Optical measuring device and optical measuring method

Abstract

The optical measuring device includes an illumination optical system, a detection optical system, and an elimination circuit. In the fluorescence detection process, the sample is used as the illumination object, and illumination light is irradiated onto the sample. The measurement object light, which includes the fluorescence generated by the sample irradiated by the illumination light and the scattered light from the sample irradiated by the illumination light, is used as the detection light for detection. Taking into account the implementation results of the calibration process in the preprocessing, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the measurement object light. In the preprocessing, based on the calibration signal in the measurement signal that is greater than the signal corresponding to the scattered light in the measurement signal, calibration processing is performed to remove the signal component corresponding to the scattered light from the measurement signal.

Description

Technical Field

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

[0002] Patent Document 1 discloses a technique for generating and subtracting an attenuation signal to remove fluorescence noise components (fluorescence components from the substrate) contained in the fluorescence of a fluorescent sample placed on a substrate when excitation light is irradiated and fluorescence is measured. Specifically, in Patent Document 1, fluorescence noise components are removed by generating an attenuation signal with the same phase as the fluorescence of the substrate and subtracting the attenuation signal from the measured fluorescence signal.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Publication No. 2010-518394 Summary of the Invention

[0006] The problem that the invention aims to solve

[0007] As described above, in Patent Document 1, fluorescence noise components generated outside the measurement area of ​​the sample are removed from the fluorescence generated by irradiating the sample with excitation light. Here, it is considered that the noise components contained when irradiating the sample with light and detecting the light from the sample are caused not only by the light generated from the sample (e.g., fluorescence), but also by the irradiation light (e.g., excitation light) itself (e.g., scattered light). In the technology of Patent Document 1, the case where the irradiation light itself becomes a noise component is not considered, and this noise component cannot be removed.

[0008] On the other hand, the inventors, focusing on the phase difference between fluorescence and scattered light (light caused by the irradiation light itself) contained in the detection light, discovered an optical measurement device that removes the signal component of scattered light, which has a phase equal to that of the irradiation light, by self-detecting the detection light, thereby removing the noise component caused by the irradiation light itself. In such an optical measurement device, for example, a calibration process is performed beforehand. In the calibration process, the irradiation light is irradiated onto a portion of the sample that is not prone to fluorescence, and the calibration process light (ideally light containing only scattered light) is detected. Based on the calibration signal corresponding to the calibration process light, the self-detection signal is used to remove only the signal component that approximately corresponds to the scattered light component.

[0009] Here, in the calibration process, the device output is processed to become 0 using the aforementioned calibration signal (the signal corresponding to the scattered light). However, it is known that in practical devices, it is difficult to process the calibration process so that the device output becomes completely 0, resulting in calibration errors. Therefore, when fluorescence detection is performed based on the calibration result, it is difficult to completely remove the scattered light component from the self-detection signal, and both fluorescence and scattered light are detected. Therefore, there is a need to improve the accuracy of fluorescence detection (by more appropriately removing scattered light from the self-detection signal).

[0010] The inventors have conducted in-depth research on methods to solve the aforementioned problems. They focused on the fact that calibration errors arise due to limitations in the control and detection accuracy of the device, and are unaffected by differences in the amount of incident light towards the photodetector and the sample; for the same device, these errors become constant. Therefore, they discovered that the larger the calibration signal itself, the smaller the calibration error.

[0011] One aspect of the present invention is made in view of the above-mentioned actual situation, and its object is to provide an optical measuring device and optical measuring method that can remove noise components caused by the irradiation light itself and perform fluorescence measurement with high precision.

[0012] Technical means to solve the problem

[0013] That is, one aspect of the optical measurement apparatus of the present invention is an optical measurement apparatus for measuring the optical properties of a measurement object, comprising: an illumination optical system that irradiates an illumination object with illumination light; a light detection unit that detects a detection light caused by the illumination light; and a signal processing unit; performing fluorescence detection processing and a preprocessing performed prior to the fluorescence detection processing; in the fluorescence detection processing, the illumination optical system irradiates the measurement object with illumination light as the illumination object; the light detection unit detects the measurement object light, which includes fluorescence generated by the measurement object irradiated with the illumination light and scattered light from the measurement object irradiated with the illumination light, as the detection light; the signal processing unit removes the signal component corresponding to the scattered light in the calibration processing of the preprocessing from the measurement signal corresponding to the measurement object light; in the preprocessing, the signal processing unit performs calibration processing based on a calibration signal for removing the signal component corresponding to the scattered light from the measurement signal, the calibration signal being the signal involved in the illumination light or the scattered light of the illumination light detected in the light detection unit, and the signal quantity being greater than the signal corresponding to the scattered light in the measurement signal.

[0014] In one aspect of the optical measurement apparatus of the present invention, fluorescence detection processing and a preprocessing performed prior to the fluorescence detection processing are implemented. In the fluorescence detection processing, based on the calibration processing results of the preprocessing, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the light being measured. Then, in the preprocessing, the signal involved in the irradiation light or scattered light, whose signal quantity is greater than the signal corresponding to the scattered light in the aforementioned measurement signal, is set as a calibration signal, and a calibration process is performed. As described above, the calibration error in the calibration process is a fixed value; the larger the calibration signal itself, the smaller the calibration error. Therefore, as in one aspect of the optical measurement apparatus of the present invention, by increasing the signal quantity of the calibration signal (greater than the signal corresponding to the scattered light in the measurement signal), the calibration error can be suppressed. Therefore, noise components caused by the irradiation light itself can be removed, and fluorescence measurement can be performed with high precision.

[0015] In the preprocessing stage, an illumination optical system can irradiate the object with illumination light; the photodetector uses the calibration processing light as the detection light, which is light containing scattered light from the illumination light of the object being irradiated, and the amount of light is greater than the scattered light contained in the object light detected in the fluorescence detection process; the signal processing unit performs calibration processing based on the calibration signal corresponding to the calibration processing light. With this structure, the calibration processing light can be detected using the optical system used in fluorescence detection, and a calibration signal can be easily generated.

[0016] In the preprocessing stage, the irradiation optical system can also use the target object as the irradiation object, irradiating it with irradiation light. Based on this structure, calibration signals can be easily generated.

[0017] In the preprocessing, the illumination optical system can also use a reference component for calibration processing, which is different from the object being measured, as the illumination target, and illuminate the reference component with illumination light. By illuminating the reference component, which is different from the object being measured, more appropriate calibration processing light can be detected. Therefore, based on the results of the calibration processing, the signal component corresponding to the scattered light can be more appropriately removed from the measured signal during fluorescence detection processing.

[0018] The reference component may also include a reflective component that reflects the incident light. With such a structure, the amount of detection light can be easily increased.

[0019] The reflective element is preferably one that fluoresces without being irradiated by illumination light. With this structure, since fluorescence caused by the reflective element is not generated even when irradiated by illumination light (or only a negligible amount of fluorescence is generated), the signal component corresponding to the scattered light component can be reliably removed from the self-measured signal.

[0020] The reflective component may also include a reflective diffuser that diffuses the incident light. With such a structure, scattered light at various angles can be easily generated through the reflective diffuser, making it easier to increase the amount of detection light.

[0021] The reflective component may also include: a reflective substrate that reflects the irradiated light, and a diffuser supported on the reflective substrate that diffuses the irradiated light. With this structure, the amount of light detected can be more easily increased through the synergy between the reflective substrate and the diffuser.

[0022] The reflecting component may also include a mirror. With such a structure, the amount of light detected can be increased more easily by adjusting the reflection angle of the mirror relative to the light detection unit.

[0023] The aforementioned optical measuring device may also include an optical component configured to move between a position on the optical path leading to the light detection unit (position 1) and a position away from the optical path (position 2), and to increase or decrease the incident light efficiency. By providing such an optical component, a simplified structure can be achieved by appropriately increasing the amount of calibration processing light detected by the light detection unit (greater than the scattered light contained in the light of the object being measured). This allows for an appropriate increase in the signal intensity of the calibration signal and further improves the accuracy of fluorescence detection.

[0024] In the preprocessing stage, the irradiation optical system can irradiate the photodetector with irradiation light; the photodetector then uses the calibration processing light as the detection light, which is the irradiation light and has a higher intensity than the scattered light contained in the light of the target object detected in the fluorescence detection process; the signal processing unit performs calibration processing based on the calibration signal corresponding to the calibration processing light. In this way, by directly detecting the irradiation light by the photodetector in the preprocessing stage, the intensity of the calibration processing light can be easily increased. Therefore, the signal intensity of the calibration signal can be appropriately increased, further improving the accuracy of fluorescence detection.

[0025] The illumination optical system can also increase the amount of illumination light used during pretreatment compared to fluorescence detection processing. Based on this structure, the amount of light used for calibration processing can be easily increased (greater than the scattered light contained in the light from the object being measured). This allows for an appropriate increase in the signal strength of the calibration signal and further improves the accuracy of fluorescence detection.

[0026] Alternatively, the illumination optical system can illuminate the light corresponding to the modulation signal. In the preprocessing, the signal processing unit generates a calibration signal by changing the phase of the modulation signal based on a delay equivalent to the time until the scattered light from the illumination optical system is detected by the photodetector. In this way, by generating a calibration signal corresponding to the delay of the illumination optical system through the modulation signal of the illumination light, the same calibration signal (with the same phase as the scattered light) can be obtained without detecting the calibration processing light, just as in the case where the scattered light is detected as the calibration processing light.

[0027] One aspect of the optical measurement method of the present invention is an optical measurement method for measuring the optical properties of a measurement object, and includes: a step of performing a calibration process; a step of irradiating the measurement object with irradiation light; a step of detecting a measurement object light including fluorescence generated by the measurement object irradiated with irradiation light and scattered light from the measurement object irradiated with irradiation light; and a step of removing the signal component corresponding to the scattered light from the measurement signal corresponding to the measurement object light; wherein, in the step of performing the calibration process, a calibration process for removing the signal component corresponding to the scattered light from the measurement signal is performed based on a calibration signal, the calibration signal being the signal involved in the detected irradiation light or the scattered light of the irradiation light, and the signal quantity is greater than the signal corresponding to the scattered light in the measurement signal.

[0028] The effects of the invention

[0029] According to one aspect of the present invention, noise components caused by the irradiation light itself can be removed and fluorescence measurement can be performed with high precision. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the optical measuring apparatus according to an embodiment of the present invention.

[0031] Figure 2 This is a diagram illustrating the phase difference between fluorescence and scattered light.

[0032] Figure 3 This diagram illustrates a method for eliminating scattered light.

[0033] Figure 4 This diagram illustrates the fluorescence measurement method.

[0034] Figure 5 This is a flowchart showing the fluorescence measurement process performed by the optical measurement device.

[0035] Figure 6 This is a schematic diagram of the optical measuring device.

[0036] Figure 7 This diagram illustrates the removal of noise components.

[0037] Figure 8 It is a schematic diagram showing the sample.

[0038] Figure 9 This diagram illustrates the definition of S / N.

[0039] Figure 10 This diagram illustrates the signals used in the calibration process.

[0040] Figure 11 This is a diagram illustrating the calibration process.

[0041] Figure 12 It is a table showing the proportion of fluorescence components at each modulation frequency.

[0042] Figure 13 This is a flowchart showing the calibration process.

[0043] Figure 14 This is a graph illustrating the effect of the calibration process.

[0044] Figure 15 This is a diagram illustrating calibration errors.

[0045] Figure 16 This is a diagram illustrating calibration errors.

[0046] Figure 17 This is a diagram illustrating the summary of fluorescence measurement in Method 1.

[0047] Figure 18 This is a diagram illustrating the overview of fluorescence measurement in the second method, and also an illustration of the acquisition of scattered light when a mirror is used as a reference component.

[0048] Figure 19 This is a diagram illustrating the overview of fluorescence measurement in the second method, and also a diagram illustrating the acquisition of scattered light when a reflective diffuser is used as a reference component.

[0049] Figure 20 This is a diagram illustrating the overview of fluorescence measurement in the second method, and also a diagram illustrating the acquisition of scattered light when frosted glass is used as a reference component.

[0050] Figure 21 This is a diagram illustrating the summary of fluorescence measurement in method 3.

[0051] Figure 22 This is a diagram illustrating the overview of fluorescence measurement in the fourth method, and it is also a diagram illustrating the acquisition of excitation light through optical path switching.

[0052] Figure 23This is a diagram illustrating the summary of fluorescence measurement in the fourth method, and also a diagram illustrating the excitation light obtained using another detection optical system.

[0053] Figure 24 This is a diagram illustrating the summary of fluorescence measurement in the fourth method, and also a diagram illustrating the excitation light obtained using another irradiation optical system.

[0054] Figure 25 This is a schematic diagram of the optical measuring device of the fifth method.

[0055] Figure 26 This is a flowchart showing the fluorescence measurement process. Detailed Implementation

[0056] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Furthermore, the same or equivalent parts are given the same reference numerals in each drawing, and repeated descriptions are omitted.

[0057] Figure 1 This is a schematic diagram of the optical measuring apparatus 1 according to this embodiment. The optical measuring apparatus 1 is a device for detecting light emitted from a sample in response to light irradiating the sample. In this embodiment, the optical measuring apparatus 1 will be described as a fluorescence measuring device that detects fluorescence emitted from a sample in response to excitation light (irradiation light) irradiating the sample. Excitation light is light that excites the sample, and fluorescence is light emitted from the sample in response to the excitation light, and is light with a wavelength different from the excitation light. Alternatively, in this embodiment, the optical measuring apparatus 1 will be described as a device for detecting fluorescence involved in an immunochromatographic assay. Immunochromatography is an immunoassay method that utilizes an antigen-antibody reaction, and is used, for example, for the detection of influenza viruses.

[0058] like Figure 1As shown, in the assay using immunochromatography, an immunochromatographic test strip 100 is prepared as the sample. The immunochromatographic test strip 100 is housed within a reagent holder 101 as an immunochromatographic membrane containing the analyte. At a specific location (analyte portion) on the immunochromatographic membrane of the immunochromatographic test strip 100, a capture antibody (e.g., an antibody against an influenza virus antigen) is immobilized relative to a specified antigen. The reagent holder 101 is provided with an opening for dropping the sample onto the immunochromatographic membrane, i.e., a sample application window, and an opening for measuring the analyte portion containing the immobilized capture antibody, i.e., a measurement window. If the sample is dropped onto the sample application window of the reagent holder 101, the antigen in the sample first binds to the detection antibody labeled with a fluorescent reagent, and then an antigen-antibody reaction occurs between the antigen and the capture antibody, resulting in its capture. An optical measuring device 1 irradiates the immunochromatographic membrane exposed from the measurement window of the immunochromatographic test strip 100 with excitation light, and detects the fluorescence intensity by measuring the antigen-antibody complex (specifically, the fluorescent reagent of the antibody) on the analyte portion. In addition, as fluorescent reagents, europium, Q-dot (registered trademark), organic pigments, etc. can be used.

[0059] Here, it is assumed that the detection light incident on and detected by the detection optical system 20 in the optical measuring apparatus 1 includes not only fluorescence but also light generated by the excitation light itself. Examples of such light include scattered light from the excitation light. This scattered light, for example, is a portion of the excitation light generated by the excitation light irradiating and scattering the immunochromatographic test strip 100, and is light with the same phase as the excitation light (no phase difference). Since the immunochromatographic membrane and reagent holder 101 of the immunochromatographic test strip 100 are generally white, they are prone to generating the aforementioned scattered light. Furthermore, there are also cases where the excitation light itself is detected by the configuration of the sample being measured and the detection optical system. Hereinafter, the description will assume that the detection light detected in the optical measuring apparatus 1 includes both fluorescence and scattered light.

[0060] like Figure 1 As shown, the optical measuring device 1 includes: an illumination optical system 10, a detection optical system 20 (light detection unit), a light source driving circuit 30, an IV conversion amplifier 40, a waveform generation circuit 50, an elimination circuit 60 (signal processing unit), a timing generator 70, an AD converter 80, and a CPU 90.

[0061] The illumination optical system 10 irradiates the immunochromatographic test strip 100 (sample), which is the object to be measured, with excitation light (irradiation light). The illumination optical system 10 includes: a light source 11, an aperture 12, an excitation light filter 13, and a collimating lens 14. The light source 11 irradiates the immunochromatographic test strip 100 (sample) with excitation light. The light source 11 is, for example, a semiconductor light-emitting element. In this embodiment, the light source 11 is described as a light-emitting diode (LED), but it is not limited to this. For example, an LD can be used 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 with a desired beam cross section. The excitation light filter 13 is a wavelength-selective filter that filters the wavelength required for excitation of the excitation light arriving through 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 bandpass filter composed of a dielectric multilayer film filter that allows only a specific wavelength band (the excitation wavelength of the fluorescent reagent) to pass through. Collimating lens 14 is a lens that images the filtered excitation light from excitation light filter 13 onto immunochromatographic test strip 100 (more specifically, the measurement object of the immunochromatographic membrane).

[0062] The detection optical system 20 detects the detection light caused by the excitation light. Specifically, the detection optical system 20 detects the fluorescence from the immunochromatographic test strip 100. However, in reality, in the detection optical system 20, in addition to the fluorescence incident from the immunochromatographic test strip 100 (the fluorescence from the analyte portion of the immunochromatographic membrane), light containing scattered light caused by the excitation light itself, i.e., the detection light, is also incident and detected. The detection optical system 20 includes: a photodetector element 21, a fluorescence filter 22, and a condenser lens 23. The detection light is focused by the condenser lens 23 and incident on the photodetector element 21 via the fluorescence filter 22. The fluorescence filter 22 is a wavelength selective filter provided to suppress light other than fluorescence from the detection light from the immunochromatographic test strip 100 from reaching the photodetector element 21. The fluorescence filter 22 is an optical filter such as a dielectric multilayer film filter or a colored glass filter, more specifically, a bandpass filter combining a dielectric multilayer film filter that allows only specific wavelength bands to pass through and a colored glass filter. However, in cases where the excitation wavelength and fluorescence wavelength are similar, it is difficult to allow fluorescence with the fluorescence wavelength to pass through the fluorescence filter 22 appropriately while effectively blocking only the scattered light with the excitation wavelength. Furthermore, the characteristics of dielectric multilayer filters, which are generally used as highly efficient wavelength-selective filters, vary depending on the incident angle of the light. Therefore, in this embodiment, the fluorescence filter 22 is constructed using a combination of a dielectric multilayer filter and a colored glass filter, and the colored glass filter effectively blocks scattered light from the oblique direction. However, it is difficult to obtain a sufficient effect solely through wavelength selection, and it is difficult to effectively prevent the entry of scattered light under various conditions. Hereinafter, it will be described assuming that even with the fluorescence filter 22, the detection light reaching the photodetector element 21 also includes scattered light.

[0063] The photodetector 21 is a photosensor that detects the detection light filtered by the fluorescence filter 22. The photodetector 21 is, for example, a semiconductor photoreceiving element. In this embodiment, the photodetector 21 is described as a photodiode (PD), but it is not limited to this; it can be an avalanche photodiode (APD) or a photomultiplier tube (PMT), as long as it is an element capable of high-speed response corresponding to the modulation frequency of the excitation light from the light source 11 described below. Specifically, the photodetector 21 detects the detection light, which includes fluorescence generated from the immunochromatographic test strip 100 (specifically, the fluorescent reagent of the antigen-antibody complex in the assay section of the immunochromatographic membrane) irradiated with excitation light, and the light caused by the excitation light, i.e., the scattered light described above which has no phase difference with the excitation light. The photodetector 21 outputs a detection signal corresponding to the detection light to the IV conversion amplifier 40.

[0064] 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.

[0065] 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.

[0066] As described above, the light source driving circuit 30 can determine the modulation frequency of the light source 11 by taking into account the fluorescence intensity. Specifically, the light source driving circuit 30 sets the modulation frequency of the light source 11 to a value lower than the fluorescence lifetime, which corresponds to the time it takes for the fluorescence intensity to drop from its peak to 1 / e. The light source driving circuit 30 sets the modulation frequency of the light source 11 to a value lower than the fluorescence lifetime and higher than commercial frequencies (50Hz, 60Hz), for example, around 100Hz, and around 110Hz, which reduces noise by avoiding harmonics of commercial frequencies. The light source driving circuit 30 can set the modulation frequency of the light source 11 to other values ​​around 100Hz, such as 90Hz, 80Hz, 70Hz, or 130Hz.

[0067] IV conversion amplifier 40 converts the current signal (detection signal) output from photodetector 21 into a voltage signal. IV conversion amplifier 40 outputs the converted voltage detection signal to waveform generation circuit 50.

[0068] The waveform generation circuit 50 is a circuit that generates a waveform of the detection signal based on the detection signal output from the self-IV conversion amplifier 40. The waveform generation circuit 50 receives a reference frequency signal from the timing generator 70. The timing generator 70 receives the reference frequency signal with the same timing as both the light source driving circuit 30 and the waveform generation circuit 50. The waveform generation circuit 50 outputs the generated waveform (detection signal) information to the cancellation circuit 60.

[0069] The elimination circuit 60 is a signal processing unit that processes the waveform (detection signal) generated by the waveform generation circuit 50. Based on the phase difference (phase difference) between the fluorescence and scattered light, the elimination circuit 60 removes the signal component corresponding to the scattered light from the detection signal. Furthermore, the elimination circuit 60 receives a reference frequency signal input from the timing generator 70 at the same timing as the light source driving circuit 30 and the waveform generation circuit 50 to obtain information about the phase of the excitation light (i.e., the scattered light). Therefore, the elimination circuit 60 can remove the signal component of the scattered light based on the phase difference between the fluorescence and scattered light. For details of the processing of the elimination circuit 60, please refer to... Figures 2-4 Please provide an explanation.

[0070] Figure 2 This is a conceptual diagram illustrating the phase difference between fluorescence and scattered light. For example... Figure 2As shown, the sinusoidal detection light (detection light detected in the photodetector D) from the sample S irradiated by the self-light source L with a sinusoidal excitation light includes sinusoidal scattered light and fluorescence. Furthermore, the excitation light from the light source L is not limited to a sinusoidal shape; it can also be a periodically modulated waveform such as a rectangular wave. In this case, the detection light (scattered light and fluorescence) also has the same periodically modulated waveform as the excitation light. Thus, the scattered light is light without phase difference from the excitation light, while the fluorescence is light generated from the sample S corresponding to the excitation light, and is detected with a phase delay of approximately 10 milliseconds to nanoseconds relative to the scattered light. The inventors, focusing on this phase difference, discovered a method for extracting only the fluorescence from the self-detection light by removing only the scattered light. Furthermore, in Figure 2 In this process, since the sample S and the photodetector D are arranged on the optical axis of the light source L, the fluorescence emitted in the direction intersecting the optical axis of the excitation light is detected. Figure 1 Unlike other methods, this method detects fluorescence emitted in a direction coaxial with the optical axis of the excitation light. In this case, the detection light may contain not only fluorescence and scattered light, but also the excitation light itself. Furthermore, the amount of light incident on the light detection unit D caused by the excitation light is more likely to increase. Therefore, the fluorescence extraction using this method is effective.

[0071] Figure 3 This diagram illustrates methods for removing (eliminating) scattered light. Figure 3 Only the waveform of the scattered light in the detection light is displayed. Furthermore, this waveform is identical to the waveform of the excitation light. Figure 3 In the diagram, the horizontal axis represents time, and the vertical axis represents amplitude. (Regarding...) Figure 3 The waveform corresponding to the phase of the scattered light, as shown, is separated into time regions, for example, 1 / 4 of a period. By integrating for each time region 1-4 separately, the output of the scattered light in each time region 1-4 can be obtained. Here, if the integral values ​​of each time region 1-4 are multiplied by a certain multiplier and then summed, the total output can be set to 0. That is, when the absolute values ​​of the outputs in each time region 1-4 are the same, and the amplitude ranges of time regions 1 and 2 are positive, and the amplitude ranges of time regions 3 and 4 are negative, such as... Figure 3As shown, for time region 1, if multiplied by a multiplier "-1" and amplified, the output of time region 1 becomes negative due to "positive × negative"; for time region 2, if multiplied by a multiplier "+1" and amplified, the output of time region 2 becomes positive due to "positive × positive"; for time region 3, if multiplied by a multiplier "+1" and amplified, the output of time region 3 becomes negative due to "negative × positive"; for time region 4, if multiplied by a multiplier "-1" and amplified, the output of time region 4 becomes positive due to "negative × negative". Therefore, if the integral values ​​of each time region 1 to 4, amplified by a specified multiplier, are added together, the values ​​cancel each other out, and the total output becomes 0. Thus, for the signal component corresponding to the scattered light, by separating it in a specified time unit corresponding to the phase of the scattered light, amplifying each separated component separately, and combining the amplified components, it can be removed (setting the output to 0).

[0072] Figure 4 This diagram illustrates the fluorescence measurement method. Figure 4 Displays the waveforms of scattered light and fluorescence contained in the detected signal. Figure 4 In the diagram, the horizontal axis represents time, and the vertical axis represents amplitude. As described above, for the signal component corresponding to the scattered light, by separating it in a predetermined time unit corresponding to the phase of the scattered light, amplifying each separated component, and then combining the amplified components, it can be removed (by setting the output to 0). Here, as... Figure 4 As shown, for fluorescence, due to the phase difference relative to scattered light, if separation is performed using a defined time unit corresponding to the phase of the scattered light, the integral values ​​of time regions 1 to 4 will not be the same. Therefore, the output multiplied by the same multiplier as the scattered light, amplified separately, and then summed will not be zero. Thus, by separating the scattered light and fluorescence into the same time regions, amplifying, and combining them, the signal component of the scattered light can be removed while the output intensity of the fluorescence is detected and output.

[0073] Thus, the elimination circuit 60 separates the detection signal into its components by dividing it into components within a predetermined time unit corresponding to the phase of the scattered light, amplifying each component separately, and then combining the amplified components. This process removes the signal component corresponding to the scattered light from the detection signal, yielding the fluorescent signal component. The elimination circuit 60 outputs the signal with the scattered light component removed (i.e., the signal that becomes only the fluorescent signal component), i.e., the fluorescence signal, to the AD converter 80. Furthermore, while 1 / 4 of a period is exemplified as a predetermined time unit, it is not limited to this; any time unit can be used as long as it allows the removal of the scattered light component after synthesis. Similarly, while "+1" and "-1" are exemplified as a multiplier, they are not limited to this; any multiplier can be used as long as it allows the removal of the scattered light component after synthesis.

[0074] The AD converter 80 performs AD conversion on the fluorescence signal output from the self-elimination circuit 60, converting 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 results to an external computer, for example, via serial communication. Furthermore, the CPU 90 can generate signals that determine the timing of various operations of the optical measuring device 1, which are output from the timing generator 70. Alternatively, an FPGA can be used to replace the CPU 90. Based on the above processing, the optical measuring device 1 can eliminate the influence of scattered light from the self-detection light, obtaining only the signal related to the fluorescence of the fluorescent reagent.

[0075] Secondly, regarding the fluorescence measurement processing (optical measurement method) performed on optical measurement device 1, refer to... Figure 5 Please provide an explanation.

[0076] Figure 5 This is a flowchart showing the fluorescence measurement process performed by the optical measuring device 1. For example... Figure 5 As shown, in the fluorescence assay process, firstly, the light source 11 of the irradiation optical system 10 (light source unit) irradiates the immunochromatographic test strip 100 (sample) with excitation light (step S1). By irradiating the immunochromatographic test strip 100 (more specifically, the antigen-antibody complex in the assay area of ​​the immunochromatographic membrane) with excitation light, fluorescence is emitted from the fluorescent reagent of the antigen-antibody complex. On the other hand, the excitation light is scattered by the immunochromatographic test strip 100, producing scattered light.

[0077] Next, the photodetector 21 of the detection optical system 20 (photodetector unit) detects the detection light containing the aforementioned fluorescence and scattered light (step S2). The photodetector 21 outputs the detection light to the IV-to-Voltage converter 40. Then, after the current signal (detection signal) output from the photodetector 21 is converted into a voltage signal in the IV-to-Voltage converter 40, and the waveform of the detection signal is generated in the waveform generation circuit 50, the elimination circuit 60 (signal processing unit) removes the signal component corresponding to the scattered light from the detection signal based on the phase difference between the fluorescence and the scattered light (step S3). Specifically, the elimination circuit 60 separates the detection signal for a predetermined time unit corresponding to the phase of the scattered light, amplifies each component of the separated detection signal, synthesizes the amplified components, and removes the signal component corresponding to the scattered light from the detection signal to obtain the fluorescence signal component. Afterwards, the fluorescence signal is converted into a digital value in the AD converter 80, and predetermined control and signal processing is performed in the CPU 90 to obtain a fluorescence-related signal.

[0078] Furthermore, in the optical measuring apparatus 1, it has been described that the signal component corresponding to the scattered light is removed from the self-detection signal in the elimination circuit 60, but it is not limited to this. That is, it can be as follows: Figure 6 As shown in the optical measurement device 1A, without the elimination circuit 60, the CPU 90A (signal processing unit) performs self-detection signal removal of the signal component corresponding to the scattered light after the A / D conversion by the AD converter 80. In this case, since the elimination circuit 60 is not required, it can help to miniaturize the device.

[0079] Secondly, regarding the removal of the specific signal components (noise) described in the above embodiments, refer to... Figures 7 to 16 To explain in more detail.

[0080] Figure 7 This diagram illustrates the removal of noise components. Figure 7 (a) shows the intensity of the detected light without removing the signal component (noise) corresponding to the scattered light caused by the excitation light. Figure 7 (b) shows the intensity of the detected light after removal of the signal component (noise) corresponding to the scattered light caused by the excitation light. Figure 7 (a) and Figure 7 In (b), the vertical axis represents the intensity of the detection light, and the horizontal axis represents the channel in the measuring section 501, which is the measuring area of ​​the sample 500. One channel is, for example, 0.02 mm. Figure 7 (c) is the display and Figure 7 (a) and Figure 7 The diagram shows the region of sample 500 corresponding to the channel position in (b). Figure 7The 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Next, the method for removing the signal component (noise) corresponding to the scattered light will be specifically explained. In the optical measuring device 1, a calibration process is performed in the elimination circuit 60, which serves as a locking circuit. Taking into account the results of this calibration process, the signal component (noise) corresponding to the scattered light is removed from the self-detection signal.

[0087] Specifically, in the optical measurement method using optical measuring device 1, firstly, a reference component (e.g., for calibration processing of sample 500 or a different sample 500) is used. Figure 18 An optical head of an illumination optical system 10 is configured to irradiate the sample 500 or the mirror 600A with excitation light. The scattered light (the excitation light component scattered in the sample 500 or the mirror 600A) caused by the excitation light from the illumination optical system 10 irradiating the sample 500 or the mirror 600A is then detected in the photodetector element 21 of the detection optical system 20. Here, the light detected by the detection optical system 20 is essentially scattered light that does not contain fluorescence from the sample 500 or the mirror 600A, and is used for calibration processing.

[0088] Next, a calibration process is performed. Specifically, the elimination circuit 60 of the optical measuring device 1 performs a calibration process based on the calibration signal corresponding to the calibration light used in the calibration process described above, to remove the signal component corresponding to the scattered light from the self-detection signal. Details of the calibration process will be described later. After the calibration process is completed, fluorescence information of the measuring unit 501 is obtained by scanning the measuring area (measuring unit 501) of the sample 500 with the optical head of the irradiation optical system 10. Specifically, the elimination circuit 60 removes the signal component corresponding to the scattered light from the self-detection signal by taking into account the implementation results of the calibration process described above, thereby obtaining fluorescence information.

[0089] Next, details of the calibration process will be explained. The elimination circuit 60 of the optical measurement device 1 utilizes a locking circuit, such as an FPGA (Field Programmable Gate Array). During the calibration process, the elimination circuit 60 generates a locking switch signal, which shifts the phase relative to a periodic signal whose period is etched with a frequency matching the modulation frequency (e.g., the frequency of a DDS (Direct Digital Synthesizer)) of the light source 11 set by the light source drive circuit 30. Thus, the elimination circuit 60, functioning as a locking circuit, takes the calibration signal (as the measurement signal) and the switch signal (as the reference signal) as inputs, and outputs a signal component corresponding to the scattered light, adjusting the phase of the switch signal so that the voltage value of the signal component corresponding to the scattered light is within a specified range (slope level) approximately 0.

[0090] 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 switch signals used for locking mentioned above, and are signals generated by triggering the reference signal. 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 actually 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. The phase of the switch signal is adjusted so that the voltage value of the signal component corresponding to the scattered light becomes a ramp level, that is, the phase of the reference signal is continuously offset relative to the periodic signal until the output from the cancellation circuit 60 becomes 0V (or a value close to it).

[0091] 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.

[0092] 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.

[0093] 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 As shown in (d), the phase of the reference signal is adjusted by delaying the phase of the switching signal.

[0094] Currently, the phase adjustment of the switching signal has been performed. Figure 11 In state (d), 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 negative value. In this case, such as Figure 11 As shown in (e), the phase of the reference signal is adjusted in a way that advances the phase of the switching signal. That is, during the calibration process, the cancellation circuit 60 adjusts the phase of the switching signal in a way that advances the phase of the signal when the voltage value of the signal component corresponding to the scattered light is negative rather than at the ramp level.

[0095] Then, the result after adjusting the phase of the switching signal, such as Figure 11 As shown in (e), the calibration process is complete when 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) becomes the ramp level (a value within a specified range that is approximately 0).

[0096] When the calibration process is completed, the elimination circuit 60 sets the detection signal corresponding to the detection light containing the fluorescence component and the scattered light component (excitation light component) and the switch signal whose phase was adjusted in the calibration process as inputs, and removes the signal component corresponding to the scattered light component from the detection signal.

[0097] Figure 13 This is a flowchart showing the calibration process. For example... Figure 13 As shown, in the calibration process, firstly, the input to the AD converter is switched to a specified compensation voltage, and a 0 level is stored (step S11). Then, by switching the switch, the signal of the cancellation circuit 60 (lock-in circuit) is input to the AD converter (step S12). In this state, the phase of the reference signal is forcibly shifted once (step S13). The output of the cancellation circuit 60 (lock-in circuit) is 0V when the phase of the reference signal relative to the periodic signal is 0 degrees and 180 degrees. However, if the phase is accidentally aligned to 180 degrees in the initial state, the positive and negative signs of the output signal are reversed due to the erroneous calibration process, resulting in a situation where the signal output by 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 beginning. In addition, by aligning the phase at the beginning as described above, the positive and negative signs of the output signal are fixed. As a result, the sign bit of the output signal does not need to be converted into a digital value, and the dynamic range of the AD converter can be used effectively. In addition, when it is intended to measure with a negative output, the phase after calibration can be set to 180 degrees instead of 0 degrees.

[0098] When step S13 is completed, the current input value of the AD converter is recorded (step S14), and the calibration loop process is performed. First, the current input value of the AD converter is compared with a 0 level to determine whether the input value of the AD converter is less than a 0 level (whether it is a negative value) (step S15). If it is determined in step S15 that the input value of the AD converter is negative, the phase advance of the switching signal (i.e., the reference signal) of the elimination circuit 60 relative to the periodic signal corresponding to the frequency of the DDS is performed (step S16). On the other hand, if it is determined in step S15 that the input value of the AD converter is positive, the phase delay of the switching signal (i.e., the reference signal) of the elimination circuit 60 relative to the periodic signal is performed (step S17).

[0099] Then, based on the input value of the AD converter, it is determined whether the sign remains unchanged at the ramp level (step S18). If it is determined in step S18 that the sign remains unchanged at the ramp level, the calibration process ends. On the other hand, if it is determined in step S18 that the condition is not met, it is determined whether the sign of the AD converter input has changed due to phase shift (step S19). If it is determined in step S19 that it has not changed, step S14 is performed again. If it is determined that it has changed, the width of the phase shift based on control is changed to half of the current value (step S20), and step S14 is performed again. This is the calibration process.

[0100] In this method, a calibration processing light containing scattered light but without fluorescence is detected. Based on the calibration signal corresponding to the calibration processing light, a calibration process is performed to remove the signal component corresponding to the scattered light from the self-detection signal. Taking into account the result of this calibration process, the signal component corresponding to the scattered light is removed from the self-detection signal. By performing a calibration process for removing the signal component corresponding to the scattered light from the self-detection signal in advance based on the calibration processing light containing scattered light, the signal component corresponding to the scattered light can be appropriately removed from the self-detection signal.

[0101] Regarding the effect of appropriately removing scattered light (noise) as described above, refer to Figure 14 Please provide an explanation. Figure 14 (a) shows the intensity of the detected light without removing the signal components (noise) corresponding to the scattered light. Figure 14 (b) shows the intensity of the detected light after the removal of the signal components (noise) corresponding to the scattered light. Figure 14 The results are shown when measurements are performed on a membrane coated with DTBTA-Eu3+ as a fluorescent reagent. For example... Figure 14As shown in (a), without noise removal, approximately 330 counts of compensation are needed to cover the background (BKG) of the excitation light (scattered light). Therefore, the noise (standard deviation) is 2.16, and the signal strength is 404 counts. In contrast, as... Figure 14 As shown in (b), when noise removal is performed, there is no need to compensate for the scattering of excitation light towards the film; only minimal compensation is performed for software processing (setting all signal values ​​to positive). Therefore, the noise (standard deviation) can be set to 0.69, and the signal strength can be set to 1475 counts. Since the compensation amount is small when noise removal is performed, the excitation light intensity from the light source and the amplification of the IV converter amplifier can be increased, thus appropriately improving the signal strength. As a result, compared to 187 without noise removal, the S / N can be set to 2140 with noise removal, increasing the S / N by more than 10 times.

[0102] In the calibration process, a locking switch signal is generated relative to the phase offset of a periodic signal whose period is characterized by the operating frequency of the elimination circuit 60, which matches the modulation frequency of the light source 11. The calibration signal and the switch signal are set as inputs, and the output is the signal component corresponding to the scattered light. The phase of the switch signal is adjusted such that the voltage value of the signal component corresponding to the scattered light is within a specified range approximately zero. The detection signal and the phase-adjusted switch signal from the calibration process are set as inputs, and the signal component corresponding to the scattered light is removed from the detection signal. Thus, by utilizing the locking circuit, the phase of the switch signal is adjusted in the calibration process such that the voltage value of the signal component corresponding to the scattered light is approximately zero. The phase-adjusted switch signal can then be set as input, and the signal component corresponding to the scattered light can be appropriately removed from the detection signal.

[0103] During calibration, if the voltage value of the signal component corresponding to the scattered light is greater than the specified range rather than outside the specified range, the phase of the switching signal can be adjusted by delaying the phase. Conversely, if the voltage value of the signal component corresponding to the scattered light is less than the specified range rather than outside the specified range, the phase of the switching signal can be advanced. Thus, during calibration, the voltage value of the signal component corresponding to the scattered light can be appropriately adjusted to a value approximately equal to 0.

[0104] The modulation frequency of light source 11 can be lower than the value corresponding to the fluorescence lifetime, which is the time it takes for the fluorescence intensity to drop from its peak to 1 / e. When the modulation frequency is increased to the level corresponding to the fluorescence lifetime, there is a situation where consecutive signals overlap, making it impossible to maximize the fluorescence intensity. This point allows for a suitable increase in fluorescence intensity by lowering the modulation frequency to the value corresponding to the fluorescence lifetime.

[0105] This allows the modulation frequency of light source 11 to be lower than the value corresponding to the fluorescence lifetime but higher than the commercial frequency. Thus, it is possible to avoid both the fluorescence intensity weakening due to the modulation frequency becoming higher than the value corresponding to the fluorescence lifetime and the increase in noise.

[0106] As a switching signal for locking, an X signal, a Y signal whose phase is offset by 90 degrees relative to the X signal, an X′ signal that inverts the X signal, and a Y′ signal that inverts the Y signal can be generated using separate dedicated circuits. By generating the inverted signals with separate dedicated circuits, the slight delay that would be problematic, such as when generating the inverted signal through the no circuit (the slight delay accompanying the passage of the no circuit), can be prevented.

[0107] The elimination circuit 60 (locking circuit) can switch between two operating frequencies at a predetermined ratio. Therefore, compared to setting only one operating frequency, it is easier to match the operating frequency of the locking circuit with the modulation frequency of the light source 11, thereby improving their synchronization.

[0108] The calibration light can be detected by irradiating the region of the sample 500 downstream of the capture antibody that is fixed to the test object 504 with excitation light. Although fluorescent components tend to remain upstream of the capture antibody, the calibration light can be appropriately detected by irradiating the region downstream of the supplementary antibody with excitation light, thus reducing the influence of fluorescent components.

[0109] In the aforementioned calibration process, the calibration signal and the switch signal are set as inputs, and the output is the signal component corresponding to the scattered light. The phase of the switch signal is adjusted so that the voltage value of the signal component corresponding to the scattered light is close to 0. In principle, the signal component corresponding to the scattered light can be completely removed from the detection signal in the subsequent fluorescence detection process. However, in practice, due to the control accuracy and detection accuracy of the device, it is difficult to adjust the phase of the switch signal using the calibration signal (the signal corresponding to the scattered light) so that the output of the device is completely 0, resulting in calibration errors.

[0110] Figure 15 and Figure 16 This is a diagram illustrating calibration errors. Figure 15 In the diagram, signal wave W1 represents the calibration signal, and square wave W2 represents the switching signal. Calibration refers to bringing the integral of the difference between square wave W2 and signal wave W1 close to zero. This is achieved by shifting square wave W2 relative to signal wave W1, while... Figure 15 The area difference between the two regions represented by the diagonal lines becomes 0 (making the integral of the difference between the square wave W2 and the signal wave W1 close to 0), but as Figure 15As shown, the area difference will not be completely zero, resulting in an offset region CE caused by the phase shift of the calibration signal and the switching signal. The area of ​​this offset region CE is determined by... Figure 15 It can be seen that the calibration error is derived by adding the amplitude (signal quantity) of the calibration signal to the phase offset of the calibration signal and the switching signal. If the area of ​​the offset region CE is defined as the calibration error, then the calibration error is expressed by the following formula.

[0111] Calibration error = Amplitude (signal quantity) of calibration signal × Phase offset of calibration signal and switching signal...(1)

[0112] Here, the calibration error arises from the limits of the device's control accuracy and detection accuracy, and is unaffected by differences in the amount of incident light towards the detection optical system and the sample. If it is the same device, it becomes a constant value. In equation (1) above, if the calibration error on the left is constant, the amplitude (signal quantity) of the calibration signal increases, and the phase shift between the calibration signal and the switching signal decreases. That is, if the calibration error is constant, then in the case of... Figure 16 As shown in (a), when the amplitude (signal quantity) of the calibration signal, represented by signal wave W1, is significantly increased, it is similar to... Figure 16 As shown in (b), compared to the case where the amplitude (signal quantity) of the calibration signal, represented by signal wave W1, is reduced, the phase shift of the calibration signal and the switching signal becomes smaller. Based on this consideration, the following explanation will address how, by maximizing the amplitude (signal quantity) of the calibration signal and minimizing the phase shift of the calibration signal and the switching signal, the signal component corresponding to the scattered light can be more appropriately removed during fluorescence detection processing.

[0113] As a specific method for increasing the amplitude (signal quantity) of the calibration signal, the following refers to... Figures 17-26 The following describes methods 1 through 5. In all methods, a pre-processing including calibration is performed before the fluorescence detection process. That is, in each method, the optical measuring device performs the fluorescence detection process and the pre-processing performed prior to the fluorescence detection process. In method 1, the amplitude of the calibration signal is increased by increasing the amount of illumination light during the pre-processing. In method 2, the amplitude of the calibration signal is increased by illuminating a reference component different from the sample during the pre-processing. In method 3, the amplitude of the calibration signal is increased by increasing the amount of scattered light detected during the pre-processing by configuring the optical system. In method 4, the amplitude of the calibration signal is increased by directly detecting the illumination light during the pre-processing. In method 5, the amplitude of the calibration signal is increased by generating a spurious signal during the pre-processing.

[0114] [Method 1]

[0115] Figure 17This is a diagram illustrating a summary of the fluorescence measurement in Method 1. (As shown...) Figure 17 As shown in (a), in the fluorescence measurement of the first method, firstly, the light source 11 of the irradiation optical system 10 irradiates the sample 500 (specifically, the area of ​​the sample 500 other than the measurement target portion 504) which is the object to be measured with irradiation light, and the photodetector 21 of the detection optical system 20 detects calibration processing light containing the scattered light from the sample 500 that has been irradiated with irradiation light. Then, the elimination circuit 60, which is the signal processing unit (see reference...) Figure 1 The calibration process is performed based on the calibration signal corresponding to the light used for calibration. The above is the preprocessing in the first method.

[0116] After that, as Figure 17 As shown in (b), the light source 11 irradiates the sample 500 (specifically, the measurement object section 504 of 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 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. The above is the fluorescence detection processing in method 1. If... Figure 17 (a) and Figure 17 If (b) is set as the front view, then Figure 17 (c) is the right-side view. For example... Figure 17 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.

[0117] Here, in the first method, the amount of light from the light source 11 illuminating the optical system 10 is increased compared to the amount of excitation light (illumination light) illuminating during fluorescence detection processing, compared to the amount of illumination light illuminating during pre-processing. By increasing the amount of illumination light during pre-processing, the amount of light used for calibration processing, which mainly includes scattered light, can be increased. This appropriately increases the amplitude (signal magnitude) of the signal corresponding to the calibration processing light, i.e., the calibration signal. Specifically, the signal magnitude of the calibration signal is greater than the signal magnitude of the measurement signal corresponding to the scattered light in the fluorescence detection processing. Therefore, the elimination circuit 60 performs calibration processing based on the calibration signal to remove the signal component corresponding to the scattered light from the measurement signal, which is the signal involved in the scattered light detected in the photodetector element 21 of the detection optical system 20, and whose signal magnitude is greater than the signal magnitude of the measurement signal corresponding to the scattered light.

[0118] Secondly, the effects of the first method will be explained.

[0119] The optical measuring apparatus 1 of the first type includes: an illumination optical system 10 that irradiates an irradiation object with illumination light (excitation light); a detection optical system 20 that detects detection light caused by the illumination light (excitation light); and an elimination circuit 60; a fluorescence detection process and a pre-processing process performed prior to the fluorescence detection process; in the fluorescence detection process, the illumination optical system 10 irradiates the sample 500 with excitation light, the detection optical system 20 detects the object light, which includes fluorescence generated from the sample 500 irradiated with excitation light and scattered light from the sample 500 irradiated with excitation light, as the detection light; the elimination circuit 60 removes the signal component corresponding to the scattered light from the measurement signal corresponding to the object light from the calibration process of the pre-processing; in the pre-processing process, the elimination circuit 60 performs a calibration process based on a calibration signal to remove the signal component corresponding to the scattered light from the measurement signal, the calibration signal being the signal involved in the illumination light or the scattered light of the illumination light detected in the detection optical system 20, and the signal quantity being greater than the signal corresponding to the scattered light in the measurement signal.

[0120] Thus, in the optical measuring apparatus 1, fluorescence detection processing and a preprocessing performed prior to the fluorescence detection processing are implemented. In the fluorescence detection processing, based on the calibration processing results of the preprocessing, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the light of the object being measured. Then, in the preprocessing, a calibration processing is performed, which sets the signal involved in the illumination light or scattered light whose signal amount is greater than the aforementioned measurement signal as the calibration signal. As described above, the calibration error of the calibration processing is a constant value, and the calibration error is derived by adding the "amplitude of the calibration signal" and the "phase shift of the calibration signal and the switch signal". Therefore, as in the optical measuring apparatus 1 of this embodiment, by increasing the signal amount (amplitude) of the calibration signal (greater than the measurement signal corresponding to the scattered light), the influence of the phase shift of the calibration signal and the switch signal can be reduced. Therefore, in the calibration processing, the output of the device, i.e., the voltage value, can be brought closer to 0 using the calibration signal (the signal corresponding to the scattered light), and in the fluorescence detection processing, the signal component corresponding to the scattered light can be appropriately removed from the measurement signal. As a result, the accuracy of fluorescence detection can be further improved.

[0121] In the first method, during preprocessing, the light source 11 of the illumination optical system 10 illuminates the irradiated object with illumination light. The photodetector 21 of the detection optical system 20 detects the calibration processing light as the detection light. This calibration processing light is light containing scattered light from the irradiated object that has been illuminated, and its intensity is greater than the scattered light contained in the light of the measured object detected in the fluorescence detection process. The elimination circuit 60 performs calibration processing based on the calibration signal corresponding to the calibration processing light. With this structure, the calibration processing light can be detected using the optical system used in fluorescence detection, and the calibration signal can be easily and appropriately generated. That is, the existing device structure can be used to appropriately generate the calibration signal with a simple structure.

[0122] Furthermore, in the first method, during pretreatment, the light source 11 of the irradiation optical system 10 irradiates the sample 500, which is the object to be measured, with irradiation light. For example, by irradiating the sample 500 with irradiation light towards the portion of the sample 500 that is not prone to fluorescence, even when the sample 500 is the irradiation object, the calibration processing light, which mainly contains scattered light (not containing much fluorescence), can be detected. According to this structure, a calibration signal can be easily and appropriately generated with a simple structure using existing device structures.

[0123] Compared to fluorescence detection processing, the light source 11 of the irradiation optical system 10 increases the amount of irradiated light used during preprocessing. With this structure, the amount of light used for calibration processing can be easily and appropriately increased (greater than the scattered light contained in the light of the object being measured). This allows for an appropriate increase in the signal intensity of the calibration signal and further improves the accuracy of fluorescence detection.

[0124] [Method 2]

[0125] Next, the fluorescence measurement in the second method will be explained. In the second method, during the pretreatment, the light source 11 of the irradiation optical system 10 irradiates a reference component used for calibration treatment, which is different from the sample 500, with irradiation light, thereby increasing the amplitude of the calibration signal. Hereinafter, a specific structural example of the reference component will be described.

[0126] Figure 18 This diagram illustrates the acquisition of scattered light using mirror 600A as a reference element. (See diagram for example.) Figure 18As shown, the reference component may include mirror 600A. Mirror 600A can be any mirror as long as it is capable of reflecting illumination light, but it is more preferable that mirror 600A is formed of a material that does not produce fluorescence upon illumination by illumination light (does not produce fluorescence caused by mirror 600A, or only produces a negligible degree of fluorescence). Mirror 600A may be, for example, a mirror whose surface is not easily soiled. Mirror 600A reflects the illumination light emanating from light source 11. The tilt angle of mirror 600A is adjusted such that the reflected illumination light (scattered light) is detected by light detection element 21. Mirror 600A is fixed at a predetermined tilt angle by an adjustment mechanism (not shown). By appropriately adjusting the tilt angle of mirror 600A relative to light detection element 21, the amount of light detected in light detection element 21 can be increased.

[0127] Figure 19 This diagram illustrates the acquisition of scattered light using a reflective diffuser 600B as a reference component. (See diagram for example.) Figure 19 As shown, the reference component may include a reflective diffuser 600B. The reflective diffuser 600B is, for example, a component made of a material that does not fluoresce upon irradiation by the irradiation light (it does not produce fluorescence caused by the reflective diffuser 600B, or only produces a negligible amount of fluorescence), or a reflective diffuser plate made of a white component with a relatively stable amount of incident light towards the light detection element 21. The reflective diffuser 600B reflects the irradiation light from the light source 11 in a manner that diffuses it at various angles. The reflective diffuser 600B is positioned at a location where the reflected irradiation light (scattered light) is detected by the light detection element 21 with a large amount of light. The reflective diffuser 600B is preferably a one-piece molded component made of, for example, resin or ceramic, but it can be formed by machining or by combining multiple components, or it can be formed by, for example, providing a film on the surface of a plate-like component that can scatter the irradiation light at a specific angle.

[0128] Figure 20 This diagram illustrates the acquisition of scattered light using mirror 611 (reflective substrate) and frosted glass 610 (diffuser) as reference components. Figure 20As shown, the reference member may include a reference member 600C, which includes frosted glass 610, which serves as a glass member through which irradiated light passes, and a mirror 611, which reflects the irradiated light. In the reference member 600C, the frosted glass 610 is configured to be supported (stacked) on the mirror 611 and is configured to receive the irradiated light before the mirror 611. The frosted glass 610 is formed of a material, for example, that transmits irradiated light of 380 nm or higher, and has unevenly shaped protrusions 610a formed on its surface (the incident surface of the irradiated light). Alternatively, a film or the like that that can scatter the irradiated light at a specific angle may be provided on the surface of the frosted glass 610. The mirror 611 only needs to reflect the irradiated light and may be, for example, a plate-shaped metal member made of aluminum or the like, or a plate-shaped member whose surface is covered with a metal film such as aluminum. It may be configured as a box-shaped member to house the frosted glass 610. Furthermore, both mirror 611 and frosted glass 610 are more preferably formed of materials that produce fluorescence without irradiation by light (i.e., do not produce fluorescence caused by mirror 611 and frosted glass 610, or produce only a negligible degree of fluorescence). Figure 20 As shown, in the reference member 600C, the incident light on the frosted glass 610 diffuses in the recessed portion 610a and passes through the interior of the frosted glass 610 to reach the mirror 611. It is reflected on the mirror 611 and then passes through the interior of the frosted glass 610 again to reach the surface of the frosted glass 610, where it diffuses in various directions and exits (generating scattered light at various angles). By generating scattered light at various angles, a sufficient amount of scattered light is detected in the light detection element 21. Furthermore, in the reference member 600C, a reflective material with a roughened surface can be used. In this case, the roughened surface of the reflective material acts as a diffuser to diffuse the incident light.

[0129] Secondly, the effects of the second method will be explained.

[0130] In the second method, during the pre-processing, the light source 11 of the irradiation optical system 10 irradiates a reference component (mirror 600A, reflective diffuser 600B, or reference component 600C) used for calibration processing, which is different from the sample 500, with irradiation light. By irradiating the reference component, which is different from the sample 500, more appropriate calibration processing light can be detected. Therefore, based on the results of the calibration processing, the signal component corresponding to the scattered light can be more appropriately removed from the self-measured signal during fluorescence detection processing.

[0131] Reference component 600 can be as follows Figures 18-20The 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), the signal component corresponding to the scattered light component can be reliably removed from the measured signal.

[0132] Reference component 600 can be as follows Figure 19 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.

[0133] Reference component 600 can be as follows Figure 20 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.

[0134] Reference component 600 can be as follows Figure 18 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.

[0135] [Method 3]

[0136] Next, the fluorescence measurement in the third method will be explained. In the third method, the amplitude of the calibration signal is increased by increasing the amount of scattered light (calibration processing light) detected in the preprocessing through the configuration of the optical system.

[0137] Figure 21 This is a diagram illustrating the summary of the fluorescence measurement in the third method. In this third method, a fluorescence filter 900 (optical component) is provided, which can be configured in the optical path from the sample 500 to the photodetector 21. The fluorescence filter 900 optically has, for example, a... Figure 1 The fluorescence filter 22 shown serves the same function, acting as a wavelength-selective filter to suppress light other than fluorescence from reaching the photodetector element 21. The fluorescence filter 900 is positioned on the optical path toward the photodetector element 21 at position 1 (see reference). Figure 21 (b) and the position outside the light path, i.e., the second position (refer to...). Figure 21The (a) can be movably constructed.

[0138] Figure 21 (a) shows the status of the fluorescence filter 900 during preprocessing. Figure 21 (b) shows the state of fluorescence filter 900 during fluorescence detection processing. For example... Figure 21 As shown in (a), during pretreatment, the fluorescence filter 900 retracts towards the second position described above. In this state, scattered light from the sample 500 can reach the photodetector 21 without being affected by the fluorescence filter 900. On the other hand, as Figure 21 As shown in (b), during fluorescence detection processing, the fluorescence filter 900 is configured in the first position described above. In this state, scattered light from the sample 500, which is light other than fluorescence, reaches the photodetector element 21 in only a small amount due to the influence of the fluorescence filter 900. Thus, by changing the configuration of the fluorescence filter 900 during preprocessing and fluorescence detection, compared with fluorescence detection, the amount of scattered light (calibration processing light) detected by the photodetector element 21 can be increased during preprocessing, thereby appropriately increasing the amplitude of the calibration signal.

[0139] Secondly, the effects of the third method will be explained.

[0140] In the third embodiment, the optical measuring apparatus 1 includes a fluorescence filter 900. During normal fluorescence detection, the fluorescence filter 900 is positioned at a first position on the optical path toward the photodetector element 21, suppressing light other than fluorescence (scattered light, etc.) from reaching the photodetector element 21. The fluorescence filter 900 is also movably positioned at a second position, where it is retracted from the optical path toward the photodetector element 21 during preprocessing. Therefore, during preprocessing, scattered light can easily reach the photodetector element 21 without being affected by the fluorescence filter 900. With this structure, the amount of calibration processing light detected by the photodetector element 21 can be appropriately increased (greater than the scattered light contained in the light of the object being measured) with a simplified structure. This appropriately increases the signal intensity of the calibration signal and further improves the accuracy of fluorescence detection.

[0141] Furthermore, the optical component used to increase the amount of light used in the calibration process is not limited to the fluorescence filter 900 described above. For example, the optical component may be an optical component that increases the detection efficiency of the illumination light. In this case, the optical component is positioned in the optical path up to the photodetector element 21 during the preprocessing, and is positioned at a position that is retracted from the optical path during the fluorescence detection process.

[0142] [Method 4]

[0143] Next, the fluorescence measurement in the fourth method will be explained. In the fourth method, the amplitude of the calibration signal is increased by directly detecting the irradiation light as calibration light during the pretreatment. That is, in the fourth method, during the pretreatment, the light source 11 of the irradiation optical system 10 irradiates the photodetector element 21 with irradiation light, and the photodetector element 21 directly detects the irradiation light as calibration light (without passing through the sample 500, etc.).

[0144] Figure 22 This is a diagram illustrating the overview of fluorescence measurement in method 4, specifically illustrating the acquisition of excitation light via optical path switching. Figure 22 In the example shown, an optical path 950 is formed from the light source 11 of the self-illuminating optical system 10 to the photodetector element 21, and a shutter 930 is provided that can be configured in the optical path 950. In such a structure, during preprocessing, such as Figure 22 The shutter 930 is positioned in a position away from the optical path 950. In this state, the illumination light emitted from the light source 11 reaches the photodetector 21 without being affected by the shutter 930. On the other hand, during fluorescence detection processing, the shutter 930 moves onto the optical path 950. In this state, the excitation light (illumination light) emitted from the light source 11 illuminates only the sample 500. Thus, by utilizing the shutter 930, the illumination light (calibration processing light) is directly detected by the photodetector 21 only during the preprocessing stage. Then, the elimination circuit 60 (see reference 60) serves as the signal processing unit. Figure 1 The calibration process is performed based on the calibration signal corresponding to the calibration processing light. The illumination light is light with the same phase as the scattered light. Therefore, by detecting the illumination light as the calibration processing light, calibration processing for removing the signal component corresponding to the scattered light from the self-detection signal can also be appropriately performed. Furthermore, during fluorescence detection processing, scattered light (light with the same phase as the excitation light) from the sample 500 is also detected in the photodetector 21, but the amount of light is less compared to the case where the illumination light is directly irradiated from the light source 11 (the pre-processed case). That is, according to Figure 22 The structure shown can increase the amplitude of the calibration signal corresponding to the calibration light by directly detecting the illumination light as the calibration light in the preprocessing stage.

[0145] Figure 23 This is a diagram illustrating the overview of the fluorescence measurement in the fourth method, and also a diagram illustrating the illumination obtained using a detection optical system 720, which is different from the photodetector element 21 (detection optical system 20). Figure 23In the illumination light acquisition method shown, the light source 11 of the illumination optical system 10 illuminates the light source 11 towards the light detection element 721 of the detection optical system 720, which is different from the light detection element 21. Therefore, the light detection element 721 directly detects the illumination light, i.e., the calibration processing light. In this structure, the detection optical system 720 is provided for acquiring illumination light for preprocessing, and the detection optical system 20 is provided for acquiring fluorescence for fluorescence detection processing. In this structure, it is also similar to... Figure 22 Similarly, by directly detecting the illumination light as calibration processing light during preprocessing, the amplitude of the calibration signal corresponding to the calibration processing light can be increased.

[0146] Figure 24 This is a diagram illustrating the overview of the fluorescence measurement in the fourth method, and also a diagram illustrating the results obtained using illumination light from an illumination optical system 910, which is different from that of the illumination optical system 10. Figure 24 In the illumination light acquisition method shown, the light source 911 of the illumination optical system 910, which is different from the light source 11 of the illumination optical system 10, illuminates the photodetector 21 of the detection optical system 20 with illumination light. Thus, the detection element 21 directly detects the illumination light, i.e., the calibration processing light. In this structure, the illumination optical system 910 is provided for acquiring illumination light for preprocessing, and the illumination optical system 10 is provided for acquiring fluorescence for fluorescence detection processing. In this structure, it is also similar to... Figure 22 and Figure 23 Similarly, by directly detecting the illumination light as calibration processing light during preprocessing, the amplitude of the calibration signal corresponding to the calibration processing light can be increased.

[0147] Secondly, the effects of the fourth method will be explained.

[0148] In the fourth method, during preprocessing, the light source 11 of the irradiation optical system 10 irradiates the photodetector element 21 of the detection optical system 20 with irradiation light. The photodetector element 21 detects the calibration processing light as the detection light. This calibration processing light is the irradiation light, and its intensity is greater than the scattered light contained in the light of the measurement object detected in the fluorescence detection process. The elimination circuit 60 performs calibration processing based on the calibration signal corresponding to the calibration processing light. In this way, by directly detecting the irradiation light by the photodetector element 21 during preprocessing, compared with the case where scattered light is detected as in fluorescence detection, the intensity of the calibration processing light can be easily and appropriately increased. As a result, the signal intensity of the calibration signal can be appropriately increased, and the accuracy of fluorescence detection can be further improved.

[0149] [Method 5]

[0150] Next, the fluorescence measurement in method 5 will be explained. In method 5, the calibration light is not detected during pretreatment; instead, a spurious signal (calibration signal) is generated to increase the amplitude of the calibration signal.

[0151] Figure 25 This is a schematic diagram of the optical measuring device 801 according to the fifth embodiment. The optical measuring device 801 of the fifth embodiment is similar to the optical measuring device 1 described above (see reference 1). Figure 1 The structure is roughly the same, but as Figure 25 As shown, the optical measuring device 1 differs from the device in that it includes a delay-amplification circuit 802. The delay-amplification circuit 802 functions as a signal processing unit together with the cancellation circuit 60. Here, as described above, the light source driving circuit 30 sets the modulation frequency of the light source 11 based on a frequency signal that serves as a reference input from the timing generator 70. The delay-amplification circuit 802 generates a pseudo-signal (calibration signal) based on the frequency signal input from the timing generator 70, causing a phase change in the signal (modulation signal) of the modulation frequency set by the light source driving circuit 30 for the light source 11.

[0152] 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 when it is detected by the detection optical system 20 as detection light. The delay amplifier circuit 802 sets the amplitude (signal quantity) of the pseudo-signal (calibration signal) such that its amplitude is at least greater than the amplitude of the measurement signal corresponding to the scattered light in the fluorescence detection process. 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 amplifier circuit for changing the amplitude.

[0153] Secondly, the effects of the fifth method will be explained.

[0154] In the fifth method, during preprocessing, the elimination circuit 60 generates a calibration signal by changing the phase of the modulation signal corresponding to a delay equivalent to the time until the scattered light from the illumination light irradiated by the self-illuminating optical system 10 is detected by the detection optical system 20 as detection light. Thus, by generating a calibration signal corresponding to the delay of the illumination optical system 10 using the modulation signal for the illumination light, a calibration signal with the same phase as the scattered light can be obtained without detecting the calibration processing light. Furthermore, since a calibration signal is generated, the signal intensity of the calibration signal can be increased more appropriately and easily. This further improves the accuracy of fluorescence detection.

[0155] Finally, refer to Figure 26 This describes the fluorescence measurement process described in methods 1 through 5. Figure 26 This is a flowchart showing the fluorescence measurement process.

[0156] like Figure 26 As shown, in the fluorescence measurement process, a calibration process is first performed (step S51). Specifically, based on the calibration signal, a calibration process is performed in the fluorescence detection process to remove the signal component corresponding to the scattered light from the self-measurement signal. This calibration signal is the signal involved in the illumination light or scattered light detected in the detection optical system 20, and the signal amount is greater than the signal of the scattered light corresponding to the fluorescence detection process. Details of the calibration process are as follows (refer to...). Figures 10-13 As mentioned above, the explanation is omitted.

[0157] Subsequently, by scanning the measurement area of ​​the sample 500 with the optical head of the irradiation optical system 10, excitation light (irradiation light) is irradiated onto the measurement target portion 504 of the sample 500 (step S52). The photodetector 21 detects the measurement target 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 (step S53). Then, in the elimination circuit 60, considering the implementation result of the calibration process in step S51, the signal component corresponding to the scattered light is removed from the measurement signal corresponding to the above-mentioned measurement target light (step S54). Specifically, as described above, the elimination circuit 60 takes the measurement signal and the switch signal whose phase is adjusted in the calibration process as inputs, and removes the signal component corresponding to the scattered light from the measurement signal.

[0158] Explanation of symbols

[0159] 1, 1A, 801… Optical measuring device, 10, 910… Irradiation optical system, 20, 720… Detection optical system, 60… Elimination circuit, 600A… Mirror, 600B… Reflector diffuser, 610… Frosted glass, 611… Mirror, 802… Delay and amplification circuit, 900… Fluorescence filter.

Claims

1. An optical measuring device, wherein, It is an optical measuring device used to measure the optical properties of an object. have: An illumination optical system that illuminates the object with illumination light corresponding to a modulation signal; A photodetector unit that detects detection light caused by the illumination light; and Signal processing unit, Perform fluorescence detection processing and pretreatment performed prior to the fluorescence detection processing; In the fluorescence detection process, The irradiation optical system uses the object to be measured as the irradiation object and irradiates the object to be measured with the irradiation light. The photodetector uses the test object light, which includes fluorescence generated by the test object irradiated by the irradiation light and scattered light from the test object irradiated by the irradiation light, as the detection light. Based on the phase difference between the fluorescence and the scattered light, the signal processing unit removes the signal component corresponding to the scattered light in the calibration process during the preprocessing from the measurement signal corresponding to the light of the object being measured. In the preprocessing, The signal processing unit performs calibration processing based on a calibration signal to remove the signal component corresponding to the scattered light from the measurement signal. The calibration signal is a signal related to the illumination light or the scattered light of the illumination light detected in the light detection unit, and the signal quantity is greater than the signal corresponding to the scattered light in the measurement signal. The unit changes the phase of the modulation signal and generates the calibration signal based on a delay equivalent to the time until the scattered light of the illumination light irradiated by the illumination optical system is detected by the light detection unit as the detection light.

2. The optical measuring device as described in claim 1, wherein, In the preprocessing, The irradiation optical system irradiates the irradiated object with the irradiation light; The photodetector uses calibration processing light as the detection light. The calibration processing light is light containing scattered light from the irradiated object that has been irradiated by the irradiated light, and the amount of light is greater than the scattered light contained in the light of the measured object detected in the fluorescence detection process. The signal processing unit performs the calibration process based on the calibration signal corresponding to the calibration processing light.

3. The optical measuring device as described in claim 2, wherein, In the preprocessing, The irradiation optical system uses the object to be measured as the irradiation object and irradiates the object to be measured with the irradiation light.

4. The optical measuring device as described in claim 2, wherein, In the preprocessing, The irradiation optical system uses a reference component used for calibration processing, which is different from the object being measured, as the irradiation object, and irradiates the reference component with the irradiation light.

5. The optical measuring device as described in claim 4, wherein, The reference member includes a reflective member that reflects the illumination light.

6. The optical measuring apparatus as described in claim 5, wherein, The reflective component does not fluoresce when irradiated by the illumination light.

7. The optical measuring apparatus as described in claim 5, wherein, The reflective member includes a reflective diffuser that diffuses the irradiated light.

8. The optical measuring apparatus as described in claim 6, wherein, The reflective member includes a reflective diffuser that diffuses the irradiated light.

9. The optical measuring apparatus as described in claim 5, wherein, The reflective component includes: A reflective substrate that reflects the irradiated light; and A diffuser, which is supported on the reflective substrate, diffuses the irradiated light.

10. The optical measuring apparatus as claimed in claim 6, wherein, The reflective component includes: A reflective substrate that reflects the irradiated light; and A diffuser, which is supported on the reflective substrate, diffuses the irradiated light.

11. The optical measuring apparatus as claimed in claim 5, wherein, The reflective component includes a mirror.

12. The optical measuring apparatus as claimed in claim 6, wherein, The reflective component includes a mirror.

13. The optical measuring apparatus according to any one of claims 2 to 12, wherein, It also includes an optical component configured to move between a first position, which is a position on the optical path leading to the light detection unit, and a second position, which is a position away from the optical path, and has the function of increasing or decreasing the incident efficiency of light.

14. The optical measuring apparatus as claimed in claim 1, wherein, In the preprocessing, The irradiation optical system uses the light detection unit as the irradiation target and irradiates the light towards the light detection unit; The photodetector uses the calibration light as the detection light, which is the illumination light, and the light intensity is greater than the scattered light contained in the light of the measurement object detected in the fluorescence detection process. The signal processing unit performs the calibration process based on the calibration signal corresponding to the calibration processing light.

15. The optical measuring apparatus according to any one of claims 2 to 12, wherein, The illumination optical system increases the amount of illumination light during the pre-processing compared to the fluorescence detection process.

16. An optical measurement method, wherein, It is an optical measurement method for measuring the optical properties of an object. Include: The procedure for performing calibration; The process of irradiating the object to be measured with irradiation light corresponding to the modulation signal; The process of detecting object light, which includes fluorescence generated by the object being measured after being irradiated by the irradiation light, and scattered light from the object being measured after being irradiated by the irradiation light; and The step of removing the signal component corresponding to the scattered light from the measurement signal corresponding to the light of the object being measured, based on the phase difference in the fluorescence and the scattered light, in the calibration process. In the process of performing the calibration procedure described above Based on the calibration signal, the calibration process is performed to remove the signal component corresponding to the scattered light from the measurement signal. The calibration signal is the signal involved in the detected illumination light or the scattered light of the illumination light, and the signal amount is greater than the signal corresponding to the scattered light in the measurement signal. The phase of the modulation signal is changed according to a delay equivalent to until the scattered light of the illumination light is detected as the detection light, and the calibration signal is generated.

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