JP2012112863A - Multiphoton excitation measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multiphoton excitation measuring device capable of identifying chemical components.SOLUTION: The multiphoton excitation measuring device of the present invention performs measurement of a sample using multiphoton absorption phenomenon by optical pulses having high luminance and comprises: a multi-wavelength light source for emitting multiple optical pulses having different wavelengths; a scanning optical system which overlaps, in the sample, light paths of the multiple optical pulses having different wavelengths generated at the multi-wavelength light source and which scans an overlapping position in the sample; and a detector for detecting signal light along with multiphoton excitation generated from the sample by radiation of the optical pulses.

Description

  The present invention relates to a multiphoton excitation measurement apparatus that irradiates a sample with ultrashort pulse light having a high peak light intensity and measures the sample using a multiphoton absorption phenomenon.

  As a means for performing fluorescence observation with a microscope, as shown in Patent Document 1, a fluorescence observation method using multiphoton excitation by ultrashort pulse light having a specific wavelength is known.

  In multiphoton excitation, an excitation phenomenon equivalent to the original absorption wavelength is caused by simultaneously irradiating the phosphor with a light beam having a wavelength that is approximately an integral multiple of the absorption wavelength. The excitation light used in multiphoton excitation is generally infrared light, and is used for the purpose of measuring the excitation phenomenon of ultraviolet light and visible light.

  In general, light with a longer wavelength is less likely to scatter (Rayleigh scattering), so in a scattering sample such as a biological sample, light can be made to enter deeper by using multiphoton excitation. . This means that it is possible to observe the deep part of the living body that could not be observed with normal visible light. Moreover, since infrared light has lower phototoxicity than ultraviolet light and visible light, it is possible to observe the form of a biological sample without damaging it as much as possible.

  For example, a brain substance called serotonin has an autofluorescent property having an absorption wavelength in the ultraviolet region. However, ultraviolet light cannot reach the deep part of the brain and is highly phototoxic. Under such circumstances, the multiphoton excitation laser scanning microscope works effectively. Since ultraviolet light and infrared light are about three times apart in wavelength, serotonin can be excited using infrared light if three-photon excitation is performed.

  As described above, fluorescence observation using multiphoton excitation has a great merit and is a very effective means in current morphological observation.

JP 2010-008989 A

  However, the microscope described in Patent Document 1 applies multiphoton excitation by a single wavelength using only light of a specific wavelength. For this reason, if the wavelength regions of the absorption bands of several molecules overlap, it is impossible to identify the chemical component of the sample (function observation).

  Therefore, the present invention has been made in view of such problems, and an object of the present invention is to provide a multiphoton excitation measurement apparatus that can identify more chemical components.

  The multiphoton excitation measurement apparatus of the present invention is a multiphoton excitation measurement apparatus that measures a sample by using a multiphoton absorption phenomenon caused by a high-intensity light pulse, and emits a plurality of light pulses having different wavelengths. A scanning optical system in which optical paths of a plurality of light pulses having different wavelengths generated by a multi-wavelength light source are overlapped in the sample and the overlapping position is scanned in the sample, and multi-photons generated from the sample by irradiation of the light pulse It has a detector which detects the signal light accompanying excitation.

  According to the microscope of the present invention, by observing an absorption image or a fluorescence image of molecules by light having a plurality of wavelengths, an effect of enabling more molecules to be distinguished can be obtained.

The figure which shows the structure of the principal part of the multiphoton excitation measuring apparatus which concerns on the 1st and 2nd embodiment of this invention. The figure which shows the principal part structure of an example of the multiwavelength light source shown in FIG. The figure which shows the structure of the principal part of another multiphoton excitation measuring apparatus which concerns on 1st and 2nd embodiment of this invention. The figure which shows the structure of the principal part of the multiphoton excitation measuring apparatus which concerns on the 3rd Embodiment of this invention. The figure which shows the structure of the principal part of the multiphoton excitation measuring apparatus which concerns on the 3rd Embodiment of this invention. Diagram showing the spectral distribution of multiple light pulses generated from a multi-wavelength light source The figure which shows the structure of the principal part of the multiphoton excitation measuring device which concerns on the 4th Embodiment of this invention. The figure which shows the structure of the principal part of the cylindrical beam former shown in FIG. The figure which shows the principal part structure of an example of the multiwavelength light source shown in FIG. The figure which shows the optical profile in the surface C in the multiwavelength excitation measuring apparatus shown in FIG. Diagram showing the transmission spectrum of distilled water

  Embodiments of the present invention will be described below with reference to the drawings.

(Embodiment 1)
FIG. 1 is a diagram showing a schematic configuration of a multiphoton excitation measurement apparatus according to Embodiment 1 of the present invention. The multiphoton excitation measurement apparatus 100 includes a multiwavelength light source 101 that emits light having a plurality of wavelengths, a laser scanning optical unit 102, and an optical fiber 103. The light pulse oscillated from the multi-wavelength light source 101 is incident on the laser scanning optical unit 102 through the optical fiber 103 and condensed on the sample 104. Here, the optical fiber 103 is connected to the multi-wavelength light source 101 and the laser scanning optical unit 102 by optical fiber connectors 105 and 106, respectively.

  The sample 104 is mitochondria in which, for example, cytochrome C (protein) is present in a living cell, and the light pulse collected on the sample 104 exhibits strong resonance Raman scattering due to multiphoton absorption in the cytochrome C.

  FIG. 2 is a diagram showing a partial configuration of an example of the multi-wavelength light source 101 shown in FIG. The multi-wavelength light source 101 includes pulse generators 201 and 202, semiconductor lasers 203 and 204, fiber amplifiers 205 and 206, pulse width compressors 207 and 208, a reflection mirror 209, a dichroic prism 210, and an optical fiber connector 105. The pulse generators 201 and 202 both generate electric pulses of several ns or less, and currents are injected into the semiconductor lasers 203 and 204, respectively, with the electric pulses.

  The semiconductor lasers 203 and 204 perform a so-called gain switch operation in which a gain is instantaneously generated or extinguished by a current injected by an electric pulse from the pulse generators 201 and 202. As a result, the semiconductor lasers 203 and 205 generate an optical pulse with a width of several tens of picoseconds chirped temporally from a short wavelength to a long wavelength. As the semiconductor lasers 203 and 204, for example, a surface emitting laser (VCSEL), a quantum well distributed feedback laser diode (QWDFBLD), a quantum dot distributed feedback laser diode (QDDFBLD), or the like can be used. Use VCSEL. Further, the wavelength of the light pulse may be any wavelength at which resonant Raman scattered light is generated by multiphoton absorption of the sample 104. For example, visible to near-infrared light and infrared light can be used. Here, it is assumed that near-infrared light in the 1064 nm band from the semiconductor laser 203 and 1100 nm band from the semiconductor laser 204 oscillates.

  Optical pulses generated from the semiconductor lasers 203 and 204 are amplified by fiber amplifiers 205 and 206. The fiber amplifiers 205 and 206 are pumped by a fiber laser or a semiconductor laser, and a single clad or double clad optical fiber doped with Nd, Yb, Tm, Er, or the like as a pumped medium, or a gap in the cross section. An optical fiber is used. Here, a single clad optical fiber doped with Yb is used as the excited medium.

  The optical pulses oscillated by the semiconductor lasers 203 and 204 are amplified by the fiber amplifiers 205 and 206 into optical pulses having an average output of several tens mW to several W.

  The two optical pulses in the 1064 nm band and the 1100 nm band after amplification are reflected by, for example, the 1100 nm band optical pulse by the reflection mirror 209 so that the optical path is perpendicular to the 1064 nm band optical pulse. Then, using the dichroic prism 210 that transmits the light of 1064 nm band and reflects the light of 1100 nm band, installed in the part where the optical path of the two optical pulses is orthogonal, the optical path of the two optical pulses is synthesized into the same propagation path. To do. The synthesized light pulse is emitted from the multi-wavelength light source 101 via the optical fiber connector 105.

  As the optical fiber connector 105, various types of connectors of SC type, FC type, ST type, MU type, and LC type can be used. In this embodiment, the FC type is used.

  The light pulse emitted from the multi-wavelength light source 101 propagates through the optical fiber 103 and enters the laser scanning optical unit 102 through the optical fiber connector 106 as shown in FIG. Here, the optical fiber connector 106 also uses the FC type similarly to the optical fiber connector 105.

  The light pulse incident on the laser scanning optical unit 102 is emitted to free space and enters the two-dimensional scanning mirror 107. Here, the scanning mirror changes the scanning direction of the light pulse, and the light propagation angle is manipulated. The two-dimensional scanning mirror 107 includes, for example, two Gullfield mirrors that can swing around two axes that are orthogonal to each other.

  The light pulse that has passed through the two-dimensional scanning mirror 107 is condensed by the pupil projection lens 108 and formed as an intermediate image, then converted into parallel light by the imaging lens 109, transmitted through the dichroic mirror 110, and the objective lens 111. Thus, the light is condensed on the sample 104. Thereby, the cytochrome C contained in the sample 104 is multiphoton excited, and Raman scattered light is emitted. Light emitted from the sample 104 passes through the objective lens 111 and enters a dichroic mirror 110 that reflects light in a specific wavelength band (the wavelength band is different from the wavelength of the light pulse collected on the sample 104). . Since this light is different from the wavelength of the optical pulse to be collected on the sample 104, it is reflected by the dichroic mirror 110, collected by the condenser lens 112 on the detector 113, and converted into an electrical signal.

  The detector 113 is more preferably a so-called spectrophotometer provided with wavelength separating means such as a diffractive optical element and a prism. Thereby, the distribution image of cytochrome C can be obtained more accurately.

  The condensing position of the light pulse is scanned in the XY direction in the sample 104 by the two-dimensional scanning mirror 107. Here, a two-dimensional multiphoton-excited Raman scattering image is obtained by associating and storing the coordinates of the condensing position of the light pulse on the sample 104 and the electric signal detected by the detector 113. Can do. For example, when the two-dimensional scanning mirror is configured by MEMS, the angle of the two-dimensional scanning mirror and the light scanning angle (or the condensing position on the sample 104 are determined from the amount of voltage applied to each electrode provided in the MEMS. Coordinates) can be calculated.

  It is more desirable to provide a means for changing the condensing position in the Z direction. For example, a three-dimensional multiphoton excitation Raman scattering image can be obtained by making the objective lens 111 a variable focus lens using PZT or KTN. It becomes possible to acquire at high speed.

  In the present invention, as described above, the sample 104 is irradiated with light pulses in the 1064 nm band and the 1100 nm band. Thus, the two-photon excitation gives the same effect as the excitation by visible light in the 532 nm band and the 550 nm band.

  Here, the oxidized protein and the reduced protein have different wavelength-dependent characteristics of Raman scattering. For example, oxidized proteins exhibit properties that increase as incident wavelength increases, and reduced proteins exhibit properties that decrease as incident wavelength increases. The characteristics of increase and decrease are not limited to those described above, and may be reversed. In any case, oxidized protein and reduced protein have Raman scattering due to photoexcitation in the 532 nm band and Raman due to photoexcitation in the 550 nm band. The scattering spectra are different, and the relational expression between the amount of Raman scattering and the wavelength is expressed by different expressions. In addition, the formula which shows the spectral characteristic of a substance is previously determined according to the substance.

  Here, the Raman scattering amount of the oxidized protein and the Raman scattering amount of the reduced protein are included in the Raman scattering amount of the 532 nm band and the Raman scattering amount of the 550 nm band, respectively. Using the values of the Raman scattering amount in the 532 nm band and the Raman scattering amount in the 550 nm band, and the wavelength dependence of the Raman scattering characteristics of the oxidized protein and the reduced protein (relational expression of the Raman scattering spectrum), the oxidized protein and the reduced protein are reduced. The amount of Raman scattering in the 532 nm band and the 550 nm band of the type protein can be calculated. Thereby, even when the wavelength regions of the Raman scattering spectra of different substances overlap, the amount of Raman scattering of the specific substance in each wavelength band can be calculated.

  As described above, component analysis by multivariate analysis can be performed by comparing the amounts of Raman scattered light having a plurality of wavelengths.

  Further, as described above, the coordinates of the condensing position of the light pulse on the sample 104 are stored in association with the electric signal detected by the detector 113, and the concentration distribution of each substance is imaged by imaging. Can be

  With the above-described configuration, it is possible to distinguish between oxidized protein and reduced protein, and to image the concentration distribution of each (function observation).

  Further, the multiphoton excitation measurement apparatus of the present invention is less invasive to the part other than the observation part (light pulse condensing position) of the sample, is inexpensive (no confocal optical system is required), and the light propagation direction The resolution in the (optical axis direction) can be increased.

  In this embodiment mode, the oscillation of the 1064 nm band light pulse and the 1100 nm band light pulse is synchronized, and the condensing position is made coincident to irradiate the sample 104, whereby the interaction between the two lights is performed. Causes multiphoton absorption. That is, it becomes possible to obtain a Raman scattering image by excitation of visible light in the 541 nm band which is the sum frequency.

  Here, the sum frequency is generated only in a very limited region including the focal point of the two-wavelength light pulse. Therefore, compared to the case where a single-wavelength light pulse having the same frequency as the sum frequency is incident on the sample 104, the region where the Raman scattering occurs is limited when the sum circumference is generated. For this reason, the resolution of measurement can be further improved by generating a sum frequency by injecting light pulses having a plurality of wavelengths.

  Thus, the two-photon absorption caused by the overlapping of two light pulses having different wavelengths, in other words, the two-photon absorption caused by the two photons having different energies meeting each other, hereinafter referred to as “Wako photon absorption”. And the resulting excitation is called “Wako photon excitation”.

  In addition, since the Raman scattered light generated in the sample has a polarization direction rotated in the sample and includes a polarization component different from the light pulse before the sample is incident, a polarization separation mirror may be used instead of the dichroic mirror 110. By using a dichroic mirror, it is possible to obtain the output of Raman scattered light of all polarization components. As a result, functional image observation with higher accuracy is possible. On the other hand, when a polarization separation mirror is used, the fixed angle likelihood of the mirror becomes wider, and the microscope has excellent vibration resistance and heat resistance. In the present invention, it is necessary to irradiate a sample with light pulses having a plurality of wavelengths and measure their Raman scattered light, and the effect of improving vibration resistance and heat resistance by using a polarization separation mirror is great.

  In addition, although this Embodiment demonstrated based on two-photon absorption, the multiphoton absorption more than three-photon absorption may be sufficient. Further, although the present embodiment has been described using light of two different wavelengths, the multi-wavelength light source 101 may use light of three or more wavelengths.

(Embodiment 2)
In the first embodiment, an example is shown in which infrared light (light pulse) in the 1030 nm band, 1064 nm band, 1100 nm band, or the like is irradiated to the mitochondria (sample) and Raman scattered light generated by cytochrome C in the sample is measured. It was. However, the multiphoton excitation measurement apparatus of the present invention can obtain the same effect by irradiating an arbitrary sample (material) with a plurality of light beams having different optical characteristics. Become.

  For example, in the present embodiment, a multi-photon excitation measurement having a configuration similar to that of FIG. 1 using a multi-wavelength light source having a configuration similar to FIG. The apparatus will be described. In the present embodiment, both the hemoglobin concentration distribution (morphological image) inside the human body and the oxygenation ratio (functional image) of hemoglobin can be acquired.

  More details are shown below.

According to the present embodiment, it is possible to obtain absorbances at two wavelengths of 700 nm band and 933 nm band using two-photon absorption of two lights of 1400 nm band and 1867 nm band. Furthermore, it is possible to determine the absorbance in the 800 nm band using the sum photon absorption in the 1400 nm band and the 1867 nm band. Moreover, the absorbance of oxygenated hemoglobin (Hb) in the wavelength band of 800 nm and that of deoxygenated hemoglobin (HbO ₂ ) are equal, the absorbance in the 700 nm band is higher in deoxygenated hemoglobin, and the absorbance in the 933 nm band is higher in oxygenated hemoglobin. Is expensive. For this reason, the concentration of hemoglobin which is the sum of oxygenated hemoglobin (Hb) and deoxygenated hemoglobin (HbO ₂ ) is determined by determining the absorbance at a wavelength of at least 800 nm band.

Furthermore, the concentration of each of oxygenated hemoglobin (Hb) and deoxygenated hemoglobin (HbO ₂ ) is calculated by measuring the absorbance of either 700 nm band or 933 nm band and comparing it with the absorbance in the 800 nm band. Can do. This makes it possible to determine the oxygenation rate of hemoglobin.

For example, suppose that the oxygenated hemoglobin concentration is x, the deoxygenated hemoglobin concentration is y, the function of the absorbance of light at 800 nm with respect to the oxygenated hemoglobin and deoxygenated hemoglobin concentrations is f, and the deoxygenated hemoglobin concentration. If the function of the absorbance of 700 nm light with respect to g is g, and the function of the absorbance of 700 nm light with respect to the concentration of deoxygenated hemoglobin is h,
f (x + y) = (800 nm absorbance)
g (x) + h (y) = (absorbance at 700 nm)
By solving the simultaneous equations as follows, it becomes possible to obtain the oxygenation rate of hemoglobin.

In the above example, the absorbance in the 800 nm band was used. However, using the absorbance amounts obtained in the 700 nm band and the 933 nm band, the concentrations of oxygenated hemoglobin (Hb) and deoxygenated hemoglobin (HbO ₂ ) were determined. It may be calculated. Moreover, although the absorbance in the 800 nm band was used for the difference in obtaining the total hemoglobin concentration, oxygenated hemoglobin (Hb) and deoxygenated hemoglobin (HbO ₂ ) obtained using the absorbance amounts in the 700 nm band and the 933 nm band were used. You may calculate the sum total of hemoglobin from each density | concentration.

  In the present invention, it is possible to use long-wavelength infrared light having a higher transmittance of the living body by using the multi-photon excitation phenomenon as compared with the case of directly irradiating the living body with light of 700 nm band, 800 nm band, and 933 nm band. This is desirable because it is possible to examine the hemoglobin concentration and oxygenation ratio in a deeper part of the living body.

  In the present embodiment, as in the first embodiment, the sample may be irradiated with a light pulse and the output of the returned light may be measured. However, the light pulse incident on the sample and the sample returned from the sample may be used. The wavelengths of the light beams are the same, and it is desirable to use a polarization separation mirror instead of the dichroic mirror 110. Thereby, the light returned from the sample is guided to the detector 113, and the attenuation amount (absorbance) in the sample is obtained.

  Further, as shown in FIG. 3, a laser pulse optical unit 302 in which the dichroic mirror 110 is omitted from the laser scanning optical unit 102 is used to irradiate the sample 104 with light pulses, and a detector on the opposite side of the laser scanning optical unit 302. A multiphoton excitation measurement apparatus 300 including 303 may be used. This configuration is desirable because it is possible to examine the absorbance more accurately and to obtain a more accurate hemoglobin concentration distribution and oxygenation ratio (a more accurate functional image observation is possible).

  In addition, it is desirable to install a condenser lens 301 between the sample 104 and the detector because the measurement accuracy is further improved.

  In the configuration of FIG. 1, functional image observation is possible even for a sample with low light transmittance or a sample with much scattering. Therefore, there is an advantage that the number of types of materials that can be observed can be increased compared to the configuration of FIG.

(Embodiment 3)
FIG. 4 is a diagram showing a schematic configuration of a multiphoton excitation measurement apparatus according to Embodiment 3 of the present invention. Similar to the multiphoton excitation measurement apparatus 100 shown in FIG. 1, this multiphoton excitation measurement apparatus 400 makes two optical pulses oscillated by a multiwavelength light source 101 incident on a laser scanning optical unit through an optical fiber 103. Thus, the sample 104 (mitochondria containing cytochrome C in living cells) is irradiated.

However, in the laser scanning optical unit 402 shown in this embodiment, the incident light pulse is emitted into free space and enters the diffraction grating 401. Here, since the diffraction grating 401 diffracts light pulses having two different wavelengths so that the reflection angles differ according to the wavelengths, the optical paths are separated as indicated by a dotted line and a broken line in FIG. 4, for example. Both optical pulses of two wavelengths whose optical paths are separated by the wavelength are incident on the sample 104 by the objective lens 404. The objective lens 404 is designed so that two light pulses whose optical paths are separated are superimposed again in the sample, and the light pulses are collected at the same position in the sample 104. For example, when the distance from the diffraction grating to the objective lens is a, the distance from the objective lens to the condensing position in the material is b, and the focal length of the objective lens is f,
1 / f = 1 / a + 1 / b
It is sufficient to select an objective lens of f that satisfies the following relationship.

  Thereby, the cytochrome C contained in the sample 104 is multiphoton excited, and Raman scattered light is emitted.

  At the position where two light pulses overlap (intersect), the two-photon absorption (excitation) of each light pulse and the sum photon absorption (excitation) phenomenon of two light pulses of different wavelengths occur as in the first embodiment. The Raman scattered light generated there is converted into an electrical signal by the detector 113.

  In the present embodiment, the objective lens 404 translates in the XY direction, so that the position where two light pulses having different wavelengths overlap each other scans the sample 104 in the XY direction.

  The detector 113 is more preferably a so-called spectrophotometer provided with wavelength separation means such as a diffractive optical element and a prism. Thereby, the distribution image of cytochrome C can be obtained more accurately.

  By storing the coordinates of the intersecting positions of the light pulses on the sample 104 and the spectrum of the Raman scattered light detected by the detector 113, a two-dimensional multiphoton excitation Raman scattering image can be obtained. it can. Thereby, the concentration distribution of cytochrome C in mitochondria can be observed.

  In the present embodiment, it is desirable to provide the condensing lens 403 so that the focal points of the two light pulses are positioned on the grating surface of the diffraction grating 401.

  As a result, the position where the two light pulses intersect in the sample 104 and the position of the focal point of each light pulse coincide with each other, so that a stronger multiphoton excitation phenomenon occurs and a density distribution image of cytochrome C with less noise is acquired. It becomes possible to do.

  In the present embodiment, since the optical paths of two optical pulses having different wavelengths are different, the Wako photon excitation phenomenon occurs only at the intersecting portion. For this reason, the number of observation points of the scattered spectrum can be increased by measuring the Raman scattering spectrum by the Wako photon excitation. Moreover, since the generation area of the Japanese circle group is limited to the vicinity of the condensing point, a higher resolution cytochrome C concentration distribution image can be obtained by generating the Japanese circle group and measuring the scattering spectrum.

  In the present embodiment, as in the first embodiment, the same effects as excitation by visible light in the 532 nm band, 541 nm band, and 550 nm band are given by making the sample 104 enter the 1064 nm band and 1100 nm band light pulses. It becomes. By comparing Raman scattering images when excited with a light pulse in the 1064 nm band and excited with a light pulse in the 1100 nm band, the oxidized protein and the reduced protein are identified, and each concentration distribution is imaged. (Function observation) becomes possible.

  In addition, the multiphoton excitation measurement apparatus of the present embodiment is also less invasive with respect to parts other than the observation part (light pulse condensing position) of the sample, and is inexpensive (without requiring a confocal optical system). It is possible to increase the resolution in the propagation direction (optical axis direction).

  In the present embodiment, it is desirable that the light pulse incident on the laser scanning optical unit 402 through the optical fiber 103 is passed through the condenser lens 403 and condensed on the reflection surface of the two-dimensional scanning diffraction grating 401.

  As a result, the reflecting surface of the two-dimensional scanning diffraction grating 401 can be made smaller, and two-dimensional scanning with lower power consumption can be achieved at a lower cost.

  Further, since the two light pulses in the sample 104 overlap at the respective condensing positions, a functional image with higher contrast can be obtained.

  Further, on two surfaces (A surface and B surface) substantially perpendicular to the optical pulse optical path before the objective lens 404, the expansion ratio of the beam diameters of the two optical pulses on the A surface and the B surface, and the two optical pulse optical paths. It is desirable that the enlargement ratios of the intervals substantially coincide. The beam diameter enlargement ratio means the beam diameter on the B surface / the beam diameter on the A surface. As a result, in the sample 104, the two light pulses overlap at the respective condensing positions. That is, it becomes possible to perform the Wako photon absorption (Wako photon excitation) at the position where the light intensity is highest, and the obtained Raman scattering spectrum is also strengthened. As a result, a functional image with higher contrast can be obtained.

  Further, this embodiment may adopt the configuration shown in FIG. That is, the Raman scattered light generated in the sample 104 travels backward in the optical pulse optical path made incident on the sample 104 and is again taken into the laser scanning optical unit from the objective lens 404 and reflected by the diffraction grating 401. The multiphoton excitation measurement apparatus 500 includes a laser scanning optical unit 501 that separates a light pulse before being incident on the sample 104 and an optical path of the light reflected by the sample 104 using the dichroic mirror 110 and makes the light incident on the detector 113. May be.

  The configuration of FIG. 4 can receive light from a larger number of samples, and there is less electrical noise, and more accurate (highly reproducible) cytochrome C concentration distribution and oxidation / reduction ratio images can be obtained. Is desirable. However, the configuration of FIG. 5 is desirable because it allows more accurate spectrophotometric measurement and increases the accuracy of component analysis.

  Needless to say, the optical configuration of the present embodiment may be used as a multiphoton excitation measurement device for examining the concentration of hemoglobin and the proportion of oxygenation in a living body, as in the second embodiment, and another sample. It may be used for functional image observation and morphological image observation.

  In the multiphoton excitation measurement apparatus of this embodiment, as shown in the first and second embodiments, the optical pulses of two wavelengths are desirable because the resolution in the Z direction is higher than that of the configuration of the same optical path. In addition, the multiphoton excitation measurement apparatus having the configuration of the first and second embodiments is desirable because the optical system can be made simpler and less expensive.

(Embodiment 4)
FIG. 7 is a diagram showing a schematic configuration of a multiphoton excitation measurement apparatus according to Embodiment 4 of the present invention. In this multiphoton excitation measurement apparatus 700, two optical pulses having different wavelengths oscillated by a multiwavelength light source 701 are incident on a laser scanning optical unit 702 through separate optical fibers 103 and 703, respectively, and a sample 104 (cytochrome C) is introduced. Mitochondria contained in living cells).

  Here, the optical fiber 103 is connected to the multi-wavelength light source 101 and the laser scanning optical unit 702 by optical fiber connectors 105 and 106, respectively.

  The sample 104 is mitochondria in which cytochrome C (protein) is present in a living cell, and the light pulse collected on the sample 104 exhibits strong resonance Raman scattering due to multiphoton absorption in the cytochrome C.

  FIG. 9 is a diagram showing a partial configuration of an example of the multi-wavelength light source 701 shown in FIG. The multi-wavelength light source 701 includes pulse generators 201 and 202, semiconductor lasers 203 and 204, fiber amplifiers 205 and 206, pulse width compressors 207 and 208, and optical fiber connectors 105 and 705.

  The pulse generators 201 and 202 both generate electric pulses of several ns or less, and currents are injected into the semiconductor lasers 203 and 204, respectively, with the electric pulses.

  The semiconductor lasers 203 and 204 perform a so-called gain switching operation in which a gain is instantaneously generated and extinguished by a current injected by an electric pulse from the pulse generators 201 and 202, thereby chirping from a short wavelength to a long wavelength in time. An optical pulse having a width of several tens of picoseconds is generated. As the semiconductor lasers 203 and 204, for example, a surface emitting laser (VCSEL), a quantum well distributed feedback laser diode (QWDFBLD), a quantum dot distributed feedback laser diode (QDDFBLD), or the like can be used. Use VCSEL. The wavelength of the light pulse may be any wavelength that causes resonance Raman scattering due to multiphoton absorption of the sample 104, and visible to near-infrared light and infrared light can be used. Here, it is assumed that near-infrared light in the 1064 nm band from the semiconductor laser 203 and the 1100 nm band from the semiconductor laser 204 oscillate.

  Optical pulses generated from the semiconductor lasers 203 and 204 are amplified by fiber amplifiers 205 and 206. The fiber amplifiers 205 and 206 are excited by a fiber laser or a semiconductor laser. As a pumped medium in the fiber laser, a single clad or double clad optical fiber doped with Nd, Yb, Tm, Er, or the like, or an optical fiber having a gap in the cross section is used. Here, a single clad optical fiber doped with Yb is used as a pumped medium, and excitation is performed using a semiconductor laser having a wavelength of 940 nm.

  Here, the optical pulses oscillated by the semiconductor lasers 203 and 204 are amplified by the fiber amplifiers 205 and 206 into optical pulses having an average output of several tens mW to several W.

  The two amplified optical pulses of the 1064 nm band and 1100 nm band are emitted from the multi-wavelength light source 701 through the optical fiber connectors 105 and 705, respectively.

  The optical fiber connector 705 is also an FC type in this embodiment.

  The light pulse emitted from the multi-wavelength light source 701 propagates through the optical fibers 103 and 703 and enters the laser scanning optical unit 702 via the optical fiber connectors 106 and 706 as shown in FIG. Here, the optical fiber connector 706 also uses the FC type as in the optical fiber connector 705.

  In the laser scanning optical unit 702 shown in the present embodiment, the two incident light pulses are both emitted into free space and become parallel light through the collimating lenses 707 and 708.

  Thereafter, one of the two optical pulses having different wavelengths oscillated by the multi-wavelength light source 701 (here, an optical pulse in the 1100 nm band) is incident on the cylindrical beam former 704. Here, as shown in FIG. 8, the cylindrical beam former 704 includes a pair of facing axico lenses (conical lenses) 801 and 802, and converts a Gaussian beam 803 incident from the plane side of the axico lens 801 into a cylindrical beam 804. It becomes composition.

  As shown in FIG. 7, the light pulse in the 1100 nm band emitted from the cylindrical beam former 704 is reflected by the reflection mirror 209 and the dichroic prism 210 and is not incident on the cylindrical beam former 704 ( (1064 nm band optical pulse) and the same propagation path.

  Two optical pulses having different wavelengths and having the same optical path are incident on the two-dimensional scanning mirror 107. Here, the propagation angle is scanned.

  The light pulse that has passed through the two-dimensional scanning mirror 107 is condensed by the pupil projection lens 108 and formed as an intermediate image, then converted into parallel light by the imaging lens 109, and condensed on the sample 104 by the objective lens 111. . Thereby, the cytochrome C contained in the sample 104 is multiphoton excited, and Raman scattered light is emitted.

  Here, FIG. 10 shows a light pulse profile (light intensity distribution) on the surface C immediately after the objective lens 111. The optical pulse profile 1001 in the 1064 nm band shows a Gaussian distribution with the optical axis as the center. Similarly, an optical pulse profile 1002 in the 1100 nm band is centered on the optical axis, but shows an annular light intensity distribution. Further, in FIG. 10, a portion of light intensity equal to or higher than e ^ −2 of the maximum intensity of light in the 1100 nm band is indicated by hatching in the light intensity in the 1100 nm band. In addition, among the light intensities in the 1064 nm band, a portion having a light intensity equal to or higher than e ^ −2 of the maximum intensity of the light in the 1064 nm band is indicated by a hatched portion.

In the optical pulse profile 1002 in the 1100 nm band, the inner diameter a and the outer diameter b can be increased by widening the distance between the axico lens 801 and the axico lens 802 in the cylindrical beam former 704. The inner diameter “a” in the optical pulse profile 1002 of the 1100 nm band is larger than the beam diameter of the light pulse of the 1064 nm band (the distance between the portion of e ⁻² times the light intensity at the highest light intensity and the optical axis). By arranging two axico lenses, it is possible to suppress overlapping of profiles of two light pulses. As described above, when the overlap of the profiles of the two light pulses on the surface C immediately after the objective lens is eliminated, the overlap of the profiles of the two light pulses is eliminated except at the position where the light pulse in the 1100 nm band is focused. However, the profile of the light pulse in the 1100 nm band is also a one-peak profile having a peak light intensity near the true center of the optical axis at the condensing position. That is, the 1100 nm band optical pulse and the 1064 nm band optical pulse overlap only at the condensing position of the 1100 nm band optical pulse.

  For this reason, the Raman scattered light by the sum photon excitation is generated only at the condensing position of the light pulse in the 1100 nm band. As a result, the measurement resolution can be improved.

  The Raman scattered light generated by the sum photon excitation is condensed on the detector 303 by the condenser lens 301 behind the sample 104 and converted into an electric signal. The two-dimensional scanning mirror 107 scans the sample 104 in the X and Y directions for the light pulse condensing position, and the coordinates of the light pulse condensing position on the sample 104 and the electrical signal detected by the detector 113 are obtained. By storing the data in association with each other, a two-dimensional sum photon excited Raman scattering image can be obtained.

  In the present invention, as described above, the sample 104 is irradiated with light pulses in the 1064 nm band and the 1100 nm band. Thus, the two-photon excitation gives the same effect as the excitation by visible light in the 532 nm band and the 550 nm band.

  Oxidized protein and reduced protein differ in Raman scattering due to photoexcitation in the 532 nm band and Raman scattering spectrum due to photoexcitation in the 550 nm band. Imaging (functional observation) becomes possible.

  Further, the multiphoton excitation measurement apparatus of the present invention is less invasive to the part other than the observation part (light pulse condensing position) of the sample, is inexpensive (no confocal optical system is required), and the light propagation direction The resolution in the (optical axis direction) can be increased.

  As in the first embodiment, it is also possible to obtain a Raman scattering spectrum image by light excitation in the 541 nm band at a condensing position where two light beams having different wavelengths overlap.

  In the present embodiment, the detector 303 is provided with a pinhole, and a confocal optical system is used to measure only the Raman scattered light generated at the condensing positions of the 1064 nm band and 1100 nm band light pulses. Is possible.

  This is desirable because it is possible to compare the absorbance of light of three wavelengths of 532 nm, 541 nm, and 550 nm at the same position.

  As described above, in the first to fourth embodiments, an example of a multi-wavelength light source including a plurality of pulse light sources that oscillate light having different wavelengths has been described. However, a multi-wavelength light source including one pulse light source having a variable wavelength is used. Even in this case, it is possible to measure the multiphoton absorption rate of a plurality of wavelengths. That is, it becomes a multiphoton excitation measurement apparatus capable of not only morphological image observation but also functional image observation.

  However, as shown in the first to fourth embodiments, the multi-photon excitation measurement apparatus of the present invention uses a multi-wavelength light source including a plurality of pulse light sources and simultaneously oscillates a plurality of lights having different wavelengths. Each photon absorption, photon absorption,..., Not only the phenomenon but also the photon absorption phenomenon can be used, so that it is possible to measure the multiphoton absorption rate of more wavelengths.

  Furthermore, as shown in Embodiments 3 and 4, it is possible to increase the resolution by changing the optical paths of a plurality of lights having different wavelengths so that they overlap each other only in a certain part.

  In addition, with respect to the first to fourth embodiments of the present invention, the resolution in the Z direction is the first and second embodiments <the fourth embodiment> the third embodiment, and the price of the optical system portion is also the first embodiment, 2 <Embodiment 4 <Embodiment 3 Therefore, it is desirable to use an embodiment suitable for the application.

  The following can be said in common with respect to the first to fourth embodiments.

  First, it goes without saying that an optical characteristic image of arbitrary wavelength excitation can be obtained by using a multi-wavelength light source that oscillates an optical pulse of an arbitrary wavelength instead of the multi-wavelength light source 101.

  For example, when the sample is cytochrome C in mitochondria and the Raman scattering spectrum of the sample is observed, a semiconductor laser that oscillates a light pulse in the 1030 nm band may be used instead of the semiconductor laser 201 in FIG. The sample can be irradiated with light pulses of 1030 nm band and 1100 nm band, and by two-photon excitation and sum photon excitation of 1030 nm band and 1100 nm band, respectively, 515 nm band, 532 nm band, It becomes possible to obtain a Raman scattering image by excitation with visible light of three wavelengths in the 550 nm band.

Cytochrome C is desirable because a strong Raman peak (753, 1127, 1314, 1583 cm ⁻¹ ) due to Raman scattering due to light pulse excitation in the 515 nm band is observed. Compared to the configuration in which the light pulses of the two wavelengths of the 1064 nm band and the 1100 nm band described above are irradiated, cytochrome C distribution image observation (morphological image observation) can be performed more accurately. This feature can be applied to all configurations of the present invention in which cytochrome C is irradiated with light pulses having a wavelength of less than 1045 nm and light pulses having a wavelength longer than 1045 nm.

  In addition, the configuration in which the light pulse with a wavelength of less than 1080 nm and the light pulse with a wavelength longer than 1080 nm are irradiated is desirable because the oxidation / reduction ratio of cytochrome C can be obtained more accurately.

  In the first to fourth embodiments, a multiwavelength light source including a semiconductor laser and a fiber amplifier is used. However, a titanium sapphire laser light source that is generally used in a multiphoton excitation measurement apparatus may be used.

Alternatively, a Q-switched short pulse solid-state laser using a laser medium such as YAG or YVO ₄ to which rare earth such as Yb or Nd is added may be used.

  However, it is desirable to use a pulsed light source composed of a semiconductor laser and a fiber amplifier because it is possible to oscillate optical pulses of various wavelengths at a lower cost, and it is possible to identify more types of chemical substances.

  Further, by using a titanium sapphire laser light source or a Q-switched short pulse solid-state laser, it is possible to irradiate a sample with a light pulse with a higher peak, which is desirable because the concentration measurement accuracy of chemical substances is improved.

  In addition, when the sample is a living body, use of a fiber laser such as a chrome forsterite laser light source that excels in light transmission properties to the living body enables morphological image observation and functional image observation to a deeper depth of the living body. Is desirable because it becomes possible.

  In the multiphoton excitation measurement apparatus shown in Embodiments 1 to 4, it is desirable to irradiate the sample with light pulses having three or more different wavelengths, and the accuracy of the morphological image and the contrast of the functional image are improved. .

However, as shown in FIG. 6, the spectrum of the light pulse applied to the sample is the shortest wavelength, and the center wavelengths are λ ₁ , λ ₂ ... Λ _n , and λ _{n + 1} , respectively, and the full width at half maximum of the wavelength. Is Δλ ₁ , Δλ ₂ ... Δλ _n , Δλ _{n + 1} , it is desirable to satisfy the following formula 1 for at least one n (n is a natural number).

  Thereby, since the accuracy of the separation of the optical characteristics by m photon excitation (m is a natural number of 2 or more) of light of adjacent wavelengths is greatly improved, it is possible to increase the contrast of the obtained functional image.

  More preferably, it is desirable to satisfy Equation 2 for at least one wavelength.

  As a result, the accuracy of separating the optical characteristics by the sum photon excitation of the light of the adjacent wavelengths is greatly improved, so that it is possible to further increase the contrast of the obtained functional image.

When the sample contains cytochrome C,
(1) Wavelength shorter than 1045 nm (2) Wavelength longer than 1045 nm and shorter than 1080 nm (3) Accuracy and function of morphological image by including at least one light pulse in three wavelength regions longer than 1080 nm This is desirable because the contrast of the image is the highest.

  In a multiphoton excitation measurement device that irradiates a sample with short optical pulses of multiple wavelengths regardless of the wavelength from the multiwavelength light source, the sample is irradiated with multiple light pulses of different wavelengths simultaneously, and the sample is irradiated separately In this case, it is desirable to measure and compare light emitted from the sample (Raman scattered light in the first embodiment; the same applies to fluorescence). Thereby, functional image observation with higher contrast becomes possible.

  For example, taking Embodiment 1 as an example, first, the sample 104 is irradiated with light pulses of 1064 nm band and 1100 nm band separately (at the timing), and Raman scattering images by visible light excitation in the 532 nm band and 550 nm band are obtained. get. Next, the sample 104 is simultaneously irradiated with light pulses in the 1064 nm band and 1100 nm band, and a Raman scattering image by visible light excitation in the 541 nm band is acquired.

  Thereby, it is possible to more accurately separate Raman scattered light according to the respective wavelengths of the 532 nm band, the 541 nm band, and the 550 nm band. That is, more accurate Raman scattering images of respective wavelengths can be acquired, and the accuracy (reproducibility) of functional image observation can be improved. Although not shown, a timing control means for controlling the oscillation timing of each optical pulse may be provided.

  More preferably, it is desirable to irradiate the sample while changing the light intensity of the light pulses of a plurality of wavelengths independently, and to measure and compare the light emitted from the sample. It is possible to classify optical characteristics (Raman scattering, light absorption characteristics, fluorescence characteristics) by three-photon excitation, and optical characteristics by multiphoton excitation.

  For example, when measuring Raman scattered light, it is possible to separate Raman scattering by two-photon excitation, Raman scattering by three-photon excitation, and further higher-order Raman scattering, so that higher-contrast functional image observation is possible. Become. Similarly, since fluorescence and Raman scattering can be separated, the contrast of the functional image is further improved.

  Similarly, it is desirable to provide means for deforming the pulse waveforms of the optical pulses having two wavelengths. By modifying the pulse waveform and adjusting the timing of the two optical pulse oscillations, the two-photon absorption, the three-photon absorption, and the sum photon absorption of each optical pulse at the position where the two optical pulse optical paths overlap can be obtained. It can be arbitrarily adjusted, and the contrast of the functional image can be improved.

  In addition, optical pulses of two wavelengths after amplification (in the first example of the first embodiment, two optical pulses in the 1064 nm band and 1100 nm band) are respectively provided with pulse width compressors as shown in FIG. 207 and 208 may be installed to shorten the pulse. Since the counter-excitation phenomenon in the sample 105 occurs more remarkably, more accurate functional image observation is possible. Although the details of the pulse width compressors 207 and 208 are not shown, it is possible to use an optical fiber exhibiting chromatic dispersion characteristics, a dispersion compensation means having a different optical path length depending on the wavelength by combining a diffraction grating and a mirror, and the like. Since an optical pulse generated by operating a gain switch of a semiconductor laser exhibits a wavelength characteristic chirped in time from a short wavelength to a long wavelength, as described above, a pulse width compressor having a configuration in which the optical distance differs depending on the wavelength is used. Use is desirable because it allows compression of the pulse width. A cheaper multi-wavelength light source is possible.

  The fiber amplifiers 205 and 206 may be multistage amplifiers in which a plurality of fiber amplifiers are connected in series. The amplification factor for each stage of the fiber amplifier is desirably 1000 times or less, and this makes it possible to perform optical pulse amplification with less noise and high efficiency, so that a more accurate form image can be obtained.

  Further, the amplification factor for each stage is desirably 50 times or more, and the multiphoton excitation of the specimen can be performed at a lower cost.

  Moreover, in the application for obtaining the hemoglobin concentration and oxygenation ratio in the living body, it is desirable to use light having a wavelength that is less absorbed by water. FIG. 11 shows a transmission spectrum of distilled water having a cell length of 1 cm. As shown in FIG. 11, it is desirable to irradiate a living body using a light pulse having a wavelength of 1440 nm or less and a light pulse having a wavelength of 1625 nm or more. This makes it possible to obtain the hemoglobin concentration and oxygenation ratio at a deeper position in the living body.

  Further, oxygenated hemoglobin and deoxygenated hemoglobin have the same absorbance with respect to light near 800 nm. For this reason, it is desirable to determine the absorbance of light in the vicinity of 800 nm, the absorbance of short waves from the 800 nm band, and the absorbance of long waves from the 800 nm band. In other words, it is desirable to obtain the absorbance of light having a wavelength of 800 nm by sum photon excitation using light pulses having two different wavelengths, and the wavelengths of two light pulses generated by a multi-wavelength light source are λ1 [nm] and λ2 When it is [nm], it is desirable to satisfy all of the following conditions (1) to (3), whereby the oxygenation rate of hemoglobin can be measured more accurately.

(1) λ1 <1440
(2) 1625 <λ2 <1925
(3) λ1 = (λ3 * λ2) / (λ2-λ3)
Here, λ3 is substantially the same as 800 nm, and in the range from 790 nm to 810 nm, it is regarded as substantially the same, and the same effect is obtained.

In order to satisfy the conditions (2) and (3),
(4) 1340 <λ1
It is necessary to satisfy.

Further, (3) is (3) ′ 790 <(λ1 * λ2) / (λ1 + λ2) <810.

  In the multiphoton excitation measurement apparatus of the present invention, it is possible to generate highly accurate multiphoton excitation by generating significant multiphoton absorption, and to improve resolution when used for morphological image observation. It becomes possible. For this reason, it is desirable that the absorption energy due to multiphoton absorption is somewhat larger than the absorption energy due to linear absorption in the specimen.

  It is preferable that the light absorption energy due to multiphoton absorption is at least 10% of the light absorption energy due to linear absorption.

  Further, the total light absorption energy due to the sum photon absorption is proportional to the light intensity of a plurality of overlapping light pulses, and the light absorption energy ratio of each light pulse is inversely proportional to the wavelength of each light pulse.

  For this reason, when a light pulse with the same pulse width and a high light intensity and a light pulse with a low light intensity are overlapped, the light pulse with a low light intensity has a higher light absorption rate due to multiphoton absorption. In other words, by monitoring the output of the light pulse with the smaller light intensity after leaving the sample, adjusting the overlap of both light pulses (adjusting the oscillation timing), the absorbance due to multiphoton absorption is more prominent. This is desirable because it can be obtained and a high-contrast functional image can be obtained.

  For this reason, in the component densitometers of the first to fourth embodiments, when the sample is irradiated with two light pulses having different wavelengths and the optical path and oscillation timing of each light pulse are adjusted so as to overlap in the sample, It is desirable that there is a difference in the light intensity of the two light pulses.

  More desirably, the light intensity of the light pulse having the shorter wavelength is desirably small.

  The same applies to a configuration in which a sample is irradiated with a plurality of light pulses having different wavelengths.

  In addition, it is desirable to oscillate the light pulse with the smaller light intensity by using a broadband pulse light source such as SC (super continuum) light source, SLD, femtosecond OPO, etc., and measure the absorbance of various wavelengths with higher accuracy. This is desirable because it can be done. Thereby, a functional image with higher accuracy can be obtained.

  In the first to fourth embodiments, it is more desirable that at least one light pulse is generated from a wavelength tunable light source, and it is possible to measure the light absorbance of a plurality of wavelengths with higher sensitivity and higher accuracy. Become. As a result, it is possible to obtain a functional image with high accuracy up to a deep position of a scatterer or a sample having a low transmittance, which is more difficult to receive light.

  In the first to fourth embodiments, the multiphoton excitation measurement device using a living body containing cytochrome C or hemoglobin as a target sample has been shown. However, the application range of the multiphoton excitation measurement device of the present invention is not limited to these, The same effect is exhibited in various applications.

  For example, it can be used as a component concentration meter for measuring blood sugar levels in the body. For example, when measuring blood sugar levels, by irradiating a human body with a far-infrared light pulse with a wavelength of 3 to 4 μm, the light absorption characteristics corresponding to the reference vibration due to the molecular vibration of glucose and the light absorption characteristics such as overtones. Both can be observed, which is desirable because it enables more accurate component concentration measurement.

  In addition, by using optical pulses of near-infrared light in the 2 to 1 μm band, it is possible to measure component concentrations applying both overtones of molecular vibrations and light absorption characteristics due to electronic vibrations. This is desirable because value measurement is possible.

  The multiphoton excitation measurement apparatus of the present invention can also be applied as a measurement apparatus that performs measurement by multiphoton excitation (nonlinear excitation) of another sample such as a semiconductor material.

  In addition, when the multi-photon excitation measurement apparatus of the present invention simultaneously oscillates a plurality of light pulses having different wavelengths and uses it as a fluorescence microscope utilizing the fluorescence of the substance by the sum photon excitation, it outputs the light pulse output from the multi-wavelength light source. It is desirable to make it variable.

  This makes it possible to identify substances using supersaturated absorption and non-linear fluorescence phenomena, and to measure the concentration (distribution) of more substances.

  In the above-described embodiment, the example in which the multiphoton excitation measurement device is used to calculate the concentration distribution of a plurality of substances has been described. However, even when the concentration of the substance is not calculated (that is, only the spectrum is observed), the number of observation points that can be measured can be increased as compared with the conventional measurement apparatus. As a result, it is possible to obtain a specific effect that the measurement accuracy can be further improved.

  In addition, when measuring a sample containing a plurality of substances, the concentration of each substance is obtained without performing subsequent calculations by performing measurements using two wavelength bands with little overlap of absorption spectra of each substance. be able to. For example, a wavelength λ1 at which Raman scattering occurs in the substance A (and a wavelength at which Raman scattering is difficult to occur at the substance B) and a wavelength λ2 at which Raman scattering occurs at the substance B (and a wavelength at which Raman scattering is unlikely to occur at the substance A). By performing the measurement using, the concentration of two different substances can be calculated in one measurement.

  In addition, when the sum frequency of λ1 and λ2 is present in the wavelength band where Raman scattering occurs in either substance A or substance B, the measurement results obtained in this sum frequency group are used. The measurement points of the substance A or the substance B can be increased. Therefore, measurement accuracy can be further improved.

  As described above, the multiphoton excitation measurement device of the present invention is not limited to the specific purpose of using the obtained measurement spectrum, and there is also a specific calculation method using the obtained measurement spectrum. It is not limited to the method.

  As mentioned above, although it showed about the measuring apparatus of this invention, the structure shown by this specification is an example, and it cannot be overemphasized that various changes are possible in the range which does not deviate from the main point of this invention.

  The multiphoton excitation measurement apparatus according to the present invention is not limited to the multiphoton excitation laser scanning microscope for measuring living cells described in the above embodiment, but multiphoton excitation (nonlinear excitation) of other samples such as semiconductor materials. The present invention can also be applied to a measuring device that measures.

  In addition, it is possible to provide a measuring apparatus that can not only observe the structure of a substance but also identify a chemical substance.

DESCRIPTION OF SYMBOLS 100 Multiphoton excitation measuring apparatus 101 Multiwavelength light source 102 Laser scanning optical unit 103 Single mode optical fiber 104 Sample 105 Optical fiber connector 106 Optical fiber connector 107 Two-dimensional scanning mirror 108 Pupil projection lens 109 Imaging lens 110 Dichroic mirror 111 Objective lens 112 Condensing lens 113 Detector 201 Pulse generator 202 Pulse generator 203 Semiconductor laser 204 Semiconductor laser 205 Fiber amplifier 206 Fiber amplifier 207 Pulse width compressor 208 Pulse width compressor 209 Reflecting mirror 210 Dichroic prism 300 Multiphoton excitation measuring apparatus 301 Condensing lens 302 Laser scanning optical unit 303 Detector 400 Multiphoton excitation measuring device 401 Diffraction grating 402 Laser scanning optical unit 403 Condensing lens 404 Objective lens 500 Multiphoton excitation measurement device 501 Laser scanning optical unit 700 Multiphoton excitation measurement device 702 Laser scanning optical unit 703 Optical fiber 704 Cylindrical beam former 705 Optical fiber connector 706 Optical fiber connector 707 Collimating lens 708 Collimating lens 801 Axico lens 802 Axico lens 803 Gaussian beam 804 Cylindrical beam 1001 Profile of light pulse in 1064 nm band 1002 Profile of light pulse in 1100 nm band

Claims (11)

A multiphoton excitation measurement device that measures a sample using a multiphoton absorption phenomenon caused by a light pulse,
A multi-wavelength light source that emits a plurality of light pulses having different wavelengths;
A scanning optical system that superimposes the optical paths of a plurality of light pulses having different wavelengths generated by the multi-wavelength light source in the sample, and scans the overlapped position in the sample;
A detector for detecting signal light accompanying multiphoton excitation generated from the sample by irradiation of the light pulse;
A multiphoton excitation measurement apparatus comprising: Separating an optical path of at least one optical pulse among the plurality of optical pulses having different wavelengths from an optical path of at least one other optical pulse after the optical pulse is emitted from the multi-wavelength light source;
The multiphoton excitation measurement apparatus according to claim 1, wherein the multiphoton excitation measurement apparatus overlaps again in the sample. Means for converting at least one of the plurality of light pulses having different wavelengths into a cylindrical beam;
A condensing optical system for condensing the cylindrical beam in the sample;
The multiphoton excitation measurement apparatus according to claim 1, wherein The optical system is designed so that the at least two optical pulses are condensed at a position where at least two optical pulses of the plurality of optical pulses having different wavelengths overlap each other. 3. The multiphoton excitation measurement apparatus according to 3. Comprising timing control means for changing the timing of oscillating a plurality of optical pulses having different wavelengths;
The timing control means oscillates at least a plurality of light pulses having different wavelengths so that optical paths of the plurality of light pulses having different wavelengths are simultaneously overlapped in the sample, and collects light having the plurality of wavelengths. 2. The light pulse is oscillated so that at least a part of the plurality of light pulses having different wavelengths is condensed in the sample at a timing different from the timing of light emission. The multiphoton excitation measurement apparatus described. The signal light obtained when the light of the plurality of wavelengths is condensed so as to overlap in the sample at the same time, and the signal light obtained when the at least some light pulses are condensed in the sample. The multiphoton excitation measurement apparatus according to claim 6, wherein comparison is performed. The means for converting the light pulse into a cylindrical beam includes a pair of conical lenses, and the intensity distribution of light emitted from a condensing optical system for condensing the cylindrical beam is made different for each light pulse. The multiphoton excitation measurement apparatus according to claim 3. The multi-wavelength light source oscillates at least a light pulse having a wavelength shorter than 1045 nm and a light pulse having a wavelength longer than 1045 nm, irradiates the sample with a light pulse having a wavelength shorter than 1045 nm, and the 1045 nm. Using a signal obtained by irradiating the sample with a light pulse having a longer wavelength, and a signal obtained at a sum frequency wavelength of the light pulse having a wavelength shorter than 1045 nm and the light pulse having a wavelength longer than 1045 nm. The multiphoton excitation measurement apparatus according to claim 1, wherein concentration distributions of oxidized protein and reduced protein in the sample are obtained. From the multi-wavelength light source,
Light pulses with wavelengths λ1 and λ2, and 790 nm <(λ1 * λ2) / (λ1 + λ2) <810 nm
Oscillate an optical pulse that satisfies
The multiphoton excitation measurement apparatus according to claim 1, wherein concentration distributions of oxygenated hemoglobin and deoxygenated hemoglobin in the sample are obtained. The λ1 and λ2 are 1340 nm <λ1 <1440 nm, respectively.
1625 nm <λ2 <1925 nm
The multiphoton excitation measurement apparatus according to claim 7, wherein: With respect to the spectrum of the plurality of optical pulses having different wavelengths generated by the multi-wavelength light source, the center wavelengths are set to λ ₁ , λ ₂ ... Λ _n , λ _{n + 1} from the shortest wavelength, respectively, and the half value of the wavelength The total width is Δλ ₁ , Δλ ₂ ... Δλ _n , Δλ _{n + 1} ,
For at least one n (n is a natural number), λ _{n + 1} −λ _n > Δλ _{n + 1} + Δλ _n
The multiphoton excitation measurement apparatus according to claim 1, wherein:

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