WO2013136891A1

WO2013136891A1 - Spectroscopic device, spectroscopic lens and spectroscopic container

Info

Publication number: WO2013136891A1
Application number: PCT/JP2013/053236
Authority: WO
Inventors: 山口　光城; 田邊　哲也
Original assignee: オリンパス株式会社
Priority date: 2012-03-12
Filing date: 2013-02-12
Publication date: 2013-09-19

Abstract

The objective of the present invention is, when photocounting light for a level of one molecule in a specimen solution, to improve an SN ratio and perform high-precision detection. Provided is a spectroscopic device provided with an objective lens (6), and wherein: a tip optical member (6a) of the objective lens (6) comprises an indentation recessed toward an image side at an endmost surface (6b) of said tip optical member (6a); and a focal point position (D) is placed within the indentation, said focal point position (D) being placed on the endmost surface (6b) or closer to the image side. Said objective lens: condenses excitation light emitted from a light source and shines said excitation light into the specimen solution (A), which is placed at the focal point position; and condenses light emitted from a light-emitting substance that diffuses in the specimen solution (A) and exists at the focal point position.

Description

Optical analysis device, optical analysis lens and optical analysis container

The present invention relates to an optical analysis device, an optical analysis lens, and an optical analysis container.

Conventionally, fluorescence correlation spectroscopy (FCS) is known as a technique for detecting weak light at the level of one photon or one fluorescent molecule (see, for example, Patent Document 1).
This FCS uses a laser confocal microscope optical system and a photocounting technique to measure the fluorescence intensity from fluorescent molecules entering or exiting a microregion in a sample solution or fluorescently labeled molecules.

Japanese Patent Laying-Open No. 2005-98766

However, since the optical system used in this FCS uses an immersion objective lens provided in a laser confocal microscope for imaging fluorescence in a sample, most of the fluorescence emitted from fluorescent molecules cannot be used. There is a disadvantage that the SN ratio is low. That is, since the particles in the solution rotate at high speed, the emitted fluorescence is emitted uniformly and isotropically in all directions, but the angle of capture of the immersion objective lens of a normal laser confocal microscope is the light. Since the angle range is 70 ° on one side with the axis as the center, the fluorescence emitted in the direction corresponding to the angle range of 110 ° on the other side is discarded without being detected.

The present invention has been made in view of the above-described circumstances. In the case of photocounting light of one molecule level in a sample solution, the light that can improve the SN ratio and perform highly accurate detection. An object of the present invention is to provide an analyzer, a lens for optical analysis, and a container for optical analysis.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention condenses excitation light emitted from a light source and irradiates a sample solution disposed at a focal position, and is dispersed in the sample solution and emitted from a light-emitting substance existing at the focal position. An objective lens for condensing light, and a tip optical member of the objective lens has a concave portion recessed on the image side on the most distal surface, and the focal position is on the image surface side or on the most image side It is an optical analyzer arrange | positioned in the said recessed part.

According to this aspect, the excitation light emitted from the light source is collected by the objective lens and irradiated to the sample solution arranged at the focal position. Since the luminescent substance is dispersed in the sample solution, the excitation light generates light such as fluorescence or phosphorescence by exciting the luminescent substance that passes through the focal position of the objective lens. The light generated in the luminescent material at the focal point is emitted isotropically in all directions, but the focal point is located on the foremost surface of the objective lens or in the concave portion on the image side, so that the light Light emitted in a direction corresponding to an angle range of about 90 ° on one side about the axis (that is, about half of the generated light) can be taken into the objective lens from the inner surface constituting the recess and detected. Become. That is, even with light of a single molecule level in the sample solution, the SN ratio in the case of photocounting can be improved and highly accurate detection can be performed.

In the above aspect, the tip optical member collects the light incident from the first surface and the first surface constituting the inner surface of the recess, on which the light emitted from the light emitting substance at the focal position is incident. And a second surface having a positive power.
By doing in this way, the light entered into the objective lens from the first surface constituting the inner surface of the concave portion can be condensed by the second surface, and all of the light can be directed to the image side.

Moreover, in the said aspect, the said 1st surface may be comprised by the inner spherical surface centering on the said focus position.
In this way, the light generated by exciting the fluorescent material by irradiating the excitation light at the focal position is emitted radially around the focal position, but the inner surface ( Spherical inner surface) makes it possible to enter the objective lens without refraction on the inner surface.

In the above aspect, the second surface may be a spherical surface that is decentered toward the image side in the optical axis direction with respect to the first surface.
By doing in this way, manufacture of a tip optical member can be made easy.

In the aspect described above, the second surface may be an ellipsoidal surface decentered toward the optical axis direction image side with respect to the first surface.
By doing in this way, a front-end | tip optical member can be comprised flatly and size reduction of the optical analyzer containing an objective lens can be achieved.

According to another aspect of the present invention, there is provided an outer surface having a shape complementary to the inner surface of the concave portion provided in the objective lens of any one of the above optical analyzers, and accommodating the sample solution therein. A container in which the sample solution can be placed at a position that coincides with the focal point of the objective lens when the container is combined with the recess.

According to this aspect, when the sample solution is accommodated in the accommodating portion and the outer surface of the accommodating portion is combined with the inner surface of the concave portion provided in the objective lens, the outer surface of the accommodating portion is brought into close contact with the inner surface of the concave portion. In this state, a part of the sample solution accommodated in the accommodating portion is arranged at a position that matches the focal position of the objective lens, so that it is dispersed in the sample solution by irradiating excitation light through the objective lens. Among the luminescent materials, the luminescent materials that pass through the focal position are excited to generate light.

The generated light passes through the wall surface of the housing portion from the inside of the housing portion, enters the optical member at the tip of the objective lens, and is collected. By doing in this way, the SN ratio in the case of carrying out photocounting can be improved in the state which put the sample solution in the container, and a highly accurate detection can be performed.

In another aspect of the present invention, the excitation light emitted from the light source is collected and irradiated to the sample solution disposed at the focal position, and the luminescent substance is dispersed in the sample solution and present at the focal position. A lens for optical analysis for condensing light emitted from the optical system, comprising a tip optical member having a recess recessed on the image side on the most distal surface, and the focal position is on the image surface or on the most image side It is the lens for optical analysis arrange | positioned in the said recessed part.

In the above aspect, the tip optical member collects the light incident from the first surface and the first surface constituting the inner surface of the recess, on which the light emitted from the light emitting substance at the focal position is incident. And a second surface having a positive power.
Moreover, in the said aspect, the said 1st surface may be comprised by the inner spherical surface centering on the said focus position.
Further, in the above aspect, the second surface may be a spherical surface that is decentered toward the optical axis direction image side with respect to the first surface, or may be an ellipsoidal surface.

According to the present invention, when photo-counting light of one molecule level in a sample solution, the SN ratio can be improved and highly accurate detection can be performed.

It is a block diagram which shows the optical analyzer which concerns on one Embodiment of this invention. It is a longitudinal cross-sectional view which shows an example of the front-end | tip optical member of the lens for optical analysis which concerns on this embodiment with which the optical analyzer of FIG. 1 was equipped. It is a figure which shows typically the optical path of the laser beam condensed on the center of the recessed part of the front-end | tip optical member of FIG. 2, and the fluorescence emitted from the center of a recessed part. It is a figure which shows the detail of the front-end | tip optical member of FIG. It is a figure which shows typically the optical path of the fluorescence when changing the relationship between the 1st surface and 2nd surface of the front-end | tip optical member of FIG. It is a figure which shows the fluorescence taken in by the front-end | tip optical member of FIG. 2 among the fluorescence generate | occur | produced in the sample solution. It is a figure explaining a solid angle. It is a figure which shows the modification of the front-end | tip optical member of FIG. It is a partial longitudinal cross-sectional view which shows an example of the container for optical analysis which concerns on this embodiment which accommodates the sample solution used in the optical analyzer of FIG. It is a figure which shows typically the optical path of the fluorescence emitted from the laser beam condensed from the center of the recessed part of the front-end | tip optical member at the time of using the container for optical analysis of FIG. 8, and the recessed part.

An optical analyzer 1, an optical analysis lens 6, and an optical analysis container 15 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the optical analyzer 1 according to the present embodiment reflects a light source 4 that emits laser light, and the laser light emitted from the light source 4 and transmits light generated in the sample solution A. Dichroic mirror 5, an objective lens (a lens for photoanalysis) 6 that collects the laser light reflected by the dichroic mirror 5 and irradiates the sample solution A, and is generated in the sample solution A and collected by the objective lens 6. The condensing lens 7 that collects the light that has passed through the dichroic mirror 5, the confocal pinhole 8 that is disposed near the focal position of the condensing lens 7, and the light that has passed through the confocal pinhole 8 And an analysis unit 10 that analyzes the intensity of light detected by the light detector 9 and sets an autocorrelation function.

In the figure, reference numeral 11 denotes a collimating lens, reference numeral 12 denotes a barrier filter, reference numeral 13 denotes a mirror, and reference numeral 14 denotes a condenser lens. The confocal pinhole 8 is disposed at a position optically conjugate with the focal position of the objective lens 6. The photodetector 9 is, for example, a photomultiplier tube.

The objective lens 6 according to the present embodiment is arranged with the optical axis directed in the vertical direction and the tip thereof directed vertically upward.
The objective lens 6 includes a tip optical member 6a shown in FIG. Although only the tip optical member 6a is shown in the figure, in practice, one or more lenses (not shown) for further collecting the light collected by the tip optical member 6a are provided on the image side.

The distal optical member 6a includes a concave portion 6c that is recessed toward the image side in the direction of the optical axis C on the frontmost surface 6b that is a plane orthogonal to the optical axis C thereof.
In the example shown in FIG. 2, the recess 6 c is configured by an inner spherical surface that is a hemispherical inner surface. The tip optical member 6a includes a first surface 6d made of an inner surface constituting the recess 6c, and a second surface 6e made of a spherical surface arranged on the image side in the optical axis C direction.

The first surface 6d is an inner spherical surface having a center D at a position where the optical axis C and the foremost surface 6b intersect.
The second surface 6e is a spherical surface having a center E (see FIG. 4) disposed at an interval on the image side in the optical axis C direction with respect to the center D of the first surface 6d.

The sample solution A is a living body such as an atom, molecule, or an aggregate thereof (hereinafter referred to as a particle) such as a protein, peptide, nucleic acid, lipid, sugar chain, amino acid, or an aggregate thereof. Particles such as molecules, viruses, cells, or non-biological particles are dispersed or dissolved. These particles may be either particles that emit light themselves, or particles to which any luminescent label or luminescent probe is added, and are hereinafter referred to as luminescent particles (luminescent materials). The light emitted from the luminescent particles is fluorescence or phosphorescence emitted by irradiation with excitation light.

In FIG. 1, the sample solution A is set in the optical analyzer 1 while being accommodated in a container 15.
As shown in FIGS. 9 and 10, the container (optical analysis container) 15 according to the present embodiment is attached to a part of the wall surface of the container 15 (a part of the bottom surface of the container 15 in FIG. 9). A convex portion (accommodating portion) 16 protruding in the direction is provided.

The outer surface 16 a of the convex portion 16 has a shape complementary to the first surface 6 d of the concave portion 6 c provided on the tip optical member 6 a of the objective lens 6. That is, since the concave portion 6c has the first surface 6d made of a hemispherical surface, the outer surface 16a of the convex portion 16 also has a spherical shape with the same or slightly smaller radius as the concave portion 6c. The wall surface of the container 15 of the convex portion 16 has a substantially uniform thickness dimension, and the sample solution A stored in the container 15 is located at the center of the spherical surface constituting the outer surface 16a of the convex portion 16. It has come to be arranged. A liquid may be interposed between the convex portion 16 of the container 15 and the concave portion 6c of the objective lens 6 as necessary, so that the two are brought into close contact with each other without any gap.

The operation of the optical analyzer 1 according to this embodiment configured as described above will be described below.
In order to detect light from the luminescent particles in the sample solution A using the optical analyzer 1 according to the present embodiment, the convex portion 16 provided on the bottom of the container 15 containing the sample solution A is shown in FIG. As shown in the drawing, the objective lens 6 is inserted into a recess 6c provided on the most distal surface 6b of the most distal member 6a, and the both are kept in close contact with each other.

In this state, laser light is emitted from the light source 4. The laser light is converted into substantially parallel light by the collimating lens 11, then reflected by the dichroic mirror 5 and incident on the objective lens 6.

In the objective lens 6 according to the present embodiment, since the focal position is disposed at the center D position of the concave portion 6c formed of the inner spherical surface provided on the foremost surface 6b, as shown in FIG. The laser light incident on 6 is condensed at the focal position. The luminescent particles dispersed in the sample solution A are excited when irradiated with laser light, and generate fluorescence.

Fluorescence generated in the sample solution A is collected by the objective lens 6 and passes through the dichroic mirror 5 as substantially parallel light. After the laser light is removed from the fluorescence transmitted through the dichroic mirror 5 by the barrier filter 12, it is condensed by the condenser lens 7, reflected by the mirror 13, and only the fluorescence that has passed through the confocal pinhole 8 is condensed. The light is collected by the lens 14 and detected by the photodetector 9.

In this case, since the confocal pinhole 8 is disposed at a position optically conjugate with the focal position of the objective lens 6, the fluorescence detected by the photodetector 9 through the confocal pinhole 8 is Only the fluorescence generated from the focal position of the objective lens 6 is obtained.
As shown in FIG. 6, the objective lens 6 is isotropically radiated in all directions as shown in FIG. 6, but the focal position is disposed on the most distal surface of the tip optical member 6 a of the objective lens 6. Therefore, as shown by the solid line in FIG. 6, the fluorescence in the range of the one-side angle θ = 90 ° with respect to the optical axis C is collected. That is, as schematically shown in FIG. 3, after being refracted to the image side by the tip optical member 6a, it is condensed by a lens (not shown) in the subsequent stage and converted into parallel light whose optical axis direction is directed to the image side. The

Here, as shown in FIG. 7, the solid angle Ω (unit: steradian) of the uptake is
Ω (θ) = 2π · (1-cosθ)
Can be described.
In the case of a general objective lens, the angle range on one side with respect to the optical axis C of fluorescent light that can be captured is 70 ° at most. Therefore, when θ can be 90 ° as in this embodiment,
Ω (90) / Ω (70) = 1.5
Thus, there is an advantage that it is possible to take in a fluorescence of 1.5 times the amount of light.

Further, in the present embodiment, the concave portion 6c provided on the foremost surface 6b of the tip optical member 6a of the objective lens 6 is constituted by the first surface 6d made of an inner spherical surface having the focal position as the center D. The fluorescence emitted from the focal position is incident on the first surface 6d perpendicularly. Therefore, the fluorescence is not refracted on the first surface 6d but is incident on the tip optical member 6a, and is further refracted on the image side on the second surface 6e on the image side.

Here, it is not difficult to match the shape of the second surface and the refractive index of the glass so that the fluorescence propagating in the tip optical member 6a is not totally reflected on the second surface 6e. For example, as shown in FIGS. 4 and 5, the first surface 6d is an inner spherical surface, the second surface 6e is a spherical surface having a radius R, and the center E position of the second surface 6e is set to the center D position of the first surface 6d. On the other hand, it is assumed that the image side is decentered by a distance L.

When the refractive index n1 of the glass is greater than the refractive index n2 of the air, the fluorescence generated from the focal point is not totally reflected by the second surface 6e under the following conditions from geometrical and optical analysis. Led to the side.
0 <L <R · (n2 / n1)

As shown in FIG. 5, when the distance L is gradually increased, all of the fluorescence in FIGS. 5 (a) and 5 (b) is guided to the image side, but when it becomes FIG. 5 (c). Thus, when the light enters the second surface 6e, total reflection occurs in the fluorescence, and the amount of fluorescence guided to the image side decreases. Therefore, if the conditions are set so that total reflection does not occur, the amount of fluorescent light can be detected to the maximum.

As described above, according to the optical analyzer 1, the objective lens 6, and the container 15 according to the present embodiment, it is possible to detect a larger amount of fluorescent light and to photocount one-level light in the sample solution. In addition, there is an advantage that the SN ratio can be improved and highly accurate detection can be performed.

In the present embodiment, the tip optical member 6a in which the second surface 6e is a spherical surface is illustrated, but instead, as shown in FIG. 8, the second surface 6e may be configured by an ellipsoidal surface. Good. By doing in this way, the dimension along the optical axis C direction of the front-end | tip optical member 6a can be shortened, and it becomes possible to achieve size reduction of the objective lens 6 whole.

In the present embodiment, the container 15 capable of performing optical analysis while containing the sample solution A has been proposed, but instead of using the container 15, the objective lens 6 can be used as the objective lens 6. The optical analysis may be performed using the immersion objective lens while the sample solution A is dropped and stored directly in the recess 6c.

A Sample solution D Center (focus position)
1 Optical Analyzer 4 Light Source 6 Objective Lens (Lens for Optical Analysis)
6a Front end optical member 6b Most advanced surface

6c Concave portion

6d First surface 6e Second surface 16a Convex portion (accommodating portion)

Claims

An objective lens that collects the excitation light emitted from the light source and irradiates the sample solution disposed at the focal position, and also disperses the light emitted from the luminescent material present at the focal position while being dispersed in the sample solution. With
The tip optical member of the objective lens has a concave portion that is recessed toward the image side on the most distal surface thereof, and the focal position is disposed on the most distal surface or within the concave portion on the image side relative to that. Analysis equipment.
The tip optical member has a first surface that constitutes the inner surface of the concave portion, on which light emitted from the light emitting material at the focal position is incident, and positive power that condenses the light incident from the first surface. The optical analyzer according to claim 1, further comprising a second surface.
3. The optical analyzer according to claim 2, wherein the first surface is constituted by an inner spherical surface centered on the focal position.
4. The optical analyzer according to claim 3, wherein the second surface is a spherical surface decentered toward the image side in the optical axis direction with respect to the first surface.
The optical analyzer according to claim 3, wherein the second surface is an ellipsoidal surface decentered toward the image side in the optical axis direction with respect to the first surface.
6. An accommodating portion that has an outer surface that is complementary to an inner surface of the concave portion provided in the objective lens of the optical analyzer according to claim 1, and that can accommodate the sample solution therein. With
A container for optical analysis in which a sample solution can be arranged at a position that coincides with a focal point of the objective lens when the accommodating portion is combined with the concave portion.
An optical analysis that collects the excitation light emitted from the light source and irradiates the sample solution arranged at the focal position, and also collects the light emitted from the luminescent substance existing at the focal position, dispersed in the sample solution. Lenses,
Provided with a tip optical member having a recess recessed on the image side on the most advanced surface,
The optical analysis lens in which the focal position is arranged on the forefront surface or in the concave portion on the image side.
The tip optical member has a first surface that constitutes the inner surface of the concave portion, on which light emitted from the light emitting material at the focal position is incident, and positive power that condenses the light incident from the first surface. The optical analysis lens according to claim 7, further comprising a second surface.
The optical analysis lens according to claim 8, wherein the first surface is constituted by an inner spherical surface centered on the focal position.
The optical analysis lens according to claim 9, wherein the second surface is a spherical surface that is decentered toward the image side in the optical axis direction with respect to the first surface.
The optical analysis lens according to claim 9, wherein the second surface is an ellipsoidal surface decentered toward the image side in the optical axis direction with respect to the first surface.