JP2009229714A - Chart for resolution evaluation of coherent raman microscope, its manufacturing method, light source device for coherent raman microscope, and method of adjusting coherent raman microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chart for resolution evaluation for objectively and quantatively evaluating imaging performance of a coherent Raman microscope. <P>SOLUTION: The chart 10 for resolution evaluation used for evaluating the imaging performance of the coherent Raman microscope includes: a substrate 11 optically smooth for illumination light and not emitting fluorescence for illumination light excitation; and a Raman active layer 12 containing a Raman active substance not emitting fluorescence for illumination light excitation by being two-dimensionally formed on the substrate 11 and having oscillation spectrum different from the substrate 11. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a resolution evaluation chart for a coherent Raman microscope, a manufacturing method thereof, a light source device for a coherent Raman microscope, and a method for adjusting a coherent Raman microscope.

  The technology of the optical microscope is old, and various types of microscopes have been developed. In recent years, more advanced microscope systems have been developed due to advances in peripheral technologies such as laser technology and electronic image technology.

  In such a background, a highly functional microscope using various spectroscopic processes has been proposed, and not only the shape of the sample but also the identification and structural analysis of the molecules contained in the sample are possible. Among them, there is a Raman spectroscopic method for analyzing the structure by observing the optical response of a biological sample or industrial material without staining it, and application to the microscope field is expected (for example, patent documents). 1).

  Raman spectroscopy is based on a kind of nonlinear optical effect called the Raman effect. When light is scattered by molecules and atoms with a strong photon flux, the quantum state of the molecules changes and the energy of the entire system changes. At that time, the changed energy shifts to scattered photons, and as a result, light having a wavelength different from that of the incident light is generated. Such a phenomenon is called Raman scattering. The Raman scattering includes (1) non-resonant Raman scattering, (2) genuine resonance Raman scattering, (3) pre-resonance Raman scattering, and (4) coherent Raman scattering. Hereinafter, these Raman scattering will be described in more detail with reference to FIGS. 5 and 6.

  FIG. 5A is an energy diagram illustrating non-resonant Raman scattering. Non-resonant Raman scattering can be explained by second-order perturbation theory from the viewpoint of atoms and molecules. That is, as shown in FIG. 5A, this is a kind of two-photon process assuming a virtual quantum level called S (imaginary). In this two-photon excitation process, a molecule that is in the lowest electronic state and in the lowest vibrational rotational level, that is, in the ground state S0, is, for example, once with a very strong laser beam and a virtual quantum level S (imaginary). And then de-excited to the high vibrational rotation level (V2) of the lowest electronic state. As a result, as is clear from FIG. 5A, the incident light gives the photon energy of (Ei-E0) to the atom or molecule, and after non-resonant Raman scattering, the light is equivalent to the photon. Energy is lost, and the apparent wavelength of light is λ1, but is changed to a longer wavelength of λ2 and scattered. In general, a two-photon process based on a virtual quantum level including non-resonant Raman scattering has a very low transition probability, and an ultrashort pulse laser on the order of femtoseconds is required to induce this process. In some cases.

  FIG. 5B is an energy diagram for explaining the true resonance Raman scattering. Authentic resonance Raman scattering is a scattering process when non-resonance Raman scattering satisfies special conditions, and when S (imaginary) matches the actual first electronically excited state S1, as shown in FIG. It is. This case corresponds to a process in which a molecule in the ground state S0 is excited to the actual first electronic excited state S1 and then deexcited to the vibrational rotational level (V2) having the lowest electronic state. Apparently, as shown in FIG. 1 (b), it is as if the fluorescence of λ2 is emitted after excitation of S0 → S1. Since this genuine resonance Raman scattering uses an existing quantum state, the scattering probability is extremely high, and Raman scattering can be expressed with a much stronger light intensity than non-resonance Raman scattering.

  FIG. 5C is an energy diagram illustrating the pre-resonance Raman scattering. Pre-resonance Raman scattering has an intermediate property between genuine resonance Raman scattering and non-resonance Raman scattering. That is, S (imaginary) exists in the vicinity of the first electronically excited state S1.

  6A and 6B are diagrams illustrating coherent Raman scattering. FIG. 6A is an energy diagram of Coherent Anti-Stokes Raman Scattering (CARS), and FIG. 6B is a coherent Stokes Raman. It is an energy diagram of scattering (Coherent Stokes Raman Scattering; CSRS). Coherent Raman Scattering is one of the means to study vibrational dynamics in the time domain, and various studies have been conducted from both experimental and theoretical sides.

  This coherent Raman scattering is one of third-order nonlinear optical processes, and generally uses two laser beams (ω1 light and ω2 light) having different angular frequencies. First, the laser light of ω1 light and ω2 light that interacts with molecules is also called pump light and Stokes light. When the angular frequency difference between these two incident lights matches the angular frequency Ω of the vibration mode of the sample molecule, the vibration modes of many sample molecules are excited in resonance and in phase, that is, coherently. The The generated vibrational polarization lasts for the phase relaxation time, so that another ω1 light, which is the probe light, interacts with the molecule in the meantime to generate coherent Raman as a polarization wave derived from the third-order nonlinear polarization. Scattered light can be extracted.

  That is, information on the phase relaxation time of molecular vibration can be obtained by changing the delay time between the pump light and Stokes light and the probe light. In particular, as shown in FIG. 6A, Raman scattered light whose frequency is increased by + Ω is called CARS. In addition, as shown in FIG. 6B, the Raman scattered light whose frequency is reduced by −Ω is called CSRS. In particular, in the case of CARS, since signal light is detected at a shorter wavelength side than excitation light, it is not easily affected by the background due to autofluorescence, and signal light can be detected with good S / N. Widely applied to analytical microscopes.

  As described above, since the CARS process is a nonlinear optical process, signal light is generated only from a specific minute three-dimensional portion where laser light, that is, pump light (probe light) and Stokes light are strongly condensed. As a result, it is not necessary to introduce a pinhole unlike a confocal microscope, and by introducing a CARS process in a scanning laser microscope, an essentially high three-dimensional spatial resolution can be realized. Moreover, since a biological sample can be observed without staining, the commercialization thereof can be strongly expected.

Japanese translation of PCT publication No. 2002-520612

  By the way, in a normal optical microscope, for example, illumination light and signal light are proportional to each other as in a fluorescence microscope or a transmission type bright field microscope. In addition, since it is not necessary to condense light of a plurality of wavelengths in the same space with high accuracy, assembly and adjustment of the microscope and fine adjustment during observation are not complicated, and a user-friendly microscope system can be provided. In addition, there is a test chart (microscope resolution chart) whose space shape is accurately calibrated. Therefore, it is possible to optimize the imaging performance by performing optical adjustment while observing the microscope resolution chart. Specifically, as shown in FIG. 7A, the fine line pattern of the wavelength order formed on the microscope resolution chart 100 is observed, and the image is measured as shown in FIG. Transfer functions and two-dimensional point spread functions can be evaluated quantitatively. Detailed system adjustment (microscope adjustment) such as selection of a microscope objective lens, optical axis adjustment, detector adjustment, and stage scanning mechanism check can be easily performed so as to optimize these evaluation physical quantities.

However, microscopy using the coherent Raman process is different. Certainly, as described above, a microscope using a coherent Raman process, in particular, a CARS microscope has an excellent function that an ordinary optical microscope does not have, but it is difficult to adjust the system to realize this. There is a face. The main reason is that the coherent Raman process uses a third-order nonlinear optical effect using two-color light sources. That is, assuming that the pump light intensity P ₁ and the Stokes light intensity P ₂ are obtained, the obtained Raman scattering signal, that is, the signal intensity I is given by the following equation (1).

  In addition, in the microscope method using the latest and more advanced CARS process, a more precise measurement method using three colors of light instead of two colors of light is being studied. In FIG. 6, the ω1 illumination light (pump light) excited from the ground state and the ω1 illumination light excited from the excitation vibration level use light of the same wavelength (probe light). The light and the probe light become the same and become a two-color light source. However, the pump light and the probe wavelength may be different. That is, assuming that the wavelength of the probe light is ω1 ′, the wavelength ω1 ′ is made variable to resonate with an arbitrary vibration level, and among the various vibration levels due to this, CARS scattered light caused by a specific vibration level It is a method of detecting. Such a measurement method is called multiplex anticoherent Raman microscopy. In this case, in order to obtain CARS scattered light efficiently, it is necessary to optically adjust the three colors of light completely coaxially.

As apparent from the equation (1), the signal intensity I of Raman scattering is the product of the power of the pump light intensity and the intensity of the Stokes light on the sample focusing surface due to the nonlinear effect of the coherent Raman scattering process. It is proportional. Therefore, the generation condition of the Raman scattering signal greatly varies depending on the overlap state of the pump light and the Stokes light in the spatial and temporal regions and the intensity of the pump light and the Stokes light. More specifically, the signal space distribution generated in the vicinity of the sample condensing surface changes greatly. For this reason, in some cases, the imaging performance is greatly degraded. For example, basically, since the pump signal and the Stokes light are not three-dimensionally overlapped, a Raman signal cannot be obtained. Therefore, not only the S / N is extremely deteriorated, but also the point spread function is widened. Spatial resolution is extremely degraded. Further, even if the intensity adjustment of the pump light and Stokes light is mistaken, the final state is saturated, the point spread function is greatly changed, and the spatial resolution is deteriorated. In multiplex CARS microscopy, this is reflected more remarkably in the adjustment conditions.

  As is clear from the above, the CARS microscope requires much more accurate and careful system adjustment than a normal microscope. Therefore, conventionally, in the CARS microscope, the system adjustment was performed by developing an image of polystyrene beads having a diameter of about 200 nm on a substrate and observing the image.

However, with this conventional adjustment method, strict quantitative evaluation is impossible for the following reasons.
(1) Basically, since polystyrene beads are monodispersed and an isolated bead image is measured, the most important two-point resolution and contrast transfer function cannot be measured as microscope performance.
(2) Although the diameter of the polystyrene beads is selected by a centrifuge or the like, there is a variation of about 20% at the maximum.

  This is due to the fact that there is no chart for resolution evaluation that is easily available for CARS microscopes. That is, there is no standard sample in which a Raman active substance is two-dimensionally or three-dimensionally arranged with a high spatial control accuracy finer than the wavelength order. This has made the work of evaluating the imaging performance as a CARS microscope system and the adjustment based on it uncertain and inefficient.

  Accordingly, a first object of the present invention made in view of such a point is to provide a resolution evaluation chart capable of objectively and quantitatively evaluating the imaging performance of a coherent Raman microscope.

  Furthermore, the second object of the present invention is to provide a manufacturing method capable of easily manufacturing the above-mentioned resolution evaluation chart.

  A third object of the present invention is to provide a light source device that is optimal for use in a coherent Raman microscope.

  A fourth object of the present invention is to provide an adjustment method that can easily optimize a coherent Raman microscope.

The invention according to claim 1, which achieves the first object, is a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
A substrate that is optically smooth with respect to illumination light and that does not emit fluorescence with respect to illumination light excitation;
A Raman active layer that is formed two-dimensionally on the substrate, has a vibration spectrum different from that of the substrate, and includes a Raman active material that does not emit fluorescence in response to illumination light excitation;
It is characterized by having.

Furthermore, the invention according to claim 2 for achieving the first object is a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
A substrate that is optically smooth with respect to illumination light and that does not emit fluorescence with respect to illumination light excitation;
A Raman active layer formed on the substrate and having a vibration spectrum different from that of the substrate and including a Raman active material that does not emit fluorescence in response to illumination light excitation;
A mask layer that is two-dimensionally formed on the Raman active layer, has a vibration spectrum different from that of the Raman active material, does not emit fluorescence in response to excitation of illumination light, and reflects or absorbs illumination light;
It is characterized by having.

The invention according to claim 3 is the resolution evaluation chart of the coherent Raman microscope according to claim 1 or 2,
The Raman active substance is composed of an organic substance.

The invention according to claim 4 is the resolution evaluation chart of the coherent Raman microscope according to claim 3,
The Raman-active substance includes a chemical group having a vibration level that resonates with a difference in frequency between illumination light of pump light and Stokes light.

The invention according to claim 5 is the resolution evaluation chart of the coherent Raman microscope according to claim 4,
The chemical group is a CH group.

The invention according to claim 6 is the resolution evaluation chart of the coherent Raman microscope according to claim 5,
The Raman-active substance includes polyethylene.

Furthermore, the invention according to claim 7 that achieves the second object is to produce a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope.
On a substrate that is optically smooth with respect to illumination light and does not emit fluorescence with respect to illumination light excitation, a Raman active substance that has a vibration spectrum different from that of the substrate and does not emit fluorescence with respect to illumination light excitation. A Raman active layer forming step of forming a Raman active layer including;
A pattern forming step of forming the two-dimensional pattern of the Raman active layer by two-dimensionally removing the Raman active layer formed on the substrate;
It is characterized by including.

Furthermore, the invention according to claim 8 that achieves the second object described above is to produce a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope.
On a substrate that is optically smooth with respect to illumination light and does not emit fluorescence with respect to illumination light excitation, a Raman active substance that has a vibration spectrum different from that of the substrate and does not emit fluorescence with respect to illumination light excitation. A Raman active layer forming step of forming a Raman active layer including;
On the Raman active layer formed on the substrate, a mask layer that has a vibration spectrum different from that of the Raman active material, does not emit fluorescence in response to excitation of illumination light, and reflects or absorbs illumination light. A mask layer forming step to form two-dimensionally;
It is characterized by including.

The invention according to claim 9 is the method for producing a chart for resolution evaluation according to claim 7 or 8,
The Raman active layer forming step is characterized in that a Raman active material made of an organic substance is spread on the substrate by a spin coating method or a dip coating method to form the Raman active layer.

The invention according to claim 10 is the method of manufacturing the resolution evaluation chart according to claim 7,
The pattern forming step is characterized in that a two-dimensional pattern of the Raman active layer is formed by a nanoimprinting method.

The invention according to claim 11 is the method for producing the resolution evaluation chart according to claim 8,
The mask layer forming step is characterized in that the mask layer is formed by a vapor deposition method.

Furthermore, the invention according to claim 12 for achieving the third object is a light source device used for a coherent Raman microscope,
A laser light source for emitting laser light;
A laser medium including an organic crystal that emits Raman light that is incident on the laser light from the laser light source and emits Raman light shifted to a longer wavelength side than the laser light together with the laser light;
It is characterized by having.

The invention according to claim 13 is the light source device for the coherent Raman microscope according to claim 12,
A spectral dispersion element that removes a high-order Raman light component having a wavelength longer than that of the primary Raman light is provided in an optical path from the laser medium.

The invention according to claim 14 is the light source device for a coherent Raman microscope according to claim 13,
The spectral dispersion element is composed of a multilayer filter or a diffraction grating.

The invention according to claim 15 is the light source device for a coherent Raman microscope according to any one of claims 12 to 14,
The organic crystal includes a CH group.

The invention according to claim 16 is the light source device for a coherent Raman microscope according to claim 15,
The organic crystal includes polyethylene.

Furthermore, the invention according to claim 17 that achieves the fourth object is to adjust a coherent Raman microscope.
Using the resolution evaluation chart according to any one of claims 1 to 6, adjusting the contrast ratio of a contrast transfer function obtained by photographing a microscope image of the resolution evaluation chart to be maximum. It is a feature.

The invention according to claim 18 is the adjustment method of the coherent Raman microscope according to claim 17,
The light source device according to any one of claims 12 to 16 is used as a light source device for microscopic observation of the resolution evaluation chart.

  According to the resolution evaluation chart of the present invention, the illumination active light has a two-dimensional pattern of a Raman active layer containing a Raman active substance. Therefore, by taking an image of the resolution evaluation chart with a coherent Raman microscope. The imaging performance of the coherent Raman microscope can be objectively and quantitatively evaluated.

  According to the method for manufacturing a resolution evaluation chart of the present invention, after forming a Raman active layer containing a Raman active material on a substrate, the Raman active layer is removed two-dimensionally to obtain a two-dimensional pattern of the Raman active layer. Or after forming a Raman active layer on the substrate, a mask layer is two-dimensionally formed on the Raman active layer to form a two-dimensional pattern of the Raman active layer. Can be easily manufactured.

  According to the light source device for a coherent Raman microscope of the present invention, a laser medium including an organic crystal is included, and the laser beam is incident on the laser medium, so that the incident laser beam and the longer wavelength side of the laser beam are incident. Since the shifted Raman light is emitted, it is possible to provide an optimum light source device for the coherent Raman microscope.

  According to the method for adjusting a coherent Raman microscope of the present invention, a resolution evaluation chart having a two-dimensional pattern of a Raman active layer containing a Raman active substance is used, and the contrast ratio is maximized by photographing the microscope image. Because of the adjustment, the coherent Raman microscope can be easily optimized.

(Outline of the embodiment)
First, an outline of an embodiment of the present invention will be described. If the present inventors can devise a standard resolution evaluation chart that can accurately and quantitatively evaluate the imaging performance of a microscope using a Raman process, the coherent Raman analysis is performed by photographing this and analyzing the microscope image. We thought that an adjustment method that can optimize the performance of the microscope is possible.

  Therefore, the inventors first developed a standard resolution evaluation chart for a coherent Raman microscope. This resolution evaluation chart is basically formed by two-dimensionally developing a Raman active layer containing a Raman active substance on a substrate and then forming a two-dimensional fine pattern. In recent years, the number of biological samples has increased, and chemical groups such as CH, OH, HN, and COOH are becoming important observation targets. For this reason, as an example of the resolution evaluation chart, an organic thin film is selected as the Raman active substance, and this organic thin film is coated on, for example, a glass substrate or a silicon substrate.

  As an organic thin film, for example, if PMMA (methyl methacrylate) which is easy to be thinned is selected, it is preferable because CH and COOH unique to biomolecules are contained abundantly. Moreover, PMMA can be easily dissolved by a chemical solvent such as ethyl acetate or heating, and a thin film having a uniform thickness of nanometer order can be developed two-dimensionally by a spin coating method. In addition, since no fluorescence is emitted on the longer wavelength side than the visible light region, a high S / N can be expected when performing Raman spectroscopy. This is a value that can be sufficiently ignored with respect to the wavelength of visible light, and is optimal for evaluation of resolution in the depth direction, for example.

  For thin film formation, a dip coating method can also be applied. In the dip coating method, the substrate is immersed in a molten organic material (medium) and pulled up at a constant speed, so that the medium is uniformly adsorbed on the substrate surface with a constant thickness by the surface tension acting between the substrate and the medium. The thickness can be controlled by the pulling speed of the substrate.

  Since both commercially available spin coating methods and dip coating methods are available, an organic thin film can be easily formed. Alternatively, a thin film can be formed by depositing a Raman active material on a substrate in a vacuum layer by a sputtering method. In the case of the sputtering method, the Raman active material is not limited to an organic material, and even if it is an inorganic material, a semiconductor, or a metal, there is an advantage that it can be developed two-dimensionally on the substrate.

  A Raman active layer containing a Raman active material formed by planar development on a substrate is processed into a fine two-dimensional pattern, for example. As this processing method, an electron beam method or a lithography method can be applied, and in any case, an etching pattern of a thin line group composed of lines and spaces of several tens of nanometers can be drawn. Since this production accuracy can be confirmed with an electron microscope, calibration can be easily performed.

  Since the thin line group formed in this way is a size smaller than the diffraction limit in a normal optical microscope, if a microscopic image of this thin line group is taken, the contrast of the obtained thin line group is the optical performance of the microscope alone. The contrast transfer function to be determined. Therefore, if this is evaluated, the imaging performance of the microscope alone can be understood, so that the microscope system can be adjusted so that the contrast ratio given by the contrast transfer function is maximized. Specifically, adjustment of excitation intensity of pump light and Stokes light, optical axis adjustment of pump light and Stokes light, noise reduction by selective adjustment of optical filter, sensitivity adjustment of detector (for example, photomultiplier tube), Adjustment of the microscope objective lens and the like can be adjusted while objectively evaluating the contrast transfer function.

  Therefore, by using the resolution evaluation chart prepared as described above, everyone can easily optimize the coherent Raman microscope as a standardized operation without relying on the subjective evaluation of the adjuster. Can do.

  In addition to the electron beam method and the lithography method described above, a nanoimprinting method can also be applied to the production of a two-dimensional fine pattern. The nanoimprinting method is a patterning method by pressing a two-dimensional fine pattern template prepared in advance against a heat-meltable thin film layer (Raman active layer). According to this method, since patterning can be performed easily, a large number of resolution evaluation charts can be supplied at low cost.

  Further, instead of directly processing the Raman active layer, it is also possible to form a fine pattern by depositing a metallic thin film on the Raman active layer. For example, chromium having a high absorptance or gold thin film having a high reflectance may be coated on the Raman active layer. In this case, since the illumination light does not reach the Raman active layer in the area masked with the metal thin film, no Raman signal is detected from this area.

  The present invention further provides a light source device suitable for a coherent Raman microscope. The coherent Raman microscope generates Raman scattered light that is shifted relative to the pump light by the vibration frequency of the chemical group to be detected. Usually, a near-infrared femtosecond: titanium (Ti) sapphire laser is used, its fundamental wave is directly used as Stokes light, and a part of it is branched to a nonlinear wavelength such as an optical parametric oscillator (OPO). Wavelength conversion is performed by a conversion optical system to generate pump light. Since this method can change the wavelength of the pump light, it can cope with chemical groups having any natural frequency, but requires a large scale and extremely high optical adjustment.

  Accordingly, the present inventors have conducted extensive studies and found that, for example, if attention is paid to a representative chemical group, pump light (probe light) and Stokes light can be generated easily and stably. For example, when it is assumed that a microscope adjustment is performed on CH groups that are abundantly contained in a biological sample, a Raman laser unit is similarly produced using an organic crystal containing CH groups, and the wavelength is converted by this unit. Use light.

  For example, when crystalline polyethylene is used, since the chemical bond group is only CH group, an extremely strong Raman laser beam shifted to the long wavelength side by about 2900 Kaiser is generated with respect to the incident laser beam. At the same time, the higher-order harmonic waves are also generated. If this higher-order harmonic wave is cut by a filter, high-intensity laser light with extremely high monochromaticity can be generated. Therefore, if this incident laser light and Raman laser light are used in a coherent Raman microscope, pump light (probe light) and Stokes light for a CH chemical group can be easily generated without wavelength tuning using OPO. it can. In addition, since the Raman laser light is basically generated in complete synchronization with the excitation laser light, operations such as timing adjustment of generated pulses, which are peculiar to the coherent Raman microscope, can be simplified.

  If this Raman laser unit is introduced as a light source device of a coherent Raman microscope, it can be utilized for observation of a biological sample containing organic matter. Furthermore, when using the resolution evaluation chart including the organic thin film such as PMMA described above in the Raman active layer at the time of adjusting the microscope, the difference in the frequency of the pump light and Stokes light from the light source device and the frequency of the sample Are particularly effective since they are perfectly matched and a very strong Raman signal is obtained. In addition, when a resolution evaluation chart in which the Raman active layer is made of a polyethylene thin film is used, a Raman signal having an extremely high S / N ratio due to CH vibration can be obtained. Therefore, the coherent Raman microscope can be easily optimized by adjusting the coherent Raman microscope using the resolution evaluation chart and the light source device. If such a resolution evaluation chart and light source device are introduced into a production line of a coherent Raman microscope, a standardized inspection method can be realized.

  Hereinafter, specific embodiments will be described with reference to the drawings.

(First embodiment)
1A and 1B show a resolution evaluation chart according to the first embodiment of the present invention. FIG. 1A is an enlarged plan view, and FIG. This resolution evaluation chart 10 has a vibration spectrum different from that of the substrate 11 on a substrate 11 that is optically smooth with respect to the illumination light used in the coherent Raman microscope and that does not emit fluorescence in response to excitation of the illumination light. In addition, a two-dimensional pattern of the Raman active layer 12 containing a Raman active substance that does not emit fluorescence in response to illumination light excitation is formed.

  FIG. 1 shows a two-dimensional pattern of the Raman active layer 12, a 5 μm pattern portion 13a in which a Raman active layer 12 having a line width of 5 μm is formed at intervals of 5 μm (space), and a Raman active layer 12 having a line width of 3 μm formed at intervals of 3 μm. 3 μm pattern portion 13b, 1 μm pattern portion 13c in which Raman active layer 12 having a line width of 1 μm is formed at intervals of 1 μm, 500 nm pattern portion 13d in which Raman active layer 12 having a line width of 500 nm is formed at intervals of 500 nm, and a line width of 250 nm The 250 nm pattern portion 13e in which the Raman active layer 12 is formed at an interval of 250 nm, the 100 nm pattern portion 13f in which the Raman active layer 12 with a line width of 100 nm is formed at an interval of 100 nm, and the Raman active layer 12 with a line width of 50 nm are formed at an interval of 50 nm. 50 nm pattern part 13g and each Raman Line sexual layer 12 indicates the case of forming in parallel. In addition, the line width and interval in each pattern part are shown schematically.

  The substrate 11 has an accuracy of about λ / 20 (accuracy not affecting the resolution in the depth direction (vertical resolution) with respect to the wavelength λ of illumination light generally used by a coherent Raman microscope, for example ( For example, when illumination light having a wavelength of 1 μm is used, a glass substrate optically polished with an accuracy of 50 nm) is used. The Raman active layer 12 is formed by using PMMA containing a CH group as shown in FIG. 2 as a Raman active material, coating this PMMA on the substrate 11 by spin coating, and then nanoimprinting.

  Here, since PMMA easily melts at about 100 ° C., it is easy to process and can be easily formed into a film. Further, the surface accuracy of the substrate 11 is sufficiently higher than the smoothness of about λ / 4 according to the general optical theory, in which the shape of the condensed two-dimensional point spread function is not greatly broken. Therefore, if the spin coating speed condition is, for example, between 10,000 rpm and 1000 rpm and the dissolved PMMA is coated, the PMMA film can be accurately formed on the glass substrate in units of 10 nm. Thereby, for example, as shown in FIG. 1A, a two-dimensional pattern of the Raman active layer 12 including the 50 nm pattern portion 13g can be formed using a commercially available apparatus.

  In particular, when a two-dimensional pattern of the Raman active layer 12 is formed by the nano-imprinting method, the nano-imprinting method can pattern both a conductor and an insulator, so that the contrast has sufficient shape accuracy for an optical microscope. A transfer function resolution evaluation chart 10 can be obtained.

  Accordingly, if this resolution evaluation chart 10 is photographed with a coherent Raman microscope and the contrast of the photographed two-dimensional pattern of the Raman active layer 12 is analyzed wave-optically, a two-point resolution, a point spread function, Distortion etc. can be known quantitatively. Moreover, the alignment state of the optical axis of pump light (probe light) and Stokes light can be grasped from the contrast transfer function. In particular, when the two-dimensional pattern of the Raman active layer 12 is formed by the nanoimprinting method, the surrounding organic material can be removed leaving only a limited point (point area: size of 100 nm). Dimensional point spread function can be measured directly. That is, since the point area has a small illumination light condensing size, the image two-dimensionally mapped with the Raman signal directly corresponds to the two-dimensional point spread function.

(Second Embodiment)
3A and 3B show a resolution evaluation chart according to the second embodiment of the present invention. FIG. 3A is an enlarged plan view and FIG. This resolution evaluation chart 20 has a vibration spectrum different from that of the substrate 21 on a substrate 21 that is optically smooth with respect to the illumination light used in the coherent Raman microscope and does not emit fluorescence in response to excitation of the illumination light. And a Raman active layer 22 containing a Raman active material that does not emit fluorescence in response to illumination light excitation, and has a vibration spectrum different from that of the Raman active material on the Raman active layer 22 for excitation of illumination light. In contrast, a two-dimensional pattern of the mask layer 23 that does not emit fluorescence and reflects or absorbs illumination light is formed.

  The substrate 21 does not affect the resolution in the depth direction (vertical resolution) with respect to the wavelength λ of the illumination light generally used by the coherent Raman microscope, for example, similarly to the substrate 11 shown in FIG. A glass substrate optically polished with an accuracy of about λ / 20 is used. The Raman active layer 22 is formed by using PMMA as a Raman active material and developing the PMMA on the substrate 21 by spin coating.

  In the present embodiment, the Raman active layer 22 is not directly processed to form a two-dimensional pattern, but a mask layer 23 is formed on the Raman active layer 22 as, for example, chromium having high absorptance or gold having high reflectivity. A two-dimensional pattern of the thin film is formed by vapor deposition, and the two-dimensional pattern of the Raman active layer 22 is indirectly formed.

  FIG. 3 shows a two-dimensional pattern of the Raman active layer 22 and the mask layer 23, a 5 μm pattern portion 24a in which a Raman active layer 22 and a mask layer 23 having a line width of 5 μm are alternately formed, a Raman active layer 22 having a line width of 3 μm, and A 3 μm pattern portion 24b in which mask layers 23 are alternately formed, a 1 μm pattern portion 24c in which line width 1 μm of Raman active layers 22 and mask layers 23 are alternately formed, a Raman active layer 22 and mask layers 23 having a line width of 500 nm, The alternately formed 500 nm pattern portions 24d, the 250 nm pattern portions 24e alternately formed with the Raman active layers 22 and the mask layers 23 having a line width of 250 nm, and the Raman active layers 22 and the mask layers 23 having a line width of 100 nm were alternately formed. 100 nm pattern part 24f and Raman activity with a line width of 50 nm 22 and a 50nm pattern portion 24g formed alternately mask layer 23, the lines of the Raman active layer 22 and mask layer 23 shows a case of forming in parallel. Note that the line width and interval in each pattern portion are schematically shown in the same manner as in FIG.

  Therefore, if this resolution evaluation chart 20 is photographed with a coherent Raman microscope and the contrast of the photographed two-dimensional pattern of the Raman active layer 22 is analyzed by wave optics, the resolution evaluation chart of the first embodiment is used. As in the case of using the chart 10, the two-point resolution, the point spread function, the distortion, and the like can be known quantitatively.

(Third embodiment)
FIG. 4 is a diagram showing a schematic configuration of a coherent Raman microscope using the light source device according to the third embodiment of the present invention. In this coherent Raman microscope, the illumination light emitted from the light source device 31 is condensed on the sample 36 held on the sample stage 35 via the reflection mirror 32, the beam splitter 33 and the microscope objective lens 34. The scattered light from the sample 36 enters the illumination light cut filter 37 through the microscope objective lens 34 and the beam splitter 33, and after the wavelength component of the illumination light is cut here, the CARS light is taken out by the spectroscope 38 to increase the photoelectrons. Light is received by the double tube 39. The illumination light condensed on the sample 36 is scanned by the sample stage 35 or a scanning optical system (not shown).

  The light source device 31 according to the present embodiment receives the pulse laser light source 41 and the pulse laser light from the pulse laser light source 41, and enters the pulse laser light of the incident light and the longer wavelength side than the pulse laser light. A laser medium 42 including an organic crystal that emits shifted Raman light and a spectral dispersion element 43 that removes a higher-order Raman light component having a longer wavelength than the first-order Raman light from the laser medium 42 are included.

  The pulse laser light source 41 uses, for example, a titanium / sapphire laser that emits a near infrared femtosecond pulse with a high peak value. As the laser medium 42, for example, C-PET (crystallized polyethylene terephthalate) having a high degree of crystallinity is used. The spectral dispersion element 43 uses a high-pass filter or a diffraction grating.

  In this way, when C-PET is used as the laser medium 42, CH-chains are arranged in C-PET with spatial regularity, so that stimulated Raman light is easily generated. For example, when an end face of a C-PET block in cm is mirror-polished and a near-infrared femtosecond pulse with a high peak value emitted from a pulse laser light source 41 made of a titanium / sapphire laser is incident, CH oscillation Intense coherent light including high-order overtones corresponding to, and having a frequency shifted to the long wavelength side by 2900 Kaiser is stimulated and emitted. Therefore, if primary Stokes Raman light including incident pulse light emitted from the laser medium 42 is extracted by the spectral dispersion element 43, pump light (probe light) and Stokes light each having excellent monochromaticity are emitted from the light source device 31, respectively. It can be made to function as a microscope light source excellent in CH group observation. Of course, similar effects can be obtained for multiplex CARS microscopy.

(Fourth embodiment)
In the fourth embodiment of the present invention, the resolution evaluation chart shown in FIG. 1 or FIG. 3 is set on the sample stage 35 instead of the sample 36 in the coherent Raman microscope shown in FIG. adjust. Here, as the resolution evaluation chart, a Raman active layer containing polyethylene is used.

  According to the adjustment method according to the present embodiment, since the resolution evaluation chart having the Raman active layer containing polyethylene is used, the Raman signal obtained from the resolution evaluation chart is from CH vibration, and the light source device Since 31 also emits illumination light that can only excite CH vibrations, the microscope can be adjusted with very high S / N and accuracy.

  Therefore, the adjustment method according to the present embodiment is very useful because the application of the coherent Raman microscope for living organisms is specialized in CH vibration observation such as observation of lipid droplets. It is a realistic combination.

  The present invention is not limited to the above-described embodiment, and various modifications or changes can be made without departing from the spirit of the invention.

It is a figure which shows the chart for resolution evaluation which concerns on 1st Embodiment of this invention. It is a figure which shows the chemical group contained in the Raman active layer shown in FIG. It is a figure which shows the chart for resolution evaluation which concerns on 2nd Embodiment of this invention. It is a figure which shows schematic structure of the coherent Raman microscope using the light source device which concerns on 3rd Embodiment of this invention. It is an energy diagram explaining non-resonance Raman scattering, genuine resonance Raman scattering, and pre-resonance Raman scattering. It is an energy diagram explaining coherent Raman scattering. It is a figure for demonstrating the adjustment method in the conventional microscope.

Explanation of symbols

10, 20 Resolution evaluation chart 11, 21 Substrate 12, 22 Raman active layer 13a, 24a 5 μm pattern part 13b, 24b 3 μm pattern part 13c, 24c 1 μm pattern part 13d, 24d 500 nm pattern part 13e, 24e 250 nm pattern part 13f, 24f 100 nm pattern portion 13 g, 24 g 50 nm pattern portion 23 Mask layer 31 Light source device 32 Reflection mirror 33 Beam splitter 34 Microscope objective lens 35 Sample stage 36 Sample 37 Illumination light cut filter 38 Spectroscope 39 Photomultiplier tube 41 Pulse laser light source 42 Laser medium 43 Spectral dispersion element

Claims (18)

A resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
A substrate that is optically smooth with respect to illumination light and that does not emit fluorescence with respect to illumination light excitation;
A Raman active layer that is formed two-dimensionally on the substrate, has a vibration spectrum different from that of the substrate, and includes a Raman active material that does not emit fluorescence in response to illumination light excitation;
A chart for evaluating the resolution of a coherent Raman microscope, comprising: A resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
A substrate that is optically smooth with respect to illumination light and that does not emit fluorescence with respect to illumination light excitation;
A Raman active layer formed on the substrate and having a vibration spectrum different from that of the substrate and including a Raman active material that does not emit fluorescence in response to illumination light excitation;
A mask layer that is two-dimensionally formed on the Raman active layer, has a vibration spectrum different from that of the Raman active material, does not emit fluorescence in response to excitation of illumination light, and reflects or absorbs illumination light;
A chart for evaluating the resolution of a coherent Raman microscope, comprising:   The chart for evaluating the resolution of a coherent Raman microscope according to claim 1, wherein the Raman active substance is made of an organic substance.   The chart for evaluating the resolution of a coherent Raman microscope according to claim 3, wherein the Raman-active substance includes a chemical group having a vibration level that resonates with a difference in frequency between illumination light of pump light and Stokes light. .   The chart for resolution evaluation of a coherent Raman microscope according to claim 4, wherein the chemical group is a CH group.   The chart for resolution evaluation of a coherent Raman microscope according to claim 5, wherein the Raman active substance includes polyethylene. In producing a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
On a substrate that is optically smooth with respect to illumination light and does not emit fluorescence with respect to illumination light excitation, a Raman active substance that has a vibration spectrum different from that of the substrate and does not emit fluorescence with respect to illumination light excitation. A Raman active layer forming step of forming a Raman active layer including;
A pattern forming step of forming the two-dimensional pattern of the Raman active layer by two-dimensionally removing the Raman active layer formed on the substrate;
The manufacturing method of the chart for resolution evaluation characterized by including these. In producing a resolution evaluation chart used for evaluating the imaging performance of a coherent Raman microscope,
On a substrate that is optically smooth with respect to illumination light and does not emit fluorescence with respect to illumination light excitation, a Raman active substance that has a vibration spectrum different from that of the substrate and does not emit fluorescence with respect to illumination light excitation. A Raman active layer forming step of forming a Raman active layer including;
On the Raman active layer formed on the substrate, a mask layer that has a vibration spectrum different from that of the Raman active material, does not emit fluorescence in response to excitation of illumination light, and reflects or absorbs illumination light. A mask layer forming step to form two-dimensionally;
The manufacturing method of the chart for resolution evaluation characterized by including these.   9. The Raman active layer forming step is characterized in that a Raman active material made of an organic material is spread on the substrate by a spin coating method or a dip coating method to form the Raman active layer. The manufacturing method of the chart for resolution evaluation of description.   The method for producing a chart for resolution evaluation according to claim 7, wherein the pattern forming step forms a two-dimensional pattern of the Raman active layer by a nanoimprinting method.   9. The method for manufacturing a resolution evaluation chart according to claim 8, wherein the mask layer forming step forms the mask layer by a vapor deposition method. A light source device used for a coherent Raman microscope,
A laser light source for emitting laser light;
A laser medium including an organic crystal that emits Raman light that is incident on the laser light from the laser light source and emits Raman light shifted to a longer wavelength side than the laser light together with the laser light;
A light source device for a coherent Raman microscope, comprising:   13. The coherent Raman microscope according to claim 12, further comprising a spectral dispersion element that removes a higher-order Raman light component having a wavelength longer than that of the first-order Raman light in an optical path from the laser medium. Light source device.   The light source device for a coherent Raman microscope according to claim 13, wherein the spectral dispersion element includes a multilayer filter or a diffraction grating.   The light source device for a coherent Raman microscope according to any one of claims 12 to 14, wherein the organic crystal includes a CH group.   The light source device for a coherent Raman microscope according to claim 15, wherein the organic crystal includes polyethylene. In adjusting the coherent Raman microscope,
Using the resolution evaluation chart according to any one of claims 1 to 6, adjusting the contrast ratio of a contrast transfer function obtained by photographing a microscope image of the resolution evaluation chart to be maximum. Adjustment method of the characteristic coherent Raman microscope.   The method for adjusting a coherent Raman microscope according to claim 17, wherein the light source device according to claim 12 is used as a light source device for observing the resolution evaluation chart with a microscope. .

Images