JP2001242083A - Light intensifying method, light intensifying device, fluorometry using it, and fluorometer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a light intensifying method stably providing fluorescence intensifying effect, a light intensifying device, a fluorometry using it, and a fluorometer. SOLUTION: A sample mounting base 1 is constructed of a base board 10 on a light incident face 1a side and a dielectric multiplayer film 11 on a sample mounting face lb side. Excitation light entering from the light incident face 1a is fully reflected on the sample mounting face 1b, while its evanescent light is fed to a sample. In construction of a low refractive index layer 12 and a high refractive index layer 13 constituting the dielectric multiplayer film 11, thickness of each layer 12, 13 is set on the basis of the wavelength of the excitation light so that light intensity of the excitation light is localized in the vicinity of the sample mounting face 1b. In this way, evanescent light fed to the sample is intensified and efficient fluorescence intensifying can be realized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a light enhancement method and a light enhancement device for enhancing excitation light applied to a sample, and a fluorescence measurement method and a fluorescence measurement device using the same.

[0002]

2. Description of the Related Art In recent years, single molecule detection based on fluorescence measurement,
And the development of imaging and spectroscopy technologies is underway.
It offers a variety of potential applications in analytical chemistry.
In such a fluorescence measurement device that performs single-molecule detection, the sample on the substrate is irradiated with excitation light, and the fluorescence detection device is set at a position other than the excitation light path to the sample and the reflection light path from the sample. By the means, fluorescence from the sample is detected.

For example, in a fluorescence measuring apparatus disclosed in Japanese Patent Application Laid-Open No. 148076/1994, a silicon wafer is used as a substrate for mounting a sample. The sample mounted on the silicon wafer is irradiated with laser light from a laser as an excitation light source from an oblique direction, and the sample is detected by fluorescence detection means including an optical microscope installed above the silicon wafer. Is detected.

[0004]

The molecules to be detected by the above-described single molecule detection by fluorescence measurement mainly have a fluorescent property having an absorption spectrum maximum in the visible region (wavelength: 400 to 800 nm), particularly in the vicinity of 500 nm. Limited to pigments. On the other hand, when single molecule detection is applied to, for example, nucleobases, the light absorption efficiency and the fluorescence quantum yield are extremely small as compared with fluorescent dyes. For this reason, the fluorescence intensity generated from the sample with respect to the excitation light is weakened, and it is difficult to detect a single molecule.

That is, fluorescent dyes have (a) a molecular extinction coefficient ε representing the efficiency of light absorption of about 10 ⁵ cm
⁻¹ (mol / l) ^−1, and (b) the condition that the fluorescence quantum yield is about 0.1 to 1.0 and close to 1. In contrast, nucleobases are (1)
The efficiency of light absorption is about 10 ⁴ cm ^-1 (mol / l) ^-1
And about 1/5 to 1/10 that of fluorescent dyes, and (2) the fluorescent quantum yield is as small as about 10 ^-4 , which is disadvantageous compared to fluorescent dyes. . In general,
It is empirically known that single-molecule detection is virtually impossible when the fluorescence quantum yield is less than 0.1.

[0006] The maximum of the light absorption spectrum of the nucleic acid base is in the ultraviolet region having a wavelength of up to 260 nm. However, when excitation is directly performed using ultraviolet light, there is a problem that (3) background light caused by contamination of the sample and surroundings, impurities, optical components, and the like is increased, and fluorescence measurement is hindered.

It is also possible to enhance the fluorescence by converting a nucleic acid base into a fluorescent derivative by a chemical reaction and further using a two-photon excitation fluorescence method. For example, guanine is known to increase the fluorescence yield by about 100 times by methylating the 7-position. In addition, a fluorescent derivative of a nucleic acid base (maximum absorption wavelength 280 to 310 n)
When the two-photon excitation fluorescence method is applied to m), the wavelength of the excitation light is in the visible region (wavelength 560 to 620 nm). At this time,
The wavelength of the background fluorescence caused by impurities and the like is always longer than the excitation light (wavelength: 560 nm or more), so it affects the fluorescence wavelength region (wavelength: 320 to 450 nm) of the nucleobase derivative by the two-photon excitation fluorescence method. No longer
The problem of background light, such as when using ultraviolet light, can be avoided.

However, when such a method is used, (4) the fluorescence quantum yield of the fluorescent derivative of the nucleobase is
There is a problem that a case where the value is less than 0.1 (for example, about 0.01) at which single-molecule measurement can be performed may occur.
It is known that the two-photon excitation process is a nonlinear optical process, and the fluorescence intensity is proportional to the square of the excitation light intensity.
In general, (5) there is a problem that the efficiency of two-photon excitation is smaller than that of one-photon excitation.

[0009] In response to such a problem of fluorescence intensity, reference 1 (Hiroaki Yokota et al., Phys. Rev. Lett. 80, pp.
4606-4609 (1998) "shows a fluorescence enhancement method. In the enhancement method of Document 1, a metal thin film is formed on a sample mounting surface of a quartz substrate on which a sample is mounted. Then, the surface plasmon in the metal thin film is excited by evanescent light from the quartz substrate side, and the fluorescence from the sample mounted on the metal thin film is enhanced.

[0010] However, it is known that a metal thin film generally causes fluorescence quenching in addition to the fluorescence enhancement effect. Further, whether the fluorescence enhancement or quenching is obtained as an effect using the metal thin film depends on the distance between the fluorescent molecule and the metal surface (for example, see US Pat. No. 4,649,280). In particular, it is considered that the quenching becomes dominant as the distance between the fluorescent molecule and the metal surface decreases.

[0011] For example, in the above-mentioned literature 1, there are several reports on the measurement of the activity of motile single protein molecules in which a fluorescence enhancing effect is obtained (fluorescence enhancement rate 13 times on silver surface, aluminum surface 2.3 times, gold surface 1.6 times)
It is thought that they happened to function as spacers. In addition, metals are often chemically active and may have undesirable effects on a sample other than fluorescence quenching, such as causing some chemical reaction with the sample. For example, in a case where silver having the highest fluorescence enhancement rate is used as the metal thin film, the result is that the enzyme activity of the protein is lost. That is, in the fluorescence enhancement method using a metal thin film, there is a problem that the fluorescence enhancement effect cannot always be obtained as a whole effect, or the property itself of the sample molecule is affected.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and provides a light enhancement method and a light enhancement apparatus capable of stably obtaining a fluorescence enhancement effect, a fluorescence measurement method using the same, and a fluorescence measurement method. It is an object to provide a measuring device.

[0013]

In order to achieve the above object, a light enhancement method according to the present invention provides a light enhancement method that localizes and enhances the intensity of excitation light of a predetermined wavelength applied to a sample. A method in which excitation light is transmitted and a substrate having one surface serving as a light incident surface and a low-refractive-index layer and a high-refractive-index layer are alternately laminated on the other surface of the substrate. And a dielectric multilayer film in which the surface opposite to the substrate serves as a sample mounting surface, and the thickness of each of the low refractive index layer and the high refractive index layer in the dielectric multilayer film is a predetermined wavelength. On the basis of the above, using the sample mounting table whose thickness is set such that the light intensity of the excitation light is localized near the sample mounting surface, the excitation light is incident on the sample mounting table from the light incident surface. , The total energy reflected on the sample mounting surface and the enhanced energy supplied to the sample mounted on the sample mounting surface. And generating a Ssento light.

Further, the optical enhancement device according to the present invention comprises a sample mounting table for mounting a sample, and enhances the light by localizing and enhancing the intensity of the excitation light of a predetermined wavelength applied to the sample. An apparatus, in which a sample mounting table transmits an excitation light and alternately laminates a substrate having one surface serving as a light incident surface and a low refractive index layer and a high refractive index layer on the other surface of the substrate. And a dielectric multilayer film having a surface on the opposite side to the substrate serving as a sample mounting surface, and a thickness of each of the low refractive index layer and the high refractive index layer in the dielectric multilayer film. However, based on the predetermined wavelength, the light intensity of the excitation light is set to a thickness that is localized near the sample mounting surface, with respect to the sample mounting table,
The excitation light is incident from the light incident surface, is totally reflected by the sample mounting surface, and generates enhanced evanescent light to be supplied to the sample mounted on the sample mounting surface.

In the light enhancement method and the light enhancement device using the sample mounting table, the excitation light is totally reflected by the sample mounting surface, and the excitation light is supplied by the evanescent illumination method. For this reason, generation of background light due to supply of excitation light to impurities and the like is suppressed. In addition, the sample mounting surface side of the sample mounting table is formed of a dielectric multilayer film, and of the low refractive index layer and the high refractive index layer constituting the multilayer film, the excitation light in the layer on the sample mounting surface side. The thickness of each layer is set based on the wavelength of the excitation light so that the light intensity is localized. Thereby, the light intensity of the evanescent light generated by the total reflection of the excitation light on the sample mounting surface and supplied to the sample is enhanced.

In particular, the dielectric multilayer film on which the sample is placed is:
Unlike metal thin films, it does not cause fluorescence quenching. Therefore, it is possible to provide a light enhancement method and a light enhancement device capable of efficiently and stably achieving fluorescence enhancement.

In the light enhancement method, the thicknesses d _L and d _H of the low-refractive index layer and the high refractive index layer are set to n _L and n _H , respectively. Assuming that the transmission angles of the light in the optical path are θ _L and θ _H , for a predetermined wavelength λ of the excitation light, the condition n _L · d _L cos θ _L = n _H · d _H c
osθ _H = λ / 4.

Further, in the light intensifying device, the low refractive index layer and the high refractive index layer have respective thicknesses d _L and d _{H and} have respective refractive indices n _L and n _H , and pass through the respective layers. Assuming that the passing angles of the excitation light in the optical path are θ _L and θ _H , for a predetermined wavelength λ of the excitation light, the condition n _L · d _L cos θ _L = n _H · d _H
It is characterized by being formed so as to satisfy cos θ _H = λ / 4.

In this way, the thickness of each layer of the dielectric multilayer film is set to form a one-dimensional photonic crystal,
By causing the layer on the sample mounting surface side to function as a defect layer, localization of the excitation light in the vicinity of the sample mounting surface and thereby enhancement of evanescent light can be realized particularly efficiently.

In the light enhancement method (light enhancement device), the flatness of the substrate is λ / λ with respect to a predetermined wavelength λ of the excitation light.
It is characterized by being 10 or less. By using a substrate having a high degree of flatness as described above, a dielectric multilayer film formed on the substrate can be made of good quality, and characteristics for localizing excitation light can be improved.

Further, the light intensifying device is characterized in that, in the sample mounting table, the layer closest to the sample mounting surface among the low refractive index layer and the high refractive index layer is made of SiO ₂ . S
iO ₂ is a chemically particularly stable substance, and it is preferable that the sample be placed on a layer equivalent to a quartz substrate or the like made of SiO _2, such as loss of chemical reaction or enzyme activity. The occurrence of unaffected effects can be suppressed.

Further, in the fluorescence measuring method according to the present invention, the excitation light output from the excitation light source is made incident on the light incident surface of the sample mounting table, and the light of the incident excitation light is emitted using the above-described light enhancement method. A sample excitation step of localizing the intensity near the sample mounting surface of the sample mounting table to supply enhanced evanescent light to the sample mounted on the sample mounting surface; and And a fluorescence detecting step of detecting fluorescence from the light source.

Further, the fluorescence measuring apparatus according to the present invention comprises: an excitation light source for outputting excitation light of a predetermined wavelength irradiated on the sample;
It is characterized by comprising the above-mentioned light intensifier and fluorescence detecting means for detecting fluorescence from a sample placed on a sample mounting table constituting the light intensifier.

Thus, the sample is excited using the evanescent light enhanced by localizing the excitation light in the vicinity of the sample mounting surface, and the fluorescence generated from the sample is efficiently and stably enhanced. Can be realized. In addition, since the excitation light is totally reflected from the sample mounting surface and the sample is mounted on the sample mounting surface, when an optical microscope is used for the fluorescence detecting means, the objective lens is applied to the sample. On the other hand, it can be freely approached to a short distance. In addition, since a substrate or the like is not disposed between the sample and the objective lens, problems such as a decrease in the amount of measured fluorescence and a limitation on a measurable fluorescence spectrum range are solved.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of a light enhancement method, a light enhancement device, a fluorescence measurement method using the same, and a fluorescence measurement device according to the present invention will be described below in detail with reference to the drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description. Also, the dimensional ratios in the drawings do not always match those described.

FIG. 1 is a side view showing the structure of a sample mounting table 1 which is an embodiment of the light enhancing device according to the present invention. The sample mounting table 1 is configured such that one surface (lower surface in the drawing) is a light incident surface 1a and the other surface (upper surface in the drawing) is a sample mounting surface 1b. In FIG. 1A, a prism 14 installed on the light incident surface 1a side of the sample mounting table 1 includes:
Optical microscope (to be described later) installed above the sample mounting surface 1b
The objective lens 42 of FIG.
4B illustrates the optical path of the excitation light supplied to the sample placed on b. On the other hand, FIG. 1B specifically illustrates the configuration of the sample mounting table 1 with the dielectric multilayer film 11 being enlarged.

The sample mounting table 1 includes a substrate 10 and a dielectric multilayer film 11 formed on the substrate 10. The substrate 10 is preferably a glass substrate or a quartz substrate, and one surface thereof is the above-mentioned light incident surface 1a. This substrate 10 transmits the excitation light of a predetermined wavelength incident from the light incident surface 1a to the other surface.

The dielectric multilayer film 11 is formed on the surface of the substrate 10 opposite to the light incident surface 1a, and the surface opposite to the substrate 10 is the above-described sample mounting surface 1b. The dielectric multilayer film 11 has a low refractive index layer 12 preferably made of SiO ₂ (silicon dioxide) and a high refractive index preferably made of HfO ₂ (hafnia dioxide) having a higher refractive index than the low refractive index layer 12. One-dimensional photonic crystal (one-dimensional photonic crystal)
photonic-crystal, hereinafter referred to as 1DPC).

In the present embodiment, as shown in FIG. 1 (b), the low-refractive index layer 12 ₁ is disposed on the substrate 10, a higher refractive index layer 13 and the low refractive index layer 12, placing most samples They are formed alternately to the low-refractive index layer 12 ₈ with a layer of surface 1b side. The number of layers, eight layers of the low refractive index layer is 12 ₁ to 12 _8, a 7-layer of the high refractive index layer is _{131-134 7.}

Here, of the layers constituting the dielectric multilayer film 11, the layer having the upper surface serving as the sample mounting surface 1b is replaced with a low-level layer made of chemically stable SiO ₂ equivalent to a quartz substrate or the like. It is the refractive index layer 12 _8. At this time, the sample mounting surface 1
Undesirable effects due to chemical reactions and the like on the sample placed on b are particularly suppressed.

The low refractive index layer 12 and the high refractive index layer 1
The thicknesses 3 are set such that the light intensity of the excitation light is localized near the sample mounting surface 1b based on the wavelength λ of the excitation light to be enhanced, as described later. It is formed. Here, the refractive indices of the low refractive index layer 12 and the high refractive index layer 13 are n _L and n _H , the thickness of each layer is d _L and d _H , and the optical path of the excitation light passing through each layer. Are represented as θ _L and θ _H.

In the light intensifying method using the sample mounting table 1 which is the above-described light intensifying apparatus, the excitation light having the wavelength λ to be subjected to light enhancement enters the sample mounting table 1 from the light incident surface 1a side. Is done. In the example shown in FIG. 1A, the excitation light is applied in the stacking direction of the dielectric multilayer film 11 (the vertical direction in FIG. 1).
At a predetermined angle (for example, 45 °) with respect to
And is incident on the sample mounting table 1 via.

Excitation light that has entered the sample mounting table 1 passes through the substrate 10 from the light incident surface 1a side, further passes through the dielectric multilayer film 11, and reaches the sample mounting surface 1b. Then, the excitation light is totally reflected by the sample mounting surface 1b, and the reflected light passes through the dielectric multilayer film 11 and the substrate 10 in the opposite direction, and the light incident surface 1b.
a. At this time, excitation light is supplied to the sample on the sample mounting surface 1b by evanescent light generated by total reflection by the sample mounting surface 1b. Here, since the excitation light is localized near the sample mounting surface 1b as described above, the evanescent light that is the excitation light supplied to the sample is enhanced.

The specific structure of the dielectric multilayer film 11 constituting the sample mounting table 1 and the obtained fluorescence enhancing effect will be further described with reference to FIG. In FIG. 2, the configuration of the sample mounting table 1 and the like is partially omitted.

The excitation light is incident at a predetermined angle to the specimen table 1 by incident light path l ₁ is guided to the specimen table 1 outside by reflected light path l ₂ it is totally reflected by the specimen mounting face 1b. At this time, the optical configuration of the sample mounting table 1 with respect to the excitation light is such that a virtual sample mounting table 2 having a mirror image relationship is continuously formed on the sample mounting table 1 as schematically shown in FIG. It is equivalent to the case where they are arranged. In this hypothetical configuration, the excitation light without being reflected by the sample mounting surface 1b, and passes through the virtual optical path l ₃ in the specimen table 2, and is emitted from the substrate 20 side.

At this time, the dielectric multilayer films 11 and 21 of the sample tables 1 and 2 which are superimposed in a mirror image form a 1DPC as a whole. Then, located across a sample mounting surface 1b at the center of the stacking direction, a layer composed of a low refractive index layer 12 ₈ and 22 ₈ thickness than the other of the low refractive index layer and the high refractive index layer Double this 1DPC
Layer in the photonic crystal structure of GaN
r) Construct 15.

In a photonic crystal having a periodic structure, a photonic band gap is formed with respect to light, so that light in a specific wavelength region cannot exist or pass inside. When a defect layer that disturbs the periodic structure is introduced to such a structure, a localized level is formed in the photonic band gap, and light having a wavelength corresponding to the level can exist in the crystal. (For example, in Reference 2, Jing
Yong Ye et al., Appl. Phys. Lett. 75, pp. 3605-360
7 (1998) "and Reference 3" Toshiaki Hattori et al.,
J. Opt. Soc. Am. B14, pp. 348-355 (1997) ”).

In the virtual 1DPC shown in FIG. 2, when the refractive index of the defect layer 15 is n _x , the thickness of the layer is d _x , and the passing angle of the optical path of the excitation light passing through the layer is θ _x. , _{L L} d _L cos θ _L = n _H d _H cos θ _H = n _X d _X cos θ _X / 2 = λ / 4 By forming each layer, localization corresponding to light of wavelength λ is achieved. Levels are formed in 1DPC. This localized level functions as a transmission window having a sharp line width in the transmission wavelength curve of light with respect to 1DPC, so that the excitation light having the wavelength λ is transmitted and the optical field is locally enhanced in the defect layer 15 (locally). Is realized.

In the configuration of the dielectric multilayer film 11 of the sample mounting table 1 for realizing this localization condition, the thickness of each of the low refractive index layer 12 and the high refractive index layer 13 is determined by the condition n.
_It is formed so as to satisfy _L · d _L cos θ _L = n _H · d _H cos θ _H = λ / 4. At this time, the thickness of the virtual defect layer 15 formed near the sample mounting surface 1b is n _x = n _L , d _x
= 2d _L , θ _X = θ _L , the condition n _X · d _X cos θ _X / 2 =
λ / 4 is automatically satisfied. Therefore, localized excitation light having a wavelength λ is in the low refractive index layer 12 ₈ sample mounting surface 1b side, the light intensity of the evanescent light to be supplied to the local light intensity and sample of the excitation light is enhanced Is obtained.

In a specific embodiment using the sample mounting table 1 shown in FIG. 1, a low refractive index layer 12 and a high refractive index layer 1 will be described.
3 is formed of SiO ₂ and HfO ₂ respectively, the respective refractive indices are n _L = 1.46 and n _H = 1.94.
It is. On the other hand, a transmission window is formed for excitation light having a wavelength of λ = 600 nm.
The passing angle of the excitation light path at 2 is set to θ _L = 45 ° (see FIG. 1A). Passage angle of the excitation light optical path of the high refractive index layer 13, from the law _{n L sinθ L = n H sinθ} H refracting, and θ _H = 32 °.

Under the above conditions, the thicknesses of the low refractive index layer 12 and the high refractive index layer 13 are n _L · d _L = 212.1n, respectively.
m (d _L = 145.3 nm) and n _H · d _H = 176.
8 nm (d _H = 91.13 nm). FIG.
3 is a graph showing a transmission wavelength curve of light obtained when the dielectric multilayer film 11 is formed by applying the above-mentioned thickness setting in the sample mounting table 1 of FIG. In this transmission wavelength curve, a photonic band gap through which light is not transmitted is formed in a wavelength region of about 540 to 680 nm. A transmission peak having a sharp line width (a transmission window for excitation light) is formed at a wavelength λ = 600 nm in the photonic band gap.

[0042] In the above mentioned optical intensifier and the optical enhancement process according to the sample table 1 has a configuration, the low refractive index layer 12 ₈ of the sample mounting surface 1b side of the dielectric multilayer film 11 functions as the defect layer As a result, a localized level corresponding to the wavelength λ is formed, and the light intensity of the excitation light having the wavelength λ is localized.
At this time, the light intensity of the evanescent light generated by the total reflection of the excitation light by the sample mounting surface 1b and supplied to the sample is enhanced, and the fluorescence of the sample is enhanced.

Here, the dielectric multilayer film 1 on which the sample is placed
1 does not cause fluorescence quenching unlike metal thin films,
Fluorescence enhancement can be realized efficiently and stably.
Particularly, the layer on the sample mounting surface 1b side is made of a stable SiO ₂
Since there is a low refractive index layer 12 ₈ made of the occurrence of undesirable effects, such as the activity of the enzyme which is a sample is lost is prevented. In addition, since the evanescent illumination method is used, generation of background light due to excitation of impurities or the like is suppressed.

If the flatness of the substrate 10 in the sample mounting table 1 is poor, the dielectric multilayer film 11 formed thereon
Similarly, the flatness also deteriorates, so that a good transmission window cannot be obtained in the light transmission wavelength curve. In order to accurately realize the above structure of the dielectric multilayer film 11 and obtain a good transmission window, the flatness of the substrate 10 is preferably λ / 10 or less with respect to the wavelength λ of the excitation light.

In order to obtain such high flatness and sufficient mechanical strength, the substrate 10 is required to have a certain thickness. For example, assuming that the material of the substrate 10 is glass or quartz, the thickness of the substrate 10 is 25 mm (1 inch) and the thickness is 2 mm.
When the thickness is about mm, the flatness is about λ / 4 due to conditions such as a polishing technique, and a sufficient flatness cannot be obtained.
When a flatness λ / 10 at which a good transmission window is obtained is required, for example, a thickness of about 5 mm is required.

Reference 2 describes the fluorescence enhancement using the localization of light in the defect layer of 1DPC. In this document 2, a quartz substrate (diameter 30 mm, thickness 5
mm) on which two dielectric multilayer films are formed to face each other, and a polymer film of a predetermined thickness to which sample molecules are added is sandwiched between the two as a defect layer to constitute a 1DPC. This configuration, in FIG. 2, a virtual specimen table 2 in a relationship of mirror image relative to the specimen table 1 actually installed, configured to pass through the optical path l ₃ without reflects the excitation light It corresponds to the case of doing.

However, in this configuration, the sample cannot be directly mounted on the sample mounting table, and a polymer film having a predetermined thickness, for example, doped with sample molecules is required to form a defect layer. Also, for example, when a polymer film is sandwiched, the positional accuracy between the dielectric multilayer films is important in forming a localized level, and in order to reduce the influence of the unevenness of the polymer film, using a vise or the like. Work such as smoothing out irregularities by applying pressure is required. In addition, since the fluorescence generated from the sample is measured after passing through the dielectric multilayer film and the substrate, the amount of fluorescence measured thereby is reduced, or the measurable fluorescence spectrum range is limited. Cause problems.

Further, the substrate and the dielectric multilayer film are disposed on both sides of the sample. As described above, the substrate has a certain thickness (for example, about 5 mm) in order to form the dielectric multilayer film well. ) Is required, there is a problem that the objective lens of the optical microscope cannot be sufficiently close to the sample due to the configuration of the apparatus. The distance between the objective lens and the sample greatly affects the efficiency of the fluorescence measurement.

In the detection of single-fluorescent molecules, it is important to collect and measure the fluorescence from the sample with the highest possible efficiency. For this reason, it is preferable to use an objective lens having the largest possible value η (this value is a measure of the fluorescence collection efficiency) obtained by dividing the aperture ratio NA by the refractive index n of the medium in which the tip of the objective lens is immersed.

FIG. 4 shows the relationship between NA and light collection efficiency. In FIG. 4, the refractive indices of the medium are all 1.
It is set to 0. In a commercially available oil-immersed or water-immersed objective lens, the value of NA is about 1.40 at the maximum, but considering the water and oil (n to 1.5) of the medium, η to
The limit is 0.93, and the light-collecting efficiency is equivalent to that of an objective lens having an NA of 0.95 using air as a medium. The NA of the objective lens having a magnification of 40 to 100 times is approximately 0.7 or more.

The distance between the objective lens and the sample is called the working distance (WD, Working Distance) of the objective lens. In general, as NA increases, WD decreases and N
When A is 1.0 or more, WD is very small, typically 0.1 mm. On the other hand, in the example of Literature 2, since a substrate having a thickness of 5 mm is provided between the sample and the objective lens, it is necessary to use an objective lens whose WD exceeds 5 mm. At this time, the NA is 0.4 or less, which is disadvantageous for fluorescence measurement.

On the other hand, when the sample mounting table 1 according to the above-described embodiment is configured so that the excitation light is totally reflected by the sample mounting surface 1b and the sample is mounted on the sample mounting surface 1b. As shown in FIG. 1A, the WD can be reduced by freely bringing the objective lens 42 of the optical microscope close to the sample to a short distance. Therefore, it is possible to perform fluorescence measurement with high efficiency by appropriately selecting various objective lenses such as those having a large NA. Further, since no substrate or the like is disposed between the sample and the objective lens 42, the amount of measured fluorescence decreases,
There is no problem that the measurable fluorescence spectrum range is limited.

FIG. 5 is a block diagram schematically showing one embodiment of a light intensifying device and a fluorescence measuring device using the light intensifying method according to the present invention. In this device, the sample A is placed on the sample mounting surface 1b.
A sample mounting table 1 mounted thereon and an excitation light source 30 for supplying excitation light incident from the light incident surface 1a side of the sample mounting table 1
And a photon counting system (detection unit 40, optical microscope 41, objective lens 42, detection control unit 44, computer 45, monitor 46, storage device 47) as fluorescence detection means for detecting fluorescence from sample A. I have.

The sample mounting table 1 is placed in a clean booth of class 1000 or less, and is placed in a clear atmosphere. The detection of fluorescence by the detection unit 40 is performed by the detection control unit 4.
4, the detection control unit 44, the computer 4
5. In the monitor 46 and the storage device 47, accumulation of signals (photon counting of fluorescence), image processing, display of images, storage of records, and the like are performed. This apparatus can use, as the sample A, various substances that emit fluorescence, such as nucleic acid bases and proteins. Further, even those which do not emit fluorescence can be used in the same manner by binding to a fluorescent substance.

The wavelength λ = 600 nm as the sample mounting table 1
The fluorescence from the sample was measured using the above-described embodiment having a transmission window. As the excitation light source 30, a light source composed of an argon ion laser-pumped femtosecond titanium sapphire laser oscillator, an argon ion laser-pumped femtosecond titanium sapphire laser regenerative amplifier, and an optical parametric amplifier is used. 150 fs, frequency 20
It was used as excitation light for irradiating an optical output with 0 kHz and an output intensity of 10 mW.

The optical microscope 41 is a Zeiss Axio Pla
n, objective lens 42 is Zeiss Fluar (magnification 20x, NA
0.75, WD2 mm), and the detection unit 40
Comprised a polychromator and a streak camera. In this configuration, the fluorescence from the sample A, which is excited by the excitation light supplied by the evanescent light, is collected and guided by the objective lens 42 and the optical microscope 41, and is introduced into the polychromator of the detection unit 40. , Measured by a streak camera.

As the sample A, a sample prepared by spin-coating the sample mounting table 1 with a fluorescent dye Nile Blue (100 μl, 1 μmol / l) was used. FIG. 6 is a graph showing the measured fluorescence intensity. In this graph,
In addition to the measurement results using the sample mounting table 1 described above, a case where a quartz substrate on which a dielectric multilayer film is not formed is used as a mounting table is also shown as a comparative example.

In FIG. 6, the vertical axis indicates a change in the measured fluorescence intensity. The horizontal axis indicates the incident angle of the excitation light from the excitation light source 30 to the sample mounting table 1.
At this time, in the comparative example using the quartz substrate, almost constant fluorescence intensity was obtained regardless of the incident angle of the excitation light. On the other hand, when the sample mounting table 1 in the above-described embodiment is used, the incident angle at which the condition that the excitation light is localized near the sample mounting surface 1b and the evanescent light is enhanced is satisfied. At 45 °, the measured fluorescence intensity was greatly enhanced, and a fluorescence intensity approximately 18 times that of the quartz substrate was obtained.

The light enhancement method and light enhancement device according to the present invention
It is not limited to the above-described embodiments and examples,
Various modifications are possible. For example, the low refractive index layer 12 and the high refractive index layer 13 of the dielectric multilayer film 11 are made of SiO ₂ and Hf.
Various substances other than O ₂ may be used, and the number of layers may be appropriately changed depending on necessary conditions such as a line width. The layer closest to the sample mounting surface 1b may not be the low refractive index layer 12, or if the influence on the sample is sufficiently small, a layer made of a material other than SiO ₂ may be used. Layer.

Further, the above-described light enhancement method, fluorescence measurement method using the light enhancement device, and fluorescence measurement device are not limited to the above-described embodiments and examples. For example,
As the detection unit 40, various devices such as a CCD camera may be used in addition to the above-described configuration. In addition to ordinary fluorescence measurement, it is also useful for enhancing various optical phenomena such as non-linear optical phenomena such as Raman scattering and two-photon excitation fluorescence.
The same can be applied.

[0061]

The light enhancing method and the light enhancing apparatus according to the present invention have the following effects as described in detail above. That is, the sample mounting table is composed of the substrate on the light incident surface side and the dielectric multilayer film on the sample mounting surface side, and the excitation light incident from the light incident surface is totally reflected on the sample mounting surface,
The evanescent light is supplied to the sample. Then, in the configuration of the low refractive index layer and the high refractive index layer constituting the dielectric multilayer film, the thickness of each layer is adjusted so that the light intensity of the excitation light is localized near the sample mounting surface. Set based on the wavelength of

At this time, the light intensity of the evanescent light supplied to the sample is enhanced by the localization of the excitation light. In addition, the dielectric multilayer film does not cause fluorescence quenching unlike the metal thin film, so that the effect of enhancing the fluorescence by increasing the excitation light intensity can be obtained efficiently and stably. Further, since the excitation light is supplied by the evanescent illumination method, generation of background light due to impurities or the like is suppressed.

In the above-described light enhancement method, the fluorescence measurement method using the light enhancement device, and the fluorescence measurement device, the excitation light is totally reflected by the sample mounting surface, and the sample is mounted on the sample mounting surface. When the optical microscope is used as the fluorescence detecting means, the objective lens can be freely brought close to the sample to a short distance. In addition, since a substrate or the like is not disposed between the sample and the objective lens, problems such as a decrease in the amount of measured fluorescence and a limitation on a measurable fluorescence spectrum range are solved.

[Brief description of the drawings]

FIG. 1 is a side view showing a configuration of a sample mounting table which is an embodiment of a light enhancement device.

FIG. 2 is a schematic diagram for describing localization of excitation light on a sample mounting table shown in FIG.

FIG. 3 is a graph showing a light transmission wavelength curve in one embodiment of the sample mounting table shown in FIG. 1;

FIG. 4 is a graph showing a correlation between NA of an objective lens and light collection efficiency.

FIG. 5 is a configuration diagram showing one embodiment of a fluorescence measurement device using the sample mounting table shown in FIG.

FIG. 6 is a graph showing fluorescence enhancement by the fluorescence measurement device shown in FIG.

[Explanation of symbols]

Reference numeral 1 denotes a sample mounting table, 1a denotes a light incident surface, 1b denotes a sample mounting surface,
10: substrate, 11: dielectric multilayer film, 12: low refractive index layer,
13: high refractive index layer, 14: prism, 15: defect layer, 3
0: excitation light source, 40: detection unit, 41: optical microscope, 42
... Objective lens, 44 detection control unit, 45 computer, 46 monitor, 47 storage device.

Continuation of the front page (72) Inventor Mitsuru Ishikawa 1126 Nomachi, Hamamatsu-shi, Shizuoka Pref. F-term in Hamamatsu Photonics Co., Ltd. 2H048 GA01 GA07 GA12 GA34 GA51 GA61

Claims (9)

[Claims] 1. A light enhancement method for localizing and enhancing the light intensity of excitation light of a predetermined wavelength applied to a sample, wherein the excitation light is transmitted and one surface is formed as a light incident surface. And a dielectric multilayer in which a low-refractive-index layer and a high-refractive-index layer are alternately laminated on the other surface of the substrate, and the surface opposite to the substrate is a sample mounting surface. A thickness of the low refractive index layer and the high refractive index layer in the dielectric multilayer film, based on the predetermined wavelength,
Using a sample stage on which the light intensity of the excitation light is localized near the sample stage, the excitation light is incident on the sample stage from the light incident surface. And generating an enhanced evanescent light to be supplied to the sample mounted on the sample mounting surface by total reflection on the sample mounting surface. 2. The respective thicknesses d _L and d _H of the low refractive index layer and the high refractive index layer are n _L and n _H , respectively, of the refractive index, and the excitation light passing through the respective layers. Assuming that the passing angles of the optical paths are θ _L and θ _H , the condition n _L · d _L cos θ _L = n _H · d _H cos θ _H = λ / 4 is satisfied with respect to the predetermined wavelength λ of the excitation light. The light enhancement method according to claim 1, wherein: 3. The optical enhancement method according to claim 1, wherein the substrate has a flatness of λ / 10 or less with respect to the predetermined wavelength λ of the excitation light. 4. An excitation light outputted from an excitation light source is made to enter from a light incident surface of a sample mounting table, and the excitation light is inputted by using the light enhancement method according to claim 1. Sample excitation for localizing the light intensity of light in the vicinity of the sample mounting surface of the sample mounting table and supplying the enhanced evanescent light to the sample mounted on the sample mounting surface And a fluorescence detection step of detecting the fluorescence from the sample that has been excited. 5. An optical enhancement device comprising a sample mounting table for mounting a sample, wherein the light enhancement device localizes and enhances the light intensity of excitation light having a predetermined wavelength applied to the sample. The mounting table is formed by transmitting the excitation light and alternately stacking a low refractive index layer and a high refractive index layer on the other surface of the substrate, and a substrate having one surface serving as a light incident surface. And a dielectric multilayer film in which a surface opposite to the substrate is a sample mounting surface, and a thickness of each of the low refractive index layer and the high refractive index layer in the dielectric multilayer film. However, based on the predetermined wavelength,
The light intensity of the excitation light is set to a thickness localized in the vicinity of the sample mounting surface. The excitation light is incident on the sample mounting table from the light incident surface, and the sample mounting is performed. A light enhancement device that generates an enhanced evanescent light to be totally reflected by a surface and supplied to the sample mounted on the sample mounting surface. 6. The low refractive index layer and the high refractive index layer,
The respective thicknesses d _L and d _{H define} the respective refractive indices as n _L
And n _H , when the passing angles of the optical paths of the excitation light passing through the respective layers are θ _L and θ _H , the condition n _L · d _L cos θ _{L is} satisfied for the predetermined wavelength λ of the excitation light. _{= n H · d H cosθ H} = λ / 4 optical intensifier according to claim 5, characterized by being formed so as to satisfy. 7. The optical enhancement device according to claim 5, wherein the flatness of the substrate is λ / 10 or less with respect to the predetermined wavelength λ of the excitation light. 8. The sample mounting table, wherein a layer closest to the sample mounting surface among the low refractive index layer and the high refractive index layer is made of SiO _2.
The light intensifier according to any one of claims 7 to 7. 9. An excitation light source for outputting excitation light of a predetermined wavelength applied to a sample, an optical enhancement device according to claim 5, and the sample constituting the optical enhancement device. Fluorescence detection means for detecting fluorescence from the sample mounted on the mounting table,
A fluorescence measurement device comprising: