated with the irradiation light; and performing a calibration process for removing a signal component corresponding to the scattered light from a measurement signal based on a calibration signal corresponding to the calibration-process light. The second process includes: irradiating the measurement target with the irradiation light; detecting measurement-target light including fluorescence generated from the measurement target irradiated with the irradiation light and scattered light from the measurement target irradiated with the irradiation light; and removing a signal component corresponding to the scattered light of the calibration process in the first process from a measurement signal corresponding to the measurement-target light.

[0026] An optical measurement method according to one embodiment of the present application is an optical measurement method of measuring an optical property of a measurement target, and performs a first process and a second process. The first process includes: irradiating a reference member for a calibration process with irradiation light; detecting calibration-process light including scattered light from the reference member irradiated with the irradiation light; and performing a calibration process for removing a signal component corresponding to the scattered light from a measurement signal based on a calibration signal corresponding to the calibration-process light. The second process includes: irradiating the measurement target with the irradiation light; detecting measurement-target light including fluorescence generated from the measurement target irradiated with the irradiation light and scattered light from the measurement target irradiated with the irradiation light; and removing a signal component corresponding to the scattered light of the calibration process in the first process from a measurement signal corresponding to the measurement-target light.

[0027] Effects of the Invention

[0028] According to one embodiment of the present application, it is possible to remove a noise component due to the irradiation light itself and perform fluorescence measurement with high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1is a schematic configuration view of an optical measurement apparatus of an embodiment of the present application.

[0030] Figure 2 is a graph illustrating a phase difference of fluorescence and scattered light.

[0031] Figure 3 is a graph illustrating a method of eliminating scattered light.

[0032] Figure 4 is a graph illustrating a method of measuring fluorescence.

[0033] Figure 5 is a flowchart showing a process of measuring fluorescence by an optical measurement apparatus.

[0034] Figure 6 is a schematic configuration view of an optical measurement apparatus.

[0035] Figure 7 is a graph illustrating a method of removing noise components.

[0036] Figure 8 is a graph schematically showing a sample.

[0037] Figure 9 is a graph illustrating a definition of S / N.

[0038] Figure 10 is a graph illustrating a signal utilized in a calibration process.

[0039] Figure 11 is a graph illustrating a calibration process.

[0040] Figure 12 is a table showing a proportion of a fluorescence component of each modulation frequency.

[0041] Figure 13 is a flowchart showing a calibration process.

[0042] Figure 14 is a graph illustrating an effect of a calibration process.

[0043] Figure 15 is a graph illustrating a sequence of measuring fluorescence.

[0044] Figure 16 is a graph illustrating an effect of a calibration in a case where a reference member that generates fluorescence is utilized.

[0045] Figure 17 is a graph illustrating an effect of a calibration in a case where a reference member that does not generate fluorescence is utilized.

[0046] Figure 18 is a graph illustrating an outline of a method of measuring fluorescence of the first embodiment.

[0047] Figure 19 is a diagram for explaining scattered light acquisition in a case where a mirror is used as a reference member.

[0048] Figure 20 is a diagram for explaining scattered light acquisition in a case where a reflection diffuser is used as a reference member.

[0049] Figure 21 is a diagram for explaining scattered light acquisition in a case where frosted glass is used as a reference member.

[0050] Figure 22 is a flowchart showing the fluorescence measurement processing of the first mode.

[0051] Figure 23 is a schematic configuration diagram of the optical measurement apparatus of the second mode.

[0052] Figure 24 is a diagram for explaining an outline of the fluorescence measurement of the second mode.

[0053] Figure 25 is a diagram for explaining irradiation light acquisition of the second mode.

[0054] Figure 26 is a flowchart showing the fluorescence measurement processing of the second mode.

[0055] Figure 27 is a schematic configuration diagram of the optical measurement apparatus of the third mode.

[0056] Figure 28 is a diagram for explaining an outline of the fluorescence measurement of the third mode.

[0057] Figure 29 is a flowchart showing the fluorescence measurement processing of the third mode. DETAILED DESCRIPTION

[0058] Hereinafter, the embodiments of the present application will be explained in detail with reference to the drawings. In addition, the same or equivalent portions are assigned with the same symbols in each drawing, and the repeated explanation is omitted.

[0059] Figure 1is a schematic configuration diagram of the optical measurement apparatus 1 of the present embodiment. The optical measurement apparatus 1 is an apparatus that detects light generated from a sample in response to light irradiated on the sample. In the present embodiment, the optical measurement apparatus 1 is described as a fluorescence measurement apparatus that detects fluorescence generated from a sample in response to excitation light (irradiation light) irradiated on the sample. The excitation light is light that excites a sample, and the fluorescence is light emitted from the sample in response to the excitation light, and is light having a wavelength different from that of the excitation light. In addition, in the present embodiment, the optical measurement apparatus 1 is described as an apparatus that detects fluorescence involved in measurement using an immunochromatography method. The immunochromatography method is an immunoassay method using an antigen-antibody reaction, and is used for, for example, detection of influenza viruses and the like.

[0060] As Figure 1 shown, in measurement using the immunochromatography method, an immunochromatography test piece 100 is prepared as a sample. The immunochromatography test piece 100 houses an immunochromatography membrane that is a measurement target in a reagent holder 101. At a specific position (measurement target portion) of the immunochromatography membrane of the immunochromatography test piece 100, a capture antibody (for example, an antibody with respect to an influenza virus antigen) with respect to a prescribed antigen is fixed. In the reagent holder 101, an opening portion, that is, a sample application window for dropping a sample on the immunochromatography membrane, and an opening portion, that is, a measurement window for measuring the measurement target portion in which the capture antibody is fixed, are provided. If the sample is dropped on the sample application window of the reagent holder 101, the antigen in the sample first binds to a detection antibody labeled with a fluorescent reagent, and secondarily causes an antigen-antibody reaction with the capture antibody and is captured. The optical measurement apparatus 1 irradiates excitation light on the immunochromatography membrane exposed from the measurement window of the immunochromatography test piece 100, and measures the intensity of fluorescence by detecting the fluorescence from the antigen-antibody complex (in detail, the fluorescent reagent of the antibody) of the measurement target portion. In addition, as the fluorescent reagent, for example, europium, Q-dot (registered trademark), an organic dye, or the like can be used.

[0061] Here, it is considered that, in the detection light incident on and detected by the detection optical system 20 in the optical measurement apparatus 1, not only fluorescence but also light caused by the excitation light itself is included. Such light can be exemplified by, for example, scattered light of the excitation light. Such scattered light is, for example, a part of the excitation light generated by scattering of the excitation light irradiated on the immunochromatography test piece 100, and is light having the same phase (no phase difference) as the excitation light. The immunochromatography membrane of the immunochromatography test piece 100 and the reagent holder 101 are generally white, and thus the above-described scattered light is easily generated. In addition, there are cases where the excitation light itself is detected by the configuration of the sample and the detection optical system of the measurement. Hereinafter, the detection light detected in the optical measurement apparatus 1 is described as including fluorescence and scattered light.

[0062] As Figure 1As shown, the optical measurement device 1 includes an irradiation optical system 10, a detection optical system 20 (light detection section), a light source drive circuit 30, an IV conversion amplifier 40, a waveform generation circuit 50, a cancellation circuit 60 (signal processing section), a timing generator 70, an AD converter 80, and a CPU 90.

[0063] The irradiation optical system 10 irradiates excitation light (irradiation light) toward the immunochromatography test sheet 100 (sample) as a measurement target. The irradiation optical system 10 includes a light source 11, an aperture 12, an excitation light filter 13, and a collimator lens 14. The light source 11 irradiates excitation light toward the immunochromatography test sheet 100 (sample). The light source 11 is, for example, a semiconductor light emitting element. In the present embodiment, the light source 11 is assumed to be a light emitting diode (LED) and will be described as such, but is not limited thereto. For example, an LD can be used in order to ensure the amount of light. The aperture 12 is a beam shaping member for shaping the light emitted from the light source 11 into light having a desired beam cross section. The excitation light filter 13 is a wavelength selection filter that filters the wavelength required for excitation with respect to the excitation light that has reached via the aperture 12. The excitation light filter 13 is an optical filter such as a dielectric multilayer film filter or a colored glass filter, and more specifically, a band pass filter composed of a dielectric multilayer film filter that transmits only a specific wavelength band (excitation wavelength of the fluorescent reagent). The collimator lens 14 is a lens that images the excitation light filtered by the excitation light filter 13 on the immunochromatography test sheet 100 (more specifically, the measurement target section of the immunochromatography membrane).

[0064] The detection optical system 20 detects the detection light due to the excitation light. Specifically, the detection optical system 20 detects the fluorescence from the immunochromatography test piece 100. However, in reality, in the detection optical system 20, in addition to the fluorescence (fluorescence from the measurement target portion of the immunochromatography membrane) from the immunochromatography test piece 100, light including the scattered light due to the excitation light itself, that is, the detection light, is incident, and this detection light is detected. The detection optical system 20 has a light detecting element 21, a fluorescence filter 22, and a condenser lens 23. The detection light is condensed by the condenser lens 23, and is incident on the light detecting element 21 via the fluorescence filter 22. The fluorescence filter 22 is a wavelength selection filter provided to suppress light other than the fluorescence from reaching the light detecting element 21 with respect to the detection light from the immunochromatography test piece 100. The fluorescence filter 22 is an optical filter such as a dielectric multilayer film filter or a colored glass filter, and more specifically, a band pass filter in which a dielectric multilayer film filter that transmits only a specific wavelength band and a colored glass filter are combined. However, in a case where, for example, the excitation light wavelength and the fluorescence wavelength are close, it is difficult to appropriately transmit the fluorescence having the fluorescence wavelength through the fluorescence filter 22 while effectively blocking only the scattered light having the excitation light wavelength. In addition, generally, the characteristics of the dielectric multilayer film filter, which is widely used as a wavelength selection filter with high efficiency, vary depending on the incident angle of light. Therefore, in the present embodiment, the fluorescence filter 22 is configured by using a combination of a dielectric multilayer film filter and a colored glass filter, and the scattered light from the oblique direction is effectively blocked by the colored glass filter. However, it is difficult to obtain sufficient effects only by wavelength selection, and it is difficult to effectively prevent the entry of scattered light having various conditions. Hereinafter, it is assumed that the description is made assuming that the scattered light is included in the detection light that reaches the light detecting element 21 even by providing the fluorescence filter 22.

[0065] The light detecting element 21 is a light sensor that detects the detection light after the filtering by the fluorescence filter 22. The light detecting element 21 is, for example, a semiconductor light receiving element. In the present embodiment, the description is made assuming that the light detecting element 21 is a photodiode (PD), but is not limited thereto, and can be an avalanche photodiode (APD) or a photomultiplier tube (PMT) or the like as long as it is an element that can respond at high speed in correspondence with the modulation frequency of the excitation light from the light source 11 described below. The light detecting element 21 specifically detects the detection light including the fluorescence generated from the immunochromatography test piece 100 (specifically, the fluorescent reagent of the antigen-antibody complex in the measurement target portion of the immunochromatography membrane) irradiated with the excitation light, and the light due to the excitation light, that is, the above-described scattered light having no phase difference with the excitation light. The light detecting element 21 outputs a detection signal corresponding to the detection light to the IV conversion amplifier 40.

[0066] The light source driving circuit 30 is a circuit that drives the light source 11 by outputting a driving current to the light source 11, which is an LED. The light source driving circuit 30 receives a sinusoidal frequency signal as a reference from the timing generator 70. Based on the input frequency signal as a reference, the light source driving circuit 30 modulates the frequency of the driving current. That is, the light source driving circuit 30 sets the modulation frequency of the light source 11 that outputs excitation light. Correspondingly, the frequency modulation of the excitation light output from the light source 11 results in a sinusoidal change in the amount of light (excitation light) from the light source 11. Furthermore, the modulation frequency can be determined based on the fluorescence lifetime of the fluorescent reagent used. For example, when using europium, which has a fluorescence lifetime of several milliseconds, as the fluorescent reagent, the modulation frequency can be set to about 1 kHz; when using Q-dot, which has a fluorescence lifetime of tens of nanoseconds, the modulation frequency can be set to about 100 MHz; and when using organic pigments with a fluorescence lifetime of several nanoseconds to tens of nanoseconds, the modulation frequency can be set to about 1 GHz.

[0067] Generally, fluorescence lifetime is defined as the time it takes for fluorescence intensity to drop from its peak to 1 / e (approximately 37%). Based on this definition of fluorescence lifetime, it is assumed that, for example, when using europium with a fluorescence lifetime of several milliseconds, the optimal modulation frequency is preferably 1 kHz, and when using organic pigments with fluorescence lifetimes of several nanoseconds to tens of nanoseconds, the optimal modulation frequency is preferably around 100 MHz to 1 GHz. However, measurements of the actual fluorescence signal output relative to the modulation frequency using europium reagents show that modulation at a frequency lower than that determined by fluorescence lifetime results in higher fluorescence intensity and a larger proportion of fluorescence signal relative to excitation light (see reference). Figure 12 ).like Figure 12 As shown, the fluorescence intensity increases at a lower frequency than 1 kHz, which is determined by the fluorescence lifetime. Specifically, the fluorescence lifetime is defined as "the time it takes for the peak fluorescence intensity to drop to 1%" rather than 1 / e. The fluorescence intensity can be increased by calculating the modulation frequency based on this time. In this case, if europium is used, the fluorescence lifetime becomes approximately 10 ms, and the modulation frequency of the light source 11 determined by it becomes approximately 100 Hz.

[0068] As described above, the light source drive circuit 30 can determine the modulation frequency of the light source 11 in consideration of the fluorescence intensity. Specifically, the light source drive circuit 30 makes the modulation frequency of the light source 11 lower than a value corresponding to the time for the fluorescence intensity to decrease from the peak value to 1 / e, that is, the fluorescence lifetime (in detail, 1 / fluorescence lifetime). The light source drive circuit 30 sets the modulation frequency of the light source 11 to be lower than the value corresponding to the fluorescence lifetime and higher than the commercial frequency (50 Hz, 60 Hz), for example, to be around 110 Hz which is near 100 Hz and reduces the influence of noise by avoiding the multiple of the commercial frequency. The light source drive circuit 30 can set the modulation frequency of the light source 11 to other values around 100 Hz, for example, 90 Hz, 80 Hz, 70 Hz, or 130 Hz, and the like.

[0069] The IV conversion amplifier 40 converts the current signal (detection signal) output from the light detecting element 21 into a voltage signal. The IV conversion amplifier 40 outputs the detection signal converted into the voltage signal to the waveform generation circuit 50.

[0070] The waveform generation circuit 50 is a circuit that generates the waveform of the detection signal based on the detection signal output from the IV conversion amplifier 40. The waveform generation circuit 50 receives the input of the frequency signal serving as a reference from the timing generator 70. The timing generator 70 inputs the frequency signal serving as a reference to the light source drive circuit 30 and the waveform generation circuit 50 with the same timing. The waveform generation circuit 50 outputs the information of the generated waveform (detection signal) to the cancellation circuit 60.

[0071] The cancellation circuit 60 is a signal processing section that processes the waveform (detection signal) generated by the waveform generation circuit 50. The cancellation circuit 60 removes the signal component corresponding to the scattered light from the detection signal based on the difference in the phase (phase difference) of the fluorescence and the scattered light. Further, the cancellation circuit 60 acquires the information of the phase of the excitation light (that is, the scattered light) by receiving the input of the frequency signal serving as a reference from the timing generator 70 with the same timing as the light source drive circuit 30 and the waveform generation circuit 50. Thus, in the cancellation circuit 60, the removal of the signal component of the scattered light based on the phase difference of the fluorescence and the scattered light can be performed. For details of the processing of the cancellation circuit 60, refer to Figures 2-4 for explanation.

[0072] Figure 2 is a conceptual diagram illustrating the phase difference of the fluorescence and the scattered light. As Figure 2As shown, the sinusoidal detection light from the sample S irradiated with the sinusoidal excitation light from the light source section L (the detection light detected in the light detection section D) contains sinusoidal scattered light and fluorescence. Further, the excitation light from the light source section L is not limited to a sinusoidal wave, but can be a periodic modulation waveform such as a rectangular wave, in which case the detection light (scattered light and fluorescence) also has the same periodic modulation waveform as the excitation light. Thus, the scattered light is light having no phase difference from the excitation light, and, in contrast, the fluorescence is light generated from the sample S in response to the excitation light, and is detected with a phase delay of about 10 milliseconds to nanoseconds with respect to the scattered light. The present inventors focused on such a phase difference, and found a method of removing only the scattered light from the detection light and extracting only the fluorescence. Further, in the present application, since the sample S and the light detection section D are disposed on the optical axis of the light source section L, the fluorescence emitted in a direction intersecting the optical axis of the excitation light is detected differently from the fluorescence emitted in a direction coaxial with the optical axis of the excitation light. Figure 2 Figure 1 In this case, the detection light can contain the excitation light itself in addition to the fluorescence and the scattered light. In addition, the amount of light of the light incident on the light detection section D due to the excitation light is also likely to increase. Thus, the extraction of the fluorescence using the present method is effective.

[0073] Figure 3 is a diagram illustrating a method of removing (eliminating) the scattered light. Figure 3 only the waveform of the scattered light in the detection light is shown. Further, this waveform is equal to the waveform of the excitation light. In Figure 3 , the horizontal axis represents time, and the vertical axis represents amplitude. With respect to the waveform shown in Figure 3 corresponding to the phase of the scattered light, the time unit is separated (the time region is separated) by, for example, 1 / 4 of 1 period, and if the integrals are performed with respect to each of the time regions 1 to 4, the outputs of the scattered light in each of the time regions 1 to 4 can be obtained. Here, if the integral values of each of the time regions 1 to 4 are each multiplied by a certain multiplier and all added up, the sum of the outputs can be set to 0. That is, when the absolute values of the outputs of each of the time regions 1 to 4 are the same, and the range of the amplitudes of the time regions 1 and 2 is positive and the range of the amplitudes of the time regions 3 and 4 is negative, as shown in Figure 3 ​As shown, for the time region 1, if amplified by multiplying the multiplier "-1", the output of the time region 1 becomes a negative value due to "positive x negative"; for the time region 2, if amplified by multiplying the multiplier "+1", the output of the time region 2 becomes a positive value due to "positive x positive"; for the time region 3, if amplified by multiplying the multiplier "+1", the output of the time region 3 becomes a negative value due to "negative x positive"; for the time region 4, if amplified by multiplying the multiplier "-1", the output of the time region 4 becomes a positive value due to "negative x negative". Therefore, if the integrated values of each of the time regions 1 to 4 amplified by multiplying the prescribed multipliers are all added, the values cancel each other out, and the total of the output becomes 0. In this way, for the signal component corresponding to the scattered light, by separating in the prescribed time unit corresponding to the phase of the scattered light, amplifying each of the separated components, and synthesizing the amplified components, the scattered light can be removed (set the output to 0).

[0074] Figure 4 is a diagram for explaining the fluorescence measurement method. Figure 4 The waveforms of the scattered light and the fluorescence contained in the detection signal are shown. In Figure 4 the horizontal axis indicates time, and the vertical axis indicates amplitude. As described above, for the signal component corresponding to the scattered light, by separating in the prescribed time unit corresponding to the phase of the scattered light, amplifying each of the separated components, and synthesizing the amplified components, the scattered light can be removed (set the output to 0). Here, as shown in Figure 4 for the fluorescence, since there is a phase difference with respect to the scattered light, if separated in the prescribed time unit corresponding to the phase of the scattered light, the integrated values of each of the time regions 1 to 4 do not become the same value, and thus the value amplified and added by multiplying the same multiplier as the scattered light is not a value of 0. In this way, for the scattered light and the fluorescence, by separating into the same time region and amplifying and synthesizing, the signal component of the scattered light can be removed while the output intensity of the fluorescence is detected and output.

[0075] Thus, the cancellation circuit 60 removes the signal component corresponding to the scattered light from the detection signal by separating the detection signal in the prescribed time unit corresponding to the phase of the scattered light, amplifying each component of the separated detection signal, and synthesizing the amplified components, and obtains the signal component of the fluorescence. The cancellation circuit 60 outputs the fluorescence signal, which is the signal in which the signal component corresponding to the scattered light has been removed, that is, the signal in which only the signal component of the fluorescence remains, to the AD converter 80. Further, as the prescribed time unit, although a time of 1 / 4 of 1 cycle is exemplified, it is not limited thereto, and can be any time unit as long as it is a time unit in which the signal component corresponding to the scattered light can be removed after the synthesis. In addition, as the multiplier for amplification, although "+1" and "-1" are exemplified, it is not limited thereto, and can be any multiplier as long as it is a multiplier in which the signal component corresponding to the scattered light can be removed after the synthesis.

[0076] The AD converter 80 performs AD conversion on the fluorescence signal output from the cancellation circuit 60 and converts it into a digital value, and outputs it to the CPU 90. The CPU 90 performs prescribed control and signal processing on the digital signal (fluorescence signal) output from the AD converter 80. The CPU 90 can transmit the signal processing result to an external computer, for example, by serial communication. In addition, the CPU 90 can generate a signal that determines the signal output from the timing generator 70, that is, the various operation timings of the optical measurement device 1, and output it to the timing generator 70. Further, the CPU 90 can be replaced with an FPGA. According to the above processing, the optical measurement device 1 can remove the influence of the scattered light from the detection light and obtain only the signal related to the fluorescence of the fluorescent reagent.

[0077] Next, the fluorescence measurement processing (optical measurement method) performed by the optical measurement device 1 will be described with reference to Figure 5 .

[0078] Figure 5 is a flowchart showing the fluorescence measurement processing performed by the optical measurement device 1. As Figure 5 indicated, in the fluorescence measurement processing, first, the light source 11 of the illumination optical system 10 (light source unit) irradiates excitation light toward the immunochromatography test piece 100 (sample) (step S1). By irradiating the excitation light on the immunochromatography test piece 100 (in detail, the antigen-antibody complex in the measurement target portion of the immunochromatography film), fluorescence is emitted from the fluorescent reagent of the antigen-antibody complex. On the other hand, the excitation light is scattered by the immunochromatography test piece 100, and scattered light is generated.

[0079] Subsequently, the light-detecting element 21 of the light-detecting system 20 (light-detecting section) detects the detection light including the above-described fluorescent light and scattered light (step S2). The light-detecting element 21 outputs the detection light to the IV conversion amplifier 40. Then, when the current signal (detection signal) output from the light-detecting element 21 is converted into a voltage signal in the IV conversion amplifier 40, the waveform of the detection signal is generated in the waveform generating circuit 50, and then the cancellation circuit 60 (signal processing section) removes the signal component corresponding to the scattered light from the detection signal on the basis of the phase difference between the fluorescent light and the scattered light (step S3). Specifically, the cancellation circuit 60 removes the signal component corresponding to the scattered light from the detection signal by separating the detection signal in a prescribed time unit corresponding to the phase of the scattered light, amplifying each component of the separated detection signal, and synthesizing the amplified components, and obtains the signal component of the fluorescent light. Thereafter, by converting the fluorescent signal into a digital value in the AD converter 80, prescribed control and signal processing are performed in the CPU 90, and a signal related to the fluorescent light can be obtained.

[0080] Further, in the optical measurement apparatus 1, it has been described that the signal component corresponding to the scattered light is removed from the detection signal in the cancellation circuit 60, but it is not limited thereto. That is, as shown in the optical measurement apparatus 1A, the cancellation circuit 60 can not be provided, and the processing of removing the signal component corresponding to the scattered light from the detection signal can be performed in the CPU 90A (signal processing section) after the A / D conversion in the AD converter 80. In this case, since the cancellation circuit 60 does not need to be provided, the downsizing of the apparatus can be facilitated. Figure 6

[0081] Next, the removal of the specific signal component (noise) described in the above-described embodiment will be described in detail with reference to Figures 7-16 .

[0082] Figure 7 is a graph for describing the removal of the noise component. Figure 7 (a) of FIG. 10 shows the intensity of the detection light in a case where the removal of the signal component (noise) corresponding to the scattered light due to the excitation light has not been performed, Figure 7 (b) of FIG. 10 shows the intensity of the detection light in a case where the removal of the signal component (noise) corresponding to the scattered light due to the excitation light has been performed. In Figure 7 (a) of FIG. 10 and Figure 7 (b) of FIG. 10, the vertical axis represents the intensity of the detection light, and the horizontal axis represents the channel indicating the position in the measurement section 501 which is the measurement region of the sample 500. One channel is, for example, 0.02 mm. Figure 7 (c) of FIG. 10 is a graph showing the region of the sample 500 corresponding to the position of the channel of Figure 7 (a) of FIG. 10 and Figure 7 (b) of FIG. 10. In Figure 7 ​The enlarged schematic diagram of sample 500 shown in (c) is as follows. Figure 8 .like Figure 8 As shown, the sample 500 is arranged from upstream to downstream as follows: a dropping section 502 for dropping the sample, a holding section 503 for holding the detection antibody labeled with a fluorescent reagent, and a measuring section 501 for immobilizing the captured antibody on the test subject section 504. The fluorescent reagent is, for example, DTBTA-Eu3+. Since the measuring section 501 is, for example, part of a white immunochromatographic membrane, it easily scatters the excitation light.

[0083] When a sample 500 is dropped onto the dropping section 502, the sample moves downstream due to capillary action. If the analyte is present in the sample, the detection antibody in the holding section 503 reacts with the analyte to form a complex, which continues to move downstream in the measuring section 501. Then, when the complex reaches the analyte section 504 on the measuring section 501, the complex is captured by the capture antibody in the analyte section 504, forming a complex consisting of the analyte, the detection antibody, and the capture antibody. By irradiating the measuring section 501, which serves as the measuring area, with excitation light while changing the focusing position (channel) in this state, an image can be obtained... Figure 7 (a) and Figure 7 The detected light intensity for each channel is shown in (b). Figure 7 (a) and Figure 7 In (b), the channel whose light intensity is greater than others is the channel corresponding to the position of the measurement object 504 that captures the complex.

[0084] like Figure 7 As shown in (a), since the detection light contains both fluorescence and scattered light without removing the signal component (noise) corresponding to the scattered light, the intensity of the detection light increases. Therefore, since this noise increases with increasing excitation light intensity, as... Figure 7 As shown in (a), when the excitation light intensity is doubled, the noise also doubles by approximately two times. Generally, as a method to improve the signal-to-noise ratio (S / N), increasing the excitation light intensity to increase the fluorescence signal intensity is considered. However, as mentioned above, in situations where… Figure 7 As shown in (a), the noise also increases with the amount of excitation light, making it difficult to improve the S / N ratio. Furthermore, there is also the problem of narrowing the dynamic range due to increasing the amount of excitation light.

[0085] On the other hand, such as Figure 7 As shown in (b), since the signal component (noise) corresponding to the scattered light has been removed, the detection light contains approximately only fluorescence, thus only the signal to be detected (the fluorescence-based signal) can be detected. In this case, since the noise is approximately zero, therefore...Figure 7 As shown in (b), even if the excitation light intensity is increased (e.g., even by a factor of 2), as long as the photodetector is not saturated, the effect of the excitation light (scattered light) can be approximately eliminated to zero, and the noise will not become extremely large. As mentioned above, when performing... Figure 7 In the noise removal structure shown in (b), since the signal component is increased while the noise component is approximately eliminated to zero when the excitation light is increased, the signal-to-noise ratio (S / N) is improved. This structure is highly effective against noise components, thus enabling an increase in the excitation light quantity and a higher multiplication rate of the IV converter amplifier.

[0086] Figure 9 This diagram illustrates the definition of S / N. Figure 9 An example showing the intensity of the detection light for each channel (the intensity of the detection light at each location in the measurement area). For example... Figure 9 As shown, there is a fluctuation component of approximately ±4 around the intensity of the detected light at 10 count. This fluctuation (standard deviation) in the basic light intensity is the value obtained by scanning the excitation light onto the measurement unit 501 (which is not coated with any fluorescent material, etc.) (or, the measurement unit 501 is set to a wet state, similar to the measurement state). Hereinafter, this fluctuation in the basic light intensity is defined as noise N. Furthermore, the signal S is defined as "the value of the peak fluorescence intensity from the measurement target unit 504 minus the average value of the noise components in all channels except for the position of the measurement target unit 504". S / N is defined as the value of dividing the signal defined above by the noise.

[0087] In addition, Figure 9 In the example shown, the noise value deviates by approximately 10 counts. Through the calibration process described below, the noise value is theoretically eliminated to approximately 0. However, although there is a background deviation corresponding to the noise value, from the viewpoint of software analysis, it is preferable that the signal is always positive; therefore, background offset processing is performed. Furthermore, the offset is set to keep the signal within the dynamic range (0 to 4096 counts). The offset is minimized based on the dynamic range viewpoint and is set such that the background signal obtained by scanning the excitation light towards the measurement unit 501 is always (approximately reliably) a positive value. Specifically, the offset can be, for example, set to the average intensity of the detection light obtained by scanning the excitation light towards the measurement unit 501 without any fluorescent material (or, the measurement unit 501 in a wet state, similar to the measurement state) plus 6σ of that average intensity. Furthermore, an appropriate margin can be added to the offset calculated above to set the final offset, in case of sudden noise intrusion into the circuit system. The offset is selected in a way that does not sacrifice the dynamic range and prevents the signal output from becoming negative; for example, it can be approximately +20 counts.

[0088] Next, the method for removing the signal component (noise) corresponding to the scattered light is described in detail. In the optical measurement apparatus 1, the calibration process is performed in the cancellation circuit 60 as a lock circuit, and the signal component (noise) corresponding to the scattered light is removed from the detection signal taking into account the implementation result of the calibration process.

[0089] Specifically, in the optical measurement method using the optical measurement apparatus 1, first, the optical head of the irradiation optical system 10 is arranged in a manner that excitation light is irradiated to the reference member 600 (refer to Figure 15 (a)) different from the test sample 500 for the calibration process. Then, the scattered light (excitation light component scattered in the reference member 600) caused by the irradiation of the excitation light from the irradiation optical system 10 to the reference member 600 is detected in the light detection element 21 of the detection optical system 20. Here, the light detected by the detection optical system 20 is basically light of only the scattered light not containing the fluorescent light in the reference member 600, and is the calibration process light for the calibration process.

[0090] Then, the calibration process is implemented. Specifically, the cancellation circuit 60 of the optical measurement apparatus 1 implements the calibration process for removing the signal component corresponding to the scattered light from the detection signal based on the calibration signal corresponding to the above-described calibration process light. Details of the calibration process are described later. Thereafter, after the calibration process is completed, the fluorescent light information of the measurement section 501 is acquired by scanning the optical head of the irradiation optical system 10 on the measurement region (measurement section 501) of the test sample 500. Specifically, the cancellation circuit 60 acquires the fluorescent light information by removing the signal component corresponding to the scattered light from the detection signal taking into account the implementation result of the above-described calibration process.

[0091] Next, details of the calibration process are described. The cancellation circuit 60 of the optical measurement apparatus 1 is a lock circuit using, for example, an FPGA (Field Programmable Gate Array). In the calibration process, the cancellation circuit 60 generates a switching signal for locking, which shifts the phase with respect to a period signal that depicts the period of the operation frequency of the cancellation circuit 60 matching the modulation frequency (for example, the frequency of the DDS (Direct Digital Synthesizer)) of the light source 11 set by the light source driving circuit 30. Then, the cancellation circuit 60 functioning as a lock circuit inputs the calibration signal as the measurement signal and the switching signal as the reference signal, and outputs the signal component corresponding to the scattered light in a manner that the voltage value of the signal component corresponding to the scattered light becomes within a prescribed range (sloping level) where it is approximately 0, and adjusts the phase of the switching signal.

[0092] Figure 10The signal used for calibration processing is shown inside the FPGA of the elimination circuit 60. Figure 10 The periodic signal shown is a clock signal that characterizes the period by matching the frequency of the DDS as described above. The reference signal is a signal with the same frequency as the periodic signal, located at an arbitrary phase (phase offset relative to the periodic signal) from the periodic signal, and is used as the trigger for the XY signals described below. The XY signals are the aforementioned lock-up switch signals, generated using the reference signal as the 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 offset by 90 degrees relative to the reference signal. In addition to the X and Y signals, the cancellation circuit 60 further generates an X′ signal (third signal) that inverts the X signal, and a Y′ signal (fourth signal) that inverts the Y signal. The X, Y, X′, and Y′ signals are generated by separate dedicated circuits. Adjusting the phase of the switch signal so that the voltage value of the signal component corresponding to the scattered light becomes a ramp level means continuously offsetting the phase of the reference signal relative to the periodic signal until the output from the cancellation circuit 60 becomes 0V (or a value approximately thereto).

[0093] Figure 11 This diagram illustrates the adjustment process where the output is set to 0V by shifting the phase of the reference signal relative to the periodic signal. Currently, the initial phase relationship between the periodic signal, the reference signal, and the switching signal is as follows: Figure 11 The state shown in (a). Therefore, based on the switching signal in Figure 11 The integration is performed on the meshed intervals, and the output (the voltage value of the signal component corresponding to the scattered light) is not a ramp level and is a positive value. In this case, such as Figure 11 As shown in (b), the phase of the reference signal is adjusted by delaying the phase of the switching signal. That is, during the calibration process, the elimination circuit 60 adjusts the phase of the switching signal by delaying the phase when the voltage value of the signal component corresponding to the scattered light is positive rather than at a ramp level.

[0094] Currently, the phase adjustment of the switching signal has been performed. Figure 11 In state (b), the result of the integration of the meshed interval is also that the output (the voltage value of the signal component corresponding to the scattered light) is not at the ramp level and is a positive value. In this case, as... Figure 11 As shown in (c), the phase of the reference signal is adjusted by further delaying the phase of the switching signal.

[0095] Currently, the phase adjustment of the switching signal has been performed. Figure 11 In state (c), the result of the integral processing of the meshed interval is that the output (the voltage value of the signal component corresponding to the scattered light) is not at the ramp level and is a positive value. In this case, as...Figure 11 (d) to adjust the phase of the reference signal in a manner to delay the phase of the switching signal.

[0096] Currently, in the state of (d) where the phase adjustment of the switching signal has been performed Figure 11 The result of the integration processing of the screened area in the state of (e) shown in FIG. 8 is that the output (voltage value of the signal component corresponding to the scattered light) becomes a ramp level (a value within a prescribed range approximating 0). In this case, the calibration processing is completed. Figure 11 (e) to adjust the phase of the reference signal in a manner to advance the phase of the switching signal. That is, the cancellation circuit 60 performs the adjustment in a manner to advance the phase of the switching signal in the case where the voltage value of the signal component corresponding to the scattered light is a negative value other than a ramp level in the calibration processing.

[0097] Then, when the result of the adjustment in a manner to advance the phase of the switching signal, the result of the integration processing of the screened area in the state of (e) shown in FIG. 8 is that the output (voltage value of the signal component corresponding to the scattered light) becomes a ramp level (a value within a prescribed range approximating 0). In this case, the calibration processing is completed. Figure 11

[0098] When the calibration processing is completed, the cancellation circuit 60 sets the detection signal corresponding to the detection light including the fluorescent component and the scattered light component (excitation light component) and the switching signal whose phase is adjusted in the calibration processing as inputs, and removes the signal component corresponding to the scattered light component from the detection signal.

[0099] Figure 13 is a flowchart showing the calibration processing. As shown in (a) of FIG. 9, in the calibration processing, first, the input to the AD converter is switched to a prescribed compensation voltage, and 0 level is stored (step Sll). Then, the signal of the cancellation circuit 60 (lock circuit) is input to the AD converter by switching of the switch (step S12). In this state, the phase of the reference signal is forcibly shifted once (step S13). The output of the cancellation circuit 60 (lock circuit) becomes 0 V when the phase of the reference signal with respect to the periodic signal is 0 degrees and 180 degrees, but in the case where the phase is accidentally aligned with 180 degrees in the initial state, since the calibration processing is completed by mistake, the positive and negative of the output signal are reversed, and thus there is a case where the signal output through the structure of the subsequent circuit cannot be detected. This point can be prevented by forcibly shifting the phase of the reference signal at the start. Further, by aligning the phase at the start as described above, the positive and negative of the output signal are fixed. As a result, the dynamic range of the AD converter can be effectively used without converting the sign bit of the output signal to a digital value. Further, when measurement with a negative output is intended, the phase at which the calibration is completed can be set to 180 degrees other than 0 degrees. Figure 13

[0100] ​​When the step S13 is completed, the current input value of the AD converter is recorded (step S14), and the loop processing of the calibration is executed. First, the current input value of the AD converter is compared with the 0 level, and it is determined whether the input value of the AD converter is less than the 0 level (whether it is a negative value) (step S15). When it is determined in the step S15 that the input value of the AD converter is a negative value, the phase of the switching signal (i.e., the reference signal) of the cancellation circuit 60 is advanced with respect to the periodic signal corresponding to the frequency of the DDS (step S16). On the other hand, when it is determined in the step S15 that the input value of the AD converter is a positive value, the phase of the switching signal (i.e., the reference signal) of the cancellation circuit 60 is delayed with respect to the periodic signal (step S17).

[0101] Then, based on the input value of the AD converter, it is determined whether the sign becomes the ramp level without change (step S18). When it is determined in the step S18 that the sign becomes the ramp level without change, the calibration processing is ended. On the other hand, when it is determined in the step S18 that the condition is not satisfied, it is determined whether the sign of the input of the AD converter is changed due to the offset phase (step S19). In the case where it is determined in the step S19 that it is not changed, the processing of the step S14 is performed again, and in the case where it is determined that it is changed, the change width of the phase based on the control is changed to the current half (step S20), and the processing of the step S14 is performed again. The above is the calibration processing.

[0102] In the present mode, the calibration processing light which does not contain fluorescence and contains scattered light is detected, the calibration processing for removing the signal component corresponding to the scattered light from the detection signal is implemented based on the calibration signal corresponding to the calibration processing light, and the signal component corresponding to the scattered light is removed from the detection signal in consideration of the implementation result of the calibration processing. By performing the calibration processing for removing the signal component corresponding to the scattered light from the detection signal in advance based on the calibration processing light containing the scattered light, the signal component corresponding to the scattered light can be appropriately removed from the detection signal.

[0103] The effects of appropriately removing the scattered light (noise) as described above will be described with reference to Figure 14 . Figure 14 (a) shows the intensity of the detection light in the case where the removal of the signal component (noise) corresponding to the scattered light is not performed, Figure 14 (b) shows the intensity of the detection light in the case where the removal of the signal component (noise) corresponding to the scattered light is performed. Figure 14 The results in the case where the film coated with DTBTA-Eu3+ as a fluorescent reagent is measured are shown. As Figure 14In the case of (a) shown in FIG. 6, about 330 counts of offset is required for the background (BKG) of the excitation light (scattered light) without removing the noise. Thus, the noise (standard deviation) is 2.16 and the signal intensity is 404 counts. In contrast, in the case of (b) shown in FIG. 6, the compensation for the scattering of the excitation light toward the film is not required and only the minimum compensation for the processing on the software (processing for making the values of the signals all positive) is performed with the removal of the noise. Thus, the noise (standard deviation) can be set to 0.69 and the signal intensity can be set to 1475 counts. Thus, since the amount of compensation is small with the removal of the noise, the amount of the excitation light from the light source and the amplification of the IV conversion amplifier can be increased and the signal intensity can be appropriately increased. As a result, the S / N with the removal of the noise is 187 and the S / N with the removal of the noise can be set to 2140, which is more than 10 times higher. Figure 14

[0104] In the calibration process, the switch signal for locking the phase of the period signal with respect to the operation frequency of the cancellation circuit 60 matching the modulation frequency of the light source 11 can be generated and the calibration signal and the switch signal can be set as inputs. The signal component corresponding to the scattered light can be outputted and the voltage value of the signal component corresponding to the scattered light can be adjusted to be within a predetermined range in which the value is approximately 0. The detection signal and the switch signal whose phase is adjusted in the calibration process can be set as inputs and the signal component corresponding to the scattered light can be removed from the detection signal. Thus, by using the locking circuit, the phase of the switch signal can be adjusted in the calibration process in such a manner that the voltage value of the signal component corresponding to the scattered light becomes a value approximately 0. The switch signal whose phase is adjusted can be set as an input and the signal component corresponding to the scattered light can be appropriately removed from the detection signal.

[0105] In the calibration process, the phase of the switch signal can be adjusted in such a manner that the voltage value of the signal component corresponding to the scattered light is greater than the value within the predetermined range. In the calibration process, the phase of the switch signal can be adjusted in such a manner that the voltage value of the signal component corresponding to the scattered light is less than the value within the predetermined range. Thus, the voltage value of the signal component corresponding to the scattered light can be appropriately adjusted to a value approximately 0 in the calibration process.

[0106] The modulation frequency of the light source 11 can be set to a value lower than the value corresponding to the lifetime of the fluorescence in which the intensity of the fluorescence decreases from the peak value to 1 / e. In the case where the modulation frequency is increased to the value corresponding to the lifetime of the fluorescence, the continuous signals can overlap with each other and the intensity of the fluorescence cannot be maximized. By setting the modulation frequency to a value lower than the value corresponding to the lifetime of the fluorescence, the intensity of the fluorescence can be appropriately increased. ​

[0107] The modulation frequency of the light source 11 can be made lower than the value corresponding to the fluorescent lifetime and higher than the commercial frequency. Thus, it is possible to avoid both the fluorescent intensity from weakening as the modulation frequency becomes higher than the value corresponding to the fluorescent lifetime and the increase in noise.

[0108] As the switching signal for locking, an X signal, a Y signal having a phase shifted by 90 degrees with respect to the X signal, an X' signal in which the X signal is inverted, and a Y' signal in which the Y signal is inverted can be generated by independent dedicated circuits. By generating the inverted signals by independent dedicated circuits, it is possible to prevent a slight delay (a slight delay accompanying the passing of the NOT circuit) that becomes a problem when the inverted signals are generated by a NOT circuit.

[0109] The lock circuit 60 (the lock circuit) can switch the operation frequency by a prescribed ratio. Thus, it is possible to more easily match the operation frequency of the lock circuit with the modulation frequency of the light source 11 and to improve the degree of synchronization thereof than when the operation frequency is set to one.

[0110] The calibration processing light can be detected by irradiating excitation light to a region of the sample 500 that is downstream of the capture antibody fixed to the measurement target portion 504. Although the fluorescent component is likely to remain in the region upstream of the capture antibody, by irradiating excitation light to a region downstream of the supplementary antibody, the calibration processing light can be detected, and the calibration processing light with the influence of the fluorescent component reduced can be appropriately detected.

[0111] Next, specific modes (1st to 3rd modes) of the calibration processing of the above-described fluorescent measurement will be described with reference to Figures 15-29 will be described. In each mode, the calibration processing method is different. Specifically, in the 1st mode, the calibration processing is performed by detecting the calibration processing light by irradiating the reference member with irradiation light. In the 2nd mode, the calibration processing is performed by detecting the irradiation light as the calibration processing light directly by the optical system. In the 3rd mode, the calibration processing is performed by generating a calibration signal as a pseudo signal. Further, the structures of the optical measurement apparatus 1 (refer to Figure 1 described above), the optical measurement apparatus 701 of the 2nd mode (refer to Figure 23 ), and the optical measurement apparatus 801 of the 3rd mode (refer to Figure 27 ) are different from each other. Hereinafter, each mode will be described in detail.

[0112] [1st Mode]

[0113] Figure 15 is a view for explaining the fluorescent measurement sequence of the 1st mode. As Figure 15As shown in (a), in the fluorescence measurement of the first method, firstly, the light source 1 of the irradiation optical system 10 irradiates the reference member 600, which is different from the sample 500 being measured, with irradiation light. The photodetector 21 of the detection optical system 20 detects the calibration processing light, which includes the scattered light from the irradiated reference member 600. Then, the elimination circuit 60 (refer to the signal processing unit) Figure 1 The calibration process is performed based on the calibration signal corresponding to the light used for calibration. This is the first process in the first method.

[0114] Subsequently, as Figure 15 As shown in (b), the light source 11 irradiates the sample 500, which is the object to be measured, with excitation light (irradiation light). The photodetector 21 detects the object light, which includes fluorescence generated from the sample 500 irradiated with the excitation light and scattered light from the excitation light of the sample 500 irradiated with the excitation light. Then, the elimination circuit 60, which is the signal processing unit (see reference...),... Figure 1 Considering the results of the calibration process, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the light of the object being measured. This is the second processing step in the first method. If... Figure 15 (a) and Figure 15 If (b) is set as the front view, then Figure 15 (c) is the right-side view. For example... Figure 15 As shown in (c), when obtaining fluorescence from the sample 500, fluorescence information of the measurement object portion 504 of the sample 500 is obtained by scanning the measurement area of ​​the sample 500 with the optical head of the irradiation optical system 10.

[0115] Such fluorescence measurements, in more detail, are derived from... Figure 18 The structure shown is implemented as follows. First, during calibration, the optical head of the irradiation optical system 10 is configured to irradiate a reference member 600, which is fixed at a position different from the sample 500 placed on the sample stage, specifically fixed at a fixed portion 601 extending substantially perpendicularly from the fixed portion 602, with irradiation light. Then, when the calibration is complete, the optical head of the irradiation optical system 10 is moved to irradiate the sample 500 with excitation light (irradiation light), and the optical head of the irradiation optical system 10 performs a scanning operation to obtain fluorescence information of the measurement target portion 504 of the sample 500.

[0116] Here, the reference member 600 of the first embodiment is preferably a member that reflects illumination light and does not itself fluoresce upon irradiation by the illumination light. Furthermore, the statement "not fluorescing upon irradiation by the illumination light" in this invention means that even when irradiated by the illumination light, no fluorescence is generated due to the reference member 600, or only a negligible amount of fluorescence is generated. On the other hand, Figure 16This figure illustrates the calibration effect when using a reference component that generates fluorescence. Figure 16 (a) shows the calibration processing light containing fluorescence detected when a reference component is irradiated. Figure 16 (b) Display based on Figure 16 The calibration results shown in (a) are the fluorescence measurement results of sample 500 involving the calibration process light. Figure 16 In (a), the horizontal axis represents wavelength, the vertical axis represents light intensity, and the dashed line represents the lowest wavelength detectable by the photodetector (photodetection element 21). Figure 16 In (b), the horizontal axis represents the channel, and the vertical axis represents the light intensity. Figure 16 In (a), it is shown that when a reference component that generates a certain fluorescence is excited by irradiation light at 380 nm, fluorescence of a wavelength detectable by the photodetector 21, specifically around 600-850 nm, is detected. Thus, when the reference component generating fluorescence is irradiated with irradiation light, both scattered light (irradiation light) and fluorescence from the reference component are detected in the photodetector 21. That is, calibration processing light containing fluorescence is detected in the photodetector 21. When calibration processing is performed based on such calibration processing light containing fluorescence, when the phase of the switching signal is adjusted using the calibration signal such that the voltage value of the device output becomes 0, the phase of the switching signal is adjusted to the phase affected by the fluorescence component (locked to the phase of the fluorescence component). Therefore, as... Figure 16 As shown in (b), if the fluorescence measurement of sample 500 is performed considering the calibration results, there is a concern that the waveform of the fluorescence from sample 500 may not be obtained due to the influence of adjusting the switching signal in a manner that removes (eliminates) the fluorescence component from the reference component during the calibration process. Figure 16 In (b), an example is shown where the waveform of fluorescence from sample 500 cannot be obtained in either channel.

[0117] Figure 17 This figure illustrates the effect of calibration when using a reference component that does not produce fluorescence. Figure 17 (a) shows the calibration processing light detected when a reference component is illuminated with illumination light. Figure 17 (b) Display based on Figure 17 The calibration results shown in (a) are the fluorescence measurement results of sample 500 involving the calibration process light. Figure 17 In (a), the horizontal axis represents wavelength, the vertical axis represents light intensity, and the dashed line represents the lowest wavelength detectable by the photodetector (photodetection element 21). Figure 17 In (b), the horizontal axis represents the channel, and the vertical axis represents the light intensity. Figure 17(a) shows that no fluorescence (or only a trace amount of fluorescence that can be ignored) is detected when the irradiation light of 380 nm is irradiated on a certain reference member that does not generate fluorescence. Thus, in a case where the calibration processing light does not include fluorescence and substantially includes only scattered light, as Figure 17 (b) shows that, if the fluorescence measurement of the sample 500 is performed while the calibration result is taken into account, only the signal component corresponding to the scattered light from the sample 500 can be removed (eliminated), and the waveform of the fluorescence from the sample 500 can be appropriately obtained. According to the above, it can be said that the reference member 600 is preferably a member that does not generate fluorescence by itself.

[0118] Next, as one example of the reference member 600, the case where Figures 19-21 is described. Figure 19 is a view for explaining the scattered light acquisition when the mirror 600A is used as the reference member 600. As shown in Figure 19 , the reference member 600 can include the mirror 600A. The mirror 600A can be any mirror as long as it is a mirror that can reflect the irradiation light, but it is more preferable that the mirror 600A is formed of a material that does not generate fluorescence by the irradiation of the irradiation light (does not generate fluorescence due to the mirror 600A or generates only a trace amount of fluorescence that can be ignored). The mirror 600A can also be, for example, a mirror whose surface is not easily soiled. The mirror 600A reflects the irradiation light irradiated from the light source 11. The mirror 600A adjusts the inclination angle so that the reflected irradiation light (scattered light) is detected by the light detecting element 21. The mirror 600A is fixed to a predetermined inclination angle by an adjustment mechanism (not shown). By appropriately adjusting the inclination angle of the mirror 600A with respect to the light detecting element 21, the amount of light detected in the light detecting element 21 can be increased.

[0119] Figure 20 is a view for explaining the scattered light acquisition when the reflection diffuser 600B is used as the reference member 600. As shown in Figure 20 , the reference member 600 can include the reflection diffuser 600B. The reflection diffuser 600B is, for example, a member formed of a material that does not generate fluorescence by the irradiation of the irradiation light (does not generate fluorescence due to the reflection diffuser 600B or generates only a trace amount of fluorescence that can be ignored), and is a reflection diffusing plate composed of a white member in which the amount of incident light to the light detecting element 21 is stable. The reflection diffuser 600B reflects the irradiation light irradiated from the light source 11 in such a manner that the reflected light is diffused toward various angles. The reflection diffuser 600B is disposed at a position at which the reflected irradiation light (scattered light) is detected by the light detecting element 21 in a large amount of light. The reflection diffuser 600B is preferably a one-piece member composed of, for example, resin or ceramic, but can be formed by cutting processing or combination of a plurality of members, or can be composed by, for example, providing a film that can diffuse the irradiation light toward a specific angle on the surface of a plate-shaped member, or the like.

[0120] Figure 21 is a diagram for explaining the acquisition of scattered light in the case where the mirror 611 (reflective base material) and the ground glass 610 (diffuser) are used as the reference member 600. As shown in Figure 21 , the reference member 600 can include a reference member 600C including the ground glass 610 as a glass member that transmits the irradiation light and the mirror 611 that reflects the irradiation light. In the reference member 600C, the ground glass 610 is provided to be supported (stacked) on the mirror 611 and is provided to be incident with the irradiation light before the mirror 611. The ground glass 610 is formed of a material that transmits the irradiation light of 380 nm or more, for example, and a convex-concave portion 610a is formed on the surface (the incident surface of the irradiation light) in a convex-concave shape. Alternatively, a film or the like that can scatter the irradiation light at a specific angle can be provided on the surface of the ground glass 610. The mirror 611 is a plate-shaped metal member formed of aluminum or the like, or a plate-shaped member in which the surface is covered with a metal film of aluminum or the like, for example, and can be a box-shaped member that can accommodate the ground glass 610, as long as the mirror 611 reflects the irradiation light. In addition, both the mirror 611 and the ground glass 610 are more preferably formed of a material that does not generate fluorescence by irradiation of the irradiation light (does not generate fluorescence caused by the mirror 611 and the ground glass 610 or generates only fluorescence of a degree that can be ignored). As shown in Figure 21 , in the reference member 600C, the irradiation light incident on the ground glass 610 is diffused in the convex-concave portion 610a and transmitted through the inside of the ground glass 610 to reach the mirror 611, is reflected on the mirror 611, and is again transmitted through the inside of the ground glass 610 to reach the surface of the ground glass 610, is diffused in various directions in the convex-concave portion 610a, and is emitted (various-angle scattered light is generated). By generating the various-angle scattered light, a sufficient amount of scattered light is detected in the light detection element 21. Further, in the reference member 600C, a reflective material whose surface is roughened can be used, in which case the roughened portion of the surface of the reflective material corresponds to a diffuser that diffuses the irradiation light.

[0121] Next, the fluorescence measurement processing of the first mode is explained with reference to Figure 22 . Figure 22 is a flowchart showing the fluorescence measurement processing of the first mode. As shown in Figure 22 , in the fluorescence measurement processing of the first mode, first, the optical head of the irradiation optical system 10 is disposed in a manner that the irradiation light is irradiated on the reference member 600 for calibration processing that is different from the sample 500 (step S31. Refer to (a) of Figure 15 ). Then, the light for calibration processing including the scattered light from the reference member 600 that is irradiated with the irradiation light is detected in the light detection element 21 (step S32. Refer to Figure 15(a)). Then, in the cancellation circuit 60, based on a calibration signal corresponding to the calibration processing light, a calibration process for removing a signal component corresponding to the scattered light from the detection signal is implemented (step S33). For details of the calibration process, since it is as described above with reference to Figures 10-13 and the like, the description is omitted.

[0122] Then, by causing the optical head of the irradiation optical system 10 to act in a scanning manner in the measurement region of the sample 500, the excitation light (irradiation light) is irradiated to the measurement target portion 504 of the sample 500 (step S34). Figure 15 (c)), and the measurement target light containing the fluorescence generated from the sample 500 irradiated with the excitation light and the scattered light from the sample 500 irradiated with the excitation light is detected in the light detecting element 21 (step S35. Refer to Figure 15 (b)). Then, in the cancellation circuit 60, based on the implementation result of the calibration process of step S33, a signal component corresponding to the scattered light is removed from the detection signal corresponding to the measurement target light (step S36). Specifically, as described above, the cancellation circuit 60 sets the detection signal and the switch signal whose phase is adjusted in the calibration process as inputs, and removes the signal component corresponding to the scattered light from the detection signal.

[0123] Next, the effects of the first mode are described.

[0124] The optical measurement apparatus 1 of the first mode includes: an irradiation optical system 10 that irradiates an irradiation target with irradiation light (excitation light); a detection optical system 20 that detects detection light caused by the irradiation light (excitation light); and a cancellation circuit 60 that processes a detection signal corresponding to the detection light. Then, in the optical measurement apparatus 1, in a first process, the irradiation optical system 10 irradiates a reference member 600 for calibration processing, which is different from the sample 500, with the irradiation light (refer to Figure 15 (a)), the detection optical system 20 detects calibration processing light containing the scattered light from the irradiation light of the reference member 600 irradiated with the irradiation light as the detection light (refer to Figure 15 (a)), the cancellation circuit 60 takes a calibration signal corresponding to the calibration processing light as the detection signal, and based on the calibration signal, implements a calibration process for removing a signal component corresponding to the scattered light from the detection signal of a second process described below. In addition, in the optical measurement apparatus 1, in the second process, the irradiation optical system 10 irradiates the sample 500 with the excitation light (irradiation light) with the sample 500 as the irradiation target (refer to Figure 15(b) of FIG. 10, the detection optical system 20 detects, as detection light, measurement object light containing fluorescence generated from the sample 500 irradiated with excitation light, and scattered light from the sample 500 irradiated with excitation light (see Figures 19-21 (b) of FIG. 10, the detection optical system 20 detects, as detection light, measurement object light containing fluorescence generated from the sample 500 irradiated with excitation light, and scattered light from the sample 500 irradiated with excitation light (see

[0125] Thus, in the optical measurement device 1, in the first process involved in the calibration process, the reference member 600 different from the sample 500 is irradiated with the irradiation light, and the calibration process light including the scattered light is detected. Therefore, by preparing the reference member 600 that generates fluorescence (or generates only fluorescence to an extent that can be ignored) by irradiation with the irradiation light, for example, the calibration process light that does not include fluorescence (or includes only fluorescence to an extent that can be ignored) can be detected. Thus, in the second process, by appropriately removing the signal component corresponding to the scattered light from the measurement signal based on the implementation result of the calibration process, the noise component due to the irradiation light itself can be removed and the fluorescence measurement can be performed with high accuracy. In addition, by using the reference member 600 for the calibration process that is different from the sample 500 (actual sample), a deviation in the result of the calibration process corresponding to the characteristics of the actual sample (characteristics determined depending on the moving speed of the fluorescent substance and the like) that becomes a problem in a case where the irradiation light is irradiated toward the actual sample and the calibration process light is detected, for example, can be prevented. More specifically, in a case where there is a deviation in the characteristics of the actual sample due to a difference in the manufacturing lot, for example, there is a case where the moving speed of the fluorescent substance and the like differs. In this case, the amount of the fluorescent substance (that is, the amount of the fluorescent light) in the region irradiated with the irradiation light at the time of the calibration process can differ for each actual sample. Therefore, the result of the calibration process is unstable, and there is a concern that sufficient reliability cannot be obtained for the determination of the result of the optical measurement, for example, but by using the reference member 600, the fluorescence measurement can be performed with higher accuracy. In addition, in a case where the result output from the device differs depending on the difference in the characteristics of each actual sample, for example, in a case of the measurement using the immunochromatography method, it is difficult to set the threshold value for determining the positivity or negativity, but in a case where the calibration is performed using the reference member 600, such a problem can also be suppressed. In addition, even in a case where a plurality of samples 500 different in shape, for example, are measured with one optical measurement device 1 (for example, a plurality of immunochromatography test kit reagents each corresponding to a different antigen), it is not necessary to set an appropriate calibration region for each of the samples 500, and one reference member 600 can be used for the calibration process, and thus the control in hardware and the control in software can be simplified. Furthermore, since the structure of the optical measurement device 1 itself does not need to be greatly changed compared to a case where the irradiation light is irradiated toward the actual sample and the calibration process light is detected, for example, the present mode has an advantage in terms of cost. In addition, since the calibration process is performed based on the measured value, the present mode can ensure the accuracy of the calibration process (correct phase matching can be performed in the calibration process). Thus, according to the optical measurement device 1 of the present mode, the calibration process can be more appropriately performed, and by more appropriately removing the noise component due to the irradiation light itself, the fluorescence measurement can be performed with high accuracy.

[0126] The reference member 600 can beFigure 20 The diagram shows reflective components (mirror 600A, mirror 611, reflective diffuser 600B) that reflect the illumination light. This structure allows for easy increase in the amount of detection light. Furthermore, the reflective components preferably fluoresce without being irradiated by the illumination light. With this structure, since fluorescence caused by the reflective components is not generated even when irradiated by the illumination light (or only a negligible amount of fluorescence is generated), in the second processing, the signal component corresponding only to the scattered light component can be reliably removed from the measured signal.

[0127] Reference component 600 can be as follows Figure 21 The diagram shows a reflective diffuser 600B that diffuses the irradiated light. With this structure, scattered light at various angles can be easily generated by the reflective diffuser 600B, making it easier to increase the amount of detection light. Furthermore, since this structure allows for a simpler reference component, it offers advantages in terms of manufacturability and cost.

[0128] Reference component 600 can be as follows Figure 19 The device includes a mirror 611 that reflects the illumination light and a frosted glass 610 supported on the mirror 611 and diffusing the illumination light. With this structure, the intensity of the detection light can be increased more easily through the cooperation of the mirror 611 and the frosted glass 610. Furthermore, since the surface of the frosted glass 610 has irregularities 610a, scattered light at various angles can be easily generated, further increasing the intensity of the detection light.

[0129] Reference component 600 can be as follows Figure 23 The image shows a mirror. With this structure, the amount of light detected can be increased more easily by adjusting the reflection angle of the mirror relative to the light detection element 21.

[0130] [Method 2]

[0131] Figure 1 This is a schematic diagram of the optical measuring device 701 according to the second embodiment. The optical measuring device 701 according to the second embodiment is the same as the optical measuring device 1 described above (see Figure 1). Figure 23 The structure is roughly the same, but as Figure 24As shown, the optical measurement apparatus 1 differs in points of the light-detecting element 721 provided with the detection optical system 720 (1st detection optical system) and the IV conversion amplifier 740. The light-detecting element 721 is of a different structure from the light-detecting element 21 of the detection optical system 20 (2nd detection optical system). In addition, the IV conversion amplifier 740 is of a different structure from the IV conversion amplifier 40. The light-detecting element 721, as a process of the calibration process, directly detects the irradiation light (light for calibration process) irradiated from the irradiation optical system 10 as the detection light. The light-detecting element 721 outputs the detection signal corresponding to the detection light to the IV conversion amplifier 740. The IV conversion amplifier 740, as a process of the calibration process, converts the electric power signal (detection signal) inputted from the light-detecting element 721 into a voltage signal. The IV conversion amplifier 740 outputs the detection signal converted into the voltage signal to the waveform generating circuit 50. In this case, the waveform generating circuit 50 generates the waveform of the detection signal based on the detection signal inputted from the IV conversion amplifier 740, and outputs the information of the generated waveform (detection signal) to the canceling circuit 60.

[0132] Figure 24 is a diagram illustrating an outline of the fluorescence measurement of the 2nd method. As shown, Figure 25 In the fluorescence measurement of the 2nd method, first, in the case where the calibration process is performed, the optical head of the irradiation optical system 10 is arranged in a manner of irradiating the irradiation light to the light-detecting element 721 fixed to a position different from the test sample 500 placed on the sample stage, specifically, to the fixing site 601 extending substantially perpendicularly from the fixing site 602. The irradiation light, i.e., the light for calibration process, is detected by performing the irradiation light acquisition as described above. Then, when the calibration process is completed, the optical head of the irradiation optical system 10 is moved in a manner of irradiating the excitation light (irradiation light) to the test sample 500, and the optical head of the irradiation optical system 10 performs the scanning operation in a manner of acquiring the fluorescence information of the measurement target portion 504 of the test sample 500.

[0133] Figure 25 is a diagram illustrating the excitation light acquisition of the 2nd method. As shown, Figure 26In the irradiation light acquisition of the second method, the light source 11 of the irradiation optical system 10 irradiates the light detection element 721 of the detection optical system 720 with the irradiation light. Thus, the light detection element 721 directly detects the irradiation light, i.e., the light for calibration processing. Then, the cancellation circuit 60 as the signal processing section performs the calibration processing based on the calibration signal corresponding to the light for calibration processing. The irradiation light is light having the same phase as the scattered light. Therefore, by the light detection element 721 detecting the irradiation light as the light for calibration processing, the calibration processing for removing the signal component corresponding to the scattered light from the detection signal can be appropriately performed. Further, the processing after the calibration processing (second processing) is the same as the first method, and thus the description thereof is omitted.

[0134] Next, the fluorescence measurement processing of the second method will be described with reference to Figure 26 . Figure 26 is a flowchart showing the fluorescence measurement processing of the second method. As shown in Figure 25 , in the fluorescence measurement processing of the second method, first, the optical head of the irradiation optical system 10 is arranged so as to irradiate the light detection element 721 fixed to a position different from the sample 500 with the irradiation light (step S41. Refer to (a) of Figure 25 ). Then, the irradiation light, i.e., the light for calibration processing is detected in the light detection element 721 (step S42. Refer to (a) of Figures 10-13 ). The irradiation light is light having the same phase as the scattered light. Then, in the cancellation circuit 60, based on the calibration signal corresponding to the light for calibration processing, the calibration processing for removing the signal component corresponding to the scattered light from the detection signal is performed (step S43). The details of the calibration processing are as described above with reference to Figure 23 , and thus the description thereof is omitted.

[0135] Next, by the optical head of the irradiation optical system 10 acting in a scanning manner on the measurement region of the sample 500, the excitation light (irradiation light) is irradiated on the measurement target portion 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 from the sample 500 irradiated with the excitation light is detected in the light detection element 21 (step S45). Then, in the cancellation circuit 60, the signal component corresponding to the scattered light of the calibration processing of step S43 is removed from the detection signal corresponding to the measurement target light (step S46). Specifically, as described above, the cancellation circuit 60 sets the detection signal and the switch signal having the phase adjusted in the calibration processing as inputs, and removes the signal component corresponding to the scattered light from the detection signal.

[0136] Next, the effects of the second method will be described.

[0137] The optical measurement device 701 of the second method (refer to Figure 25 ) includes a light detection section having an irradiation optical system 10 that irradiates irradiation light (excitation light), a detection optical system 20 that detects detection light due to the irradiation light (excitation light), and a detection optical system 720, and a cancellation circuit 60 that processes a detection signal corresponding to the detection light. Thus, in the optical measurement device 701, in a first process, the irradiation optical system 10 irradiates the light detection element 721 of the detection optical system 720 with irradiation light, and the light detection element 721 detects the irradiation light, i.e., a light for calibration process, as detection light (refer to (a) of Figure 25 ), and the cancellation circuit 60 takes a calibration signal corresponding to the light for calibration process as a detection signal, and based on the calibration signal, performs a calibration process for removing a signal component corresponding to scattered light from a detection signal of a second process described below. In addition, in the optical measurement device 701, in the second process, the irradiation optical system 10 irradiates the sample 500 with excitation light (irradiation light), the detection optical system 20 detects, as detection light, measurement target light including fluorescence generated from the sample 500 irradiated with the excitation light and scattered light from the sample 500 irradiated with the excitation light, and the cancellation circuit 60 takes a measurement signal corresponding to the measurement target light as a detection signal, and removes a signal component corresponding to the scattered light of the calibration process of the first process from the measurement signal.

[0138] Thus, in the optical measurement device 701, in the first process of the calibration process, the light-detecting element 721 of the detection optical system 720 is directly irradiated with the irradiation light, and the irradiation light is detected as the calibration-process light. The irradiation light is light having the same phase as the scattered light. Therefore, by the detection optical system 720, the irradiation light is detected as the calibration-process light, and light not containing the fluorescent light and having the same phase as the scattered light can be appropriately detected as the calibration-process light. Thus, in the second process, by the implementation result of the calibration process, the signal component corresponding to the scattered light is appropriately removed from the measurement signal, and the noise component due to the irradiation light itself is removed and the fluorescent light measurement is performed with high accuracy. Further, since the calibration-process light is detected without irradiating the sample 500 with the irradiation light, a deviation of the result of the calibration process corresponding to the characteristics of the sample 500, which is a problem in a case where, for example, the sample 500 is irradiated with the irradiation light and the calibration-process light is detected, does not occur. More specifically, there is a case where the characteristics actually vary due to, for example, a difference in manufacturing lot, and, for example, there is a case where the moving speed of the fluorescent substance or the like varies. In this case, the amount of the fluorescent substance (that is, the amount of the fluorescent light emission) in the region irradiated with the irradiation light at the time of the calibration process can vary for each actual sample. Therefore, the result of the calibration process is unstable, and, for example, there is a concern that sufficient reliability cannot be obtained for the determination of the result of the optical measurement, but since the calibration-process light is detected without irradiating the sample 500 with the irradiation light, the fluorescent light measurement can be performed with higher accuracy. Further, in a case where the result output from the device varies depending on the difference in the characteristics of each actual sample, for example, in a case of measurement using the immunochromatography method, it is difficult to set a threshold value for determining positivity or negativity, but in a case where the calibration-process light is detected without irradiating the sample 500 with the irradiation light, such a problem can also be suppressed. Further, even in a case where, for example, a plurality of samples 500 having different shapes (for example, a plurality of immunochromatography test kit reagent cases each corresponding to each of various antigens) are measured with one optical measurement device 1, it is not necessary to set an appropriate calibration region for each of the plurality of samples 500, and for the calibration process, the calibration-process light can be detected without irradiating the sample 500 with the irradiation light, and thus the control in hardware and the control in software can be simplified. Further, since the irradiation light is directly detected and set as the calibration-process light, the amount of light for the calibration process can be sufficiently ensured. Thus, according to the optical measurement device 701 of the present embodiment, the calibration process can be more appropriately performed, and by more appropriately removing the noise component due to the irradiation light itself, the noise component due to the irradiation light itself can be removed and the fluorescent light measurement can be performed with high accuracy.

[0139] Further, in the above description, the optical measurement device 701 is described as having the detection optical system 720 as the first detection optical system and the detection optical system 20 as the second detection optical system, but, for example, the optical measurement device 701 can be configured to have the detection optical system 20 as the first detection optical system and the detection optical system 720 as the second detection optical system. Figure 27As shown in (b), the optical measurement apparatus has one detection optical system 820 that functions as the first and second detection optical systems. The detection optical system 820 has a light detecting element 821. In this case, the irradiation optical system includes, for example, an irradiation optical system 910 that irradiates the detection optical system 820 with irradiation light when the detection optical system 820 functions as the first detection optical system (i.e., a detection optical system that detects irradiation light as calibration processing light), and an irradiation optical system 10 that irradiates the sample 500 with excitation light (irradiation light) when the detection optical system 820 functions as the second detection optical system (i.e., a detection optical system that detects measurement target light including fluorescence and scattered light from the sample 500). Furthermore, the light source 911 of the irradiation optical system 910 and the light source 11 of the irradiation optical system 10 in this case both need to be modulated with respect to the reference signal.

[0140] [3rd Mode]

[0141] Figure 1 is a schematic configuration diagram of an optical measurement apparatus 801 of the 3rd mode. The optical measurement apparatus 801 of the 3rd mode has substantially the same structure as the optical measurement apparatus 1 (refer to Figure 28 ) described above, but as shown in Figure 29 , differs from the optical measurement apparatus 1 in that it has a delay / amplification circuit 802. The delay / amplification circuit 802 is a structure that functions as a signal processing section together with the cancellation circuit 60. Here, as described above, the light source drive circuit 30 sets the modulation frequency of the light source 11 based on the frequency signal that becomes the reference that is input from the time series 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 by the light source drive circuit 30 based on the frequency signal input from the time series generator 70.

[0142] Specifically, the delay-amplifier circuit 802 considers a delay equivalent to the time until the scattered light from the illumination light irradiated by the light source 11 of the self-illuminating optical system 10 is detected by the detection optical system 20 as detection light, and generates a pseudo-signal (calibration signal) that changes the phase of the aforementioned modulation signal. Thus, the pseudo-signal simulates the signal of the scattered light (illumination light) detected by the detection optical system 20 during the calibration process. Furthermore, the delay-amplifier circuit 802 sets the amplitude of the pseudo-signal (calibration signal) based on the amplitude of the scattered light from the illumination light irradiated by the light source 11 of the self-illuminating optical system 10 being detected by the detection optical system 20 as detection light. In this way, the delay-amplifier circuit 802 obtains a calibration signal without detecting the calibration processing light (without irradiation and detection of light for calibration processing) by generating a pseudo-signal that processes the phase and amplitude of the modulation signal set for the light source 11. The delay-amplifier circuit 802 specifically includes a delay circuit for changing the phase and an amplification circuit for changing the amplitude. For example... Figure 29 As shown, since no light is used for calibration processing, the position of the optical head of the irradiation optical system 10 during calibration processing is not particularly limited. Furthermore, the processing after calibration (second processing) is the same as in the first method, so a description is omitted.

[0143] Secondly, refer to Figure 29 This explains the fluorescence measurement process of method 3. Figures 10-13 This is a flowchart showing the fluorescence measurement process of method 3. (Example) Figure 27 As shown, in the fluorescence measurement process of the third method, firstly, in the delay-amplification circuit 802, a pseudo-signal (calibration signal) is generated, which causes a phase change in the modulation signal equivalent to the delay amount until the scattered light from the illumination light irradiated by the light source 11 of the self-illuminating optical system 10 is detected by the detection optical system 20 as detection light (step S51). Then, in the elimination circuit 60, based on the pseudo-signal (calibration signal), a calibration process is performed to remove the signal component corresponding to the scattered light from the self-detection signal (step S52). Details of the calibration process are as described in the reference... ​ As mentioned above, the explanation is omitted.

[0144] Next, the measurement target portion 504 of the sample 500 is irradiated with excitation light (irradiation light) by moving the optical head of the irradiation optical system 10 in a scanning manner over the measurement region 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 from the sample 500 irradiated with the excitation light is detected in the light detecting element 21 (step S54). Then, in the cancellation circuit 60, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the measurement target light, taking into account the implementation result of the calibration process of step S52 (step S55). Specifically, as described above, the cancellation circuit 60 sets the measurement signal and the switch signal whose phase is adjusted in the calibration process as inputs, and removes the signal component corresponding to the scattered light from the measurement signal.

[0145] Next, the effects of the third method will be described.

[0146] The optical measurement apparatus 801 (refer to ​ ) of the third method includes the irradiation optical system 10 that irradiates the irradiation light (excitation light) corresponding to the modulation signal, the detection optical system 20 that detects the detection light due to the irradiation light (excitation light), and the delay / amplification circuit 802 and the cancellation circuit 60 that function as a signal processing section. In the optical measurement apparatus 801, in the first process, the delay / amplification circuit 802 generates a pseudo signal (calibration signal) in which the phase of the modulation signal is changed taking into account the delay equivalent to the time from the irradiation of the irradiation light from the irradiation optical system 10 to the detection of the scattered light as the detection light by the detection optical system 20, and the cancellation circuit 60 implements the calibration process for removing the signal component corresponding to the scattered light from the detection signal for the second process, based on the pseudo signal. In the optical measurement apparatus 801, in the second process, the irradiation optical system 10 irradiates the sample 500 with the excitation light (irradiation light), the detection optical system 20 detects the measurement target light including the fluorescence generated from the sample 500 irradiated with the excitation light and the scattered light from the sample 500 irradiated with the excitation light as the detection light, and the cancellation circuit 60 removes the signal component corresponding to the scattered light of the aforementioned calibration process of the first process from the measurement signal corresponding to the measurement target light, as the detection signal.

[0147] Thus, in the optical measurement device 801, in the first process of the calibration process, a pseudo signal (calibration signal) is generated in which the phase of the modulation signal input to the irradiation optical system 10 corresponds to the delay variation of the irradiation optical system 10. Thus, by the modulation signal for the irradiation light, a calibration signal corresponding to the delay of the irradiation optical system 10 is generated, so that the same calibration signal as that in the case where scattered light is detected as the calibration process light (a calibration signal having the same phase as that of the scattered light) can be actually obtained without detecting the calibration process light. That is, according to such a configuration, a calibration signal of only the signal component of the scattered light, which does not include the signal component of the fluorescent light, can be obtained. Thus, in the second process, by appropriately removing the signal component corresponding to the scattered light from the measurement signal on the basis of the result of the calibration process, the noise component due to the irradiation light itself can be removed and the fluorescent light measurement can be performed with high accuracy. Further, since the calibration process light is detected without irradiating the irradiation light to the sample 500, a deviation in the result of the calibration process corresponding to the characteristics of the sample 500, which is a problem in the case where the irradiation light is irradiated to the sample 500 and the calibration process light is detected, for example, does not occur. More specifically, there is a case where a deviation in the characteristics due to, for example, a difference in manufacturing lot exists in actual samples, for example, there is a case where a difference in the moving speed of the fluorescent substance or the like occurs. In this case, the amount of the fluorescent substance (that is, the amount of the fluorescent light emission) in the region irradiated with the irradiation light at the time of the calibration process can be different from each other for each actual sample. Therefore, the result of the calibration process is unstable, and there is a concern that, for example, sufficient reliability cannot be obtained for the determination of the result of the optical measurement, but by the modulation signal for the irradiation light, a calibration signal corresponding to the delay of the irradiation optical system 10 is generated, so that the fluorescent light measurement can be performed with higher accuracy. Further, in the case where the result output from the device is different depending on the difference in the characteristics of each actual sample, for example, in the case of the measurement using the immunochromatography method, it is difficult to set a threshold value for determining positivity or negativity, but in the case where, by the modulation signal for the irradiation light, a calibration signal corresponding to the delay of the irradiation optical system 10 is generated, such a problem can also be suppressed. Further, even in the case where, for example, a plurality of samples 500 having different shapes (for example, a plurality of immunochromatography test kit reagent cases each corresponding to each of various antigens) are measured by one optical measurement device 1, it is not necessary to set an appropriate calibration region for each of the plurality of samples 500, by the modulation signal for the irradiation light, a calibration signal corresponding to the delay of the irradiation optical system 10 is generated, so that the control in the hardware aspect and the control in the software aspect can be simplified. Further, since the calibration signal is actually generated electrically without detecting the calibration process light, a detection optical system or the like for the calibration process is not required, so that a simple and low-cost device configuration can be provided. Thus, according to the optical measurement device 801 of one embodiment of the present application, the calibration process can be more appropriately performed, the noise component due to the irradiation light itself can be more appropriately removed, and the fluorescent light measurement can be performed with higher accuracy.

[0148] Further, in the above-mentioned first processing, the delay-amplification circuit 802 can set the amplitude of the calibration signal in consideration of the amplitude of scattered light of the irradiation light irradiated from the irradiation optical system 10 as the detection light detected by the detection optical system 20. Thus, a calibration signal more approximate to the case where the scattered light is actually detected as the light for calibration processing can be obtained.

[0149] For the above-mentioned calibration processing, in order to ensure higher reproducibility in actual measurement, it is preferable to perform the calibration processing with the same irradiation light amount each time. Therefore, in the case of the member (reference member) described in the first mode and the like, it is preferable that the member can be mechanically and stably fixed. Further, the member is preferably a member that is not easily changed over time. Further, in the case where the irradiation light is irradiated to the light detection element with respect to the calibration processing described in the second mode, in order to keep the light amount of the light source constant, feedback is preferably performed on the circuit. Furthermore, in the mode where the calibration processing is performed with the signal generated by the suspected manner as in the third mode, it is considered that the calibration can be performed with the same light amount each time.

[0150] Explanation of symbols

[0151] 1, 701, 801... optical measurement apparatus, 10... irradiation optical system, 20... detection optical system (light detection section, second detection optical system), 60... cancellation circuit (signal processing section), 600... reference member, 600A... mirror, 600B... reflective diffuser, 610... ground glass, 611... mirror, 720... detection optical system (light detection section, first detection optical system), 802... delay-amplification circuit (signal processing section), 820... detection optical system (first detection optical system and second detection optical system), 910... irradiation optical system.

Claims

1. An optical measurement apparatus, wherein is an optical measurement apparatus that measures an optical property of a measurement object, is provided with: an irradiation optical system that irradiates an irradiation object with irradiation light; a light detection section that detects detection light caused by the irradiation light; and a signal processing section that processes a detection signal corresponding to the detection light, in a first process, the irradiation optical system irradiates a reference member for calibration processing, which is different from the measurement object, with the irradiation light, the light detection section detects, as the detection light, calibration processing light that includes scattered light of the irradiation light from the reference member irradiated with the irradiation light, the signal processing section, as the detection signal, a calibration signal corresponding to the calibration processing light, and based on the calibration signal, implements calibration processing for removing a signal component corresponding to scattered light from the detection signal in a second process, in the second process, the irradiation optical system irradiates the measurement object with the irradiation light, the light detection section detects, as the detection light, measurement object light that includes fluorescence generated from the measurement object irradiated with the irradiation light and scattered light from the measurement object irradiated with the irradiation light, the signal processing section, as the detection signal, a measurement signal corresponding to the measurement object light, and removes a signal component corresponding to the scattered light in the calibration processing in the first process from the measurement signal, the reference member includes a reflection member that reflects the irradiation light, the reflection member does not generate fluorescence by irradiation of the irradiation light. 2.The optical measurement apparatus according to claim 1, wherein the reflection member includes a reflection diffuser that diffuses the irradiation light. 3.The optical measurement apparatus according to claim 1, wherein the reflection member includes: a reflection base material that reflects the irradiation light; and a diffuser that is supported on the reflection base material and diffuses the irradiation light. 4.The optical measurement apparatus according to claim 1, wherein the reflection member includes a mirror. 5.An optical measurement apparatus, wherein is an optical measurement apparatus that measures an optical property of a measurement object, is provided with: an irradiation optical system that irradiates irradiation light; a light detection section that has a first detection optical system that detects detection light caused by the irradiation light and a second detection optical system; and a signal processing section that processes a detection signal corresponding to the detection light, in a first process, the irradiation optical system irradiates the first detection optical system with the irradiation light, the first detection optical system of the light detection section detects, as the detection light, calibration processing light that is the irradiation light, the signal processing section, as the detection signal, a calibration signal corresponding to the calibration processing light, and based on the calibration signal, implements calibration processing for removing a signal component corresponding to scattered light from the detection signal in a second process, in the second process, the irradiation optical system irradiates the measurement object with the irradiation light, The light detection section detects, as the detection light, measurement target light including fluorescence generated from the measurement target object irradiated with the irradiation light and scattered light from the measurement target object irradiated with the irradiation light, The signal processing section removes, from the measurement signal, a signal component corresponding to the scattered light in the calibration processing in the first processing.

6. An optical measurement apparatus, wherein is an optical measurement apparatus that measures an optical property of a measurement target object, includes: an irradiation optical system that irradiates irradiation light corresponding to a modulation signal; a light detection section that detects detection light caused by the irradiation light; and a signal processing section, in a first processing, the signal processing section generates a calibration signal based on which calibration processing for removing a signal component corresponding to scattered light from a measurement signal in a second processing is performed, the calibration signal changing a phase of the modulation signal in accordance with a delay corresponding to a time period until the scattered light from the irradiation light irradiated from the irradiation optical system is detected as the detection light by the light detection section, in the second processing, the irradiation optical system irradiates the measurement target object with the irradiation light, the light detection section detects, as the detection light, measurement target light including fluorescence generated from the measurement target object irradiated with the irradiation light and scattered light from the measurement target object irradiated with the irradiation light, the signal processing section removes, from the measurement signal, a signal component corresponding to the scattered light in the calibration processing in the first processing.

7. The optical measurement apparatus according to claim 6, wherein in the first processing, the signal processing section sets an amplitude of the calibration signal in accordance with an amplitude at a time when the scattered light from the irradiation light irradiated from the irradiation optical system is detected as the detection light by the light detection section.

8. An optical measurement method, wherein is an optical measurement method that measures an optical property of a measurement target object, and performs a first processing and a second processing, the first processing includes: irradiating an irradiation light to a reference member for calibration processing that is different from the measurement target object; detecting calibration processing light including scattered light from the irradiation light irradiated to the reference member; and based on a calibration signal corresponding to the calibration processing light, performing calibration processing for removing a signal component corresponding to scattered light from a measurement signal, the second processing includes: irradiating the irradiation light to the measurement target object; detecting measurement target light including fluorescence generated from the measurement target object irradiated with the irradiation light and scattered light from the measurement target object irradiated with the irradiation light; and removing, from the measurement signal corresponding to the measurement target light, a signal component corresponding to the scattered light in the calibration processing in the first processing, the reference member includes a reflection member that reflects the irradiation light, ​ The reflection member does not generate fluorescent light by irradiation of the irradiation light.

9. An optical measurement method, wherein is an optical measurement method that measures an optical property of a measurement object, and performs a first process and a second process, the first process includes: irradiating irradiation light; detecting a calibration process light that is the irradiation light; and based on a calibration signal corresponding to the calibration process light, performing a calibration process for removing a signal component corresponding to scattered light from a measurement signal, the second process includes: irradiating the irradiation light to the measurement object; detecting a measurement object light that includes fluorescent light generated from the measurement object irradiated with the irradiation light and scattered light from the measurement object irradiated with the irradiation light; and removing a signal component corresponding to the scattered light in the calibration process in the first process from the measurement signal corresponding to the measurement object light.

10. An optical measurement method, wherein is an optical measurement method that measures an optical property of a measurement object, and performs a first process and a second process, the first process includes: generating a calibration signal based on which a calibration process for removing a signal component corresponding to scattered light from a measurement signal is performed, the calibration signal changing a phase of a modulation signal input to an irradiation optical system in accordance with a delay until scattered light from irradiation light irradiated from the irradiation optical system is detected by a light detection section, the second process includes: irradiating the irradiation light to the measurement object; detecting a measurement object light that includes fluorescent light generated from the measurement object irradiated with the irradiation light and scattered light from the measurement object irradiated with the irradiation light; and removing a signal component corresponding to the scattered light in the calibration process in the first process from the measurement signal corresponding to the measurement object light. ​ ​

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