JP3600867B2 - Tunable laser and Raman spectrometer - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a laser, particularly a tunable laser useful as a light source for spectroscopic analysis, and a Raman spectrometer using the tunable laser.
[0002]
[Prior art]
Near-infrared Raman spectroscopy is widely used for analyzing samples in the fields of chemistry and biology. The application of Raman spectroscopy to biomedical medical analysis has attracted many researchers for three reasons.
[0003]
(1) Raman spectroscopy is a non-invasive optical measurement method. The recently developed fiber optic probe method has enabled in situ measurement of biological tissue. Bushman et al. Demonstrated the utility of a microscopic Raman fiber optic probe in endoscopic Raman analysis (Anal. Chem., 72 , 3771 (2000)).
(2) The Raman spectrum contains much information.
(3) Real-time analysis is possible.
[0004]
For the last decade, near-infrared Fourier transform Raman spectroscopy using an Nd: YAG laser (1064 nm) as the excitation light source has been used as a powerful tool in biomedical research (Appl. Spectrosc., 46 , 533). (1992); Spectrochim. Acta A, 55 , 1691 (1999); J. Mol. Struc., 480-481 , 21 (1999)). In recent near-infrared excitation Raman in situ studies, a dispersion type Raman spectrometer with a CCD has become popular as a Raman spectrum measurement method that is faster and more sensitive than the Fourier transform Raman method.
[0005]
[Problems to be solved by the invention]
When the Raman spectrum is measured while sweeping the excitation wavelength, a Raman excitation profile is measured. Information such as the moment of electronic transition in the excited state of a molecule can be obtained from the Raman excitation profile. In statistical analysis of a biomolecule spectrum or analysis by a two-dimensional analysis method, a change in excitation wavelength can be used as a non-invasive external perturbation. Conventionally, a combination of ordinary Ti: sapphire laser, dye laser, and various fixed wavelength lasers has been used as the wavelength tunable laser required for the measurement, but the spectrum acquisition by impure light such as background radiation is used. There are problems such as restriction of the excitation wavelength by a band-pass filter used for removing interference and impurity light.
[0006]
Another problem is that the laser optical axis must be readjusted every time the laser wavelength is changed. Many tunable lasers use angle tuning devices such as birefringent filters, diffraction gratings, or prisms for wavelength selection. Not only the laser wavelength, but also the stability of the beam irradiation position cannot be so high, because it depends on the mechanical accuracy of these tuning devices. The measured intensity of the Raman spectrum depends on the laser light irradiation position on the sample.
On the other hand, an electronically controlled tunable laser (hereinafter referred to as an ETT (Electronically Tuned Tunable) laser) has been developed by the group of the present inventors as a tunable laser capable of high-speed wavelength sweeping.
[0007]
As shown schematically in FIG. 2, the ETT laser has a laser resonator including an emission side mirror 112 having a predetermined transmittance and a total reflection mirror 110, and a wavelength tunable laser medium ( (E.g., titanium sapphire, dye, etc.) 14, a birefringent acousto-optic element 100 for wavelength selection, and a prism 28 as a wavelength dispersion correction element for the diffraction angle are disposed between the output side mirror 112 and the total reflection mirror 110. Have been. A piezoelectric element 22 driven by an RF power supply 20 is attached to the birefringent acousto-optic element 100 as acoustic wave input means. When the piezoelectric element 22 is driven by the RF power supply 20, an acoustic wave propagates through the birefringent acousto-optic element 100. The total reflection mirror 110 is configured to vertically reflect only the diffracted light 106 diffracted in a predetermined direction by the birefringent acousto-optic element 100.
[0008]
The laser medium 14 is excited by the excitation laser light 24. Further, the frequency of the RF power supply is controlled according to the frequency (wavelength) of the laser light to be oscillated. In this way, the light of the next frequency ωo corresponding to the frequency ωa of the RF power supply 20 is diffracted in the light of the wide wavelength band emitted from the laser medium 14 and incident on the birefringent acousto-optic element 100. The light 106 is diffracted from the birefringent acousto-optic device 100 toward the total reflection mirror 110.
[0009]
ωo = ωi + ωa (1)
[0010]
In this manner, only the light having the frequency ωi can reciprocate in the laser medium 14, is amplified by the laser medium, generates laser oscillation, and emits the laser light from the laser resonator.
The chromatic dispersion correcting prism 28 is designed so that the diffracted light 106 emitted from the birefringent acousto-optic element 100 is always perpendicularly incident on the total reflection mirror 110 regardless of the wavelength of the diffracted light. In this case, the light beam diffracted by the birefringent acousto-optic element 100 to become the diffracted light 106 is reflected by the total reflection mirror 110 at any wavelength, and can follow the same optical path in reverse. The laser medium 14 can efficiently amplify the laser and oscillate the laser.
[0011]
Although this ETT laser has an advantage that the laser oscillation wavelength can be switched at high speed, background noise derived from broadband auto-fluorescence emitted from the laser medium 14 is harmful to high-sensitivity Raman spectroscopy. In other words, the broad spontaneous fluorescence (650 to 1100 nm in the case of titanium sapphire) emitted from the laser medium 14 is mixed with the laser oscillation light as the background radiation, so that the Raman scattered light has an intensity of about one hundred millionth of the excitation light. , It is necessary to use a band-pass filter together to remove background radiation. Even if the bandpass filter is rotated, the transmission wavelength changes only a few nm, so when trying to measure the Raman excitation profile using an ETT laser, it is necessary to prepare many bandpass filters for each excitation wavelength, which is very practical. It was not a target.
The present invention has been made in view of the above problems, and has as its object to provide a tunable laser having no background radiation. Another object of the present invention is to provide a Raman spectrometer capable of performing high-accuracy Raman spectrometry and quick measurement of a Raman excitation profile.
[0012]
[Means for Solving the Problems]
In the present invention, a wavelength tunable laser without a fluorescent background suitable for near-infrared Raman spectroscopy has been developed using the diffraction effect of a birefringent acousto-optic element.
The wavelength tunable laser according to the present invention has a wavelength tunable laser medium and a birefringent acousto-optic element for wavelength selection in a laser resonator configured by an emission side mirror having a predetermined transmittance and a total reflection mirror, In a wavelength tunable laser in which a prism as a wavelength dispersion correction element for a diffraction angle by a birefringent acousto-optic element is arranged, a wavelength tunable laser medium is arranged near the total reflection mirror, and birefringence is near the emission side mirror. And an acousto-optic element and a prism are arranged.
[0013]
It is preferable to set the distance between the birefringent acousto-optic element and the prism so that the fluctuation of the optical axis of the outgoing laser light when the wavelength is changed is one-tenth or less of the beam diameter of the outgoing laser light. If the optical axis fluctuation of the emitted laser light during wavelength tuning is within one-tenth of the beam diameter, the light irradiation point on the sample whose optical system converges it to about one-tenth of the effective beam diameter moves. It is estimated that there will be no effect on sample measurement.
The Raman spectrometer according to the present invention is a wavelength tunable laser, a spectroscope, an optical system for converging light emitted from the wavelength tunable laser to a sample, and scattered light from the sample to an entrance slit of the spectrometer. An optical system for condensing light is provided.
[0014]
When the wavelength tunable laser of the present invention is used, a background radiation light removing effect equal to or higher than that obtained when a bandpass filter is used without using a bandpass filter can be obtained. The major difference from the case where a bandpass filter is used is that the wavelength tunable laser of the present invention uses the wavelength selection element as it is as a filter for removing background radiation, and is therefore completely compatible with wavelength tunability.
[0015]
This tunable laser also has a very high pointing stability. In the Raman measurement, a laser beam is focused on a region of about 10 μm to irradiate the sample, and scattered light emitted from the region is analyzed. In the conventional Raman measurement using a wavelength tunable laser, the optical axis needs to be adjusted every time the wavelength is changed. However, when the wavelength tunable laser of the present invention is used, it is not necessary to readjust the optical axis. Therefore, Raman spectroscopy can be performed while freely changing the excitation wavelength.
[0016]
Furthermore, Raman spectra can be measured by measuring the Raman spectrum while sweeping the excitation wavelength.However, when using a conventional tunable laser, some internal standard is used to quantitatively analyze the relative band intensity. Was needed. With the tunable laser of the present invention, the error of the focal position due to the change of the excitation wavelength is very small, so the quantitative analysis of the relative band intensity by only correcting the laser intensity and the spectrometer device coefficient without using an internal standard Is possible.
The advantages of the tunable laser of the present invention in spectrum measurement are as follows.
[0017]
1. 1. No background noise 2. Easy operation and control 3. Stability of irradiation position and beam profile of TEM ₀₀ 4. The wavelength and intensity can be controlled accurately. Solid state laser (easy maintenance)
6. It has a wide tunable wavelength range (eg, 700-1000 nm when using titanium sapphire as the laser medium) suitable for Raman spectroscopy applications.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic diagram showing an example of an optical system of an ETT laser without background radiation according to the present invention. The configuration of the resonator of the ETT laser according to the present invention is the same as the configuration of the resonator of the conventional ETT laser except that the positions of the output coupling mirror and the total reflection mirror are switched. The wavelength and intensity of the oscillating laser light are controlled by a programmed computer.
[0019]
In this example, a so-called Z-hold type laser resonator is used, in which the optical path of light reciprocating in the laser resonator has a Z-shape of an alphabet. The Z-hold type laser resonator includes an emission mirror 112 and a total reflection mirror 110 having a predetermined transmittance (for example, a reflectance of 98% and a transmittance of 2%). Further, the first intermediate mirror 37 that receives the excitation laser light A and reflects the light B that reciprocates between the emission side mirror 112 and the total reflection mirror 110 and the space between the emission side mirror 112 and the total reflection mirror 110 There is provided a second intermediate mirror 38 for reflecting the reciprocating light B, and the optical path of the light B reciprocating in the laser resonator has a Z-shape of an alphabet.
[0020]
Between the first intermediate mirror 37 and the second intermediate mirror 38 on the optical path of the laser resonator, a laser medium 14 having a Brewster-cut incident end face of incident light as a wavelength tunable laser medium, and an incident end face of which is incident light. Are arranged so as to have a Brewster's angle at which the reflection of light becomes zero, and laser oscillation is generated by the excitation laser light A by longitudinal coaxial excitation. A birefringent acousto-optic element 100 made of TeO ₂ or the like is disposed as a wavelength selecting means between the second intermediate mirror 38 and the emission side mirror 112 on the optical path of the laser resonator.
[0021]
A piezoelectric element 22 driven by an RF power source 20 whose frequency is controlled by a personal computer 26 is attached to the birefringent acousto-optic element 100 as acoustic wave input means. Therefore, by driving the piezoelectric element 22 by the RF driver 20 set to an arbitrary frequency under the control of the personal computer 26 and exciting the acoustic wave corresponding to the frequency to the birefringent acousto-optical element 100, the birefringence is achieved. The acousto-optic device 100 diffracts the light D having the frequency ωo represented by the above equation (1). The piezoelectric element 22 diffracts only the light corresponding to the light B (frequency ωiｏωo) having the wavelength of the outgoing laser light C to be emitted from the emission side mirror 112 by the birefringent acousto-optic element 100 diffracted in a predetermined direction. The personal computer 26 controls the frequency ωa of the acoustic wave input to the birefringent acousto-optic element 100 so that the light is emitted as light D and laser resonance occurs.
[0022]
Between the birefringent acousto-optic element 100 and the exit side mirror 112, a prism 28 as a wavelength dispersion correction element for correcting the dispersion of the diffracted light D is provided. By using the wavelength dispersion correcting prism 28 having this diffraction angle, the direction of the emitted laser light C can be made constant. A pulse laser or a continuous wave laser (CW laser) can be used as the excitation laser 32 for injecting the excitation laser light A into the laser resonator. In this example, the laser medium was titanium sapphire, and 532 nm pulsed light (1.5 kHz) from a double frequency CW-Q switch Nd: YAG laser was used as the pumping light. The excitation laser light A generated by the excitation laser 32 is reflected by the total reflection mirror 36 by the total reflection mirror 34, collected by the total reflection mirror 36, and condensed by the laser medium 14 through the first intermediate mirror 37. Is input so as to excite the longitudinal coaxial excitation.
[0023]
In the above configuration, in order to obtain the output laser light C, the laser medium 14 is excited using the excitation laser light A incident by the excitation laser 32. In addition, the frequency ωa of the RF driver 20 is controlled by the personal computer 26 in accordance with the wavelength (frequency ωo) of the emission laser light C to be emitted from the emission side mirror 112, and the piezoelectric element 22 is driven. In this way, of the light in the wide wavelength band emitted from the laser medium 14 and incident on the birefringent acousto-optic element 100, light having a wavelength corresponding to the frequency of the RF driver 20 is converted into a birefringent acoustic wave. The light is diffracted by the optical element 100 as diffracted light D (frequency ωo). The diffracted light D is vertically incident on the output side mirror 112 via the wavelength dispersion correction prism 28 of the diffraction angle, is reflected by the output side mirror 112, and reciprocates in the laser resonator along a Z-shaped optical path. (Angle frequency ωi at the position of the laser medium 14). Therefore, only light having a wavelength corresponding to the frequency of the RF power supply 20 is amplified and laser oscillates, and the emitted laser light C (frequency ωo) having the wavelength is emitted from the laser resonator.
[0024]
The birefringent acousto-optic device was controlled by a double radio frequency driving (DRD) method in order to stabilize the laser output and the frequency. The first RF channel (RF1) of the birefringent acousto-optic driver supplies an RF signal to the birefringence acousto-optic 100 and changes the wavelength and intensity of the laser radiation. The second RF channel (RF2) is controlled to compensate for output fluctuations of RF1 and optimize at each frequency. As a result, the sum of the energy supplied from RF1 and RF2 to the birefringent acousto-optic device 100 is kept constant. The frequency of RF2 was fixed to 85 MHz corresponding to outside the laser oscillation wavelength range. The DRD method provides high reliability for wavelength (<0.1 nm) and intensity (<± 2% on average of 10 pulses; this stability is limited by the stability of the pump laser). The FWHM (Full Width at Half Maximum) of the spectrum was typically 0.2 nm or less.
[0025]
FIG. 3 is a schematic diagram of a Raman spectrometer using the tunable laser of the present invention shown in FIG. 1 as a light source. The light emitted from the wavelength tunable laser 150 is convergently applied to the sample 215 by the lens 210. The scattered light generated from the sample 215 is focused on the entrance slit of the triple polychromator 223 by the lenses 221 and 222. The spectral signal separated by the triple polychromator 223 was detected by the liquid nitrogen cooled CCD detector 224. The triple polychromator 223, the CCD detector 224, and the tunable laser 150 were all controlled simultaneously by the computer 26. The setting parameters of the spectrometry program for the spectrometer are an excitation wavelength, a measurement range, a slit width, an exposure time, and an integration count.
[0026]
FIG. 4 is a comparison diagram of a scattering spectrum measured under the same conditions using a conventional wavelength tunable laser and the wavelength tunable laser of the present invention by disposing an aluminum foil as a scatterer in a sample cuvette. The horizontal axis represents the wavelength difference from the light source wavelength (700 nm), and the vertical axis represents the count number of the CCD (signal intensity at the CCD detector, referred to as CCD count in this specification).
[0027]
As shown in FIG. 2, the spectrum a shows the case where a wavelength tunable laser in which a total reflection mirror is arranged on the side of the wavelength dispersion correcting prism 28 and an emission side mirror is arranged on the side of the laser medium 14 as shown in FIG. It is a scattering spectrum. As shown in FIG. 1, the spectrum b used the wavelength tunable laser of the present invention in which an emission side mirror was arranged on the side of the wavelength dispersion correcting prism 28 and a total reflection mirror was arranged on the side of the laser medium 14. It is a scattering spectrum in a case. The spectrum b 'is an enlarged view of the spectrum b.
[0028]
Comparing the intensity of the spectrum a with the Raman band of the maximum intensity of acetone put in the sample cuvette which is 0.3 CCD count when measured under the same conditions as the measurement of FIG. It can be seen that the level of the emitted light is two orders of magnitude higher than the signal level of a standard Raman material. On the other hand, when the wavelength tunable laser of the present invention is used, it can be seen that almost no noise caused by the spontaneous fluorescence of the laser medium 14 is observed. This indicates that the fluorescent component was removed by the birefringent acousto-optic device. The sharp peak and the increase in the baseline appearing on the smaller side of the Raman shift of the spectrum b 'are based on stray light. The stepwise intensity changes seen at 1900 cm ^-1 and 3300 cm ^-1 are artifacts created when combining a narrow range of spectral components to cover the entire frequency range.
[0029]
From the viewpoint of a Raman excitation light source, particularly from the viewpoint of resonance Raman excitation profile measurement, in addition to having no noise in the spectrum, stability of the irradiation point and high beam quality over the entire wavelength variable range are desired. FIG. 5 compares the beam profiles at the laser oscillation wavelength of 700 nm and 850 nm measured at a position 300 mm from the output side mirror of the variable wavelength laser of the present invention. The variation of the beam pattern and the beam center when the wavelength was changed was measured by a beam profiler mounted on a CCD head.
[0030]
The diffraction angle of the beam emitted from the birefringent acousto-optic element depends on the wavelength, and the difference between the diffraction angle for a wavelength of 700 nm and the diffraction angle for a wavelength of 1000 nm is theoretically 0.35 °. The beam separation due to the wavelength dependence of the diffraction angle can be reduced by bringing the prism closer to the birefringent acousto-optic element. In this example, the measurement was performed with the distance between the birefringent acousto-optic element and the prism being 2 cm. When the wavelength changed from 700 nm to 850 nm, the movement of the center of the beam was only 2 pixels (0.046 mm). Since the beam diameter is 56 pixels (1.29 mm) at 700 nm and 63 pixels (1.45 mm) at 850 nm, the displacement of the center point is practically negligible. The measured beam pattern indicated the TEM ₀₀ mode. From these results, the beam profile from the wavelength tunable laser of the present invention is comparable to that of a normal titanium sapphire laser using a birefringent filter, a diffraction grating or a prism as a wavelength selection element, and the deviation of the optical axis is the same as the conventional one. It was confirmed that there was no inferiority to the ETT laser.
[0031]
FIG. 6 is a diagram showing an excitation Raman profile of deoxyhemoglobin in an aqueous solution, measured using the Raman spectrometer shown in FIG. FIG. 6 shows 17 Raman spectra measured by changing the excitation wavelength in the range of 700 to 860 nm in steps of 10 nm. The top spectrum has an excitation wavelength of 700 nm, the excitation wavelength becomes longer as going down, and the bottom spectrum has an excitation wavelength of 860 nm.
[0032]
No realignment of the optical axis was required throughout all measurements. Despite the long exposure time of 30 minutes, no background radiation from the laser was observed in the Raman spectrum. This result indicates that the tunable laser of the present invention has high performance as a light source for Raman spectroscopy.
[0033]
In order to compare resonance effects in spectra, the intensity of all measured data at different excitation wavelengths must be calibrated. In a normal Raman excitation profile measurement, a non-resonant internal standard is added to a sample for intensity calibration. However, enhancement effect in the case of measurement of the resonance sample also reached 10 ⁶ times at the maximum must be added a large amount of internal standard. Internal standards cannot be used for in situ measurements because they can alter biological activity. Therefore, a theoretical method was used here to calibrate the intensity of the obtained spectrum. The parameters describing the intensity (Ir) of the Raman spectrum are as follows.
[0034]
Ir∝α (v) I _O (ve) T (v) v ⁴ A (v) ⁻¹ c
α (v): enhancement effect of spectrum intensity due to resonance effect I ₀ : excitation light intensity T (v): throughput of spectrometer and sensitivity of detector v: absolute wave number of Raman spectrum A (v): caused by sample Self-absorption c: sample concentration
T (v) is obtained by recording the spectrum of blackbody radiation with the present system. A (v) can be regarded as a constant because deoxyhemoglobin has a very weak absorption in the 700-1000 nm region. Since the Raman spectra of deoxyhemoglobin in FIG. 6 are all normalized by the above equation, it is possible to compare the intensity of the resonance effect depending on the excitation wavelength. The Raman spectrum excited at the maximum absorption wavelength of deoxyhemoglobin of 760 nm does not show a characteristic resonance enhancing effect. Due to the continuous decrease in Raman intensity as the excitation wavelength becomes longer, these resonance Raman spectra are more affected by the electronic transition called the Q band than the electronic transition observed in the 700 to 1000 nm region of deoxyhemoglobin. Suggests that it has been enhanced.
[0036]
【The invention's effect】
According to the present invention, a tunable laser without background radiation is obtained. Further, according to the present invention, a Raman spectrometer capable of performing high-accuracy Raman spectroscopic measurement and rapid measurement of a Raman excitation profile is obtained.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of an optical system of an ETT laser without background radiation according to the present invention.
FIG. 2 is a schematic configuration diagram of a conventional ETT laser.
FIG. 3 is a schematic diagram of a Raman spectrometer using a tunable laser of the present invention as a light source.
FIG. 4 is a comparison diagram of a scattering spectrum measured under the same conditions using a conventional tunable laser and the tunable laser of the present invention.
FIG. 5 is a diagram showing a beam profile of the tunable laser of the present invention measured at 700 nm and 850 nm.
FIG. 6 is a diagram showing a result of Raman spectrum measurement of hemoglobin in an aqueous solution.
[Explanation of symbols]
14 wavelength variable laser medium, 20 RF power source, 22 piezoelectric element, 26 computer, 28 wavelength compensating prism, 32 excitation laser, 100 birefringent acousto-optical element, 110 total reflection mirror, 112 ... Exit mirror, 150 ... Tunable laser, 215 ... Sample, 223 ... Triple polychromator, 224 ... CCD detector

Claims (3)

A laser resonator having an emission side mirror having a predetermined transmittance and a total reflection mirror, forming a light path for reciprocating light between the emission side mirror and the total reflection mirror, and provided on the optical path; a wavelength tunable laser medium was a birefringent acousto-optic device for wavelength selection, a wavelength tunable laser having a prism as a wavelength dispersion compensation device of the diffraction angle by the birefringent acousto-optic element, the total reflection It said wavelength tunable laser medium disposed closer to the mirror and placed the birefringent Hibiki optical element and the prism side closer to the output mirror to serve as a wavelength selection element and the background emission light removal filter wavelength With a tunable laser,
A first optical system for converging outgoing light from the tunable laser to a sample,
A second optical system for condensing scattered light from the sample on the entrance slit of the spectroscope;
A spectroscope that splits the light condensed by the second optical system,
A Raman spectroscopic measurement device comprising: A laser resonator having an emission side mirror having a predetermined transmittance and a total reflection mirror, forming a light path for reciprocating light between the emission side mirror and the total reflection mirror, and exciting laser light. A first intermediate mirror provided at a position for reflecting the light in the optical path, and a second intermediate mirror provided at a position for reflecting the light in the optical path and the excitation laser light incident on the optical path. a wavelength tunable laser medium disposed between the second intermediate mirror and the first intermediate mirror of said optical path, is provided between the second intermediate mirror and the output mirror on the optical path wavelength A birefringent acousto-optic element for selection, and a prism as a wavelength dispersion correction element for the diffraction angle of the birefringent acousto-optic element, wherein the first intermediate mirror and the second intermediate mirror on the optical path before between Place a wavelength tunable laser medium, the diffraction angle by the birefringent acousto-optical element and the birefringent acousto side closer to the output mirror to serve as a wavelength selection element and the background emission light removal filter A wavelength tunable laser , wherein a prism as a wavelength dispersion correction element is arranged ,
A first optical system for converging outgoing light from the tunable laser to a sample,
A second optical system for condensing scattered light from the sample on the entrance slit of the spectroscope;
A spectroscope that splits the light condensed by the second optical system,
A Raman spectroscopic measurement device comprising: In the tunable laser,
The light on the sample focused on the distance between the birefringent acousto-optic element and the prism so that the fluctuation of the optical axis of the emitted laser light when the wavelength is changed is less than 1/10 of the beam diameter of the emitted laser light. 3. The Raman spectrometer according to claim 1, wherein the irradiation is set so as not to move .

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