WO2014188622A1

WO2014188622A1 - Examination method

Info

Publication number: WO2014188622A1
Application number: PCT/JP2013/081669
Authority: WO
Inventors: 三津夫川▲崎▼; 哲郎高松; 義規原田; 丈夫南川; 昂司竹田; 祐山崎; 昌博川▲崎▼
Original assignee: ウシオ電機株式会社; 国立大学法人京都大学; 京都府公立大学法人
Priority date: 2013-05-20
Filing date: 2013-11-25
Publication date: 2014-11-27
Also published as: JP2014228321A

Abstract

This invention provides an examination method whereby a section of body tissue can be examined using a simple device. Said method, which is used to examine specimens containing sections of body tissue, has the following steps: a step (a) in which a light-intensifying element that has a substrate, an intensifying-electromagnetic-field formation layer in which a large number of separate metal microparticles are dispersed across the surface of the substrate, and a protective layer formed on top of the substrate and the intensifying-electromagnetic-field formation layer is prepared; a step (b) in which a specimen to be examined is placed on the top surface of the protective layer; a step (c) in which the light-intensifying element is exposed to excitation light from either above or below; and a step (d) in which light emitted from the specimen is received and the emission spectrum thereof is measured.

Description

Inspection method

The present invention relates to an inspection method, and more particularly to an inspection method of a test piece including a biological tissue section, which can analyze an emission spectrum derived from a biological tissue with high sensitivity.

As a method for medical application of biomolecule imaging using light, a method of staining with a fluorescent dye and measuring fluorescence from the dye specifically accumulated in a target cell or biological tissue section is performed.

In addition, instead of fluorescence, Raman spectroscopy of cells and biological tissue sections can be measured and mapped for each site by selecting the wavelength of the spectrum, so that it is not necessary to stain with a fluorescent dye and tissue can be cut off. It becomes possible to directly evaluate the state of cells.

As a method for measuring Raman spectroscopy with high sensitivity, methods such as TERS (Tip-Enhanced Raman Scattering) method, stimulated Raman spectroscopy, resonance Raman method and the like are known (for example, see Non-Patent Document 1).

The examination of living tissue is often used to determine whether or not there is any abnormality in the part. For example, during surgery, the target region is taken out and sliced to a size that can be examined, and then stained with a fluorescent dye, irradiated with excitation light, and the pathologist observes the light emission state from the dye. . At this time, the examination of whether or not there is an abnormality in the part requires a tissue diagnosis by a pathologist who is experienced and skilled in judgment, and the number of examinations is limited. Therefore, there is a need for a more rapid, simple and accurate diagnostic method using spectra.

In measurement using ordinary non-resonance Raman spectroscopy, the signal is weak because of low sensitivity, and a long measurement time is required to obtain a necessary and sufficient signal for obtaining an image. Therefore, when obtaining a two-dimensional image within a limited measurement time, it is necessary to narrow the area to be measured. That is, the person who performs the inspection specifies the measurement target area in a narrow range in advance, and then irradiates the region with excitation light and detects the Raman spectrum. For this reason, when a lesion location exists outside the specified measurement target area, there is a high possibility that the lesion state is overlooked.

The above-mentioned TERS method exists as a method for measuring Raman spectroscopy with high sensitivity. However, in this method, the measurement target area becomes extremely narrow, and thus the above-described problem occurs. In addition, according to the stimulated Raman spectroscopy and the resonance Raman method, an apparatus required for inspection is increased in size and an expensive apparatus is required. There are also limitations on the method such that only a specific spectrum is amplified.

In all of the stimulated Raman spectroscopy, resonance Raman method, and TERS method, the signal is weak to measure a wide range. Therefore, it is necessary to set the measurement target area very narrow and its limited area (□ 1 mm In some cases, it takes a long time of several hours. Since it is necessary to observe an area of several centimeters or more for tissue diagnosis, this method takes a very long time to be practically unusable. In nonlinear Raman spectroscopy such as stimulated Raman spectroscopy, a pulse laser or a high NA lens is required to induce efficient stimulated Raman scattering, which increases the size of the device and limits the measurement area that can be acquired at one time. Become. Therefore, there has been a demand for a method capable of measuring a Raman spectrum spectrum with high sensitivity and quickly measuring a wide area.

In addition to the above method, there is a conventional method for measuring the Raman spectrum and fluorescence spectrum of the outermost surface using the localized surface plasmon effect. In this method, a device for realizing a localized surface plasmon effect is prepared, light is irradiated in a state where a test piece to be inspected is placed on the device, and light emission from the test piece is spectrally analyzed. It is. The localized surface plasmon effect enhances light emission from the specimen.

However, according to this method, silver fine particles or the like are coated on the surface of the element for realizing the localized surface plasmon effect. Therefore, when a biological material is to be inspected, if this biological material is placed on the device, halide ions contained in the biological material and silver fine particles etc. are chemically reacted and altered, and the silver fine particles are detached and dissolved. Occurs, and the light enhancement effect cannot be obtained. In other words, conventionally, it has been thought that a biological material cannot be inspected using the localized surface plasmon effect.

In view of the above problems, an object of the present invention is to provide an inspection method capable of inspecting a biological tissue section with a simple apparatus.

The inspection method of the present invention is an inspection method of a test piece including a biological tissue section,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Preparing step (a),
Placing the test piece to be inspected on the upper surface of the protective layer (b),
Irradiating excitation light to the light enhancement element from the upper surface or from the back surface (c),
And (d) measuring light emission spectrum by receiving light emitted from the test piece.

In the method of the present invention, the localized surface plasmon effect can be used because the structure includes an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently from each other on the surface of the substrate. For this reason, unlike the surface plasmon effect using evanescent waves, there is no strict restriction on the incident angle of light with respect to the substrate, and the degree of freedom is ensured.

Further, in the method of the present invention, a test piece to be inspected is placed on the upper surface of the protective layer formed on the enhanced electromagnetic field forming layer. For this reason, even when a biological material such as a biological tissue slice is included as a test piece, the test piece is only in direct contact with the protective layer, and the metal fine particles are not kept in direct contact with the test piece for a long time. . For this reason, when the metal fine particles are continuously exposed to the halide ions for a long time, the halide ions and the metal fine particles do not undergo chemical reaction and change in quality. As a result, Ag having a high light enhancement effect can be used as the metal fine particles. In the configuration of the present invention, Ag is preferably used as the metal fine particles, but other metal materials (for example, Au) may be used.

When utilizing the localized surface plasmon effect as in the present invention, the light enhancement effect acts only in the vicinity of a large number of metal fine particles constituting the enhanced electromagnetic field forming layer. Although the test piece is placed on the upper surface of the protective layer formed on the upper layer of the enhanced electromagnetic field forming layer, the strong enhancing effect reaches the surface through the protective layer formed on the upper layer of the enhanced electromagnetic field forming layer. A light enhancement effect can be exerted on the test piece placed on the upper surface of the protective layer far from the metal fine particles. Therefore, strong luminescence derived from a living tissue as a test piece can be obtained.

Thereby, since spectrum analysis using Raman scattered light can be performed, it is possible to diagnose a living tissue from a Raman spectrum. That is, according to this method, spectrum data in the case where an abnormality exists and spectrum data in the case where no abnormality exists are prepared in advance, and by comparing these with the measured spectrum data, Since the tissue can be diagnosed, tissue diagnosis can be performed even by a pathologist who is not experienced and skilled in judgment.

The method of the present invention can be used not only for Raman scattered light from a test piece but also for fluorescence spectrum analysis.

In this case, since the localized surface plasmon effect can be used, the light emission enhancement effect is extremely high. As a result, the fluorescence enhancement effect of a substance that has not conventionally been lit in a normal state and has not been recognized as a fluorescent substance can be used. Therefore, a fluorescence spectrum analysis of a living tissue can be performed without introducing a fluorescent substance.

Moreover, the inspection method of the present invention includes:
The step (c) is a step of irradiating the light enhancement element with the excitation light while scanning in a predetermined direction.
The method includes a step (e) of two-dimensional mapping the intensity distribution of the emission spectrum obtained in the step (d).

According to this method, since the light emission enhancement effect is extremely high, the emission spectrum can be detected in a short time. As a result, in observation of a biological section sample, a wide range of two-dimensional mapping can be performed at high speed using a low-magnification objective lens using a microscopic Raman measurement apparatus. In addition, it is possible to map a wider area than before. In other words, because the sensitivity is high, the signal is strong, and it takes a short measurement time to obtain a signal that is necessary and sufficient to obtain an image of a wide observation area. Therefore, the measurement area can be widened even when a two-dimensional image is obtained within a limited measurement time.

Therefore, a mapping image in the case where an abnormality exists and a mapping image in the case where no abnormality exists are prepared in advance, and by comparing these with a mapping image based on the measured spectrum data, Since diagnosis is possible, tissue diagnosis can be performed even if the pathologist is not an experienced and skilled in judgment.

The protective layer may be composed of an inorganic substance having an orientation or an organic polymer having an orientation in connection with the numerous metal fine particles.

With this configuration, it is possible to transmit the enhanced electromagnetic field due to the localized surface plasmon effect in the enhanced electromagnetic field forming layer to the surface of the protective layer with high efficiency, and the test piece placed on the surface of the protective layer can be highly efficient. Can emit light.

The protective layer may contain a halogen element.

With this configuration, it is possible to transmit the enhanced electromagnetic field caused by the localized surface plasmon effect in the enhanced electromagnetic field forming layer to the surface of the protective layer with higher efficiency. Note that this configuration is different from the case in which halide ions are brought into direct contact with the exposed metal fine particles in the configuration having no protective layer, and therefore, a material containing a halogen element is used as the protective layer. Similarly, the function of protecting the metal fine particles from damage by halide ions is secured.

According to the present invention, a biological tissue section can be inspected with a simple device. In particular, a wide range of imaging can be performed in a short time, and a living tissue section can be diagnosed even without a skilled pathologist.

It is drawing which shows the structure of the sensor of this invention typically. It is a conceptual diagram for demonstrating the inspection method of this invention. It is a conceptual diagram of the measuring apparatus for verifying the performance of the sensor of this invention. It is a graph which shows the result of having plotted the relationship between the thickness of a protective layer, and photointensity | strength when a rhodamine 6G pigment | dye is carry | supported with respect to a sensor. It is a graph which shows the result of having plotted the relationship between the thickness of a protective layer when a fuchsin pigment | dye is carry | supported with respect to a sensor, and photointensity. 6 is a graph comparing spectral distributions of light received by the light receiving units of Example 2 and Comparative Example 1. FIG. It is the graph which compared the spectral distribution of the light received by the light-receiving part when each sensor chip of Example 3 and Comparative Example 2 was irradiated with excitation light. It is the graph which compared the spectrum distribution of the light received by the light-receiving part when each sensor chip of Example 4 and Comparative Example 3 was irradiated with excitation light. It is a graph which shows the Raman spectrum light-received in the light-receiving part when each sensor of Example 5 and Example 1 was irradiated with excitation light. It is a microscope image of the rat heart tissue section as a test piece. In Example 6 and Comparative Example 4, it is a graph which shows the Raman spectrum light-received by the light-receiving part. It is a Raman mapping image obtained in Example 6. It is a microscope image of the vagus nerve section | slice of the esophagus as a test piece. 7 is a fluorescence mapping image obtained in Example 7. It is a graph which shows the Raman spectrum light-received by the light-receiving part when each sensor chip of Example 8 and Example 9 was irradiated with excitation light.

[First Embodiment]
A first embodiment of the present invention will be described with reference to the drawings. In each figure, the dimensional ratio in the drawing does not necessarily match the actual dimensional ratio.

<Sensor structure>
FIG. 1 is a drawing schematically showing the structure of a sensor used in the inspection method of the present invention. The sensor 1 includes a substrate 7, an enhanced electromagnetic field forming layer 9, and a protective layer 11 to constitute a light enhancement element. Then, the test piece 5 to be inspected is placed on the upper surface of the protective layer 11 and inspected.

The material of the substrate 7 is not particularly limited, and for example, glass, ceramics, resin, or the like can be used. As will be described later, when heat treatment (for example, heating at 100 ° C. or higher) is performed in the manufacturing process of the sensor 1, it is preferable to have heat resistance such as glass or polyimide resin.

The enhanced electromagnetic field forming layer 9 is configured by a large number of metal fine particles 10 being dispersed and arranged on the surface of the substrate 7 independently of each other. The arrangement method of the metal fine particles 10 on the surface of the substrate 7 may be a two-dimensional random arrangement or a regular arrangement.

As the metal fine particles 10 constituting the enhanced electromagnetic field forming layer 9, for example, Ag can be suitably used, but Au, Al, and the like can be used as long as they can be excited by irradiation with excitation light to realize the localized surface plasmon effect. Other materials such as Cu can also be used. In addition, as the shape of the metal fine particles 10, for example, those having shape anisotropy such as a flat hemispherical shape or a flat plate shape can be suitably used. Note that it is desirable that all the metal fine particles 10 have a uniform size and shape, but there may be some variation in size and shape.

Further, the particle diameter of the metal fine particles 10 is preferably smaller than the wavelength of the excitation light. Here, in the present specification, “particle size” refers to a projected area equivalent circle diameter measured by microscopy. Specifically, it is obtained as follows. Field of view of a scanning electron microscope (for example, 1.5 μm × 2 μm) in which a 2 μm-long line segment is observed to be enlarged to a length of 6 cm (magnification: 30000 times) in an arbitrarily selected region on the surface of the sensor 1 As an imaging region, a secondary electron image of the region in the sensor 1 is obtained. At this time, for each of the metal fine particles having a brightness index (256 steps) of about 100 or more, the diameter of a perfect circle having the same area as the metal fine particle 10 is obtained as the particle diameter of the metal fine particle 10.

The particle size of the metal fine particles 10 is in the range of 5 to 300 nm, for example, and the thickness is in the range of 5 to 70 nm, for example. Further, the density of the metal fine particles 10 constituting the enhanced electromagnetic field forming layer 9 can be, for example, about 10 ⁸ to 10 ¹¹ particles / cm ² .

As an example of a method for forming such an enhanced electromagnetic field forming layer 9, a method in which a dispersion liquid in which a precursor of metal fine particles 10 is dispersed in an appropriate solvent is applied to the surface of the substrate 7 by a spin coating method and heated. be able to. Further, as another method, a method of dipping the precursor of the metal fine particles 10 on the surface of the substrate 7 and heating, a method of vacuum-depositing the metal fine particles 10 on the surface of the substrate 7, and a method of depositing the metal fine particles 10 on the surface of the substrate 7 A sputter deposition method or the like can be used.

In the sensor 1, a protective layer 11 is formed on the enhanced electromagnetic field forming layer 9 including the surface of the substrate 7 exposed between adjacent metal fine particles 10. Examples of the material constituting the protective layer 11 include silicon oxide, titanium oxide, cerium oxide, boron oxide, phosphorus oxide, magnesium oxide, calcium oxide, aluminum oxide, gallium oxide, germanium oxide, and zinc oxide.

The average thickness of the protective layer 11 is preferably 50 to 250 nm, for example. In particular, since the thickness of the protective layer 11 is 85 nm or more, even when the test piece 5 contains a halide ion (for example, Cl ⁻ ) such as a biological material, sufficient resistance to the metal fine particles 10 ( Protection function).

Such a sensor 1 is manufactured by the following method, for example. First, a metal nanoparticle film is formed on the surface of the substrate 7, and the graininess is changed by heat-treating the film, whereby an enhanced electromagnetic field forming layer 9 made of metal fine particles 10 having a particle diameter within a predetermined range is formed. Form. At this time, the particle diameter of the metal fine particles 10 to be formed can be adjusted by appropriately changing the heat treatment conditions.

Next, a protective layer is grown in the thickness direction from the metal fine particle 10 as a starting point on the surface of the enhanced electromagnetic field forming layer 9 including the surface portion of the substrate 7 exposed between the adjacent metal fine particles 10 by vapor deposition. Thus, the protective layer 11 having a columnar structure is formed. The thickness of the protective layer 11 can be adjusted by appropriately changing the growth conditions and time. Moreover, as a formation method of the protective layer 11, methods, such as a radio frequency (RF) sputter vapor deposition method, an electron beam vapor deposition method, or an electron cyclotron resonance (ECR) sputter vapor deposition method, can be selected suitably and can be utilized.

<How to use sensor 1>
Next, a method for inspecting the test piece 5 using the sensor 1 of the present embodiment will be described. FIG. 2 is a conceptual diagram for explaining the inspection method. Here, a method for performing spectrum analysis by irradiating the sensor 1 on which the test piece 5 is placed with excitation light and receiving Raman scattered light emitted from the test piece 5 will be described.

In the present embodiment, at the time of inspection, a light source unit 41 for irradiating excitation light from above the sensor 1, that is, a side on which the test piece 5 is placed, and a light receiving unit 43 for receiving light emitted from the test piece 5. Is used. The light source unit 41 includes an excitation light source 50, a filter 51, and a mirror 61. The light receiving unit 43 includes a filter 53, a spectroscope 55, and a photodetector 56. In FIG. 2, a half mirror 62 that transmits light from the light source unit 41 and reflects light emitted from the test piece 5 is provided. The condensing lens 52 is installed for the purpose of condensing excitation light from the light source unit 41 and light emission from the test piece 5. The apparatus configuration shown in FIG. 2 is merely an example, and the present invention is not limited to this configuration.

In a state where the test piece 5 is placed on the protective layer 11, excitation light is incident from the light source unit 41 toward the sensor 1. Due to the incident light, the plasmon electric field generated in the enhanced electromagnetic field forming layer 9 of the sensor 1 propagates to the test piece 5 placed on the upper surface of the protective layer 11. Thereby, strong excitation light is exerted on the test piece 5, and high-output Raman scattered light is emitted from the test piece 5. The Raman scattered light is reflected by the half mirror 62 and received by the light receiving unit 43. Wavelength decomposition is performed by the spectroscope 55 of the light receiving unit 43, and a spectral distribution of the light received by the photodetector 56 is obtained.

Suppose a biological material including a biological tissue section as the test piece 5. The enhanced electromagnetic field formed by the enhanced electromagnetic field forming layer 9 selectively excites the specimen 5, that is, the molecular species of the biological tissue, via the protective layer 11 having electromagnetic field propagating properties, thereby the Raman signal derived from the biological tissue. Is selectively obtained. The enhanced Raman scattered light generated in this way is optically strongly coupled to the enhanced electromagnetic field forming layer 9, so that the enhanced electromagnetic field forming layer 9 efficiently guides this scattered light to the light receiving portion 43, and thus, due to the synergistic effect thereof. Gives a strong Raman signal.

<Description of sensor 1 performance>
Hereinafter, the fact that the light enhancement effect can be realized by the enhanced electromagnetic field forming layer 9 provided in the sensor 1 will be described with reference to the examples.

(Example 1)
First, a method for manufacturing the sensor 1 of Example 1 will be described. A glass slide having a size of several centimeters was used as the substrate 7, and an Ag film for forming the metal fine particles 10 was formed on the surface of the glass slide by depositing Ag with a thickness of about 10 nm. Thereafter, the substrate 7 on which the Ag film is formed is heated for several minutes on a hot plate at about 100 ° C., thereby changing the granularity of the Ag film and thereby increasing the number of metals (Ag) as the enhanced electromagnetic field forming layer 9. An Ag fine particle monolayer film of fine particles 10 was formed. The particle diameter of the metal fine particles 10 in the obtained Ag fine particle monolayer film is in the range of 50 to 150 nm, the thickness is about 20 nm on average, and the density of the metal fine particles 10 is approximately 5 × 10 ⁹ particles / cm 3. ² .

Thereafter, using an RF sputtering apparatus “RFS-200 type” (manufactured by Ulvac), sputtering is performed under the following conditions using silicon oxide (SiO ₂ ) as a target, thereby exposing the substrate 7 exposed between the adjacent metal fine particles 10. The sensor 1 was produced by forming the protective layer 11 on the surface of the enhanced electromagnetic field forming layer 9 including the surface portion of the above. The thickness of the protective layer 11 was adjusted by appropriately changing the sputtering time.

The sputtering conditions are as follows.
-Distance from the target to the surface of the enhanced electromagnetic field forming layer 9: 45 mm
・ Atmosphere: Ar 3.0Pa (during discharge)
・ Discharge output: 100W
・ RF frequency: 13.6MHz
-Growth rate of the protective layer 11: 8.5 nm / min

Next, a performance verification method for the sensor 1 manufactured as Example 1 will be described. Here, in order to show that the sensor 1 has a light enhancement effect, the fluorescence enhancement effect is verified. That is, the dye molecule is coated on the surface of the protective layer 11 by spin-coating a diluted ethanol solution of rhodamine 6G dye (Rh6G: emission quantum yield of about 1) at 3000 revolutions on the surface of the protective layer 11 on the sensor 1. It was made to carry on. The relationship between the density of the dye molecules carried on the surface of the sensor 1 and the dye concentration of the solution used for spin coating is that the amount of dye molecules carried is 3 × 10 ¹¹ when the concentration of rhodamine 6G dye is 1 μM. / Cm ² .

The emission quantum yield Φ is defined by the ratio between the number of photons absorbed by the molecule and the number of photons emitted by fluorescence (see Equation 1). Here, k _f is a fluorescence transition rate constant of a molecule in an electronically excited state, and _knr is a non-radiative transition rate constant (speed at which quenching occurs per unit time).
(Equation 1)
_{_{Φ = k f / (k f}} + k nr)

If all excited molecules return to the ground state by fluorescence, the emission quantum yield becomes 1, and a substance having an emission quantum yield close to this value is called a “substance with a high emission quantum yield”. However, in practice, it is not 1 due to non-radiative transition. The non-radiative transition is a transition that returns to the ground state without emitting fluorescence. A dye having a low emission quantum yield is a dye having k _f <kn _nr . Note that a dye that hardly emits light under the condition of k _f << k _nr is called a non-light emitting dye.

The intensity of light emitted from the sample (dye molecule) by irradiating the sensor 1 with excitation light was measured by a measuring apparatus shown in FIG. FIG. 3 is a conceptual diagram of a measuring device for verifying the performance of the sensor 1. A light source unit 41 is configured by the diode laser as the excitation light source 50 and the filter 51, light is incident on the sensor 1 from the light source unit 41, and light from the dye molecules carried on the surface of the protective layer 11 is received by the light receiving unit. 43 receives light. The light receiving unit 43 is composed of a condenser lens 52, a filter 53, a light receiving head 54, and a photodetector 55 (electronically cooled diode array detector).

More specifically, a green diode laser (wavelength of 532 nm) having an output of less than 1 mW is used as the excitation light source 50, and light emitted from the excitation light source 50 is not condensed through the filter 51 (energy density is about 30 mW / cm). ² ) or anti-condensation (energy density of about 10 mW / cm ² or less) is made incident on the sensor 1 at an angle of about 45 ° as excitation light. Then, the light scattered in the 90 ° angle direction by the dye molecules carried on the sensor 1 was condensed by the condenser lens 52 onto the light receiving head 54 of the photodetector 55 through the filter 53.

FIG. 4 shows the measurement results of the measurement apparatus shown in FIG. In FIG. 4, the vertical axis represents the increase in luminescence intensity (unit: times), corresponding to the relative ratio to the luminescence intensity measured by the same method for the same amount of dye supported on the glass substrate without the enhancement effect. To do.

In addition, a non-luminescent fuchsine dye (luminescence quantum yield << 0.01) is used instead of rhodamine 6G dye as a sample, and the sample is supported on the surface of the protective layer 11 at a density of 3 × 10 ¹² particles / cm ^2. The emission intensity was measured by the same method as described above. The measurement results are shown in FIG.

4 and 5, it can be seen that a high light emission enhancement rate is maintained even when the thickness of the protective layer 11 is 200 nm or more regardless of the light emission quantum yield of the dye itself. In particular, it can be seen that a light enhancement factor of several thousand times or more is ensured for a dye having an emission quantum yield of less than 0.01, that is, a non-luminescent pigment. Thereby, even if the protective layer 11 having a predetermined thickness is formed on the upper layer of the metal fine particles 10, it can be seen that the light emission enhancing effect can be propagated to the upper surface.

That is, according to the sensor 1 of the present invention, due to the interaction between the molecular emission dipole and the dipole-type surface plasmon (localized surface plasmon), the radiation transition speed k _f of the dye (see the above formula 1) is increased as a result. Become. Thereby, since k _f >> kn _nr , the above-mentioned light emission quantum yield becomes large, and originally, it is a non-light emitting substance (k _f << k _nr ) whose light emission quantum yield is 0.01 or less. It can be seen that even pigments emit light strongly.

As another verification, the Raman scattering intensity of rhodamine 6G dye with the same arrangement as in FIG. 3 except that a He—Ne laser (wavelength 632.8 nm) with an output of less than 1 mW was used as the excitation light source 50 instead of the diode laser. Was measured as a function of the thickness of the protective layer 11. As a result, even if the film thickness of the protective layer 11 exceeds 200 nm, an enhanced Raman signal (intensity is about 10) which is not different from the signal obtained when the dye molecules are directly adsorbed on the surface of the metal fine particles 10. ⁵ times) was obtained.

The following points can be considered as one of the reasons why the enhancement effect propagates as much as the thickness of the protective layer 11 as described above. In the columnar protective layer 11 formed of a dielectric having orientation by vapor deposition, the plasmon electric field is not disturbed and reaches the surface of the protective layer 11 without loss. Therefore, even when the thickness of the protective layer 11 is increased to some extent, the electromagnetic field (localized surface plasmon) generated in the metal fine particles 10 is transmitted to the surface of the protective layer 11.

That is, as shown in FIG. 2, by placing the test piece 5 made of a biological material on the surface of the protective layer 11 and irradiating it with excitation light, the test piece 5 is influenced by an enhanced electromagnetic field. The substance that constitutes the test piece 5, that is, light that strongly contains a spectrum derived from living tissue is emitted. The light receiving unit 43 receives this light emission and obtains a spectrum distribution, whereby the biological tissue can be analyzed.

(Example 2)
An aqueous solution of antibody IgG labeled with a fuchsin dye was added dropwise to the sensor 1 produced by the same method as in Example 1. More specifically, an aqueous solution of antibody IgG labeled with a fuchsin dye was dropped onto the upper surface of the protective layer 11.

(Comparative Example 1)
An aqueous solution of antibody IgG labeled with a fuchsin dye was dropped onto the substrate 7 in the same manner as in Example 2.

Then, FIG. 6 shows the result of examining the spectral distribution of the light obtained by the light receiving unit 43 by causing excitation light to enter the respective elements of Example 2 and Comparative Example 1 using the measuring device shown in FIG. . In FIG. 6, the horizontal axis represents the wavelength of light, and the vertical axis represents the emission intensity.

Referring to FIG. 6, according to the configuration of Example 2, the fuchsin dye, which is a non-luminescent labeling substance, emits luminescence quantum by the interaction through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the yield is improved and the amount of light emitted by the fuchsin dye is significantly increased. On the other hand, according to the configuration of Comparative Example 1, there is no light emission enhancement effect. This shows that the sensor 1 of the present invention has a very high light emission enhancement effect.

(Example 3)
An aqueous solution of a self-luminous biopolymer collagen having a low luminescence quantum yield was spin-coated on the upper surface of the protective layer 11 of the sensor 1 produced by the same method as in Example 1.

(Comparative Example 2)
Similarly to Example 3, the substrate 7 was spin-coated with an aqueous solution of self-luminous biopolymer collagen having a low luminescence quantum yield.

Example 4
An aqueous solution of self-luminous riboflavin having a low emission quantum yield was spin-coated on the upper surface of the protective layer 11 of the sensor 1 produced by the same method as in Example 1.

(Comparative Example 3)
Similarly to Example 4, the substrate 7 was spin-coated with an aqueous solution of self-luminous riboflavin having a low emission quantum yield.

FIG. 7 shows the results of examining the spectral distribution of the light obtained by the light receiving unit 43 by causing excitation light to enter the elements of Example 3 and Comparative Example 2 using the measuring device shown in FIG. Similarly, FIG. 8 shows the result of examining the spectral distribution of the light obtained by the light receiving unit 43 by causing excitation light to enter the elements of Example 4 and Comparative Example 3 using the measuring apparatus shown in FIG. Show. 7 and 8, the horizontal axis represents the wavelength of light, and the vertical axis represents the emission intensity.

Referring to FIG. 7, according to the configuration of Example 3, the self-luminous biopolymer collagen having a low luminescence quantum yield passes through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the quantum yield of light emission is improved by the interaction, and the amount of light emitted from the biopolymer collagen is significantly increased. On the other hand, according to the structure of Comparative Example 2, there is no effect of enhancing luminescence, and it is suggested that analysis by receiving light emitted from self-luminous biopolymer collagen is difficult.

Similarly, referring to FIG. 8, according to the configuration of Example 4, the self-luminous riboflavin having a low emission quantum yield passes through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the quantum yield of light emission is improved by the interaction, and the amount of light emitted by riboflavin is significantly increased. On the other hand, according to the configuration of Comparative Example 3, there is no light emission enhancement effect, which suggests that analysis by receiving light emitted from self-luminous riboflavin is difficult.

<Description of Performance of Sensor 1 with Different Configuration>
The protective layer 11 of the sensor 1 may contain a halogen element in advance. As a method of containing the halogen element in the protective layer 11, a method of immersing the sensor 1 in which the protective layer 11 is formed in an aqueous solution of a halide salt can be used. For example, when the concentration of the halide salt aqueous solution is 0.1 to 0.3 mol / L, the maximum effect can be obtained by room temperature immersion for about 5 to 30 minutes, but the concentration of the aqueous solution is higher than that. On the other hand, if it is low, the same effect can be obtained by adjusting the immersion time according to the concentration. The halogen element content in the protective layer 11 is preferably 0.002% by mass to 0.05% by mass.

Specific examples of the halide salt include alkali metal salts such as sodium chloride (NaCl), potassium chloride (KCl), sodium bromide (NaBr), and potassium iodide (KI), and calcium chloride (CaCl ₂ ). And alkaline earth metal salts.

Thus, even when the sensor 1 is immersed in the halide salt aqueous solution in advance, since many metal fine particles 10 are covered with the protective layer 11 around and above the metal fine particles 10, halide ions directly contact the metal fine particles 10. Thus, the metal fine particles 10 do not react with the halide ions and leave the substrate 7. Then, by adding a halogen element to the protective layer 11 in advance as described above, the light enhancement effect can be further enhanced as shown in Examples described later.

(Example 5)
Using the same method as in Example 1, the processing time by the RF sputtering apparatus was adjusted, and the sensor 1 having the protective layer 11 having a thickness of 100 nm was produced. Thereafter, the substrate 7 on which the enhanced electromagnetic field forming layer 9 and the protective layer 11 are formed is immersed in a sodium chloride aqueous solution having a chloride ion concentration of 0.2 to 0.3 mol / L for about 30 minutes. By washing with water and drying, halide ions were contained in the protective layer 11 to obtain the sensor 1 of this example. Although it is difficult to accurately determine the content ratio of the halogen element contained in the protective layer in the light enhancement element obtained by this method, it is estimated to be about 0.01% by mass. Then, a thin ethanol solution of rhodamine 6G dye is spin-coated at 3000 revolutions on the upper surface of the protective layer 11 of the sensor 1, so that the dye molecules have a density of 3 × 10 ¹¹ particles / cm ² on the surface of the protective layer 11. It was made to carry by.

For comparison, the sensor 1 was also produced in the same manner except that it was not immersed in an aqueous sodium chloride solution. The sensor 1 corresponds to the element of the first embodiment.

Then, the intensity of Raman scattered light emitted from the sample (dye molecule) by irradiating excitation light to the sensor 1 of Example 5 and Example 1 is changed to a He—Ne laser having an output of less than 1 mW instead of the diode laser 50. The measurement was performed with the same measuring apparatus as in FIG. 3 except that (wavelength 632.8 nm) was used as the excitation light source. The result is shown in FIG. In the graph of FIG. 9, the vertical axis indicates the Raman scattering intensity (cps), and the horizontal axis indicates the Raman shift (cm ⁻¹ ).

In FIG. 9, the intensity of Raman scattered light was compared with each sensor 1 of Example 5 and Example 1. This is because the intensity of Raman scattered light itself is smaller than the intensity of fluorescence, so that it is easy to compare in comparing the light enhancement effects of both. In Example 5 in which the halide ions were immersed, a remarkable Raman signal was observed compared to Example 1 in which the halide ions were not immersed, suggesting that the light enhancement effect was higher.

In the sensor 1 of Example 1 in which the halide ions in FIG. 9 are not immersed, as described above with reference to FIGS. 4 and 5, the effect of extremely increasing the emission intensity is obtained. In addition, it is suggested that Example 5 in which halide ions are immersed has a higher light emission enhancing effect than Example 1.

In Example 5, a sodium chloride aqueous solution having a chloride ion concentration of 0.2 to 0.3 mol / L was immersed, but instead, the chloride ion concentration was 0.2 mol / L. An element formed by immersing a certain potassium chloride aqueous solution, an element formed by immersing a sodium bromide aqueous solution having a bromide ion concentration of 0.2 mol / L, an iodine having a iodide ion concentration of 0.2 mol / L As a result of performing the same measurement on the element formed by immersing the potassium chloride aqueous solution and the element formed by immersing the calcium chloride aqueous solution having a chloride ion concentration of 0.2 mol / L, the same results as in Example 5 were obtained. High Raman scattered light was received. On the other hand, when the same measurement was performed on an element formed by immersing a sodium sulfate aqueous solution having a sulfate ion concentration of 0.2 mol / L instead of a sodium chloride aqueous solution, the intensity of the received Raman scattered light was low. there were. This shows that the light enhancement effect can be further enhanced when a halogen element is added to the protective layer 11.

<Inspection example using sensor 1>
Hereinafter, the test result of the test piece 5 performed based on the usage method of the sensor 1 mentioned above is demonstrated.

(Example 6)
Using the sensor 1 of the present invention, a rat heart tissue (8 weeks old, female, tissue section thickness 5 μm) as a test piece 5 is placed on the upper surface of the protective layer 11, and a light source unit is formed in the same manner as in FIG. 41 was irradiated with excitation light, and the Raman spectrum from the test piece 5 was measured. More specifically, a tissue section obtained by freezing and fixing rat heart tissue with OCT-compound was used as a test piece 5 and placed on the upper surface of the protective layer 11 of the sensor 1. FIG. 10 is a microscopic image of a rat heart tissue section as the test piece 5.

The measurement conditions are as follows.
・ Measurement device: Raman-11 (manufactured by Nanophoton)
Excitation light: wavelength 532 nm, output 91.1 mW / mm ² (73 mW / Line)
Excitation light irradiation area: Measurement area 30 in FIG. 10 (scanning range is about 740 μm × 370 μm)
Objective lens: X10, NA 0.3 (Olympus)

In addition, the excitation light irradiated from the light source part 41 was made into a line shape by the cylindrical lens and the objective lens, and focused on the surface of the test piece 5 and irradiated. The Raman scattered light from the test piece 5 irradiated in a linear shape is condensed through the same objective lens, and a one-dimensional Raman image is converted into a two-dimensional CCD image sensor (Pixis 400BR, electronic cooling-70 ° C., 1340 × 400 pixels; acquired by Princeton Instruments, Inc., Trenton, NJ, USA. A two-dimensional Raman scattering image was acquired by scanning excitation light having a linear shape. One one-dimensional image has a width of about 740 μm and a slit width of 70 μm, and requires a measurement time of 1 second. By scanning 200 lines, a Raman spectrum image (two-dimensional mapping image) in a range of about 740 μm × 370 μm was obtained. .

The structure of the line irradiation type Raman scattering spectroscopic microscope is described in, for example, Harada. Y. et al., 2008, Proc. SPIE, vol.536853, 685308.

(Comparative Example 4)
A rat heart tissue (8 weeks old, female, tissue section thickness 5 μm) as a test piece 5 is placed on the upper surface of the substrate 7, and a Raman spectrum is obtained by irradiating excitation light from the light source unit 41 as in Example 6. Obtained.

The spectrum results in Example 6 and Comparative Example 4 are shown in FIG. According to FIG. 11, in comparison with Comparative Example 4 in which the test piece 5 is directly placed on the upper surface of the substrate 7, Example 6 in which the test piece 5 is placed on the upper surface of the sensor 1 including the light enhancement element is It can be seen that a clear Raman spectrum can be measured. As described above, by using the sensor 1, a spectrum clearer than a normal Raman spectrum can be obtained, so that measurement in a short time becomes possible.

In addition, the peak shown by the arrow in FIG. 11 is a peak attributed to cytochrome C contained in the mitochondria of cardiomyocytes. FIG. 12 shows the result of Raman mapping of the cardiomyocytes on the sensor 1 with a contrast difference given by the peak intensity of 754 cm ⁻¹ . From the comparison with the optical microscope image of FIG. 10, it can be seen that the Raman mapping image of FIG. 12 reflects the shape of the tissue.

That is, according to the present method, since a clear Raman spectrum is measured, a clear contrast is also obtained in the Raman mapping image obtained by scanning the excitation light. Based on this Raman mapping image, A living tissue as the test piece 5 can be diagnosed. In addition, since the Raman spectrum can be detected by the excitation light irradiation in an extremely short time as described above, a wide-range Raman mapping image can be obtained in a short time. As a result, even when a two-dimensional image is obtained within a limited measurement time, the measurement target area can be widened.

(Example 7)
Using the sensor 1 of the present invention, the vagus nerve of the esophagus (myelinated fiber: tissue section thickness 5 μm) as the test piece 5 was placed on the upper surface of the protective layer 11, and the same method and apparatus as in Example 4 were used. Excitation light was irradiated from the light source part 41, and the fluorescence spectrum from the test piece 5 and the two-dimensional fluorescence mapping image were measured. FIG. 13 is a microscopic image of a vagus nerve section of the esophagus as the test piece 5.

The measurement conditions are as follows.
・ Measurement device: Raman-11 (manufactured by Nanophoton)
Excitation light: wavelength 532 nm, output 300 mW / mm ² (50 mW / Line)
Excitation light irradiation area: Measurement area 32 in FIG. 13 (scanning range is about 1000 μm × 500 μm)
Objective lens: X10, NA 0.3 (Olympus)

A two-dimensional fluorescence image was acquired by scanning a line of excitation light. One one-dimensional image has a width of about 740 μm and a slit width of about 70 μm, and requires a measurement time of 10 seconds. By scanning 100 lines, a fluorescence spectrum image in a range of about 740 μm × 460 μm (two-dimensional mapping image) Got. More specifically, in the measurement region 32 in FIG. 13, two-dimensional mapping was performed by scanning the excitation light, integrating the fluorescence spectrum having a wavelength of 560 to 630 nm, and adding a contrast difference depending on the intensity. The fluorescence mapping image obtained as a result is shown in FIG. From the comparison with the optical microscope image of FIG. 13, it can be seen that the fluorescence mapping image of FIG. 14 reflects the shape of the tissue.

According to Example 7, it is understood that a wide range of two-dimensional mapping images can be obtained in a short time not only by Raman scattered light but also by spectral analysis using fluorescence. In particular, because of the high light emission enhancement effect of the sensor 1, it is possible to obtain fluorescence by irradiating with excitation light even for substances that have not been recognized as fluorescent substances in the past. The site expands dramatically.

However, the method of the present invention does not exclude use of the test piece 5 in a method of measuring a fluorescence spectrum by labeling a dye and irradiating excitation light. Also in this case, since the light emission enhancing effect is high, as described above with reference to FIGS. 4 and 5, a non-light-emitting dye can be used as the labeling dye.

[Second Embodiment]
A second embodiment of the present invention will be described with reference to the drawings.

This embodiment is different only in that the protective layer 11 provided in the sensor 1 described in the first embodiment is composed of an organic polymer having crystallinity (orientation), and the others are common.

As the organic polymer, an acrylic polymer such as polymethyl methacrylate, polyvinyl alcohol, or the like can be used. In this case, the protective layer 11 is formed by dropping or applying the protective layer forming liquid onto the substrate 7 using a spin coat method, a dip coat method, a spray coat method, a slit coat method, a bar coat method, or the like. However, the spin coating method can be suitably used as a method for forming the protective layer 11 having the most uniform thickness.

When the protective layer 11 contains a halogen element, the protective layer forming liquid may be prepared by dissolving a predetermined polymer and a metal salt of a halogen in a solvent, or dissolving a predetermined polymer in the solvent. Can be prepared by mixing a polymer solution obtained by mixing a metal salt solution in which a halogen metal salt is dissolved in a solvent.

The solvent for preparing the protective layer forming liquid is appropriately selected according to the polymer and metal salt used. Specifically, any polymer that can dissolve the polymer and metal salt used may be used. For example, when a water-soluble polymer such as polyvinyl alcohol is used as the polymer, water can be used as the solvent. When a polymer insoluble in water such as polymethyl methacrylate is used as the polymer, for example, cyclohexanone is used as a solvent for preparing the polymer solution, and water and acetone are used as the solvent for preparing the metal salt solution. A protective layer forming solution can be prepared by mixing a polymer solution and a metal salt solution using a mixed solvent.

The content ratio of the polymer in the protective layer forming liquid is determined by the combination of the above coating method and the target protective film thickness. For example, when a polyvinyl alcohol film is formed from the aqueous solution by using a spin coating method (3000 rpm), the content of the polymer necessary for adjusting the film thickness to 100 nm is about 4.5% by mass. The ratio of the metal salt in the protective layer forming liquid is set according to the content ratio of the halogen element in the target protective layer 11 and the content ratio of the polymer in the protective layer forming liquid.

(Example 8)
In the same manner as in Example 1, an Ag fine particle monolayer film composed of a large number of metal (Ag) fine particles 10 as the enhanced electromagnetic field forming layer 9 was formed on the substrate 7.

Also, a protective layer forming solution was prepared by dissolving 5% by mass of polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., degree of polymerization of about 500) and 0.2 mmol / L of sodium chloride in pure water. . Thereafter, the prepared protective layer forming solution is applied to the surface of the substrate 7 by spin coating, and dried at about 60 ° C., and crystallization is promoted, thereby enhancing the electromagnetic field forming layer 9 including the surface portion of the substrate 7. The sensor 1 was produced by forming the protective layer 11 on the surface. The protective layer 11 had a crystallinity of 50% or more and a thickness of 110 nm.

The upper surface of the protective layer 11 is spin-coated with a diluted ethanol solution of rhodamine 6G dye at 3000 revolutions so that the dye molecules are supported on the surface of the protective layer 11 at a density of 3 × 10 ¹¹ particles / cm ^2. It was.

Example 9
A light enhancement element was produced in the same manner as in Example 6 except that the protective layer forming solution was changed to one prepared as follows. That is, a polymer solution in which 3% by mass of polymethyl methacrylate (Wako Pure Chemical Industries, Ltd.) was dissolved in cyclohexanone (Wako Pure Chemical Industries, Ltd.) was prepared. On the other hand, a metal salt solution in which 20 mmol / L of sodium chloride was dissolved in a mixed solvent in which pure water and acetone were mixed at a mass ratio of 1: 1 was prepared. Then, a protective layer forming solution was prepared by mixing the polymer solution and the metal salt solution at a volume ratio of 9: 1.

Then, the intensity of Raman scattered light emitted from the sample (dye molecule) was measured by irradiating the sensor 1 of Example 8 and Example 9 with excitation light by the same method as in FIG. The result is shown in FIG. Similarly to FIG. 7, in the graph of FIG. 15, the vertical axis indicates the Raman scattering intensity (cps), and the horizontal axis indicates the Raman shift (cm ⁻¹ ). From the graph of FIG. 15, even when the protective layer 11 is composed of an organic polymer, a strong Raman scattering signal can be confirmed, and it can be seen that the light enhancement effect can be propagated to the upper surface of the protective layer 11. .

That is, as in the present embodiment, the sensor 1 is configured to have a protective layer 11 made of an organic polymer, and a biological tissue as the test piece 5 is placed on the upper surface of the protective layer 11 to place the light source unit 41. By irradiating with excitation light from and analyzing the spectrum of Raman scattered light or fluorescence from the test piece 5, it is possible to diagnose the test piece 5 as in the first embodiment.

In each of Examples 8 and 9, the protective layer 11 contains a halogen element. However, the protective layer 11 may be made of an organic polymer without containing a halogen element. .

[Another embodiment]
In each of the above embodiments, the sensor 1 may be configured to have a multilayer structure further including a highly reflective layer and a dielectric layer. In such a structure, the highly reflective layer and the dielectric layer are formed in this order on the surface of the substrate 7, and the enhanced electromagnetic field forming layer 9 is formed on the surface of the dielectric layer.

Further, as described with reference to FIG. 2, in the above embodiment, the test piece 5 is placed on the upper surface of the protective layer 11 of the sensor 1 and the excitation light is irradiated from the test piece 5 side. 1 may be irradiated with excitation light from the back surface side (substrate 7 side) to obtain a Raman scattered light or fluorescence spectrum from the test piece 5.

1: Sensor 5: Test piece 7: Substrate 9: Enhanced electromagnetic field forming layer 10: Metal fine particle 11: Protective layer 30: Excitation light irradiation region 31: Spectral peak attributed to cytochrome C 32: Excitation light irradiation region 41: Light source part 43: Light receiving unit 50: Excitation light source 51: Filter 52: Condensing lens 53: Filter 54: Light receiving head 55: Spectrometer 56: Photo detector 61: Mirror 62: Half mirror

Claims

A method for inspecting a specimen including a biological tissue section,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Preparing step (a),
Placing the test piece to be inspected on the upper surface of the protective layer (b),
Irradiating excitation light to the light enhancement element from the upper surface or from the back surface (c),
And an inspection method comprising the step (d) of measuring light emission spectrum by receiving light emitted from the test piece.
The step (c) is a step of irradiating the light enhancement element with the excitation light while scanning in a predetermined direction.
The inspection method according to claim 1, further comprising a step (e) of two-dimensional mapping the intensity distribution of the emission spectrum obtained by the step (d).
3. The inspection method according to claim 1, wherein light emitted from the test piece is Raman scattered light or fluorescence.
4. The protective layer according to claim 1, wherein the protective layer is composed of an inorganic substance having an orientation or an organic polymer having an orientation in association with the large number of metal fine particles. Inspection method described in 1.
4. The inspection method according to claim 1, wherein the protective layer contains a halogen element.