Light detection device, light detection method, density estimation system, and density estimation method

Abstract

This light detection device comprises: an optical path difference adjustment unit that adjusts the relative optical path difference between excitation light, first probe light, and second probe light; a phase modulation unit that performs phase modulation on at least one of the first probe light and the second probe light; a first detection unit that irradiates a sample with multiplexed light obtained by multiplexing the excitation light, the first probe light, and the second probe light, and detects a first stimulated Raman scattering signal that is generated; a second detection unit that detects the relative phase difference between the first probe light and the second probe light; and a derivation unit that derives, as a stimulated Raman scattering signal, a second stimulated Raman scattering signal obtained by subtracting the phase component of the phase difference detected by the second detection unit from the phase component of the first stimulated Raman scattering signal detected by the first detection unit.

Description

Light detection device, light detection method, concentration estimation system, and concentration estimation method

[0001] The present disclosure relates to a light detection device, a light detection method, a concentration estimation system, and a concentration estimation method.

[0002] Coherent Raman scattering microscopes are known as optical detection devices for detecting Raman scattering. Coherent Raman scattering microscopes analyze substances within a sample by irradiating the sample with two or more pulsed laser beams and observing the resulting Raman scattered light. In particular, time-resolved coherent Raman scattering microscopes can image the signals of small molecule drugs with specific molecular vibrations with high contrast without using labels, while avoiding background light from cells and biological tissues. This type of technology is generally referred to as Raman spectroscopy. Detection of trace substances using Raman spectroscopy is an important fundamental technology in analytical instruments, and many technical developments have been made. Meanwhile, with recent advances in medical technology, attempts have been made to apply trace substance detection technology to medical diagnostics, and there is a growing demand for improved trace substance detection sensitivity in this field as well.

[0003] Conventionally, coherent Raman scattering microscopes have generally been amplitude-modulated coherent Raman scattering microscopes, which modulate the amplitude of the incident laser light used for observation before irradiating it on the sample, detect the transmitted light or reflected and scattered light of the incident light with a photodetector, and demodulate it to detect a Raman signal. This amplitude-modulated coherent Raman scattering microscope can rapidly image the morphological information of cells and tissues by detecting strong Raman signals with short durations of molecular vibrations contained in living organisms, such as water and lipids.

[0004] As a conventional technique related to an amplitude-modulated coherent Raman scattering microscope, for example, a microscopic imaging system disclosed in Japanese Patent Application Laid-Open No. 2010-048805 is known. The microscopic imaging system according to Japanese Patent Application Laid-Open No. 2010-048805 is a photodetector using stimulated Raman scattering (SRS), which is known as one of the coherent Raman effects. The microscopic imaging system 10 according to Japanese Patent Application Laid-Open No. 2010-048805 uses a pump (excitation) beam with a center frequency ω １ and a laser pulse train 20 having a center frequency ω ２ The Stokes beam is amplitude-modulated by a modulator, and the excitation beam and the Stokes beam are combined in a combiner 25 and then irradiated onto a sample 22. The light transmitted through the sample 22 is sent to a photodetector 36, and an image based on stimulated Raman scattering is obtained by detecting the amplitude-modulated component from the detection signal from the photodetector 36.

[0005] With recent technological advances, there is a demand for improved sensitivity to detect extremely minute amounts of substances, even finer than conventional methods for detecting trace substances. For example, there is a need to detect extremely small amounts of drugs contained in biological samples. However, when observing Raman scattered light using the above-mentioned device, background signals with a signal intensity stronger than the signal intensity of the Raman signal of drug molecules at low concentrations (e.g., several tens of mM or less) may be generated from the biological tissue itself. For this reason, quantitative measurement of drugs at low concentrations (e.g., several tens of mM or less) has been difficult.

[0006] In addition, examples of background signals associated with stimulated Raman scattering include cross-phase modulation due to pulse excitation, thermal lensing, and four-wave mixing. Furthermore, the molecular vibrational modes of the biological sample itself can all contribute to the background signal. Because of these background signals, it is difficult to detect extremely small amounts of drugs contained in biological samples, and there is a need to remove the background signals in order to detect the signals of drug molecules.

[0007] The present disclosure has been made in consideration of the above facts, and makes it possible to provide a light detection device, a light detection method, a concentration estimation system, and a concentration estimation method that, when a stimulated Raman scattering signal is obtained by irradiating a sample with first probe light and second probe light, can more effectively reduce a background signal relative to a stimulated Raman scattering signal compared to a case in which the phase difference between the first probe light and the second probe light is not taken into consideration.

[0008] a first detection unit that detects a relative phase difference between the first probe light and the second probe light by irradiating a sample with combined light obtained by combining the excitation light, the first probe light, and the second probe light, and detects a generated first stimulated Raman scattering signal; a second detection unit that detects a relative phase difference between the first probe light and the second probe light; and a derivation unit that derives, as a stimulated Raman scattering signal, a second stimulated Raman scattering signal obtained by subtracting a phase component of the phase difference detected by the second detection unit from a phase component of the first stimulated Raman scattering signal detected by the first detection unit.

[0009] The photodetector according to a second aspect is the photodetector according to the first aspect, wherein the second detection unit includes a phase measurement branching unit that branches a part of the combined light before irradiating the sample into a first phase measurement light for measuring the phase of a probe light on the long wavelength side and a second phase measurement light for measuring the phase of a probe light on the short wavelength side, and a phase measurement unit that includes the first phase measurement unit that measures the phase of the probe light on the long wavelength side and the second phase measurement unit that measures the phase of the probe light on the short wavelength side.

[0010] The photodetector according to a third aspect is the photodetector according to the first aspect, wherein the second detector detects a relative phase difference between the first probe light and the second probe light by using the first probe light and the second probe light before being multiplexed with the excitation light.

[0011] A fourth aspect of the photodetector is the photodetector of the first aspect, wherein the second detection unit includes a spectroscopic unit that disperses a portion of the combined light before irradiating the sample into a probe light on a longer wavelength side and a probe light on a shorter wavelength side, and a spectroscopic phase measurement unit that measures the phases of the probe light on the longer wavelength side and the probe light on the shorter wavelength side.

[0012] A photodetector according to a fifth aspect is the photodetector according to the second or fourth aspect, wherein the second detector includes, upstream of the phase measurement branching unit, an optical member that adjusts a relative phase difference between the excitation light, and the first and second probe lights in the combined light so that an interference amplitude between the excitation light and the first and second probe lights is increased.

[0013] The photodetector may further include an optical path length modulation unit that performs optical path length modulation to modulate a relative optical path length difference between the excitation light and the first and second probe lights. The photodetector may further include a storage unit that stores the relative phase difference detected by the second detection unit, and the derivation unit may derive the second stimulated Raman scattering signal as the stimulated Raman scattering signal based on the stored information indicating the relative phase difference.

[0014] Furthermore, an optical detection method according to a sixth aspect includes adjusting a relative optical path difference between the excitation light and the first and second probe lights, phase-modulating at least one of the first and second probe lights, irradiating a sample with combined light obtained by combining the excitation light, the first and second probe lights, and detecting a generated first stimulated Raman scattering signal, detecting a relative phase difference between the first and second probe lights, and deriving, as the stimulated Raman scattering signal, a second stimulated Raman scattering signal obtained by subtracting a phase component of the detected phase difference from a phase component of the detected first stimulated Raman scattering signal.

[0015] A concentration estimation system according to a seventh aspect includes: a calibration curve derivation unit that derives a calibration curve indicating a correspondence relationship between the concentration of the material contained in the sample and the complex signal, based on a complex signal determined from the signal intensity and phase of the second stimulated Raman scattering signal derived by the photodetector according to any one of the first to fifth aspects; and an estimation unit that acquires a complex signal determined from the signal intensity and phase of the second stimulated Raman scattering signal derived by the photodetector for the sample containing the material at an unknown concentration, and estimates, based on the calibration curve, the concentration corresponding to the acquired complex signal as the unknown concentration.

[0016] Furthermore, a concentration estimation system according to an eighth aspect is the concentration estimation system according to the seventh aspect, further including a complex amplitude component detection unit that detects a complex amplitude component in the complex signal of a sample containing the material at a predetermined concentration, wherein the estimation unit derives a subtraction complex signal by subtracting the complex amplitude component detected by the complex amplitude component detection unit from the second stimulated Raman scattering signal derived for the sample containing the material at an unknown concentration, and estimates the concentration corresponding to the subtraction complex signal as the unknown concentration based on the calibration curve.

[0017] A concentration estimation method according to a ninth aspect includes deriving a calibration curve showing a correspondence relationship between the concentration of a predetermined material contained in the sample and the complex signal, based on a complex signal determined from the signal intensity and phase of the second stimulated Raman scattering signal derived by the photodetector according to any one of the first to fifth aspects for a sample containing the material; acquiring a complex signal determined from the signal intensity and phase of the second stimulated Raman scattering signal derived by the photodetector for the sample containing an unknown concentration of the material; and estimating, based on the calibration curve, the concentration corresponding to the acquired complex signal as the unknown concentration.

[0018] According to the present disclosure, when a stimulated Raman scattering signal is obtained by irradiating a sample with first probe light and second probe light, it is possible to more effectively reduce the background signal relative to the stimulated Raman scattering signal compared to a case where the phase difference between the first probe light and the second probe light is not taken into consideration.

[0019] 9A is a block diagram showing an example of the configuration of a photodetector according to an embodiment. FIG. 9B is a diagram for explaining characteristics on the time axis of pulsed light in a photodetector according to an embodiment. FIG. 9C is a diagram for explaining characteristics on the frequency axis of pulsed light in a photodetector according to an embodiment. FIG. 9D is a conceptual diagram showing a signal on a complex plane in a photodetector according to an embodiment. FIG. 9E is a conceptual diagram showing the relationship between signal intensity and concentration in a photodetector according to an embodiment. FIG. 9F is a flowchart showing the flow of background signal reduction processing executed in a photodetector according to an embodiment. FIG. 9G is a flowchart showing the flow of concentration estimation processing executed in a concentration estimation system according to an embodiment. FIG. 9H is a diagram showing a first modified example of a phase detection unit in a photodetector according to an embodiment. FIG. 9H is a diagram showing a second modified example of a phase detection unit in a photodetector according to an embodiment. FIG. 9I is a diagram showing the spectral distribution of a sample. FIG. 9J is a diagram showing the relationship between an SRS signal from a sample and the concentration of a target component. FIG. 9I is an enlarged view of a low concentration region in FIG. 9A.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the drawings. The following describes an example of a photodetection device and a photodetection method according to the present disclosure, in which the device and the method are applied to a phase-modulation coherent Raman scattering microscope that uses phase modulation as a means for detecting stimulated Raman scattering. The phase-modulation coherent Raman scattering microscope splits the output of a pulsed laser light source into three components: pump light, phase-modulated probe light, and reference probe light. The relative phase between the pump light, which comes first in time, and the subsequent, overlapping phase-modulated probe light and reference probe light is modulated. The resulting light intensity modulation due to interference between the Raman signal light and the reference probe light is detected and demodulated by a photodetector. By utilizing the relative time difference between the pump light and the probe light, this phase-modulation coherent Raman scattering microscope can selectively detect small molecules with relatively long molecular vibration durations with high contrast. As a result, the usefulness of coherent Raman scattering microscopes, which visualize concentration distributions while identifying molecular species without labeling, can be dramatically enhanced, for example, in biological samples.

[0021] The present disclosure also describes an example of a concentration estimation system and method applied to a concentration estimation system and method using a phase-modulated coherent Raman scattering microscope. In this disclosure, the phase of a phase-modulated probe light, which is probe light before irradiating a sample, and a reference probe light is detected as a reference phase, and the phase of a combined light obtained by combining a pump light, which is light that has passed through the sample, and two probe lights is detected as a signal phase. By calculating the difference between these reference phases and the signal phase, phase information of the sample can be obtained. The concentration of a substance, such as a drug, contained in a sample can be quantified based on a complex signal using the phase information of the sample and the signal amplitude derived from the sample.

[0022] A photodetector 10 according to the present embodiment and a concentration estimation system 60 using the photodetector 10 will be described with reference to Fig. 1 . First, the photodetector 10 will be described. As shown in Fig. 1 , the photodetector 10 includes a light source 11, a waveform shaping unit 12, a PBS (Polarizing Beam Splitter) 13, an excitation pulsed light adjuster 40, a reference probe pulsed light adjuster 41, a phase-modulated probe pulsed light adjuster 42, a microscope 25, a light-receiving unit 43, and a control unit 44. The light-receiving unit 43 is an example of a first detection unit of the present disclosure.

[0023] The light source 11 is a laser light source that generates excitation light Le and probe light (reference probe light Lr and phase-modulated probe light Lp) for generating an SRS signal. In this embodiment, pulsed excitation light (excitation pulse light Pe) and probe light (reference probe pulse light Pr and phase-modulated probe pulse light Pp) are used, and the light source 11 generates light source pulse light Ps, which is the source of these pulsed lights. Therefore, the laser light from the light source 11 is split into three: excitation light Le, reference probe light Lr, and phase-modulated probe light Lp. The light source pulse light Ps and excitation pulse light Pe according to this embodiment are ultrashort pulsed lights (femtosecond pulsed lights). The light source pulse light Ps according to this embodiment has a center wavelength of 790 nm and a pulse repetition frequency of 80 MHz, for example.

[0024] The excitation pulse light adjuster 40 adjusts the incident excitation pulse light Pe, the reference probe pulse light adjuster 41 adjusts the incident reference probe pulse light Pr, and the phase-modulated probe pulse light adjuster 42 adjusts the incident phase-modulated probe pulse light Pp. The microscope 25 irradiates the sample with the excitation light and the probe light. The light-receiving unit 43 receives Raman signal light generated in the sample, and the control unit 44 performs overall control of the entire photodetector 10. The phase-modulated probe pulse light adjuster 42 is an example of a phase modulation unit of the present disclosure. Each of these components will be described in detail below.

[0025] The photodetector 10 according to this embodiment uses an ultrashort pulse laser as the light source 11, which generates a broadband light source pulse light Ps. In this embodiment, a near-infrared broadband femtosecond laser is used as an example of the light source 11. More specifically, as shown in FIG. 1 , a titanium sapphire laser with a center wavelength of 790 nm and a pulse width of 15 fs (femtoseconds) or less is used as an example of the light source 11. However, the wavelength and pulse width of the light source 11 are not limited thereto and may be set to appropriate values ​​depending on the design details of the photodetector 10, etc. In this embodiment, the light source pulse light Ps emitted from the light source 11 is linearly polarized in a predetermined direction. However, the polarization state of the light source pulse light Ps is not limited thereto and may be, for example, circularly polarized, elliptically polarized, etc.

[0026] The waveform shaping unit 12 is a component that compensates for the light source pulsed light Ps to achieve desired characteristics. Specifically, the waveform shaping unit 12 includes, for example, a dispersion compensation optical element (not shown), an SLM (Spatial Light Modulator), and the like, and appropriately compensates for dispersion so that the pulse width of the irradiated light under the objective lens 32 (described later) is, for example, 15 fs. In this embodiment, a chirp mirror that provides high reflectivity and negative second-order dispersion compensation using a dielectric multilayer film is used as an example of the dispersion compensation optical element, and a liquid crystal spatial light modulator is used as an example of the SLM.

[0027] The PBS 13 is an optical element that splits the laser light generated by the light source 11 into excitation light Le and probe light (reference probe light Lr, phase-modulated probe light Lp).

[0028] The excitation pulse light adjuster 40 includes a λ / 4 wavelength plate 14 and an end mirror 15. In FIG. 1 , the optical path of the excitation light Le is as follows: light source 11 → waveform shaping unit 12 → PBS 13 → ¼ wavelength plate 14 → end mirror 15 → ¼ wavelength plate 14 → PBS 13 → mirror 29. The ¼ wavelength plate 14 converts the excitation light Le into circularly polarized light, and then reflects it off the end mirror 15, changing the polarization direction of the light source pulse light Ps to a direction different from the polarization direction of the light source pulse light Ps, thereby allowing it to be reflected off the PBS 13. The end mirror 15 is movable along the optical axis of the excitation light Le to adjust the delay time imparted to the excitation pulse light Pe and set the time difference between it and the probe pulse light. The end mirror 15 according to this embodiment is further equipped with a piezoelectric element (not shown), which allows for minute movement along the optical axis. Details of this minute movement will be described later. The configuration including the end mirror 15 is an example of an optical path difference adjuster according to the present disclosure.

[0029] The reference probe pulse light adjuster 41 includes a dispersion compensator 36 and a wavelength scanner 24, and the wavelength scanner 24 includes a bandpass filter 18, a quarter-wave plate 19, and an end mirror 20. The optical path of the reference probe light Lr is as follows: light source 11 → waveform shaping unit 12 → PBS 13 → bandpass filter 16 (denoted as "DM" in FIG. 1 ) → dispersion compensator 36 → bandpass filter 18 → quarter-wave plate 19 → end mirror 20 → quarter-wave plate 19 → bandpass filter 18 → dispersion compensator 36 → bandpass filter 16 → PBS 13 → mirror 29. The PBS 13 and the bandpass filter 16 are examples of a branching unit in the present disclosure.

[0030] The quarter-wave plate 19 functions similarly to the quarter-wave plate 14. As shown in FIG. 1 , the band-pass filter 18 is a tunable filter that is rotatable about a predetermined rotation axis and sets the center frequency of the reference probe pulse light Pr. In this embodiment, the variable range of the band-pass filter 18 is configured so that the center wavelength of the reference probe pulse light Pr can be selected from a range of, for example, 790 nm to 870 nm. The end mirror 20 mainly compensates for fluctuations in the delay of the reference probe pulse light Pr that accompany the rotation of the band-pass filter 18. The wavelength scanning unit 24 is connected to a control unit 44 (described later), and the band-pass filter 18 and the end mirror 20 are controlled by the control unit 44. The dispersion compensator 36 is an optical element that imparts dispersion to the reference probe pulse light Pr that corresponds to the dispersion of the optical modulator 21 (described later).

[0031] The phase-modulated probe pulse light adjusting unit 42 includes an optical modulator 21 (denoted as "EOM1" in FIG. 1), a bandpass filter 35 (denoted as "BPF" in FIG. 1), a quarter-wave plate 22, and an end mirror 23. The optical path of the phase-modulated probe light Lp is as follows: light source 11 → waveform shaping unit 12 → PBS 13 → bandpass filter 16 → optical modulator 21 → bandpass filter 35 → quarter-wave plate 22 → end mirror 23 → quarter-wave plate 22 → bandpass filter 35 → optical modulator 21 → bandpass filter 16 → PBS 13 → mirror 29. That is, in this embodiment, the PBS 13 splits the light into excitation light Le and probe light (reference probe light Lr, phase-modulated probe light Lp), and the bandpass filter 16 splits the light into reference probe light Lr and phase-modulated probe light Lp.

[0032] The optical modulator 21 is a phase modulator that modulates the phase of the phase-modulated probe light Lp. In this embodiment, an EOM (Electro-Optic Modulator) is used as an example. The optical modulator 21 is connected to a driving circuit (not shown), which is connected to the control unit 44. In this embodiment, the phase modulation by the optical modulator 21 is performed using a modulation signal with a sawtooth waveform (sawtooth wave) of 65 kHz as an example. The bandpass filter 35 is a tunable filter that sets the center frequency of the phase-modulated probe pulse light Pp. In this embodiment, the wavelength of the phase-modulated probe light Lp is set to 758 nm as an example. The quarter-wave plate 22 functions in the same way as the quarter-wave plate 14. The end mirror 23 adjusts the temporal positional relationship between the reference probe pulse light Pr and the phase-modulated probe pulse light Pp. In this embodiment, an example of adjusting the delay using end mirrors 15, 20, and 23 is described, but this is not limited to this, and any other optical element, such as an optical delay line, may be used as long as it has a mechanism that can make the optical delay variable.

[0033] Here, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp have asymmetric waveforms with fast rising edges and slow falling edges, and are arranged so as to be delayed by a predetermined time from the excitation pulse light Pe ( FIG. 2A ). In this embodiment, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp have substantially the same waveform (waveforms similar to each other) but different center wavelengths, and are superimposed in time.

[0034] 1, the above-mentioned excitation light Le, reference probe light Lr, and phase-modulated probe light Lp are reflected by a mirror 29 and then coaxially combined to form combined light Lg, which is introduced into the microscope 25 via mirrors 37 and 38. At this time, the relative delay time between the excitation pulse light Pe and the probe pulse light can be adjusted by the optical axis direction position of the end mirror 15 of the excitation pulse light adjusting unit 40. Here, the mirrors 29, 37, and 38 are elements for converting the optical path, and are not limited to the configuration shown in FIG.

[0035] The microscope 25 is an optical microscope and includes an objective lens 32, a stage 34, and folding mirrors 30 and 31. A sample 33 is placed on the stage 34, and the combined light Lg incident on the objective lens 32 is irradiated onto the sample 33. The sample 33 is, for example, a biological cell permeated with a drug. When the combined light Lg is irradiated onto the sample 33, an SRS signal is generated based on the SRS process, for example, due to molecular vibrations of drug molecules.

[0036] The light receiving section 43 includes a polarizer 26 , a long-pass filter 27 , and a light receiver 28 .

[0037] The polarizer 26 has a polarization axis in a direction different from (e.g., perpendicular to) the polarization direction of the excitation light Le and removes the excitation light Le from the combined light Lg. The long-pass filter 27 is a filter that removes the excitation light Le and removes the phase-modulated probe light Lp from the combined light Lg containing the reference probe light Lr and the phase-modulated probe light Lp. This is because, in this embodiment, the wavelength of the reference probe light Lr is set longer than the wavelength of the phase-modulated probe light Lp. The photoreceiver 28 receives the reference probe light Lr and converts it into an electrical signal. For example, a silicon photodiode is used for the photoreceiver 28. The photoreceiver 28 is connected to the control unit 44, and the received light signal from the photoreceiver 28 is sent to the control unit 44. The long-pass filter 27 is used in this embodiment because, as described above, the wavelength of the reference probe light Lr is set longer than the wavelength of the phase-modulated probe light Lp. There is no particular limitation on the relationship between the wavelength of the reference probe light Lr and the wavelength of the phase-modulated probe light Lp, so if the wavelength of the reference probe light Lr is set to be shorter than the wavelength of the phase-modulated probe light Lp, a short-pass filter can be used instead of the long-pass filter 27.

[0038] That is, after passing through the microscope 25, of the laser pulses of the excitation pulse light Pe, the reference probe pulse light Pr, and the phase-modulated probe pulse light Pp, the excitation pulse light Pe is blocked by the polarizer 26, and the phase-modulated probe pulse light Pp is blocked by the long-pass filter 27, while only the reference probe pulse light Pr passes to the photodetector 28. The light intensity of the reference probe pulse light Pr is converted into a current by a photodetector, and the light intensity modulated component superimposed on the reference probe pulse light Pr is detected by a lock-in amplifier. The light intensity modulated component detected by the lock-in amplifier is a signal component derived from Raman scattering in the sample 33, i.e., an SRS signal. The SRS signal is a signal proportional to the concentration of the sample (in this embodiment, a drug as an example). Note that while FIG. 1 illustrates an example in which the polarizer 26 and the long-pass filter 27 are arranged in this order, this order may be reversed. Note that the lock-in amplifier is configured by a control unit 44.

[0039] In this embodiment, a sampling mirror that samples a portion of the combined light Lg is applied to the mirror 38 that guides the combined light Lg to the microscope 25. That is, a portion of the combined light Lg is guided to the phase detection unit 50, and the remaining combined light Lg is introduced into the microscope 25. The phase detection unit 50 is a component that detects the relative phase difference (reference phase) between the reference probe light Lr and the phase-modulated probe light Lp. The phase detection unit 50 is an example of the second detection unit and phase measurement unit of the present disclosure.

[0040] The phase detection unit 50 according to this embodiment includes a mirror 51, a birefringent crystal 52 (denoted as "BBO" in FIG. 1), a half-wave plate 53 (denoted as "HWP" in FIG. 1), a PBS 54, a short-pass filter 55 (denoted as "SPF" in FIG. 1), a photodetector 56 (denoted as "PD" in FIG. 1), a long-pass filter 57 (denoted as "LPF" in FIG. 1), and a photodetector 58 (denoted as "PD" in FIG. 1). In FIG. 1, a portion of the combined light Lg sampled by the mirror 38 (sampling mirror) passes through the mirror 51, the birefringent crystal 52, and the half-wave plate 53 in that order, and is then guided to the PBS 54. The birefringent crystal 52 is a crystal that has birefringence and adjusts the optical path length. For example, an α-BBO crystal is used to adjust the relative delay between the excitation light Le and the probe light. The birefringent crystal 52 imparts a relative delay to each of the P-polarized and S-polarized components of light. While FIG. 1 illustrates the use of a birefringent crystal 52 such as an α-BBO crystal to adjust the relative delay between the excitation light Le and the probe light, the use of a birefringent crystal 52 such as an α-BBO crystal is not limited. For example, an optical system or other optical element capable of adjusting the relative delay between the excitation light Le and the probe light may be used. The half-wave plate 53 is an optical element that converts the P-polarized and S-polarized components of light into linearly polarized light at 45 degrees and 135 degrees. The PBS 54 is an example of a phase measurement splitter of the present disclosure, the photodetector 56 is an example of a first phase measurement unit of the present disclosure, and the photodetector 58 is an example of a second phase measurement unit of the present disclosure. The birefringent crystal 52 is an example of an optical member of the present disclosure.

[0041] The PBS 54 passes one of the P-polarized and S-polarized light components and is then irradiated onto a photodetector 56 (PD) via a short-pass filter 55 (SPF). The short-pass filter 55 is, for example, a filter that passes short-wavelength probe light (e.g., phase-modulated probe light Lp) of less than 775 nm. The photodetector 56 receives the short-wavelength probe light that has passed through the short-pass filter 55 and converts it into an electrical signal. The photodetector 56 may be, for example, a silicon photodiode. The photodetector 56 is connected to the control unit 44, and the received light signal from the photodetector 56 is sent to the control unit 44. The phase of the short-wavelength probe light is detected based on the received light signal received by the photodetector 56. Meanwhile, the other of the P-polarized and S-polarized light components is reflected by the PBS 54 and is then irradiated onto a photodetector 58 (PD) via a long-pass filter 57 (LPF). The long-pass filter 57 is a filter that passes, for example, long-wavelength probe light (e.g., reference probe light Lr) of 775 nm or more or exceeding 775 nm. The photoreceiver 58 receives the long-wavelength probe light that has passed through the long-pass filter 57 and converts it into an electrical signal. For example, a silicon photodiode is used for the photoreceiver 58. The photoreceiver 58 is connected to the control unit 44, and the light reception signal at the photoreceiver 58 is sent to the control unit 44. The phase of the long-wavelength probe light is detected based on the light reception signal received by the photoreceiver 58. Note that, although the above example shows an example in which the PBS 54 splits the light into two beams toward the photoreceivers 56 and 58, the transmission and reflection of light by the PBS 54 may be reversed. For example, the PBS 54 may be configured to reflect short-wavelength probe light of less than 775 nm and pass long-wavelength probe light of 775 nm or more or exceeding 775 nm. Furthermore, the light beams directed from the PBS 54 to the photodetectors 56 and 58 may be split by independent dedicated reflecting members. For example, the PBS 54 may include a first reflecting member that reflects the probe light beams on the short wavelength side less than 775 nm and a second reflecting member that reflects the probe light beams on the long wavelength side equal to or greater than 775 nm.

[0042] The wavelengths of the excitation light and the probe light partially overlap, and by measuring the spectral interference at each wavelength, it is possible to measure the phase offset of the probe light relative to the excitation light. For example, since the polarizations of the excitation light Le and the probe light are orthogonal, when they pass through a birefringent crystal, the relative delay between the excitation light Le and the probe light can be adjusted (for example, to about 2 ps) due to the optical path difference between the polarizations. This brings the relative delay between the excitation light Le and the probe light closer to zero, and as will be described later, the spectral interference amplitude detected by each photodetector can be increased. Furthermore, when the phase of the excitation light or the probe light is modulated, the spectral interference intensity is modulated, and the phase offset of the probe light can be measured from the interference intensity waveform.

[0043] The control unit 44 is a part that controls the photodetector 10 and includes a central processing unit (CPU), a read-only memory (ROM), a random access memory (RAM), etc. (not shown). The control unit 44 also performs a photodetection process to extract the frequency component of SRS light from the reference probe light Lr containing SRS light generated in the sample 33. The control unit 44 also performs a background signal reduction process according to this embodiment, which will be described later.

[0044] The control unit 44 is further connected to the drive circuit (including a signal generator, not shown) of the optical modulator 21, the photodetector 28, the wavelength scanning unit 24, etc. A signal generator or a high-voltage amplifier that generates an electrical signal for changing the drive voltage of the optical modulator 21 to perform phase modulation may be provided inside or externally to the control unit 44. In this case, the control unit 44 controls the signal generator to perform waveform control of the drive voltage for modulating the optical modulator 21, etc. The control unit 44 can be configured using a general personal computer, etc.

[0045] The control unit 44 drives the optical modulator 21 via a drive circuit and receives the amplitude-modulated signal from the photodetector 28 to form a lock-in amplifier, which extracts the SRS signal from the reference probe pulse light Pr that has been amplitude-modulated by heterodyne interference. More specifically, as a result of the heterodyne interference between the reference probe light Lr and the phase-modulated probe light Lp, the amplitude-modulated signal applied to the reference probe pulse light Pr is lock-in detected as a signal corresponding to the SRS signal.

[0046] The control unit 44 includes a derivation unit 45. The derivation unit 45 is a component that derives a stimulated Raman scattering signal by reducing background signals that affect low drug concentration regions, such as those contained in the sample 33, when observing a drug. The derivation unit 45 receives the SRS signal detected by the photodetector 28 and a signal indicating the phase component of the phase difference between the reference probe light Lr and the phase-modulated probe light Lp detected by the phase detection unit 50. The derivation unit 45 converts the input SRS signal into a first stimulated Raman scattering signal, and derives a signal obtained by subtracting the phase component of the phase difference of the input probe light from the phase component of the first stimulated Raman scattering signal, which is the SRS signal, as a second stimulated Raman scattering signal corresponding to the SRS signal with reduced background signals. A detailed description of the reduction of background signals that affect low drug concentration regions will be provided later.

[0047] The control unit 44 is further connected to the wavelength scanning unit 24, and the control unit 44 controls the rotation of the bandpass filter 18 included in the wavelength scanning unit 24 and the movement of the end mirror 20. The control unit 44 is also connected to a piezoelectric element attached to the end mirror 15, and drives the piezoelectric element to slightly move the end mirror 15 in the optical axis direction.

[0048] As described above, the photodetector and photodetection method according to this embodiment converts a material response (instantaneous refractive index change) generated by molecular vibration excited by excitation light Le into a time-delayed intensity modulation of a probe light, and detects the light intensity modulation component synchronized with the modulation frequency using a demodulator such as a lock-in amplifier. Then, a signal is obtained that reduces the background signal affecting low-concentration regions while taking into account the phase difference of the probe light.

[0049] Next, a method for reducing background signals in the photodetector and photodetection method according to this embodiment will be described with reference to Figures 2A to 2B and Figures 3A to 3B. Figure 2A is a diagram illustrating the characteristics of pulsed light on the time axis in the photodetector 10, and Figure 2B is a diagram illustrating the characteristics on the frequency axis. Figures 3A to 3B are explanatory diagrams illustrating the reduction of background signals that affect low drug concentration regions in the photodetector 10, taking into account the phase difference of the probe light.

[0050] In the photodetector 10, the relative delay between the excitation light and the probe light is modulated with an amplitude approximately corresponding to the wavelength of the light to reduce background signals (e.g., non-resonant background signals, etc.). To this end, in the photodetector 10, the end mirror 15 is driven by a piezoelectric element attached to the end mirror 15, and the optical path length of the excitation light Le is periodically varied. This modulation is performed, for example, with a sawtooth wave having a frequency of approximately 300 Hz. The modulation amplitude of the optical path length is set to a movement amount equivalent to a delay of one wavelength of the phase-modulated probe light Lp (e.g., wavelength 758 nm).

[0051] In this embodiment, a configuration in which the optical path length of the excitation light Le is changed by periodically varying the position of the end mirror 15 in the optical axis direction is described as an example. However, since the relative optical path lengths (delay times) of the excitation light Le and the probe pulse light are changed, the optical path length on the excitation light Le side may be fixed and the optical path length on the probe light side may be changed. Changing the relative optical path lengths of the excitation light Le and the probe light is referred to as "optical path length modulation." Note that in this embodiment, a piezoelectric element is described as an example of a mechanism for performing optical path length modulation. However, this is not limited to this. For example, a mirror holding mechanism equipped with a stepping motor or a mechanism driving multiple optical deflection elements (galvanometer mirrors) in conjunction with each other may also be used. Furthermore, the modulation waveform of the optical path length modulation is not limited to a sawtooth wave, and any periodic waveform, such as a sine wave, may be used. However, since a sawtooth wave can be configured so that the phase difference at discontinuous points is 2π, from this perspective, a sawtooth wave is preferably used. In this embodiment, the modulation frequency of the optical path length modulation is set to approximately 300 Hz, but a modulation frequency of several kHz or more is more preferable. However, the modulation frequency of the optical path length modulation is preferably equal to or lower than the modulation frequency of the phase modulation by the optical modulator 21.

[0052] 2A shows the relationship between the time waveform of each pulse and the molecular vibration amplitude, and Fig. 2A<1> shows the time relationship between the excitation pulse light Pe, the reference probe pulse light Pr, and the phase-modulated probe pulse light Pp, i.e., the incidence timing. As shown in Fig. 2A<1>, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp overlap in time, and are separated from the excitation pulse light Pe by a delay time τ ｐｒ That is, in this embodiment, a plurality of molecular vibrations are excited by the pump light Le having a short pulse width, and the vibrations are detected by heterodyne detection using two probe lights. ｐｒ However, the delay time τ ｐｒ is not limited to 500 fs, and may be set appropriately depending on the detection target, etc.

[0053] Here, in FIG. 2A<1>, for convenience, the excitation pulse light Pe and the probe pulse light are depicted as being sufficiently separated in time, but in reality, the excitation pulse light Pe and each probe pulse light have tails, and a non-resonant background signal is generated in the region where the tails overlap.

[0054] 2A<1>, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp are represented by their respective envelopes. Meanwhile, the pulse-like waveforms shown inside the envelopes conceptually represent beat signals resulting from the frequency difference between the reference probe pulse light Pr and the phase-modulated probe pulse light Pp. As shown in FIG. 2A<1>, in this embodiment, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp use waveforms that are asymmetric in time, and the reference probe pulse light Pr and the phase-modulated probe pulse light Pp have rise times of, for example, 0.4 ps and fall times of, for example, 1.9 ps.

[0055] On the other hand, Figures 2A<2> and <3> show molecular vibration amplitudes, with Figure 2A<2> showing the molecular vibration of a sample (in this embodiment, a drug is used as an example), and Figure 2A<3> showing the background molecular vibration. As shown in Figure 2A<2>, the vibration of the sample is a relatively long-lived vibration that starts from the position of the excitation pulse light Pe and continues almost to the position where the probe pulse light ends. In contrast, as shown in Figure 2A<3>, the background molecular vibration has a short life compared to the molecular vibration of the sample, and almost disappears near the rising position of the probe pulse light. In other words, the delay time τ between the excitation pulse light Pe and the probe pulse light ｐｒ is set so that the probe pulse light rises just as the background molecular vibration subsides.

[0056] Next, the frequency (wavelength) characteristics of the excitation pulse light Pe, the reference probe pulse light Pr, and the phase-modulated probe pulse light Pp will be described with reference to Fig. 2B. Fig. 2B <1> shows the optical output frequency characteristics of the excitation pulse light Pe, <2> shows the optical output frequency characteristics of the reference probe pulse light Pr and the phase-modulated probe pulse light Pp, and <3> shows the group delay frequency characteristics of the reference probe pulse light Pr and the phase-modulated probe pulse light Pp. Note that the scales of the horizontal axes (angular frequency ω) in Fig. 2B <1> to <3> are the same.

[0057] 2B<1>, in this embodiment, an ultrashort pulsed light is used as the excitation pulsed light Pe, so the excitation pulsed light Pe has a wide bandwidth. As described above, the excitation pulsed light Pe has a central wavelength of approximately 790 nm and a pulse width of approximately 15 fs.

[0058] In contrast, the reference probe pulse light Pr is filtered by the bandpass filter 18, and the phase-modulated probe pulse light Pp is filtered by the bandpass filter 16, respectively, so that the optical output frequency characteristics of the reference probe pulse light Pr and the phase-modulated probe pulse light Pp are band-limited as shown in FIG. 2B<2>. According to this embodiment, the reference probe pulse light Pr has a center wavelength of 790 nm to 870 nm and a bandwidth of approximately 20 nm, for example, while the phase-modulated probe pulse light Pp has a center wavelength of approximately 758 nm and a bandwidth of approximately 18 nm, for example. That is, in this embodiment, the bandwidth of the reference probe pulse light Pr and the bandwidth of the phase-modulated probe pulse light Pp are set to be approximately equal. Here, the difference between the center wavelength range of 790 nm to 870 nm of the reference probe pulse light Pr and the center wavelength of 758 nm of the phase-modulated probe pulse light Pp constitutes the wavelength scanning range, which corresponds to the detection range of molecular vibrations of the sample. In other words, the photodetector 10 according to this embodiment has a wavelength scanning (spectroscopic scanning) range of 80 nm. Of course, the center wavelengths and wavelength scanning ranges of the reference probe pulse light Pr and the phase-modulated probe pulse light Pp are merely examples, and may be set to appropriate values ​​depending on the design conditions of the photodetector 10. Note that the scanning of the center wavelength of the reference probe pulse light Pr is performed by rotating the band-pass filter 18, as described above.

[0059] On the other hand, as shown in FIG. 2B<3>, the delay frequency characteristics of the reference probe pulse light Pr and the phase-modulated probe pulse light Pp have a fixed delay time τ ｐｒ are commonly applied. In this embodiment, furthermore, a nonlinear chirp resulting from passing through each of the bandpass filters 18 and 16 is imparted to the reference probe pulse light Pr and the phase-modulated probe pulse light Pp. In this embodiment, the shape of the nonlinear chirp of the reference probe pulse light Pr and the shape of the nonlinear chirp of the phase-modulated probe pulse light Pp are set to be substantially the same.

[0060] Here, as described above, the center frequency of the reference probe pulse light Pr is different from the center frequency of the phase-modulated probe pulse light Pp. Hereinafter, the difference between the center frequency of the reference probe pulse light Pr and the center frequency of the phase-modulated probe pulse light Pp will be referred to as the "frequency difference Ω Ｒ "

[0061] With the above configuration, the reference probe pulse light Pr and the phase-modulated probe pulse light Pp each have a time-stretched asymmetric waveform, as shown in Fig. 2A<1>. When pulses of approximately equal bandwidths are chirped by approximately the same amount, the difference frequency of the time-stretched asymmetric pulses becomes a constant frequency regardless of time. This difference frequency is determined by the difference in the center frequencies of the probe lights, i.e., the frequency difference Ω Ｒ Among the molecular vibrations excited by the excitation light Le, this constant frequency difference Ω Ｒ Only the vibration component that matches the frequency is converted into the optical intensity modulation of the probe light.

[0062] That is, when the sample 33 is irradiated with three pulsed laser beams, namely, the excitation pulsed beam Pe, the reference probe pulsed beam Pr, and the phase-modulated probe pulsed beam Pp, first, multiple molecular vibrations are simultaneously excited by the excitation pulsed beam Pe, and then energy is exchanged between the probe beams through the stimulated Raman scattering process caused by the reference probe pulsed beam Pr and the phase-modulated probe pulsed beam Pp. At this time, the phase modulation applied to the phase-modulated probe pulsed beam Pp is converted into optical intensity modulation of the reference probe pulsed beam Pr (and the phase-modulated probe pulsed beam Pp). The detected frequency is the frequency difference Ω between the reference probe pulsed beam Pr and the phase-modulated probe pulsed beam Pp. Ｒ On the other hand, since the frequency resolution is determined by the reciprocal of the pulse width, applying a stronger chirp will improve the ability to distinguish molecules.

[0063] Next, the principle of reducing background signals (e.g., signals including non-resonant background signals) in low-concentration regions in the photodetector 10 will be described. In the following description, the relative delay time τ between the excitation pulse light Pe and the probe pulse light is ｐｒNote that the delay of the pump pulse light Pe is set to 0.

[0064] In the conventional signal detection by time-resolved phase-modulated SRS, it is common to output only the absolute value of the signal without using information on the phase offset of the lock-in amplifier output. However, in the case of drug signals in the sub-millimolar (millimolar) range (for example, with an intensity modulation factor of 10 －７ In some cases, the linearity of the SRS signal relative to concentration is not maintained when the signal is small relative to noise or residual background light, as in the signal shown below. Numerous experiments and simulations have revealed that this phenomenon of the SRS signal not maintaining linearity relative to concentration is caused by optical phase fluctuations originating from the device. Therefore, by detecting the optical phase fluctuations originating from the device and applying them to the SRS signal, a signal that maintains linearity relative to concentration can be obtained even in low-concentration regions. Specifically, the phase detection unit 50 detects the optical phase fluctuations originating from the device and obtains a phase-locked complex signal. This phase-locked complex signal corresponds to obtaining an SRS signal based on the optical phase of the device, and the signal's linearity can be ensured on the complex plane. If signal linearity can be ensured in this way, quantitative detection of materials in low-concentration regions is not impossible.

[0065] First, the electric field e of the excitation pulse light Pe ｐｕｍｐ is shown below (Equation 1), and the electric field e of the phase-modulated probe pulse light Pp ＰＭ is shown below (Equation 2), and the electric field e of the reference probe pulse light Pr ＬＯ are expressed by the following (Equation 3). Here, in (Equation 1) to (Equation 3), ω represents frequency and φ represents phase. In the following explanation, italicized letters in the formulas will be written as non-italicized letters.

[0066] The phase φ of the SRS signal based on the above equations ＳＲＳ is expressed by the following (Equation 4), and the phase φ of the photodetector is Ｍ is expressed by the following (Equation 5).

[0067] As shown in the above (Equation 4) and (Equation 5), the phase offset component (φ ＰＭ -φ ＬＯ ) is the phase φ of the SRS signal ＳＲＳ and the phase φ of the photodetector Ｍ Therefore, by taking the difference between the two as shown in (Equation 6) below, the fluctuating phase offset component can be cancelled.

[0068] From the above, the phase offset component (φ ＰＭ -φ ＬＯ ) from the SRS signal, the fluctuating phase offset component can be cancelled.

[0069] Next, the reduction of the background signal will be further explained with reference to FIGS. 3A and 3B.

[0070] 3A is a conceptual diagram showing a complex time-resolved SRS signal on a complex plane. As shown in FIG. 3A, the complex time-resolved SRS signal shows noise, background signals, and drug signals in a distinguishable manner on the complex plane. As described above, these noise and background signals are considered to be caused by optical phase fluctuations originating from the device. Therefore, a phase offset component (φ ＰＭ -φ ＬＯ ) is subtracted from the signal (second stimulated Raman scattering signal), a signal with linearity ensured on the complex plane can be obtained as a phase-locked complex signal. FIG. 3B is a conceptual diagram showing the relationship between time-resolved SRS signal intensity and concentration for a drug in a low concentration range. As shown in FIG. 3B, it can be seen that conventional signal detection that outputs only the absolute value of the signal does not maintain the linearity of the SRS signal relative to concentration in the low concentration range. On the other hand, it can be seen that the linearity of the SRS signal relative to concentration is maintained by using a signal (second stimulated Raman scattering signal) obtained by subtracting a phase offset component indicating optical phase fluctuations originating from the SRS signal.

[0071] Next, the background signal reduction process including the low density region executed by the control unit 44 of the light detection device 10 will be described with reference to Fig. 4. Fig. 4 is a flowchart showing the processing flow of the background signal reduction process program. This background signal reduction process program is stored in a storage device such as a ROM (not shown), and the CPU reads it from the ROM or the like, loads it into a RAM or the like, and executes it.

[0072] In step S100, the power supply to the light source 11 is turned on.

[0073] In step S102, a drive circuit (not shown) for the optical modulator 21 is operated to turn on the phase modulation by the optical modulator 21, thereby adjusting the phase.

[0074] In step S104, the drive circuit (not shown) for the piezoelectric element attached to the end mirror 15 is operated to turn on the optical path length modulation. Note that, although the present embodiment will be described by way of example with the phase modulation and then the optical path length modulation turned on in this order, this order may be reversed.

[0075] In step S106, the optical signals received by the photodetectors 56 and 58 in the phase detection unit 50 are detected, and the phase φ of the probe light from the device is detected. Ｍ That is, the phase offset of the probe light relative to the excitation light is detected by measuring the spectral interference between the excitation light and the probe light.

[0076] In step S108, the stimulated Raman signal (SRS signal) is sampled based on lock-in detection. It is preferable that the detection of the SRS signal continues until a predetermined signal detection time has elapsed (until sampling at all sampling points has been completed). In step S108, the phase φ of the SRS signal is detected. ＳＲＳ In this embodiment, the phase difference of the probe light (the phase difference φ Ｍ ) and the phase of the stimulated Raman signal φ ＳＲＳ However, this order may be reversed, or the processes may be performed simultaneously.

[0077] In step S110, the phase offset is removed from the SRS signal. That is, the phase φ of the detected SRS signal is removed. ＳＲＳ A signal in which the phase offset of the detected probe light relative to the excitation light is subtracted from the phase of the detected probe light is derived, thereby obtaining a signal in which linearity is ensured on the complex plane as a phase-locked complex signal. The signal in which linearity is ensured on the complex plane corresponds to an SRS signal (second stimulated Raman scattering signal), which is a signal component derived from Raman scattering in the sample 33. In this case, the complex amplitude component on the complex plane can be associated with the optical intensity modulation component.

[0078] In step S112, signal processing is performed using the detection signals sampled up to that point, and the spectrum of the stimulated Raman signal is calculated using signals that have ensured linearity on the complex plane. After that, this processing routine is terminated.

[0079] Next, a concentration estimation system 60 using the above-described light detection device 10 will be described.

[0080] The concentration estimation system 60 estimates the concentration of a sample using the photodetector 10 described above. Specifically, the concentration estimation system 60 measures a sample (e.g., a standard sample) containing a target component (e.g., a drug) whose concentration has been adjusted in advance using the photodetector 10, and derives a calibration curve using a complex signal (the second stimulated Raman scattering signal described above) determined by the signal intensity and phase. The calibration curve indicates the correspondence between the signal intensity (complex amplitude) obtained by measuring the sample and the concentration of the sample. Then, a sample containing an unknown concentration of the target component is measured using the photodetector 10, and the concentration corresponding to the signal intensity is determined based on the derived calibration curve. The determined concentration is then estimated as the unknown concentration of the sample. Note that the sample whose concentration is to be estimated may contain a background signal, such as a biological sample. In this case, the complex signal of a reference sample not containing the target component is measured in advance, and the concentration of the target component can be estimated based on the difference between the complex signal of the reference sample and the complex signal of the sample containing the target component. Furthermore, the calibration curve may be obtained by storing data indicating the calibration curve derived in advance through measurements in a memory, and the stored data may be read out and used.

[0081] 1, the concentration estimation system 60 includes a concentration estimation device 62 connected to the control unit 44 of the photodetector 10. The concentration estimation device 62 is a component that estimates the unknown drug concentration contained in a sample based on the SRS signal detected by the photodetector 10, and is configured to include a CPU (Central Processing Unit), ROM (Read Only Memory), RAM (Random Access Memory), etc. (not shown). The control unit 44 may also serve as the concentration estimation device 62.

[0082] The concentration estimation device 62 is functionally configured to operate as each of a calibration curve derivation unit 64, a background light measurement unit 66, and a concentration estimation unit 68. The concentration estimation system 60 and the concentration estimation device 62 are an example of a concentration estimation system of the present disclosure. The calibration curve derivation unit 64 is an example of a calibration curve derivation unit of the present disclosure, the background light measurement unit 66 is an example of a complex amplitude component detection unit of the present disclosure, and the concentration estimation unit 68 is an example of an estimation unit of the present disclosure.

[0083] The calibration curve derivation unit 64 operates to derive a calibration curve showing the correspondence relationship between the drug concentration and the complex signal at the Raman frequency of the estimation target. The derived calibration curve usually exhibits a linear change in amplitude with respect to the drug concentration, but may become curved when the drug concentration is high or due to its relationship with the surrounding solvent. Specifically, the calibration curve derivation unit 64 acquires the Raman spectrum of the drug contained in the sample by measurement using the photodetector 10, and derives a calibration curve (see FIG. 3B ) using a specific Raman frequency corresponding to one of the peaks in the acquired Raman spectrum. This calibration curve shows the correspondence relationship between the signal intensity, which is the complex amplitude, and the drug concentration. That is, the calibration curve derivation unit 64 measures a standard sample containing the estimation target component and derives a calibration curve from a complex signal (second stimulated Raman scattering signal) determined by the signal intensity and phase. It is preferable that the sample (standard sample) used for measurement in the calibration curve derivation unit 64 has a known drug concentration, the drug concentration of which has been adjusted in advance.

[0084] The background light measurement unit 66 operates to measure the complex amplitude of a reference sample at the Raman frequency of the estimation target. That is, the background light measurement unit 66 measures the complex amplitude using a sample with a predetermined drug concentration as a reference sample relative to the estimation target sample, and acquires information to be subtracted as a background light component when estimating the unknown drug concentration. Specifically, the background light measurement unit 66 measures the complex amplitude of the reference sample at the specific Raman frequency using the photodetector 10. It is desirable that the drug concentration of the reference sample is known, and more preferably, the drug concentration is zero.

[0085] The concentration estimation unit 68 operates to estimate the unknown drug concentration in a sample having an unknown drug concentration based on the calibration curve. The concentration estimation unit 68 can also operate to reduce the background light component. Specifically, the concentration estimation unit 68 measures the complex signal of the target sample using the light detection device 10, and estimates the drug concentration based on the complex signal obtained by subtracting the complex amplitude of the background light measurement unit 66 from the measurement result and the calibration curve obtained by the calibration curve derivation unit 64. That is, the concentration estimation unit 68 determines the concentration of the calibration curve corresponding to the complex signal obtained by subtracting the complex amplitude of the background light from the complex signal of the target sample, and estimates the determined concentration as the drug concentration of the target sample. The concentration estimation unit 68 can omit the operation of subtracting the complex amplitude of the background light measurement unit 66 to obtain the complex signal. In this case, the complex signal of the target sample can be used as is.

[0086] Next, the concentration estimation process executed by the concentration estimation device 62 of the concentration estimation system 60 will be described with reference to Fig. 5. Fig. 5 is a flowchart showing the flow of processing of a program for causing a CPU (not shown) to execute the concentration estimation process. The concentration estimation process program is stored in a storage device such as a ROM (not shown), and the CPU reads it from the ROM or the like, loads it into a RAM or the like, and executes it.

[0087] In step S200, the calibration curve deriving unit 64 derives a calibration curve that indicates the correspondence between the drug concentration of the standard sample and the complex signal at a specific Raman frequency.

[0088] In step S210, the background light measuring unit 66 measures the complex amplitude of the reference sample at a specific Raman frequency.

[0089] In step S220, the concentration estimation unit 68 measures the complex signal of the target sample with an unknown drug concentration, and estimates the drug concentration based on the complex signal obtained by subtracting the complex amplitude of the background light measurement unit 66 from the measurement result and the calibration curve obtained by the calibration curve derivation unit 64. Thereafter, this processing routine ends.

[0090] In the above-described photodetector 10, an example has been described in which a portion of the combined light Lg is guided by the mirror 38 (sampling mirror) to the phase detection unit 50 for use, but the phase detection unit 50 is not limited to using the combined light Lg sampled by the mirror 38 (sampling mirror). For example, the phase detection unit 50 may be configured to guide a portion of the combined light Lg to the phase detection unit 50 at any position in the optical path where the combined light Lg can be used before being irradiated onto the sample.

[0091] Furthermore, in the above-described photodetector 10, an example has been described in which a portion of the combined light Lg is guided to and used by the phase detector 50, but the phase detector 50 is not limited to using the combined light Lg. The phase detector 50 only needs to be able to detect the phase difference (reference phase) between the reference probe light Lr and the phase-modulated probe light Lp, and may use the reference probe light Lr and the phase-modulated probe light Lp before being combined with the excitation light Le.

[0092] FIG. 6 is a diagram illustrating a first modified example of the phase detector in the photodetector 10 described above. The first modified example illustrated in FIG. 6 illustrates a configuration example in which a phase detector is applied to the photodetector 10. As illustrated in FIG. 6 , the photodetector 10 of the first modified example includes a phase detector 70 as a phase detector that detects the phase difference between the reference probe light Lr and the phase-modulated probe light Lp. In the first modified example, the phase detector is configured using light passing through a bandpass filter 16 (DM). Specifically, the phase detector 70 includes a PBS 72, a light source 73 (denoted as "Laser" in FIG. 6 ), and a photodetector 74 (denoted as "PD" in FIG. 6 ). The phase detector 70 configures an interferometer using a light source 73, which is a laser light source that emits light near the cutoff wavelength of the bandpass filter 16 (DM). The phase detection unit 70 is configured such that laser light generated by a light source 73 is guided to the reference probe pulse light adjusting unit 41 and the phase-modulated probe pulse light adjusting unit 42 via a PBS 72, a mirror 71, and a band-pass filter 16 (DM) in this order, and return light (Lr and Lp) from both units reaches a photodetector 74 via the band-pass filter 16 (DM), the mirror 71, and the PBS 72. The photodetector 74 is configured to detect the reference probe light Lr and the phase-modulated probe light Lp. The photodetector 74 may also be configured to detect the reference probe light Lr and the phase-modulated probe light Lp separately. The phase detection unit 70 can detect the relative phase difference in the same way as the phase detection unit 50 described above, and therefore a detailed description thereof will be omitted.

[0093] Although the above description has been given of an example in which the phase difference between the reference probe light Lr and the phase-modulated probe light Lp is detected by the phase detection unit 50 (or 70) configured as an interferometer, the technology disclosed herein is not limited to configuring an interferometer. For example, the above-described relative phase difference may be detected by using a spectrometer.

[0094] FIG. 7 is a diagram showing a second modified example of a phase detection unit in the photodetector 10. In the second modified example, the phase detection unit is configured using a spectroscope. Note that the second modified example shown in FIG. 2 illustrates a configuration example in which a phase detection unit is applied to the photodetector 10, similar to the first modified example. As shown in FIG. 7 , the photodetector 10 of the second modified example includes a phase detection unit 80 configured as a spectroscope as a phase detection unit. Specifically, the phase detection unit 80 includes a birefringent crystal 81 (denoted as "BBO" in FIG. 7 ) having a configuration similar to the birefringent crystal 52, a diffraction grating 82 (denoted as "Grating" in FIG. 7 ), a lens 83, and a photoreceiver 84 such as a camera (denoted as "Camera" in FIG. 7 ). The diffraction grating 82 is an example of a spectroscopic unit of the present disclosure, and the photoreceiver 84 is an example of a spectroscopic phase measurement unit of the present disclosure.

[0095] 7 , the phase detection unit 80 is configured so that a portion of the combined light Lg sampled by the mirror 38 (sampling mirror) passes through a birefringent crystal 81, a diffraction grating 82, and a lens 83 in this order, and is guided to a photodetector 84. In the phase detection unit 80, the relative delay between the excitation light Le and the probe light is adjusted by the birefringent crystal 81, and the interference light with an increased interference amplitude is irradiated onto the diffraction grating 82, dispersed by the diffraction grating 82, collimated by the lens 83, and received by the photodetector 84. That is, in the phase detection unit 80, the spectral interference light of the combined light Lg is detected by the photodetector 84, such as a camera. The spectral interference light detected by the photodetector 84, such as a camera, can be used to detect the relative phase difference between the reference probe light Lr and the phase-modulated probe light Lp.

[0096] Furthermore, in the above-described photodetector 10, the birefringent crystal 52 (or 81) of the phase detection unit 50 (or 80) adjusts the relative delay between the excitation light Le and the probe light, but the birefringent crystal may be omitted. When the birefringent crystal is omitted in the phase detection unit, the optical path length of the combined light Lg to be combined can be adjusted so that the relative delay between the excitation light Le and the probe light is approximately zero. When the birefringent crystal is omitted in the phase detection unit, a birefringent crystal can be placed downstream of the mirror 38 and in the optical path of the eye through which the combined light Lg is irradiated onto the sample, so that the desired delay occurs on the microscope. When the birefringent crystal is placed downstream of the mirror 38, it is preferable to adjust the thickness of the birefringent crystal so that the desired delay occurs on the microscope between the excitation light Le and the probe light.

[0097] Next, examples of the photodetector, photodetection method, concentration estimation system, and concentration estimation method according to the present embodiment will be described with reference to FIGS. 8 , 9A, and 9B. FIG. 8 illustrates the spectral distribution of a sample obtained by the photodetector when cyclohexane is used as a target component sample. FIG. 8 illustrates a comparative example obtained using the absolute value of the SRS signal with a conventional photodetector that does not consider the phase difference of the probe light, and a practical example obtained by the photodetector 10 according to the embodiment after phase locking is applied, using the X component (the 0-degree direction (real axis direction) component of the signal amplitude on a complex plane where the phase angle of the peak wavenumber is zero) based on the X component. In FIG. 8, comparative examples are indicated by squares, and practical examples are indicated by black circles. FIG. 9A illustrates the relationship between the SRS signal obtained by the photodetector described above and the concentration of the target component when a dimethyl sulfoxide (DMSO) aqueous solution is used as a sample containing the target component. FIG. 9B is an enlarged view of the low-concentration region (0.50 mM or less) in FIG. 9A. In FIG. 9B, a square is dotted at the center point of the measurement result in the comparative example, and a black circle is dotted in the example.

[0098] As shown in FIG. 8 , compared to when the phase difference of the probe light is not taken into account, the photodetector according to the embodiment exhibits multiple peaks in the spectral distribution of the measurement results, making it possible to set a specific frequency as the Raman frequency. Furthermore, the appearance of multiple peaks in the spectral distribution makes it possible to obtain a signal waveform faithful to the optical modulation waveform. Furthermore, as shown in FIGS. 9A and 9B , by using the photodetector according to the embodiment, the correspondence between the SRS signal and the concentration of the target component in the sample becomes linear. In particular, by taking the phase difference of the probe light described above into account, it can be seen that the concentration characteristics become linear, even in the low concentration range of a drug, for example.

[0099] As described above, the photodetector and photodetection method according to this embodiment are configured to detect the relative phase difference between probe pulse lights and subtract it from the SRS signal. This reduces the influence of phase fluctuations caused by the phase offset between the two probe lights on the SRS signal. Therefore, even if the drug concentration in the sample, which is affected by the phase offset, is in a low concentration range, an SRS signal with a linear concentration characteristic can be obtained. Furthermore, the concentration estimation system and concentration estimation method according to this embodiment can obtain an SRS signal with a linear concentration characteristic by using the above-mentioned photodetector and photodetection method, and can quantify the drug concentration in the sample from a calibration curve with reduced phase fluctuations caused by the phase offset. For example, it becomes possible to detect and quantify low concentrations of drugs below mM (millimolar), which was previously difficult.

[0100] In the above embodiment, a configuration in which one excitation pulse light Pe is used has been described as an example, but the present invention is not limited to this, and a configuration in which a plurality of excitation pulse light Pe are used may also be used. When a plurality of excitation pulse light Pe are used, the excitation power to the sample can be gradually increased.

[0101] All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.

[0102] The disclosure of Japanese Patent Application No. 2024-009670, filed on January 25, 2024, is incorporated herein by reference in its entirety.

Claims

1. A light detection device, comprising: a laser light source that generates pulsed light of a light source; a branching unit that branches the pulsed light of the light source into excitation light, first probe light, and second probe light; an optical path difference adjustment unit that adjusts a relative optical path difference between the excitation light, the first probe light, and the second probe light; a phase modulation unit that phase-modulates at least one of the first probe light and the second probe light; a first detection unit that irradiates a sample with combined light in which the excitation light, the first probe light, and the second probe light are combined, and detects a generated first induced Raman scattering signal; a second detection unit that detects a relative phase difference between the first probe light and the second probe light; and a derivation unit that derives a second induced Raman scattering signal obtained by subtracting a phase component of the phase difference detected by the second detection unit from a phase component of the first induced Raman scattering signal detected by the first detection unit, as an induced Raman scattering signal.

2. The light detection device according to claim 1, wherein the second detection unit includes: a phase measurement branching unit that branches a part of the combined light before irradiating the sample into first phase measurement light for measuring the phase of the probe light on the long wavelength side and second phase measurement light for measuring the phase of the probe light on the short wavelength side; and a phase measurement unit including a first phase measurement unit that measures the phase of the probe light on the long wavelength side and a second phase measurement unit that measures the phase of the probe light on the short wavelength side.

3. The light detection device according to claim 1, wherein the second detection unit detects a relative phase difference between the first probe light and the second probe light using the first probe light and the second probe light before combining the excitation light.

4. The light detection device according to claim 1, wherein the second detection unit includes: a spectroscopic unit that spectroscopically divides a part of the combined light before irradiating the sample into probe light on the long wavelength side and probe light on the short wavelength side; and a spectroscopic phase measurement unit that measures the phases of the probe light on the long wavelength side and the probe light on the short wavelength side.

5. The second detection unit includes an optical member that adjusts, upstream of the phase measurement branching unit, the relative phase difference between the excitation light and the first probe light and the second probe light in the combined light so that the interference amplitude by the excitation light, the first probe light, and the second probe light becomes large. The optical detection device according to claim 2.

6. For the excitation light, the first probe light, and the second probe light into which the light source pulse light is branched, adjust the relative optical path difference between the excitation light, the first probe light, and the second probe light, phase-modulate at least one of the first probe light and the second probe light, irradiate a sample with the combined light in which the excitation light, the first probe light, and the second probe light are combined, detect the generated first induced Raman scattering signal, detect the relative phase difference between the first probe light and the second probe light, and derive, as an induced Raman scattering signal, a second induced Raman scattering signal obtained by subtracting the phase component of the detected phase difference from the phase component of the detected first induced Raman scattering signal. A light detection method.

7. A calibration curve derivation unit that derives a calibration curve showing the correspondence between the concentration of the material contained in the sample and the complex signal based on the signal intensity and phase in the second induced Raman scattering signal derived by the optical detection device according to claim 1 for a sample containing a predetermined material, and obtains a complex signal determined from the signal intensity and phase in the second induced Raman scattering signal derived by the optical detection device for the sample containing the material with an unknown concentration, and estimates, as the unknown concentration, the concentration corresponding to the obtained complex signal based on the calibration curve. A concentration estimation system comprising: an estimation unit.

8. The concentration estimation system according to claim 7, further including a complex amplitude component detection unit that detects a complex amplitude component in the complex signal of the sample containing the material with a predetermined concentration, wherein the estimation unit derives a subtracted complex signal obtained by subtracting the complex amplitude component detected by the complex amplitude component detection unit from the second induced Raman scattering signal derived for the sample containing the material with an unknown concentration, and estimates, as the unknown concentration, the concentration corresponding to the subtracted complex signal based on the calibration curve.

9. Based on the complex signal determined from the signal intensity and phase in the second induced Raman scattering signal derived by the photodetection device according to claim 1 for a sample containing a predetermined material, a calibration curve showing the correspondence between the concentration of the material contained in the sample and the complex signal is derived. For the sample containing the material with an unknown concentration, a complex signal determined from the signal intensity and phase in the second induced Raman scattering signal derived by the photodetection device is obtained, and based on the calibration curve, the concentration corresponding to the obtained complex signal is estimated as the unknown concentration. A concentration estimation method.

Images