JP2015059800A - Raman spectroscopic measuring method and raman spectroscopic measuring apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately and spectroscopically measure Raman scattered light by removing fluorescence in spectroscopically measuring the Raman scattered light, and to execute the spectroscopic measurement using a single light source.SOLUTION: The Raman spectroscopic measuring method includes: a first measurement of irradiating a measuring object with an excitation laser beam 10 having a narrow line width, spectroscopically detecting the light released from the measuring object, and acquiring a spectrum 11; and a second measurement of irradiating the measuring object with an excitation laser beam 20 having a line width wider than that in the first measurement, spectroscopically detecting the light released from the measuring object, and acquiring a spectrum 21. At this time, a laser diode is controlled so that the wavelength band of the excitation laser beam in the first measurement is included in the wavelength band of the excitation laser beam in the second measurement. By taking the difference between the spectrum 11 by the first measurement and the spectrum 21 by the second measurement, a second derivative Raman spectrum of the measuring object is acquired approximately.

Description

  The present invention relates to a Raman spectroscopic measurement method and a Raman spectroscopic measurement apparatus for spectroscopically measuring Raman scattered light.

Observation and diagnosis of a body lumen using an electronic endoscope is a diagnostic method that is currently widely used. Since this diagnostic method directly observes the body tissue, it is not necessary to excise the lesioned part and is characterized by a small burden on the subject. On the other hand, such a method for directly observing a body lumen is considered to be less accurate and accurate than pathological examination after biopsy, and efforts to improve imaging image quality are continuously made. .
Recently, in addition to the so-called videoscope, diagnostic devices utilizing various optical principles and ultrasonic diagnostic devices have been proposed, and some of them have been put into practical use. Also in these fields, in order to improve the diagnostic accuracy, a new measurement principle is introduced or a plurality of measurement principles are combined.
In particular, it is known that information that cannot be obtained simply by looking at an image of a tissue can be obtained by observing and measuring fluorescence from the tissue or fluorescence from a fluorescent material applied to the tissue. A fluorescence image endoscope system has also been proposed in which a fluorescence image is acquired and displayed so as to overlap a normal visible image. Such a system is highly promising because it leads to early detection of malignant tumors.
There is also known a method for determining the state of a tissue by acquiring fluorescence intensity information without forming a fluorescence image. In many of these methods, fluorescence is acquired without using an image sensor mounted on an electronic endoscope.
Attempts have also been made to use not only fluorescence but also various spectroscopic measurements such as scattered light and Raman scattered light for the same purpose.

The Raman effect or Raman scattering is a phenomenon in which light having a wavelength different from the wavelength of incident light is included in the scattered light when light is incident on a substance.
The difference between the frequency of Raman scattered light and the frequency of incident light (Raman shift) takes a value peculiar to the structure of the material, so the Raman effect is used to know the structure and state of molecules as in infrared spectroscopy. It is used as a nondestructive analysis method.
On the other hand, fluorescence is emitted when X-rays, ultraviolet rays, or visible rays are irradiated and absorbs the energy to excite electrons, and when it returns to the ground state, excess energy is emitted as electromagnetic waves.
In Raman spectroscopy, a sample is generally irradiated with laser light having a narrow line width (narrow wavelength band) as excitation light, and the wavelength and intensity of the measurement light generated from the sample having a wavelength different from the excitation light is measured. . In general, measurement light having a wavelength longer than that of excitation light is measured. Therefore, when measuring the Raman scattered light, fluorescence is also generated by the excitation light, and the Raman scattered light and the fluorescence are often measured simultaneously. Raman scattered light is often weaker than fluorescence, and there is a problem that weak Raman scattered light is buried by fluorescence. Moreover, even if it is not buried, there are problems that the S / N ratio of Raman scattering measurement deteriorates due to the background due to fluorescence, and that it is difficult to separate fluorescence and Raman scattered light.
In order to solve this problem, (1) the wavelength of the excitation light is set to the near-infrared region, and the generation of fluorescence is greatly suppressed. (2) As described in Patent Document 1, two excitation lights having close wavelengths are used. (3) Patent Literature 1 is used to perform Raman scattering measurement with each excitation light (see Fig. 2 of Patent Literature 1), and to remove the fluorescence background from the calculation using the two obtained data. As described in Fig. 2, Raman scattering measurement was performed on each excitation light using two excitation light sources, a narrow-line-width laser and a broadband LED whose center wavelengths are almost the same wavelength, and the two obtained A method of removing the fluorescence background from the calculation using the data is employed.
The above (1) is a very effective technique. However, when the wavelength of the excitation light is increased, the intensity of the generated Raman light is greatly reduced. , Not necessarily enough.
Therefore, the above methods (2) and (3) are implemented.

U.S. Pat. No. 7,864,311 US Patent Application Publication No. 2012/0019818

However, the above conventional techniques have the following problems.
In the above method (2), the data from which the fluorescence background has been removed is observed as a spectrum shape similar to the first-order differential shape of the Raman spectrum (see FIG. 3 of Patent Document 1). Since the peak shape of the first derivative spectrum disappears, it is difficult to directly read the original peak position from the first derivative spectrum, which is inconvenient for analysis such as identification of Raman peaks, and the Raman spectrum before differentiation is obtained by integrating. It is often converted numerically.
In the above method (3), since two excitation light sources of a narrow line width laser and a broadband LED are used, it is necessary to switch the optical path. In addition, there is a problem that a concave spectrum shape appears by drawing a spectrum by LED excitation (see Fig. 2 of Patent Document 2).

The present invention has been made in view of the problems in the prior art described above, and an object of the present invention is to remove fluorescence and perform spectroscopic measurement of Raman scattered light with high accuracy when performing spectroscopic measurement of Raman scattered light.
It is another object of the present invention to enable implementation using a single light source and to simplify the apparatus.

According to the first aspect of the present invention for solving the above-described problems, a first object of obtaining a spectrum by irradiating a measurement object with an excitation laser beam having a narrow line width and spectrally detecting light emitted from the measurement object. Measurement and
Irradiating the object to be measured with excitation laser light having a wider line width than that at the time of the first measurement, and performing a second measurement to obtain a spectrum by spectroscopically detecting the light emitted from the object to be measured,
It is a Raman spectroscopic measurement method that approximately obtains a second-order differential Raman spectrum of the measurement object by taking a difference between the spectrum of the first measurement and the spectrum of the second measurement.

  The invention according to claim 2 is characterized in that the wavelength band of the excitation laser light at the time of the first measurement is included in the wavelength band of the excitation laser light at the time of the second measurement. Is the method.

  The invention according to claim 3 is characterized in that the center wavelength of the excitation laser beam at the time of the first measurement is substantially equal to the center wavelength of the excitation laser beam at the time of the second measurement. 2. The Raman spectroscopic measurement method according to 2.

  According to a fourth aspect of the present invention, a semiconductor laser is used as a light source for the excitation laser light during the first measurement, a semiconductor laser is used as a light source for the excitation laser light during the second measurement, and the measurement is performed during the second measurement. 4. The measurement object is irradiated with excitation laser light having a wider line width than that during the first measurement by controlling the wavelength shift of the excitation laser light applied to the object. The Raman spectroscopic measurement method according to any one of the above.

  The invention described in claim 5 is characterized in that the semiconductor laser used for the light source of the excitation laser light at the time of the first measurement and the semiconductor laser used for the light source of the excitation laser light at the time of the second measurement are the same. The Raman spectroscopic measurement method according to claim 4.

  The invention according to claim 6 is characterized in that the semiconductor laser is any one of a distributed feedback laser diode, a distributed reflection laser diode, and an external cavity laser diode. It is a described Raman spectroscopic measurement method.

  The invention according to claim 7 is characterized in that the wavelength shift control is performed by controlling a temperature of a semiconductor laser used as a light source of excitation laser light at the time of the second measurement. It is a described Raman spectroscopic measurement method.

  The invention according to claim 8 is characterized in that the wavelength shift control is performed by controlling a driving current of a semiconductor laser used as a light source of excitation laser light at the time of the second measurement. The Raman spectroscopic measurement method described in 1.

  According to a ninth aspect of the present invention, a semiconductor laser used as a light source of the excitation laser light at the time of the second measurement is an external resonator type laser diode, and the wavelength shift control is performed in an external resonator of the external resonator type laser diode. The Raman spectroscopic measurement method according to claim 4, wherein the method is performed by controlling an angle of the diffraction grating.

  According to a tenth aspect of the present invention, the wavelength shift control is performed by controlling a center wavelength to a plurality of different set wavelengths, and the plurality of set wavelengths are set with respect to the center wavelength of the excitation laser light in the first measurement. 10. The Raman spectroscopic measurement method according to claim 4, wherein any one is on a short wavelength side and the other is on a long wavelength side.

  The invention according to claim 11 is the Raman spectroscopic measurement method according to claim 10, wherein the plurality of set wavelengths are two types.

  According to a twelfth aspect of the invention, the wavelength shift control is performed by continuously changing a center wavelength among a plurality of different set wavelengths. This is a measurement method.

  The invention according to claim 13 is characterized in that the wavelength shift control is performed by repeatedly changing the center wavelength so as to be the same wavelength by repeating it twice or more times. The Raman spectroscopic measurement method described in 1.

  The invention according to claim 14 is the Raman spectroscopic measurement method according to claim 10 or 11, wherein the second measurement is performed by obtaining a spectrum by time division for each set wavelength. is there.

  The invention according to claim 15 is that the full width at half maximum of the excitation laser beam at the time of the second measurement is 2 w or less, where w is the full width at half maximum of the narrowest peak among the Raman peaks in the spectrum by the first measurement. The Raman spectroscopic measurement method according to any one of claims 1 to 14, wherein:

In the invention according to claim 16, the full width at half maximum of the excitation laser light at the time of the first measurement is 0.1 [nm] or less,
15. The Raman according to claim 1, wherein the full width at half maximum of the excitation laser light in the second measurement is 0.2 [nm] or more and 2.0 [nm] or less. It is a spectroscopic measurement method.

According to a seventeenth aspect of the present invention, there is provided a first measuring means for performing a first measurement for obtaining a spectrum by irradiating an object to be measured with an excitation laser beam having a narrow line width, and spectrally detecting light emitted from the object to be measured. When,
Second measurement means for irradiating the measurement object with an excitation laser beam having a wider line width than that during the first measurement, and performing a second measurement to obtain a spectrum by spectroscopically detecting the light emitted from the measurement object; ,
An arithmetic means for approximately obtaining a second derivative Raman spectrum of the measurement object by taking a difference between the spectrum by the first measurement and the spectrum by the second measurement;
Is a Raman spectroscopic measurement device.

The invention according to claim 18 is a laser diode;
A spectroscope,
An excitation light optical system that guides light emitted from the laser diode to a measurement object;
A measurement light optical system that guides the light emitted from the measurement object to the spectrometer;
Using the laser diode as a light source, a wavelength control device that controls the wavelength of excitation laser light irradiated to the measurement object;
A detector for detecting the intensity of light split by the spectroscope and outputting spectral data;
Giving a wavelength control command to the wavelength control device, receiving the input of the spectrum data from the detector, and a computer functioning as the computing means;
With
The first measurement unit and the second measurement unit are configured to share the laser diode, the spectrometer, the excitation light optical system, the measurement light optical system, the wavelength control device, and the detector. Item 18. The Raman spectroscopic measurement device according to Item 17.

  According to a nineteenth aspect of the present invention, the computer controls the wavelength control device so that the wavelength band of the excitation laser light at the time of the first measurement is included in the wavelength band of the excitation laser light at the time of the second measurement. 19. The Raman spectroscopic measurement apparatus according to claim 18, which gives a command.

According to the present invention, a measurement object is obtained by taking a difference between a first measurement performed by irradiating an excitation laser beam having a narrow line width and a second measurement performed by irradiating an excitation laser beam having a wider line width. It is possible to obtain approximately the second derivative Raman spectrum of an object, thereby obtaining a spectrum in which the fluorescence is removed and the peak structure of the Raman spectrum is emphasized, and the Raman scattered light can be spectroscopically measured with high accuracy. .
Even in the case of excitation laser light having a narrow line width, the line width is pseudo-widened by controlling the wavelength shift during the second measurement, and thus the light source is switched between the first measurement and the second measurement. It can be shared and thus implemented with a single light source, and the apparatus can be simplified.

According to an embodiment of the present invention, (a) is a graph showing an example of a spectrum of excitation laser light at the time of the first measurement, (b) is a graph showing an example of a spectrum obtained by the first measurement, (c) Is a graph showing an example of the spectrum of the excitation laser light at the time of the second measurement, (d) is a graph showing an example of the spectrum obtained by the second measurement, and (e) is a curve by the difference of (d)-(b). It is a graph which shows an example (the recent curve of a secondary differential Raman spectrum). 1A is a graph showing an example of a spectrum of excitation laser light during a first measurement, and FIG. 4B is a graph showing an example of a spectrum of excitation laser light during a second measurement according to an embodiment of the present invention. . It is a graph which shows an example of the time change of the temperature of the semiconductor laser at the time of 2nd measurement concerning one Embodiment of this invention. 1A is a graph showing an example of a spectrum of excitation laser light during a first measurement, and FIG. 4B is a graph showing an example of a spectrum of excitation laser light during a second measurement according to an embodiment of the present invention. . It is a graph which shows an example of the time change of the temperature of the semiconductor laser at the time of 2nd measurement concerning one Embodiment of this invention. 1A is a graph showing an example of a spectrum of excitation laser light during a first measurement, and FIG. 4B is a graph showing an example of a spectrum of excitation laser light during a second measurement according to an embodiment of the present invention. . It is a graph which shows an example of the time change of the temperature of the semiconductor laser at the time of 2nd measurement concerning one Embodiment of this invention. 4 is a graph showing an example of a time change of the temperature of the semiconductor laser at the time of the second measurement according to an embodiment of the present invention, where (a) shows an example of a linear change, and (b) shows a curve change. An example is shown. (a) is a graph showing a curve with a slight peak at the center (broken line) and its baseline curve (solid line), (b) is a peak structure after removing the solid line from the broken line in (a) Is a graph showing a curve (broken line) indicating, and a curve (solid line) obtained by secondarily differentiating the curve. 1 is a configuration block diagram of a Raman spectroscopic measurement apparatus according to an embodiment of the present invention. It is a flowchart which shows the process sequence by the Raman spectrometer which concerns on embodiment of this invention.

  An embodiment of the present invention will be described below with reference to the drawings. The following is one embodiment of the present invention and does not limit the present invention.

First, an embodiment of the Raman spectroscopic measurement method of the present invention will be described with reference to FIGS.
In this method, first, an excitation laser beam having a narrow line width such as spectrum 10 shown in FIG. 1 (a) is irradiated to the measurement object, and the light emitted from the measurement object is detected by spectral detection. The measurement object is irradiated with the first measurement for obtaining the spectrum 11 as shown in b) and the excitation laser beam having a wider line width than the first measurement as in the spectrum 20 shown in FIG. 1 (c). A second measurement is performed in which the light emitted from the object is spectrally detected to obtain a spectrum 21 as shown in FIG. 1 (d). The order of the first measurement and the second measurement does not matter.
As shown in FIG. 1, the center wavelength of the excitation laser light at the time of the first measurement is λ ₁ , the full width at half maximum is Δλ ₁ , the center wavelength of the excitation laser light at the time of the second measurement is λ ₂ , and the full width at half maximum is Δλ ₂ . The center wavelength λ ₁ and the center wavelength λ ₂ are substantially equal, and Δλ ₁ <Δλ ₂ , and the relationship that the wavelength band of the spectrum 10 is included in the wavelength band of the spectrum 20 is applied.

The spectrum 11 shown in FIG. 1B includes a spectrum of Raman scattered light, that is, a fluorescence spectrum in addition to the Raman spectrum. The base portion 11F is mainly a fluorescent component, and a peak 11R due to a Raman spectrum appears thereon (four peaks are shown as an example in the figure).
Similarly, the spectrum 21 shown in FIG. 1 (d) includes a fluorescence spectrum in addition to the Raman spectrum. The base portion 21F is mainly a fluorescent component, and a peak portion 21R due to a Raman spectrum appears thereon.
When the first measurement and the second measurement are completed, the second differential Raman spectrum of the measurement object is obtained as shown in FIG. 1 (e) by taking the difference between the spectrum 11 by the first measurement and the spectrum 21 by the second measurement. Get approximately. A curve R shown in FIG. 1 (e) is a recent curve of the second derivative Raman spectrum obtained by executing (Spectrum 21)-(Spectrum 11).

As described above, a narrow linewidth laser is used as the light source for the first measurement. In particular, a distributed feedback (DFB) laser diode, a distributed reflection (DBR) laser diode, and an external cavity laser diode that can easily change the wavelength are preferable.
The result of the first measurement can be evaluated as a Raman spectrum by itself, but since the Raman scattered light and the fluorescence are superimposed, it is necessary to remove the contribution of the fluorescence from the result of the first measurement. Therefore, the second measurement is performed. As described above, in the second measurement, the line width Δλ ₂ of the excitation light is set larger than the line width Δλ ₁ of the first measurement. Thereby, of the measurement light to be detected, the Raman scattered light (peak 21R) increases in line width and gives a broad spectrum shape.
The central wavelength λ ₁ of the excitation light of the first measurement and the central wavelength λ ₂ of the second measurement are set so that the difference is reduced to such an extent that the influence due to the difference cannot be measured and can be regarded as substantially the same wavelength. .
On the other hand, the fluorescence is not affected by the change in the line width of the excitation light, and gives the same spectrum shape (base portion 11F and base portion 21F) in the first measurement and the second measurement. At this time, the change in the overall intensity can be corrected later and does not affect the identity of the shape.

In order to simplify the apparatus configuration, it is desirable to perform the first measurement and the second measurement using the same light source. However, since laser light sources generally used for Raman spectroscopic measurements are required to have a narrow line width, the laser line width can be freely changed by using a gas laser with a narrow line width or a semiconductor laser with some wavelength selection structure. It is difficult to do.
Therefore, the second measurement is performed using a light source having a pseudo-wide line width. That is, a semiconductor laser is used as the light source of the excitation laser light during the first measurement, a semiconductor laser is used as the light source of the excitation laser light during the second measurement, and the excitation laser light irradiated to the measurement object is used during the second measurement. By performing the wavelength shift control, the measurement object is irradiated with excitation laser light having a wider line width than that in the first measurement.
In DFB laser diodes and DBR laser diodes, the oscillation wavelength changes with temperature, so it is possible to create pseudo-wide-line excitation light by sweeping the temperature of the laser diode during measurement. That is, the above-described wavelength shift control is performed by controlling the temperature of the semiconductor laser used as the light source for the excitation laser light during the second measurement. In general, the oscillation wavelength shifts to the longer wavelength side as the temperature increases.
Further, in an external laser having a grating, the oscillation wavelength can be changed by temperature or mechanical operation. That is, the semiconductor laser used as the light source of the excitation laser light at the time of the second measurement is an external resonator type laser diode, and the above wavelength shift control is performed by changing the angle of the diffraction grating in the external resonator of the external resonator type laser diode. This is done by controlling.
Further, it is possible to apply a semiconductor laser capable of controlling the driving current and shifting the wavelength. That is, the above-described wavelength shift control is performed by controlling the drive current of the semiconductor laser used as the light source of the excitation laser light during the second measurement.
Therefore, it is possible to create pseudo-wide line excitation light by controlling the temperature, the angle of the diffraction grating, or the driving current.

  By subtracting the result of the first measurement from the result of the second measurement, the fluorescence having a small difference between the first measurement and the second measurement is removed, and only the contribution of the Raman scattered light as shown in FIG. It can be taken out. At this time, by setting the excitation light of the second measurement as described above, the contribution of the Raman scattered light has a positive-negative-positive spectral shape as shown in FIG.

  In general, the second-order difference of the function f (λ) is expressed by the following equation (1).

Here, α and δλ are constants.
Λ of the function f (λ) corresponds to the center wavelength λ ₁ of the excitation light in the first measurement in FIG. 1, and f (λ ₁ + δλ) + f (λ ₁ −δλ) + αf (λ ₁ ) is the second. (2 + α) f (λ ₁ ) corresponds to the spectrum obtained by the first measurement.
Since there are degrees of freedom of α and δλ, several measurement conditions can be considered for the second measurement.
Since δλ needs to be able to approximate the differential equation (1) above, it is desirable that δλ be several times as large as the measurement resolution of the apparatus. In the Raman spectroscopic measurement, in many cases, δλ is about 0.1 [nm] to 1 [nm]. Therefore, in order to obtain a recent curve R of a good second-order differential Raman spectrum, the full width at half maximum Δλ ₁ of the excitation laser beam at the time of the first measurement is set to 0.1 [nm] or less, and at the time of the second measurement. It is preferable that the full width at half maximum Δλ ₂ of the excitation laser light is 0.2 [nm] or more and 2.0 [nm] or less. .. Among the Raman peaks 11R, 11R,... In the spectrum 11 by the first measurement, where w is the full width at half maximum of the peak 11R having the narrowest line width, the full width at half maximum Δλ ₂ of the excitation laser light at the second measurement. Is preferably 2 w or less.

When α = 0, the spectrum of the second measurement corresponds to f (λ ₁ + δλ) + f (λ ₁ −δλ), and the spectrum 10 of the excitation laser light at the time of the first measurement shown in FIG. In the second measurement, an excitation laser beam whose center wavelength is controlled to the set wavelength (λ ₁ + δλ) on the long wavelength side of the center wavelength λ ₁ as shown in the spectrum 20 (solid line portion) shown in FIG. This corresponds to the measurement with the excitation laser beam whose center wavelength is controlled to the set wavelength (λ ₁ −δλ) on the wavelength side. That is, this is a case where a plurality of set wavelengths at the time of the second measurement are two types.
For this reason, two values are mainly selected for the temperature of the semiconductor laser in order to produce pseudo wide-line excitation light by controlling the temperature during the second measurement. That is, T ₁ the temperature of the semiconductor laser that emits excitation laser light during the first measurement as shown in FIG. 3, the temperature T of the same semiconductor laser used during the second measurement, comprising the temperature T _{2 <T 1 2} and a temperature T ₃ at which T ₃ > T ₁ is controlled. Typical examples include, but are not limited to, T ₁ = 27 ° C., T ₂ = 20 ° C., T ₃ = 34 ° C., or T ₁ = 25 ° C., T ₂ = 15 ° C., T ₃ = 35 ° C. .
Between T ₂ and T ₃ during the measurement of the second measurement may be moved several times. That is, the wavelength shift control at the time of the second measurement may be performed by repeatedly changing the center wavelength so as to be the same wavelength by repeating twice or more.
In the second measurement, one spectrum may be obtained by passing through two temperatures within one measurement, but the spectrum at T ₂ and the measurement at T ₃ are obtained by time division. A second measurement can also be performed. In the latter case, a spectrum 21 by the second measurement as shown in FIG. 1 (d) is obtained by adding a plurality of spectra obtained by dividing. In this way, the second measurement can be performed by obtaining the spectrum in a time division manner for each set wavelength. Further, the temperature of the semiconductor laser can be similarly changed in place of the angle of the diffraction grating or the driving current of the semiconductor laser.

When α = 1, the spectrum of the second measurement corresponds to f (λ ₁ + δλ) + f (λ ₁ −δλ) + f (λ ₁ ), and the excitation laser light at the time of the first measurement shown in FIG. , The set wavelength λ ₁ , the set wavelength (λ ₁ + δλ), and the set wavelength (λ ₁ −δλ) as in the spectrum 20 (solid line portion) shown in FIG. Since the measurement is performed using excitation laser light whose center wavelength is controlled, the measurement corresponds to measurement using excitation laser light having approximately uniform intensity within the distributed wavelength range.
Also in this case, the same semiconductor laser is used for the first measurement and the second measurement, and the temperature, the angle of the diffraction grating, or the drive current is controlled so that the set wavelength λ ₁ at the first measurement is raised and lowered at the second measurement. This can be implemented by controlling the wavelength shift. FIG. 5 is an example of a temperature change graph of the semiconductor laser when the temperature is controlled by the temperature of the semiconductor laser.
In the range of 0 <α <1, this corresponds to the fact that the center spectrum (dashed line portion) having the center wavelength λ ₁ in FIG.

When α> 1, the spectrum of the second measurement corresponds to f (λ ₁ + δλ) + f (λ ₁ −δλ) + αf (λ ₁ ) (where α> 1), and the spectrum shown in FIG. In contrast to the spectrum 10 of the excitation laser light at the time of one measurement, the setting wavelength λ ₁ , the setting wavelength (λ ₁ + δλ), and the setting wavelength as in the spectrum 20 (solid line part) shown in FIG. Since the central wavelength value is controlled to (λ ₁ −δλ) and the measurement is performed with the excitation laser light that is controlled to the set wavelength λ ₁ for the longest time, the excitation laser light having one intensity peak at the center in the distributed wavelength region It corresponds to measuring with.
Also in this case, the same semiconductor laser is used for the first measurement and the second measurement, and the temperature, the angle of the diffraction grating, or the drive current is controlled so that the set wavelength λ ₁ at the first measurement is raised and lowered at the second measurement. This can be implemented by controlling the wavelength shift. FIG. 7 is an example of a temperature change graph of the semiconductor laser when the temperature is controlled by the temperature of the semiconductor laser.

The above shows the outline of the measurement when the excitation laser light in the second measurement is swept with the typical three wavelengths. Actually, it is not necessary to use three discrete wavelengths, and measurement may be performed while continuously sweeping the wavelength between (λ ₁ −δλ) and (λ ₁ + δλ). That is, the wavelength shift control at the time of the second measurement may be performed by continuously changing the center wavelength among a plurality of different set wavelengths. FIG. 8 is an example of a temperature change graph of the semiconductor laser when the continuous change of the center wavelength of the semiconductor laser is controlled by the temperature of the semiconductor laser. The temperature of the semiconductor laser can be similarly changed in place of the angle of the diffraction grating or the driving current of the semiconductor laser. Moreover, you may implement the continuous change of the center wavelength of this semiconductor laser as a change which repeats periodically or aperiodically.

Using the results (spectrums) of the first measurement and the second measurement obtained as described above, it is possible to remove fluorescence as described above. That is, a recent curve R of the second derivative Raman spectrum as shown in FIG.
The recent curve R of the second derivative Raman spectrum approximately indicates the second derivative (second order difference) shape of the Raman spectrum of the measurement object. FIG. 9A shows a curve having a slight peak at the center (dashed line) and its baseline curve (solid line). The difference obtained by subtracting the solid baseline curve from the dashed curve in FIG. The corresponding curve is shown by a broken line in FIG. A curve obtained by second-order differentiation of the difference curve indicated by the broken line in FIG. 9B is also indicated by the solid line in FIG. 9B. As shown in FIG. 9, the second-order differential spectrum generally has the following characteristics.
(A1) The superposed spectral structure is separated so that the number and position of peak structures can be easily detected.
(A2) The baseline is removed and the peak structure is emphasized.
(A3) The intensity of the secondary differential shape is proportional to the peak intensity of the original spectrum.
(A4) The peak position of the secondary differential shape matches the peak position of the original spectrum.
Therefore, only by taking the difference between the results of the two measurements of the first measurement and the second measurement by the Raman spectroscopic measurement method of the present embodiment,
(B1) removing fluorescence superimposed on the Raman signal;
(B2) Emphasizing the peak structure of the Raman signal and making it easy to detect the peak position and the number of peaks,
Thus, the Raman scattered light can be spectroscopically measured with high accuracy.

Next, a Raman spectroscopic measurement apparatus that performs the above-described Raman spectroscopic measurement method will be described with reference to FIGS.
As shown in FIG. 10, the Raman spectroscopic measurement device 50 includes a laser diode 51, a spectroscope 52, an excitation light optical system 53, a measurement light optical system 54, a wavelength control device 55, a detector 56, and a computer 57. With.
The computer 57 controls the other units to function as first measurement means for executing the first measurement and second measurement means for executing the second measurement. The wavelength control device 55 includes a temperature control device 55a and a Peltier element module (TEC) 55b. The laser diode 51 is mounted so that the temperature of the laser diode 51 is changed by the Peltier element module (TEC) 55b and the oscillation wavelength of the laser diode 51 is shifted.
The computer 57 gives a wavelength control command to the wavelength control device (in this case, a temperature control command to the temperature control device 55a) according to the temperature control protocol 57a, thereby performing wavelength shift control of the excitation laser light output from the laser diode 51, and 2. Create excitation laser beam with wide line width during measurement. Regardless of this embodiment, a device for controlling the angle or driving current of the diffraction grating is provided as the wavelength control device 55, and the excitation laser beam having a wide line width at the time of the second measurement is controlled by controlling the angle or driving current of the diffraction grating. You may make it.

The excitation light optical system 53 is an optical system that guides the light emitted from the laser diode 51 to the measurement target 60, and is configured by optical components having functions such as reflection, refraction, and condensing. The wavelength control device uses the laser diode 51 as a light source and controls the wavelength of the excitation laser light irradiated to the measurement object 60, and is controlled until the measurement object 60 is irradiated regardless of the present embodiment. It's enough. The measurement light optical system 54 is an optical system that guides the light emitted from the measurement object 60 to the spectroscope 52, and is configured by optical components having functions such as reflection, refraction, and light collection.
A portion 61 of the excitation light optical system 53 and the measurement light optical system 54 that is close to the measurement target 60 may be configured to guide light by an optical fiber, and may be a probe that can be inserted into the body by covering it with a flexible tube. You may incorporate in an endoscope. In the former case, the probe can be a small diameter that can be inserted into a channel formed in an endoscope or catheter.
In each of the first measurement and the second measurement, the detector 56 detects the intensity of light dispersed by the spectrometer 52 and outputs spectrum data. The computer 57 receives spectral data from the detector 56. The computer 57 functions as data calculation means 57b that executes a data calculation process that takes the difference between the spectrum data from the first measurement and the spectrum data from the second measurement. By this data calculation process, a second-order differential Raman spectrum of the measurement object 60 from which fluorescence has been removed is approximately obtained.

Next, a procedure of Raman spectroscopy measurement performed by the Raman spectrometer 50 will be described with reference to the flowchart of FIG.
(First measurement)
First, the computer 57 applies a drive current to the laser diode 51 via a drive device (not shown) to emit excitation laser light from the laser diode 51 (step S1).
Next, the computer 57 sets the temperature of the laser diode 51 to the set value of the first measurement according to the temperature control protocol 57a (step S2). The set value here is a temperature value corresponding to setting the center wavelength λ ₁ of the excitation laser light at the time of the first measurement to a predetermined value.
Next, the computer 57 starts exposure of the detector 56 (step S3). As a result, the light emitted from the measurement object 60 and dispersed by the spectroscope 52 is input to the detector 56 and detection is started.
Next, the computer 57 ends the exposure of the detector 56 on condition that the predetermined exposure time has been reached (step S4).
Next, the computer 57 reads the data detected by the detector 56 into the computer 57 and stores it as spectrum data of the first measurement (step S5).

(Second measurement)
Next, the computer 57 sets the temperature of the laser diode 51 to the start setting value of the second measurement according to the temperature control protocol 57a (step S6). The start set value here is a temperature value corresponding to setting the center wavelength λ ₂ of the excitation laser light at the time of the second measurement to a predetermined value at the time of the start of the second measurement.
Next, the computer 57 starts exposure of the detector 56 (step S7). As a result, the light emitted from the measurement object 60 and dispersed by the spectroscope 52 is input to the detector 56 and detection is started.
Next, the computer 57 changes the temperature of the laser diode 51 in accordance with the temperature control protocol 57a of the second measurement (step S8). Thereby, an excitation laser beam having a wide line width at the time of the second measurement is created.
Next, the computer 57 ends the exposure of the detector 56 on condition that a predetermined exposure time has been reached (step S9).
Next, the computer 57 reads the data detected by the detector 56 into the computer 57 and stores it as spectrum data of the second measurement (step S10).

(Data calculation processing)
Next, the computer 57 performs general data correction such as removal of the baseline derived from the detector 56 and correction of wavelength sensitivity from the spectrum data of the first measurement and the spectrum data of the second measurement (step S11). ).
Next, the computer 57 normalizes the intensities of the first measurement spectrum data and the second measurement spectrum data (step S12).
Next, the computer 57 takes the difference between the spectrum data of the first measurement and the spectrum data of the second measurement, and stores this as second-order difference data (step S13). Based on the second-order difference data, the recent curve R of the second-order differential Raman spectrum described above is constructed.

In order to carry out the Raman spectroscopic measurement method described above, the computer 57 uses the wavelength control device 55 so that the wavelength band of the excitation laser light at the first measurement is included in the wavelength band of the excitation laser light at the second measurement. Is given a wavelength control command. In addition, the processes executed by the Raman spectroscopic measurement apparatus 50 and applicable modifications are as described in the Raman spectroscopic measurement method.
In addition, the normalization in step S12 aims to align the intensities of the base portions of the first measurement spectrum data and the second measurement spectrum data having different intensities. The specific method is as follows, for example.
(1) The maximum value of spectrum data is 1 and the minimum value is 0, respectively. At this time, the maximum value is a wavelength region where no Raman peak appears.
(2) The average value is 0 and the standard deviation is 1, respectively.

10 Spectrum 11 of excitation laser light at the first measurement 11 Spectrum 11R obtained by the first measurement 20 Raman peak 20 Spectrum 21 of the excitation laser light at the second measurement 21 Spectrum obtained by the second measurement 21R Raman peak 50 Raman spectroscopic measurement Device 51 Laser diode 52 Spectrometer 53 Excitation light optical system 54 Measurement light optical system 55 Wavelength control device 55a Temperature control device 56 Detector 57 Computer 57a Temperature control protocol 57b Data calculation means 60 Measurement object R Second-order differential Raman spectrum Time curve

Claims (19)

A first measurement that obtains a spectrum by irradiating an object to be measured with an excitation laser beam having a narrow line width, and spectrally detecting light emitted from the object to be measured;
Irradiating the object to be measured with excitation laser light having a wider line width than that at the time of the first measurement, and performing a second measurement to obtain a spectrum by spectroscopically detecting the light emitted from the object to be measured,
A Raman spectroscopic measurement method that approximately obtains a second-order differential Raman spectrum of the measurement object by taking a difference between the spectrum of the first measurement and the spectrum of the second measurement.   2. The Raman spectroscopic measurement method according to claim 1, wherein a wavelength band of the excitation laser light at the time of the first measurement is included in a wavelength band of the excitation laser light at the time of the second measurement.   3. The Raman spectroscopic measurement method according to claim 1, wherein a center wavelength of the excitation laser light at the time of the first measurement is substantially equal to a center wavelength of the excitation laser light at the time of the second measurement. .   A semiconductor laser is used as the light source of the excitation laser light at the time of the first measurement, a semiconductor laser is used as a light source of the excitation laser light at the time of the second measurement, and the measurement object is irradiated at the time of the second measurement. 4. The measurement object is irradiated with excitation laser light having a wider line width than that during the first measurement by controlling wavelength shift of light. Raman spectroscopy measurement method.   5. The Raman spectroscopy according to claim 4, wherein a semiconductor laser used as a light source of the excitation laser light at the time of the first measurement is the same as a semiconductor laser used as a light source of the excitation laser light at the time of the second measurement. Measuring method.   6. The Raman spectroscopic measurement method according to claim 4, wherein the semiconductor laser is any one of a distributed feedback laser diode, a distributed reflection laser diode, and an external resonator laser diode.   6. The Raman spectroscopic measurement method according to claim 4, wherein the wavelength shift control is performed by controlling a temperature of a semiconductor laser used as a light source of excitation laser light at the time of the second measurement.   6. The Raman spectroscopic measurement method according to claim 4, wherein the wavelength shift control is performed by controlling a driving current of a semiconductor laser used as a light source of excitation laser light at the time of the second measurement.   The semiconductor laser used as the light source of the excitation laser light during the second measurement is an external resonator type laser diode, and the wavelength shift control is performed to control the angle of the diffraction grating in the external resonator of the external resonator type laser diode. The Raman spectroscopic measurement method according to claim 4, wherein the method is performed.   The wavelength shift control is performed by controlling the center wavelength to a plurality of different set wavelengths, and any one of the plurality of set wavelengths is on the short wavelength side with respect to the center wavelength of the excitation laser light at the time of the first measurement. The Raman spectroscopic measurement method according to any one of claims 4 to 9, wherein any one of them is on the long wavelength side.   The Raman spectroscopic measurement method according to claim 10, wherein the plurality of set wavelengths are two types.   12. The Raman spectroscopic measurement method according to claim 10, wherein the wavelength shift control is performed by continuously changing a center wavelength among a plurality of different set wavelengths.   13. The Raman spectroscopic measurement method according to claim 10, wherein the wavelength shift control is performed by repeatedly changing the center wavelength so that the center wavelength is the same wavelength by repeating it twice or more.   12. The Raman spectroscopic measurement method according to claim 10, wherein the second measurement is performed by obtaining a spectrum by time division for each set wavelength.   The full width at half maximum of the excitation laser beam at the time of the second measurement is 2 w or less, where w is the full width at half maximum of the peak having the narrowest line width among the Raman peaks in the spectrum of the first measurement. The Raman spectroscopic measurement method according to any one of Items 1 to 14. The full width at half maximum of the excitation laser light during the first measurement is 0.1 [nm] or less,
15. The Raman according to claim 1, wherein the full width at half maximum of the excitation laser light in the second measurement is 0.2 [nm] or more and 2.0 [nm] or less. Spectroscopic measurement method. A first measuring means for irradiating a measurement object with an excitation laser beam having a narrow line width and performing a first measurement for obtaining a spectrum by spectrally detecting light emitted from the measurement object;
Second measurement means for irradiating the measurement object with an excitation laser beam having a wider line width than that during the first measurement, and performing a second measurement to obtain a spectrum by spectroscopically detecting the light emitted from the measurement object; ,
An arithmetic means for approximately obtaining a second derivative Raman spectrum of the measurement object by taking a difference between the spectrum by the first measurement and the spectrum by the second measurement;
A Raman spectrometer. A laser diode,
A spectroscope,
An excitation light optical system that guides light emitted from the laser diode to a measurement object;
A measurement light optical system that guides the light emitted from the measurement object to the spectrometer;
Using the laser diode as a light source, a wavelength control device that controls the wavelength of excitation laser light irradiated to the measurement object;
A detector for detecting the intensity of light split by the spectroscope and outputting spectral data;
Giving a wavelength control command to the wavelength control device, receiving the input of the spectrum data from the detector, and a computer functioning as the computing means;
With
The first measurement unit and the second measurement unit are configured to share the laser diode, the spectrometer, the excitation light optical system, the measurement light optical system, the wavelength control device, and the detector. Item 18. The Raman spectroscopic measurement device according to Item 17.   19. The computer according to claim 18, wherein the computer gives a wavelength control command to the wavelength controller such that a wavelength band of the excitation laser light at the time of the first measurement is included in a wavelength band of the excitation laser light at the time of the second measurement. Raman spectrometer.

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