JPH09145619A - Method and instrument for spectroscopic measurement of scattered light and so on - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect anti-Stokes Raman scattered light by utilizing a conventional Raman scattering system intactly. SOLUTION: An excited light beam 4 from a laser 2 is made to pass through a bandpass filter 6 and cast on a sample 10 through a collimator lens 8. For receiving the scattered light from the sample 10, an optical regulating part 16 having a camera lens 12 and a condenser lens 14 is provided. The camera lens 12 receives the scattered light generated from the sample 10 and forms the parallel light. The condenser lens 14 is the lens having chromatic aberation and receives and condenses the parallel light from the cameral lens 12. At the light condensing position of the condenser lens 14, an input slit 19 of a spectroscope 18 is provided as the light receiving port. The input slit 19 has the width of about 20μm. The input slit 19 is arranged at a position, where anti-Stokes Raman scattered light is condensed by the chromatic aberation of the condenser lens.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention measures a substance to be analyzed in a mixture sample, particularly a biological substance such as urine or plasma by a light scattering method, and qualitatively / quantitatively measures its components such as glucose, acetone and urea. Anti-Stokes Raman scattering as light scattering.
The present invention relates to a measurement method using (Anti-Stokes-Raman) scattering and its apparatus.

[0002]

2. Description of the Related Art There are many methods for quantitatively measuring the concentration of a substance by the method of light scattering. In the method utilizing Raman scattering, a method of nondestructively measuring by utilizing the fact that the Raman scattering intensity is proportional to the glucose concentration in the sample (Japanese Patent Laid-Open No. 7-
No. 49309). In that method, in order to suppress the fluorescence from the sample, near infrared light is used as the excitation light, the scattered light from the sample is collected by a lens, and the Rayleigh scattered light is shielded using a notch filter, and then the spectrum is separated. The Raman scattering spectrum of the target substance in the sample is obtained by spectroscopic analysis, and the target substance is quantified.

Conventionally, in order to quantify a target substance by avoiding fluorescence emitted from a mixture of a biological substance, a substance derived from a living body, or other fluorescent substance, a laser having a near infrared wavelength or a longer wavelength than that is used as an excitation light source. Used (“Laser Raman spectroscopy and clinical medicine” by Yukihiro Ozaki et al .: O Plus E 1992 4
92-99). However, since the intensity of Raman scattering weakens in inverse proportion to the fourth power of the excitation wavelength, when the excitation light is in the near infrared, the intensity of the detected light becomes significantly weaker than the excitation by the visible light source.

Further, when the excitation light has a wavelength longer than that of visible light, it is necessary to make the detector and the spectroscope associated therewith suitable for a longer wavelength. And, near-infrared surface elements and array elements are currently very expensive. Further, there is an FT-Raman spectroscopic method that uses Fourier transform as a method for measuring Raman scattering using near-infrared wavelength light, but the FT-Raman spectroscopic device is large in size, and there is a drawback in that measurement also takes time. .

[0005]

When a sample is measured by using the above Raman scattering or light scattering methods, the target scattered light spectrum from the sample is often erased by fluorescence from the sample. . The fluorescence is particularly strong when the sample is a living body itself, when it is separated and collected from a living body such as blood, urine, feces, saliva, and tear fluid, or when it is a food, fruit, agricultural product, or the like.

In the Raman scattering spectrum, the spectrum of a region where photon energy is large with respect to the excitation light (hereinafter,
An anti-Stokes line) and a small region spectrum (hereinafter referred to as Stokes line) are observed. Stokes line is when light is irradiated on a specific molecule,
Because a small part of the molecule that holds the photon does not return to the original vibrational level even after releasing the held photon, but returns to the different vibrational level of the electron ground state (higher than the ground state) Appear in. The Anti-Stokes line is
Originally, electrons that were in a higher energy level than the ground state emitted most of the irradiated photons, and then did not return to the original level, but transitioned to the ground level with some of the irradiated photons. Appear to do. In other words, anti
Since the energy width of Stokes line and Stokes line is the energy difference between the ground state and the vibrationally excited state,
Stokes lines and Stokes lines generally appear at symmetrical positions in terms of shift wave numbers with respect to the excitation light.

On the other hand, fluorescence has a spectrum in a region having a small quantum energy with respect to the excitation light, that is, a region having a long wavelength, unless the excitation spectrum and the fluorescence spectrum significantly intersect. Considering this quantumally, the energy of the irradiated light excites the electrons existing in the ground state or other levels only by the irradiated energy, and during the transition from the excited energy level to the ground state It occurs by staying in the level of for a moment. That is, fluorescence generally occurs in a region (long wavelength side) smaller than the quantum energy of excitation light.

Therefore, by observing the anti-Stokes line in Raman scattering analysis, the component spectrum of the target substance can be obtained from the sample sample by the method of Raman spectroscopic analysis avoiding the influence of fluorescence, and the spectrum of the target substance changes. It is considered that the analyte can be identified and quantified by observing the intensity and the intensity.

As a study of anti-Stokes Raman scattering, there is coherent anti-Stokes Raman spectroscopy (CARS) which measures anti-Stokes Raman scattering by injecting pump light and probe light into a substance to be measured. The principle of this spectroscopic method is to force two spectra with only the transition frequency of Raman scattering when the wavelengths of two different frequencies are incident on the sample and the difference in the quantum energy of the incident light matches the transition frequency of Raman scattering. It is utilized that the forced oscillation inside the substance produces a non-linear oscillation having a uniform phase. Therefore, in the anti-Stokes Raman scattering spectrum by the CARS method, unlike the relationship between the concentration and the Raman scattering intensity of the target substance in the usual linear Raman scattering spectrum, the nonlinear interaction between light and the substance becomes remarkable. Furthermore, non-linear phenomena such as multiphoton absorption transitions, scattering due to higher-order coherent Raman processes, stimulated Raman scattering, sum frequency and harmonic generation coexist, and their spectra do not necessarily reflect the concentration of the target sample linearly. is not. Therefore, CARS has not been used as a quantitative analysis method. Furthermore, the spectroscopic system requires several laser light source devices, which makes the device large.

The intensity ratio between the anti-Stokes Raman scattering spectrum and the Stokes Raman scattering spectrum is approximated by the following Boltzmann distribution formula. Anti-Stokes intensity / Stokes intensity = exp (−hν / kT) h: Planck's constant k: Boltzmann's constant T: Absolute temperature ν: Raman shift wavenumber

FIG. 1 shows calculated values of the intensity ratio of the anti-Stokes Raman line and the Stokes Raman line based on the Boltzmann distribution, assuming an absolute temperature of 300 ° K. When the sample temperature is 300 ° K, the intensity ratio is -100.
It is 0.008 at 0 cm ^-1 (the negative sign of the wave number indicates that anti-Stokes Raman scattering occurs by shifting to the shorter wavelength side than the excitation wavelength), and 0.007 at -1500 cm ^-1 .
Therefore, it has been considered impossible to detect the anti-Stokes Raman scattered light by the usual linear Raman spectroscopy method.

The first object of the present invention is to set the detection wavelength on the shorter wavelength side than the excitation wavelength, which results in an expensive FT-
It is possible to detect anti-Stokes Raman scattered light by using a conventional Raman spectroscopic system as it is without using a Raman spectroscope or using a long wavelength CCD element as a photodetector. . A second object of the present invention is to enable accurate qualitative and quantitative analysis by avoiding fluorescence generated when a sample is excited when this method is used as a sample analysis method. That is.

[0013]

The present invention is a method of irradiating a sample with excitation light and measuring an analyte in a sample using anti-Stokes Raman scattered light among scattered light generated from the sample. is there. When this method is applied to a quantitative analysis method, the shift wave number with good correlation between the concentration of the analyte in the sample and the anti-Stokes Raman scattering intensity is selected as the measured shift wave number specific to the analyte. Then, the anti-Stokes Raman scattering intensity at the measured shift wave number is detected, and the analyte in the sample is quantitatively analyzed using the calibration curve.

The measured shift wave number is a shift wave number in which the correlation coefficient R of the correlation between the concentration of the analyte and the anti-Stokes Raman scattering intensity is 0.6 or more, preferably 0.8 or more. The correlation coefficient R is a value calculated by the following formula. R = Σ [(xi−X) (yi−Y)] / {[Σ (xi−X) ² ] [Σ (yi
-Y) ² ]} ^1/2 xi: Concentration at each point of each analyte, yi: Raman scattering spectrum intensity with respect to xi X: Average value of concentration of each analyte Y: Average value of Raman scattering spectrum intensity An example of a sample suitable for applying the method of 1 is a biological substance such as urine or plasma, and an example of an analyte is a component contained in the biological substance such as glucose, acetone (ketone body), or urea.

The measuring apparatus of the present invention is provided with an excitation light source for irradiating a sample with excitation light, an excitation light source for irradiating the sample with excitation light, and an excitation light for irradiating the sample with the excitation light. Condensing optical adjustment unit that collects scattered light, and detects scattered light with light scattering light with the received density of anti-Stokes Raman scattered light increased from the scattered light collected by the condensing optical adjustment unit And a light receiving section for As a specific preferable example, the condensing optical adjustment unit is provided with a lens having chromatic aberration as a condensing system, and the light receiving unit has a light receiving port at an image forming position of light having a wavelength shorter than the excitation light wavelength, The scattered light incident from the light receiving port is detected.

The photodetector of the light receiving section is anti-Stokes
In order to be able to detect the Raman scattering intensity, the light-receiving unit may be provided with a mechanism such as a diffraction grating that disperses the scattered light incident from the light-receiving port, for example, a spectroscope, or it is intended to detect it. A bandpass filter that transmits only the shift wave number of anti-Stokes Raman scattered light is provided in the condensing optical adjustment unit, and even if the photodetector detects the scattered light incident from the light receiving unit without being separated, Good.

It is preferable that the excitation light source section includes a bandpass filter that transmits only the excitation light wavelength with which the sample is irradiated. The condensing optical adjustment unit may include a lens having less chromatic aberration on the light incident side of the lens having chromatic aberration, which collects scattered light generated from the sample and guides it as parallel light to the lens having chromatic aberration.

The condensing optical adjustment unit is provided with a holographic notch filter including the pumping light wavelength in the notch region in order to remove the pumping light wavelength component, or shields the pumping light wavelength and the longer wavelength side. It is preferable to provide a cut filter for In the case of the holographic notch filter, it is preferable that the notch region of the holographic notch filter is symmetrical with the bandpass region of the pumping light source unit.

The light receiving port of the light receiving section may be the entrance slit of the spectroscope, or may be one end face of the single core optical fiber. When using an optical fiber, the other end face of the optical fiber may be guided to a spectroscope or a photodetector.
In order to be able to change the wavelength at which the light receiving density is maximized, it is preferable that the distance between the lens having chromatic aberration of the condensing optical adjustment unit and the light receiving port is variable.

In order to increase the generation efficiency of the anti-Stokes Raman scattered light, the sample portion is provided with a spherical cell and an integrating sphere cell holder in which the portion holding the cell is a reflecting surface. Is preferred. Further, in order to correct the intensity change of the light source light and enhance the reproducibility of the measurement data, an optical system for the reference light for extracting and detecting a part of the excitation light is further provided, and the detection light intensity of the reference light causes・
It is preferable to correct the detection intensity of the Stokes Raman scattered light.

Further, the Boltzmann distribution indicating the existence probability of anti-Stokes Raman scattering is multiplied by the effect of increasing the received light density of the anti-Stokes Raman scattered light according to the present invention, and the result is corrected to make the excitation light wavelength. It is preferable to further include a data processing unit that performs calculation so that the light receiving sensitivity of anti-Stokes Raman scattered light becomes constant over a predetermined range on the shorter wavelength side, because the accuracy of quantitative analysis is improved.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 2 shows a schematic block diagram of the device of the present invention. Reference numeral 2 is an excitation light source, and a high power laser device such as an argon ion laser (514.5 nm, 100 mW) is suitable. The excitation light beam 4 from the laser 2 passes through the bandpass filter 6 to remove the side band, and is irradiated onto the sample in the sample chamber 10 by the collimator lens 8.
The sample is contained in the cell in the case of gas or liquid, but the cell is not necessary in the case of solid.

A condensing optical adjustment unit 16 is provided to receive and adjust scattered light from the sample. The condensing optical adjustment unit 16 includes, for example, a camera lens 12 and a condensing lens 14. The camera lens 12 is for receiving scattered light generated when the sample is excited by the excitation light 4 in the direction of 90 degrees and making it parallel light, and is a compound lens made of a material having little chromatic aberration. Generally, the camera lens is designed so that chromatic aberration is as small as possible, and such a general camera lens can be used here. As the camera lens 12, when scattered light generated from the sample is generated from one point, 450 to 650n
It is preferable that the light of all wavelengths of m can be collimated. The condenser lens 14 is a lens having chromatic aberration, and is formed of, for example, BK7 which is a general lens optical material. The condenser lens 14 receives the collimated light from the camera lens 12 and condenses the collimated light on a focus corresponding to the wavelength.

An entrance slit 19 of the spectroscope 18 is provided as a light receiving opening at the focal position of the desired wavelength by the condenser lens 14, and the entrance slit 19 has a width of about 20 μm. The position where the entrance slit 19 is provided is a position where the anti-Stokes Raman scattered light to be detected is condensed.

The spectroscope 18 is a single spectroscope using a diffraction grating 20 of 300 G / mm and a blaze wavelength of 500 nm, and its resolution is 6.8 cm ^-1 (0.18 nm / pixel). In the spectroscope 18, reference numerals 22, 24, and 26 are mirrors, respectively. The detector 28 is a liquid nitrogen cooled type (EEV element)
256 × 1024 pixel CCD image sensor (US EG & G
(Manufactured by the company, wavelength sensitivity of 200 to 1100 nm), and simultaneously receives and detects multiple wavelengths separated by the spectroscope 18. The spectroscope 18 and the detector 28 constitute a polychromator. The detection signal of the detector 28 is transferred through an optical fiber 30 to a personal computer 32 as a data processing device for data processing.

In FIG. 2, the camera lens 12 and the condenser lens 1
Between 4 and 4, a holographic image containing the excitation light wavelength in the notch region in order to remove the excitation light component from the scattered light.
A notch filter 34 is arranged. The holographic notch filter is, for example, KAISER OPTICAL SYSTE
It can be obtained from MS. INC. (USA). The holographic notch filter 34 has a characteristic of completely blocking wavelength light included in the notch region and transmitting 80% or more of light in the wavelength region other than the notch region.

FIG. 3 is a diagram for explaining the function of the condenser lens 14. 2A corresponds to the optical system of FIG. 2, in which scattered light generated from the sample is collimated by the camera lens 12, and the collimated light 40 is incident on the condenser lens 14. When the parallel light 40 of white light is incident on the condenser lens 14 having chromatic aberration, it forms an image at a position different depending on the wavelength. In the present invention, the shift of the focal position due to the wavelength in the condenser lens 14 is used, and a minute light receiving port is arranged at a position where the anti-Stokes Raman scattered light is condensed, whereby the anti-Stokes Raman scattered light is Increase the light receiving density of.

In the optical adjusting section, the camera lens 12 may be omitted, and the scattered light generated from the sample may be directly received by the condenser lens 14 having chromatic aberration. FIG. 3B shows the function of the condenser lens 14 when the camera lens 12 is not provided. The condenser lens 14 serves as a scattered light generation point 10 of the sample.
The image of the scattered light generating point 10a is formed at a position that is twice or more than the focal length away from a, and forms an image at a different position depending on the wavelength. Also in this case, similarly to the case of FIG. 3A, a minute light receiving port is arranged at a position where the anti-Stokes Raman scattered light is condensed by utilizing the shift of the focal position due to the wavelength in the condenser lens 14. By doing so, the light receiving density of the anti-Stokes Raman scattered light can be increased.

As the material of the condenser lens 14 having chromatic aberration, optical glass such as BK7, synthetic quartz, sapphire or SF11 can be used. As an example, BK7 which is a general lens optical material is used. The change in refractive index with wavelength is shown in FIG. The refractive index changes greatly in the range of 300 to 700 nm. Not only BK7 but also other optical glasses have a common point that the refractive index greatly changes in the range of 300 to 700 nm.

When measuring a Raman scattering spectrum using an argon ion laser as an excitation light source, -4000 to 400
The region of 0 cm ⁻¹ (the negative region is the anti-Stokes Raman scattering spectrum) corresponds to a wavelength of 420 to 650 nm. That is, the region of the anti-Stokes line is a region that is greatly influenced by the chromatic aberration of the condenser lens,
Chromatic aberration can be effectively utilized.

By utilizing the chromatic aberration of the lens, the light receiving density of light having a wavelength longer than that of the excitation light is reduced and the light enters the entrance slit, and the light density of light having a wavelength shorter than that of the excitation light is increased. Can be made incident on the entrance slit. If expressed by Raman scattering spectrum, it is possible to make the light receiving density of the anti-Stokes line higher than that of the Stokes line so that the light enters the entrance slit.

In Table 1, the focal length of the condenser lens is 10 mm.
The distance of the focal point shift due to the wavelength aberration of the incident light when (the value at the wavelength of 500 nm) is shown. The numerical values in the table represent the focal position (mm).

[0033]

[Table 1]

Calculation of the light density of the focal point from the distance of the shift length due to the aberration in Table 1 will be as shown in FIGS. In (A), it is assumed that light with a wavelength of 500 nm forms a circular image with a diameter of 200 μm at the focal point, and image formation with light of another wavelength (expressed by Raman shift wave number) at the image formation position is performed. Calculate the size (area) and set the density of light with a wavelength of 500 nm to 1
Is calculated as the density ratio. (B) and (C) are the results when the same calculation is performed at the positions of the focal points of light having shift wave numbers of -2000 cm ^-1 and -4000 cm ^-1 , respectively, and the density of each wavelength light is 1. The wave number 0 cm ⁻¹ is the excitation light wavelength, the − side is the anti-Stokes Raman region, and the + side is the Stokes Raman and fluorescence region. In the example of (B), the density ratio is 1/2 or less on the long wavelength side where the shift wave number is 1000 cm ^-1 or more. So, like this,
By placing the light receiving port at the focal point position on the shorter wavelength side than the excitation wavelength, it is possible to lower the fluorescence density and increase the anti-Stokes Raman scattered light density for incidence.

Further, as can be seen from FIG. 5, by making the distance between the condenser lens 14 and the light receiving port such as the entrance slit 19 variable, the wavelength with the highest density can be arbitrarily selected. Become.

In the personal computer 32 which realizes the data processing section, the Boltzmann distribution of FIG. 1 showing the existence probability of anti-Stokes Raman scattering and the excitation light wavelength by adjusting the position of the light receiving port to the spectroscope are set. Anti-Stokes Raman scattering over a predetermined range on the shorter wavelength side than the excitation light wavelength by multiplying by the light receiving density enhancement effect on the shorter wavelength side (Figs. 5 (B) and (C)) and modifying the result It is possible to have a function of performing calculation so that the light receiving sensitivity of light is constant. The process for realizing that function is shown in FIG.

FIG. 6A shows a process of keeping the light receiving sensitivity of anti-Stokes Raman scattered light constant. The light receiving port to the spectroscope is arranged on the side of the focal point of light having a wavelength shorter than the excitation light wavelength, and the standard substance is measured to obtain the anti-Stokes Raman scattered light spectrum. The corrected inverse function is calculated and stored so that the sensitivity of the spectrum is constant over a predetermined range. The measured anti-Stokes Raman scattered light spectrum is corrected using its correction inverse function and output.

Next, as shown in FIG. 6 (B), the light receiving port to the spectroscope is arranged at the same position as when measuring the standard substance,
An unknown sample is measured and an anti-Stokes Raman scattered light spectrum is obtained. The correction inverse function is called, the correction calculation is performed on the spectrum, and the anti-Stokes Raman scattered light spectrum of the unknown sample corrected so that the photosensitivity is constant is obtained. In this way, by correcting the light receiving sensitivity to be constant, it becomes easy to perform a quantitative analysis using the spectrum intensity and the peak area of the spectrum.

In the measuring apparatus of FIG. 2, the light receiving section is equipped with a spectroscope for separating scattered light incident from the light receiving port and guiding it to the photodetector. However, the shift wave number of the anti-Stokes Raman scattered light to be detected is provided. The condensing optical adjustment unit 16 may be provided with a bandpass filter that transmits only the light, and the light detector may detect the scattered light that has entered from the light receiving port by the photodetector without separating it.

[0040]

EXAMPLE A measuring apparatus used for actually measuring is shown in FIGS. The same parts as those in FIG. 2 are designated by the same reference numerals. In FIG. 7, the optical path of the excitation light beam 4 from the laser device as the excitation light source 2 is bent by the prism 40,
A thin beam is passed through the bandpass filter 6 and the collimator lens 8 to irradiate the sample in the sample chamber 10. As will be described later in detail with reference to FIG. 8, the sample chamber 10 includes a spherical quartz flow cell 42 and an integrating sphere cell holder 44 that holds the flow cell 42.

The condensing optical system for condensing the scattered light generated by irradiating the sample in the flow cell 42 with the excitation light includes a camera lens 12 having a small chromatic aberration as an objective lens, a holographic notch filter 34, and a chromatic aberration. It has a condenser lens 14 having Spectroscope 18 and detector 2
8 is as shown in detail in FIG.

The condenser lens 14 is movably supported along the optical axis thereof, and is provided with an actuator 48 whose drive is controlled by a position fine adjustment device 46 controlled by the personal computer 32, and an entrance slit of the spectroscope 18. You can move in and out of the direction.

In this measuring apparatus, a correction optical system is provided so that the light source intensity can be measured and the scattered light intensity can be corrected. The correction optical system is arranged obliquely in the optical path across the optical path between the bandpass filter 6 and the collimator lens 8 and a slide glass 50 of transparent plate glass that extracts a part of the excitation light beam 4 as the reference light 40r. In order to make the slide glass 52 that bends the optical path of the reference light 40r extracted by the slide glass 50, a neutral density filter 54 that adjusts the light amount, and the reference light 40r that has passed through the neutral density filter 54 enter the spectroscope 18. A slide glass 56 is provided across the optical path between the holographic notch filter 34 and the condenser lens 14 and obliquely arranged in the optical path. The reference light 40r is incident on the spectroscope 18 together with the scattered light from the sample, is dispersed together with the scattered light, and is detected by the detector 28. By dividing the detected intensity of the scattered light by the detected intensity of the reference light 40r, the scattered light output in which the fluctuation of the light source intensity is corrected can be obtained.

FIG. 7 shows a driving device 60 for the light source and a scanning controller 62 for the spectroscope, which are controlled by the personal computer 32. The personal computer 32 also inputs the detection output of the detector 28,
It also performs the data processing and also functions as a data processing device. Output device 6 for outputting measurement data, etc.
4 and the power supply 66 of the detector 28 are also shown.

Cell 4 used as a sample chamber in this measuring apparatus
2 and a cell holder 44 for holding the cell 42 are shown in FIG.
Shown in The cell 42 is a spherical quartz flow cell,
A cylindrical inlet 60a and outlet 60b are provided on both sides of the spherical flow cell body. The cell holder 44 is in the shape of an integrating sphere and consists of two parts 44a, 44b that hold the cell 42 in combination with each other.
Integrating sphere portion 60 that holds the spherical portion of
And holding portions 62a and 62b for holding the inlet portion 60a and the outlet portion 60b of the cell 42, the entrance hole 64 into which the excitation light beam is incident, and the scattered light generated in the cell 42 in a direction 90 degrees from the incident light direction. And an emission hole 66 that expands outward in order to take it out.

By using such an integrating sphere-shaped cell holder, the excitation light beam incident from the entrance hole 64 is multiple-reflected by the integrating sphere portion 60 to enhance the excitation efficiency, and the scattered light generated is collected. Since the light is emitted from one of the emission holes 66, scattered light is enhanced and high-sensitivity measurement becomes possible.

Next, a measurement example will be shown. The measurement was performed by using the measuring apparatus shown in FIGS. 7 and 8 and irradiating the sample with the argon ion laser beam (wavelength 514.5 nm, output 100 mW) of the light source as the excitation light.

9A and 9B show Raman scattering spectra of 99% acetone measured by a conventional system.
The exposure time of the photodetector is 5 seconds. (B) is an enlarged view. C = O stretching vibration near 790 cm- ¹ is also observed on the anti-Stokes side. However, the intensity ratio is about 0.024.
And the theoretical value expected from the Boltzmann distribution (0.023
It agrees well with 7). 10 and 11 as well as FIG.
In the spectrum of, the spectrum near the pumping light wavelength (wave number 0 cm- ¹ ) is 0, which means that the pumping light intensity is not measured by the reference light in these spectra and the pumping light component is removed by the notch filter. This is because

FIGS. 10A and 10B are measurement examples using the apparatus shown in FIGS. The exposure time of the photodetector is 5 seconds. However, in both this embodiment and the following embodiments, the end face of a single-core optical fiber with a diameter of about 200 μm is placed at the position of the light receiving port that receives the scattered light condensed by the condenser lens 14 and guides it to the spectroscope. It is arranged so that the other end face is guided to the spectroscope 18. In the measurements of these examples, the position of the light receiving port is brought closer to the condenser lens 14 side than the image formation position of the excitation light wavelength by the condenser lens 14, and the desired reception density of the anti-Stokes Raman scattered light is the highest. The position of the condenser lens 14 is adjusted so that (B) is a partially enlarged view of (A), and the intensity ratio between the anti-Stokes Raman line and Stokes Raman line of C = O stretching vibration near 790 cm- ¹ is about 12 times that of the result in Fig. 9. Has become.

FIGS. 11 (A) and 11 (B) are Raman scattering spectra of 1 M glucose aqueous solution measured by the apparatus of the embodiment of FIGS. 7 and 8, (A) showing the whole image and (B) showing -2000.
This is an enlarged view of the range of up to 2000 cm- ¹ . The exposure time of the photodetector is 5 seconds. From the result of FIG. 11, it is understood that the anti-Stokes Raman scattering of the glucose aqueous solution can be measured.

FIG. 12 is a scattering spectrum of human urine, most of which is fluorescence. In the measurement for obtaining the measurement data shown in FIG. 12 and thereafter, the exposure time of the photodetector is 1 second. FIG.
14 is a scattering spectrum of a sample prepared to contain 2 M glucose in human urine, which is measured by the apparatus of the embodiment of FIGS. 7 and 8. FIG. 13 is a wavelength side longer than the excitation light wavelength, and FIG. 14 is a short wavelength thereof. It is an anti-Stokes Raman scattering spectrum showing a range of -200 to -2500 cm- ¹ on the side. FIG.
The Stokes Raman scattering peak of is almost buried in fluorescence, but a clear peak is observed in the anti-Stokes Raman scattering spectrum of FIG.

FIG. 15 shows an anti-Stokes Raman scattering spectrum of a urine sample in which the glucose concentration is changed, measured by the apparatus of the embodiment shown in FIGS. Samples are human urine 250 μl 11 kinds of glucose aqueous solution with different concentration 2
Mix 50 μl to give a urine glucose concentration of 32-1000
It was prepared so as to be mg / dl (glucocard value: (Kyoto Daiichi Kagaku Co., Ltd.)). Figure 16 shows that
2 shows the correlation between anti-Stokes Raman scattering intensity near -1131 cm ^-1 and glucose concentration. In this case, the correlation coefficient R = 0.997, which shows that the correlation is very good. The glucose concentration in urine can be quantified using the correlation of FIG. 16 as a calibration curve.

FIG. 17 shows the correlation coefficient R at -2000 to -100 cm ^-1 between the anti-Stokes Raman scattering and the glucose concentration of the mixed sample of urine and glucose aqueous solution measured above. The range where the correlation coefficient R is 0.9 or more covers a wide range, and a calibration curve can be prepared in these ranges to quantify the glucose concentration in urine. Similar to FIG. 17, FIG. 18 shows the dependence of the correlation coefficient R on the anti-Stokes Raman shift wave number, but the data concentration range is narrowed to 32 to 514 mg / dl. The range where R is 0.9 or more is narrow.

FIG. 19 shows an anti-Stokes Raman scattering spectrum of a mixed solution of human plasma and an aqueous glucose solution. (B) is an enlarged view. FIG. 20 shows the correlation between the anti-Stokes Raman scattering intensity near -1130 cm ^-1 in the spectrum and the glucose concentration. The sample for this measurement was prepared by mixing 250 μl of human plasma with 250 μl of 12 kinds of aqueous glucose solutions having different concentrations, and the plasma glucose concentration was 41, 53, 66, 70, 85, 95, 126, 265, 52.
0, 756, 1036mg / dl (Glucocard value: (manufactured by Kyoto Daiichi Kagaku Co., Ltd.) and adjusted to be 0.5M. In this case, the correlation coefficient is R = 0.995, and the correlation is very high. The glucose concentration in plasma can be quantified using the correlation curve of Fig. 20 as a calibration curve.

FIG. 21 is a graph showing the correlation coefficient between glucose concentration in human plasma and anti-Stokes Raman scattering from -2000 to ⁰ cm ^-1 , where the correlation coefficient R is 0.9 or more. It can be seen that the range is wide, and a calibration curve can be prepared in these ranges to quantify the glucose concentration in plasma.

22 and 23 are scattering spectra of human urine containing acetone in the sample measured by the apparatus of the embodiment of FIGS. 7 and 8. FIG. 22 shows a longer wavelength side than the excitation light wavelength, and FIG. It is an anti-Stokes Raman scattering spectrum showing a range of -200 to -2500 cm ^-1 on the wavelength side. FIG.
The Stokes-Raman scattering peak of is almost buried in the fluorescence, but a clear peak is observed in the anti-Stokes Raman scattering spectrum of FIG.

24 and 25 are scattering spectra obtained by measuring the sample of human urine containing urea with the apparatus of the embodiment of FIGS. 7 and 8. FIG. 24 is a wavelength side longer than the excitation light wavelength, and FIG. It is an anti-Stokes Raman scattering spectrum showing a range of -200 to -2500 cm ^-1 on the short wavelength side. The Stokes Raman scattering peak of FIG. 24 is almost buried in the fluorescence, but the Stokes Raman scattering peak of FIG.
A clear peak is observed in the Raman scattering spectrum.

FIGS. 26 and 27 are scattering spectra of human urine containing glucose, acetone and urea both measured by the apparatus of the embodiment of FIGS. 7 and 8, and FIG. , Fig. 27 shows 0 to -2500 on the short wavelength side.
It is an anti-Stokes Raman scattering spectrum showing a range of cm ^-1 . 0 cm ⁻¹ is the excitation light wavelength, and the reference light is detected at the same time in order to correct the fluctuation of the light source intensity. The Stokes Raman scattering peak of FIG. 26 is almost buried in fluorescence, but a clear peak is observed in the anti-Stokes Raman scattering spectrum of FIG.

The results of measuring the correlation between the peak intensity and the concentration of each of 10 types of samples prepared by adding glucose, acetone and urea to human urine are shown below. Samples were prepared as shown in Table 2.

[0060]

[Table 2]

FIG. 28 shows the correlation between the peak intensity near -1130 cm ^-1 and the glucose concentration in the anti-Stokes Raman scattering spectrum. In this case, the correlation coefficient R = 0.92, indicating that the correlation is good. Using the correlation shown in FIG. 28 as a calibration curve, the glucose concentration in urine containing a plurality of types of components can be quantified.

FIG. 29 shows the correlation between the peak intensity near -789 cm ^-1 and the acetone concentration in the anti-Stokes Raman scattering spectrum. In this case, the correlation coefficient R = 0.95, indicating that the correlation is good. Using the correlation of FIG. 29 as a calibration curve, the acetone concentration in urine containing a plurality of types of components can be quantified.

FIG. 30 shows the correlation between the peak intensity near -1016 cm ^-1 and the urea concentration in the anti-Stokes Raman scattering spectrum. In this case, the correlation coefficient R = 0.93, indicating that the correlation is good. Using the correlation shown in FIG. 30 as a calibration curve, the urea concentration in urine containing a plurality of types of components can be quantified.

[0064]

Since the measuring method of the present invention measures biological substances and other substances using the anti-Stokes Raman line, even if the sensitivity of the detector is only up to 1000 nm, anti-Stokes Since the Raman line appears on the shorter wavelength side than the excitation light, Raman scattering measurement of the substance can be performed by using, for example, 1064 nm laser light as the excitation light. In this way, the usable range of the detector is widened. When the measurement method of the present invention is used to measure a sample such as a biological sample that easily emits fluorescence, it is not necessary to select the wavelength of the laser light source in order to avoid fluorescence, and a relatively inexpensive laser with large oscillation energy Even in this case, Raman scattering measurement can be performed while avoiding fluorescence.
The range of excitation wavelengths that can be used is widened because it is not necessary to consider fluorescence avoidance and there is less restriction imposed by the sensitivity of the detector. Since a high-power laser light source has a limited oscillation wavelength, widening the selection range of the excitation wavelength is a great advantage when using a high-power laser light source. Also,
Since fluorescence can be avoided, the S / N (signal-to-noise) ratio is improved and a small amount of sample can be measured. Since biological material samples may be available only in small amounts, they are particularly useful for biological material measurement. The measuring device of the present invention irradiates a sample with excitation light of a single wavelength, collects scattered light generated from the sample with a lens having chromatic aberration, and combines light of the excitation light wavelength on the optical axis of the lens. By receiving light at a position closer to the lens than the image position, the density of received anti-Stokes Raman scattered light with a wavelength shorter than the excitation light wavelength was increased, so it was difficult to use conventional Raman measurement devices. Enables the measurement of anti-Stokes Raman scattered light.

[Brief description of the drawings]

[Figure 1] Absolute temperature is 300 ° K based on Boltzmann distribution
FIG. 4 is a diagram showing a calculated value of an intensity ratio between an anti-Stokes Raman line and a Stokes Raman line under the assumption that

FIG. 2 is a perspective view schematically showing the device of the present invention.

3A and 3B are diagrams showing the function of a condenser lens in the optical adjustment section, where FIG. 3A shows the case where the incident light to the condenser lens is parallel light, and FIG.

4A and 4B show changes in the refractive index of a lens optical material depending on the wavelength of BK7, in which (A) is a chart and (B) is a graph.

5A to 5C are diagrams showing light density ratios at respective wavelength positions when light receiving positions are changed.

6A and 6B are flowcharts showing a process of making the light receiving sensitivity of the anti-Stokes Raman scattering spectrum constant, FIG. 6A is a process of calculating and storing a corrected inverse function using a standard substance, and FIG. 6B is an unknown sample. Shows the process of measuring.

FIG. 7 is a block diagram showing a part of a measuring device used for measurement as a block diagram.

FIG. 8 is an exploded perspective view showing a cell and a cell holder used in the measuring apparatus of FIG.

9A is a diagram showing a Raman scattering spectrum of 99% acetone measured by a conventional system, and FIG. 9B is an enlarged view thereof.

FIG. 10A is 99 measured using the apparatus of the example.
It is a figure showing a Raman scattering spectrum of% acetone,
(B) is an enlarged view thereof.

FIG. 11A is 1M measured using the apparatus of the example.
It is a figure which shows the Raman scattering spectrum of the glucose aqueous solution of above, and (B) is the enlarged view.

FIG. 12 is a waveform diagram showing a scattering spectrum of human urine.

FIG. 13 is a waveform diagram showing a scattering spectrum of a sample prepared so that human urine contains 2 M of glucose.

FIG. 14 is a waveform diagram showing an anti-Stokes Raman scattering spectrum of the same sample.

FIG. 15 is a diagram showing anti-Stokes Raman scattering spectra of urine samples with varying glucose concentrations.

FIG. 16 is a diagram showing a correlation between anti-Stokes Raman scattering intensity and glucose concentration near -1131 cm ^-1 .

FIG. 17 is a diagram showing a correlation coefficient R between anti-Stokes Raman scattering and glucose concentration (32 to 1000 mg / dl) at −2000 to −100 cm ⁻¹ .

FIG. 18 is a diagram showing a correlation coefficient R between anti-Stokes Raman scattering and glucose concentration (32 to 514 mg / dl) at −2000 to −100 cm ⁻¹ .

FIG. 19 (A) is a diagram showing a Raman scattering spectrum of a sample of human plasma adjusted to contain 1 M glucose, which was measured using the apparatus of Example, and FIG. 19 (B) is an enlarged view thereof. .

FIG. 20 is a diagram showing a correlation between anti-Stokes Raman scattering intensity and glucose concentration in the vicinity of −1130 cm ⁻¹ .

FIG. 21: Glucose concentration in human plasma and anti-sto
Cous Raman scattering -2000 to 0 cm ^-1Diagram showing the correlation coefficient of
It is.

FIG. 22 is a diagram showing a scattering spectrum of a sample in which human urine contains acetone.

FIG. 23 is a diagram showing an anti-Stokes Raman scattering spectrum of the same sample.

FIG. 24 is a diagram showing a scattering spectrum of a sample in which human urine contains urea.

FIG. 25 is a diagram showing an anti-Stokes Raman scattering spectrum of the same sample.

FIG. 26 is a diagram showing a scattering spectrum of a sample of human urine containing glucose, acetone and urea.

FIG. 27 is a diagram showing an anti-Stokes Raman scattering spectrum of the same sample.

FIG. 28 is a diagram showing a correlation between glucose concentration and spectrum intensity in an anti-Stokes Raman scattering spectrum of a sample in which human urine contains glucose, acetone and urea.

FIG. 29 is a view showing a correlation between acetone concentration and spectrum intensity in an anti-Stokes Raman scattering spectrum of a sample in which human urine contains glucose, acetone and urea.

FIG. 30 is a diagram showing a correlation between urea concentration and spectrum intensity in an anti-Stokes Raman scattering spectrum of a sample in which human urine contains glucose, acetone and urea.

[Explanation of symbols]

 2 Argon ion laser as an excitation light source 4 Excitation light beam 6 Bandpass filter 10 Sample chamber 12 Camera lens 14 Condensing lens 16 Optical adjustment unit 18 Spectrometer 19 Light receiving port 20 Diffraction grating 28 Detector 32 Personal computer 34 Notch filter

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Haruzo Uenoyama 57, Higashikujo Nishiamita-cho, Minami-ku, Kyoto-shi, Kyoto Prefecture Kyoto Daiichi Kagaku Co., Ltd.

Claims (19)

[Claims] 1. A method for measuring an analyte in a sample by irradiating the sample with excitation light and using anti-Stokes Raman scattered light among scattered light generated from the sample. 2. A shift wave number having a good correlation between the concentration of the analyte in the sample and the anti-Stokes Raman scattering intensity is selected as the measurement shift wave number specific to the analyte, and the measured shift wave number is selected. 2. The measuring method according to claim 1, wherein the anti-Stokes Raman scattering intensity of is detected, and the analyte in the sample is quantitatively analyzed using a calibration curve. 3. The measured shift wave number is such that the correlation coefficient R of the correlation between the concentration of the analyte and the anti-Stokes Raman scattering intensity is 0.6 or more, preferably 0.8 or more. Item 2. The measuring method according to Item 2. 4. The measuring method according to claim 1, wherein the sample is a biological substance. 5. An excitation light source unit having an excitation light source for irradiating the sample with excitation light, a sample unit for irradiating the sample with the excitation light, and a sample for collecting scattered light generated by irradiating the sample with the excitation light. And a light receiving unit for detecting the scattered light with a photodetector in a state in which the light receiving density of the anti-Stokes Raman scattered light is increased from the scattered light collected by the light collecting optical adjustment unit. And a measuring device. 6. The condensing optical adjustment section is provided with a lens having chromatic aberration as a condensing system, and the light receiving section has a light receiving port at an image forming position for light having a wavelength shorter than the excitation light wavelength and receives the light. The measuring device according to claim 5, which detects scattered light incident from a mouth. 7. The measuring device according to claim 6, wherein the light receiving unit includes a spectroscope that disperses scattered light incident from the light receiving port and guides the scattered light to the photodetector. 8. The condensing optical adjustment unit includes a bandpass filter that transmits only the shift wave number of the anti-Stokes Raman scattered light to be detected, and the light receiving unit receives the scattered light incident from the light receiving port. The measuring device according to claim 6, which is detected by the photodetector without being spectrally separated. 9. The measuring device according to claim 5, wherein the excitation light source section includes a bandpass filter that transmits only the excitation light wavelength with which the sample is irradiated. 10. The condensing optical adjustment unit includes, on the light incident side of the lens with chromatic aberration, a lens with less chromatic aberration that collects scattered light generated from a sample and guides it as parallel light to the lens with chromatic aberration. The measuring device according to any one of claims 5 to 9. 11. The condensing optical adjustment unit includes a holographic notch filter including a pumping light wavelength in a notch region in order to remove a pumping light wavelength component.
The measuring device according to 6, 7, 9 or 10. 12. The measuring device according to claim 11, wherein the notch region is symmetrical with the bandpass region of the excitation light source unit. 13. The condensing optical adjustment section includes a cut filter for blocking the pumping light wavelength and a longer wavelength side thereof in order to remove the pumping light wavelength component.
The measuring device according to 7, 9, or 10. 14. The measuring device according to claim 7, wherein the light receiving port of the light receiving unit is an entrance slit of a spectroscope. 15. The measurement according to claim 6, wherein the light receiving port of the light receiving unit is one end face of a single core optical fiber, and the other end face of the optical fiber is guided to a spectroscope or a photodetector. apparatus. 16. A distance between a lens having chromatic aberration of the condensing optical adjustment section and a light receiving port is variable.
16. The measuring device according to any one of 1 to 15. 17. The sample part according to claim 5, further comprising a spherical cell and an integrating sphere cell holder in which a portion holding the cell is a spherical reflecting surface. measuring device. 18. The measuring device according to claim 5, further comprising an optical system for reference light that extracts and detects a part of the excitation light. 19. A Boltzmann distribution showing the existence probability of anti-Stokes Raman scattering and an anti-Stokes
Multiply by the effect of increasing the received density of Raman scattered light, modify the result, and calculate so that the photosensitivity of anti-Stokes Raman scattered light becomes constant over a specified range on the shorter wavelength side than the pumping light wavelength. The measuring device according to any one of claims 5 to 18, further comprising a data processing unit to perform.