阅读说明：本技术 共聚焦显微镜单元和共聚焦显微镜 (Confocal microscope unit and confocal microscope ) 是由 山下慈郎 田边康行 松田俊辅 寺田浩敏 于 2020-03-26 设计创作，主要内容包括：实施方式的共聚焦显微镜单元(1A)包括：第1子单元(6a),其具有光源(10a)、针孔板(12a)、和光检测器(13a)；第2子单元(6b),其具有光源(10b)、针孔板(12b)、和光检测器(13b)；扫描镜(4),其使从第1和第2子单元(6a、6b)输出的激发光经由显微镜光学系统在试样(M)上扫描,并将根据激发光而从试样(M)产生且由显微镜光学系统成像的荧光向第1和第2子单元(6a、6b)引导；和主壳体(2),其构成为能够安装于连接端口(P1),且固定有扫描镜(4)、第1子单元(6a)、和第2子单元(6b),第1子单元(6a)和第2子单元(6b)以向扫描镜(4)的2个激发光的入射角错开规定的角度(θ-(S))的方式配置于主壳体(2)内。(A confocal microscope unit (1A) of an embodiment includes: a 1 st subunit (6a) having a light source (10a), a pinhole plate (12a), and a light detector (13 a); a 2 nd subunit (6b) having a light source (10b), a pinhole plate (12b), and a photodetector (13 b); a scanning mirror (4) that scans the excitation light output from the 1 st and 2 nd subunits (6a, 6b) on the sample (M) via the microscope optical system, and guides fluorescence generated from the sample (M) according to the excitation light and imaged by the microscope optical system to the 1 st and 2 nd subunits (6a, 6 b); and a main housing (2) which is configured to be attachable to the connection port (P1), and to which the scan mirror (4), the 1 st sub-unit (6a), and the 2 nd sub-unit (6b) are fixed, the 1 st sub-unit (6a), and the 2 nd sub-unit (6b)) The angle of incidence of the 2 excitation lights to the scanning mirror (4) is deviated by a predetermined angle (theta) S ) Is arranged in the main casing (2).)

1. A confocal microscope unit characterized in that,

is a confocal microscope unit which is mounted on a connection port of a microscope having a microscope optical system to constitute a confocal microscope,

the confocal microscope unit includes:

a 1 st subunit having a light source that outputs 1 st excitation light, a 1 st aperture member that restricts a light beam of 1 st fluorescence generated from a sample to be observed based on the 1 st excitation light, and a 1 st photodetector that detects the 1 st fluorescence that has passed through the 1 st aperture member;

a 2 nd subunit having a light source that outputs a 2 nd excitation light, a 2 nd aperture member that restricts a light beam of a 2 nd fluorescence generated from the sample according to the 2 nd excitation light, and a 2 nd photodetector that detects the 2 nd fluorescence that has passed through the 2 nd aperture member;

a scanning mirror that scans the excitation light output from the 1 st and 2 nd subunits over the sample via the microscope optical system and guides fluorescence generated from the sample according to the excitation light to the 1 st and 2 nd subunits; and

a main housing configured to be attachable to the connection port, and to which the scan mirror, the 1 st sub-unit, and the 2 nd sub-unit are fixed,

the 1 st subunit and the 2 nd subunit are disposed in the main casing such that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror.

2. The confocal microscope unit of claim 1,

the 1 st subunit has a 1 st optical mirror that reflects the 1 st excitation light and the 1 st fluorescence and transmits the 2 nd excitation light and the 2 nd fluorescence,

the 2 nd subunit has a 2 nd optic that reflects the 2 nd excitation light and the 2 nd fluorescence,

the 1 st optical mirror and the 2 nd optical mirror are disposed in the main housing such that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror.

3. The confocal microscope unit of claim 1,

the 1 st subunit has a 1 st optical mirror that reflects the 1 st excitation light and the 1 st fluorescence and transmits the 2 nd excitation light and the 2 nd fluorescence,

the 2 nd subunit has a 2 nd optic that reflects the 2 nd excitation light and the 2 nd fluorescence,

in the 1 st subunit and the 2 nd subunit, an incident angle of the 1 st excitation light to the 1 st optical mirror and an incident angle of the 2 nd excitation light to the 2 nd optical mirror are set so that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror.

4. Confocal microscope unit according to any one of claims 1 to 3,

the predetermined angle is an angle at which the 1 st excitation light airy disk and the 2 nd excitation light airy disk are separated from each other on the sample.

5. Confocal microscope unit according to any one of claims 1 to 4,

the scanning mirror is a MEMS mirror.

6. Confocal microscope unit according to any one of claims 1 to 5,

the 1 st sub-unit and the 2 nd sub-unit are fixed to the main casing in a state of being arranged in the order of the 1 st sub-unit and the 2 nd sub-unit along a light guiding direction based on the fluorescent light of the scanning mirror.

7. A confocal microscope, characterized in that,

the method comprises the following steps:

the confocal microscope unit of any one of claims 1 to 6; and

a microscope having a connection port to which the microscope optical system and the confocal microscope unit are mounted.

Technical Field

The present invention relates to a confocal microscope unit and a confocal microscope constituting a confocal microscope.

Background

A confocal microscope capable of obtaining an optical tomographic image of a specimen to be observed at a high resolution is known in the related art. For example, patent document 1 below discloses a microscope connection unit including a microscope connection port connected to a microscope, a stimulation unit that irradiates a specimen with light, an observation unit that detects light emitted from the specimen, and an optical path combining unit that combines optical paths optically connecting the microscope, the stimulation unit, and the observation unit. The microscope attachment unit has, in the same observation unit, an optical system for guiding light emitted from a plurality of light sources, a dichroic mirror for detecting fluorescence generated therefrom for each of a plurality of wavelengths, a confocal pinhole, and a photomultiplier tube. In such a configuration, by using excitation light of a plurality of wavelengths and detecting fluorescence generated in accordance therewith, imaging in a plurality of wavelength regions is realized by the same device.

As an apparatus for imaging a fluorescent sample using excitation light of different wavelengths, a laser scanner apparatus described in patent document 2 below is also known. According to this laser scanner device, 2 laser beams having different wavelengths and focused on a sample are spatially separated, and 2 light emission beams excited by these laser beams are also spatially separated and guided toward 2 detectors.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2011-90248

Patent document 2: japanese laid-open patent publication No. 2009-104136

Disclosure of Invention

Problems to be solved by the invention

Since the wavelength distribution of fluorescence emitted from a fluorescent substance is generally broad, when a sample containing a plurality of fluorescent substances is observed, the wavelength distribution of fluorescence emitted from each fluorescent substance may overlap. When fluorescence emitted from such a sample is simultaneously detected, there is a problem that not only fluorescence emitted from a target fluorescent substance but also fluorescence from another fluorescent substance is detected by the same detector. In general, such a problem is called infiltration (Bleed Through).

In the laser scanner device described in cited document 2, a plurality of fluorescent lights are spatially separated and detected by using a dichroic mirror having a wedge shape, thereby reducing the penetration, but it is difficult to apply the laser scanner device to a confocal microscope using a scanning mirror.

The embodiments have been made in view of such problems, and it is a technical problem to provide a confocal microscope unit that enables formation of a fluorescence image with less penetration in a plurality of wavelength regions by a simple structure.

Means for solving the problems

A confocal microscope unit according to an aspect of the present invention is a confocal microscope unit that is attached to a connection port of a microscope having a microscope optical system to constitute a confocal microscope, and includes: a 1 st subunit having a light source that outputs 1 st excitation light, a 1 st aperture member that restricts a light beam of 1 st fluorescence generated from a sample to be observed based on the 1 st excitation light, and a 1 st photodetector that detects the 1 st fluorescence that has passed through the 1 st aperture member; a 2 nd subunit having a light source that outputs a 2 nd excitation light, a 2 nd aperture member that restricts a light beam of a 2 nd fluorescence generated from the sample based on the 2 nd excitation light, and a 2 nd photodetector that detects the 2 nd fluorescence that has passed through the 2 nd aperture member; a scanning mirror that scans the excitation light output from the 1 st and 2 nd subunits over the sample via a microscope optical system and guides fluorescence generated from the sample according to the excitation light to the 1 st and 2 nd subunits; and a main housing configured to be attachable to the connection port, and to which the scanning mirror, the 1 st subunit, and the 2 nd subunit are fixed, the 1 st subunit and the 2 nd subunit being disposed within the main housing such that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror.

According to the above-described one aspect, the 1 st excitation light output from the 1 st subunit is scanned on the sample via the scanning mirror, the 1 st fluorescence generated from the sample by the scanning mirror enters the 1 st subunit via the scanning mirror, and the 1 st aperture member in the 1 st subunit forms an image thereof and is detected by the 1 st photodetector. In addition, the 2 nd excitation light output from the 2 nd subunit is scanned on the sample via the scanning mirror, the 2 nd fluorescence generated from the sample according to the scanning is incident into the 2 nd subunit via the scanning mirror, and the 2 nd aperture member in the 2 nd subunit forms an image thereof and is detected by the 2 nd photodetector. Here, since the scanning mirror and the 1 st and 2 nd sub-units are fixed to the main body case and the incident angles of the 1 st and 2 nd excitation lights from the 1 st and 2 nd sub-units to the scanning mirror are shifted from each other by a predetermined angle, the light spots of the 1 st and 2 nd excitation lights scanned on the sample can be separated, and as a result, the light beams of the 1 st and 2 nd fluorescence guided to the 1 st and 2 nd sub-units can be separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation. In addition, by adopting a configuration in which the sample is scanned by the scanning mirror, the entire apparatus configuration can be simplified.

Alternatively, another aspect of the present invention is a confocal microscope including the above-described confocal microscope unit and a microscope having a connection port to which the microscope optical system and the confocal microscope unit are attached. Such a confocal microscope can easily form a confocal image at a desired excitation wavelength and fluorescence wavelength.

ADVANTAGEOUS EFFECTS OF INVENTION

According to one aspect of the present invention, it is possible to realize fluorescence imaging using a plurality of fluorescence in a state where permeation is reduced with a simple configuration.

Drawings

Fig. 1 is a schematic configuration diagram of a confocal microscope 101 according to embodiment 1.

FIG. 2 is a graph showing a range S of Airy spots of a 1 st excitation light spot on a sample M formed by a confocal microscope 101₁And the range S of Airy spots of the spot of the 2 nd excitation light₂A graph of the distribution of (c).

Fig. 3 is a graph showing the one-dimensional light intensity distribution in the X axis direction of each of the 1 st excitation light and the 2 nd excitation light irradiated onto the sample M by the confocal microscope 101.

Fig. 4 is a schematic configuration diagram of a confocal microscope unit 1B according to embodiment 2.

Fig. 5 is a perspective view showing an example of a structure for moving the arrangement of the 3 rd and 4 th subunits 6c and 6d in the Y axis direction.

Fig. 6 is a diagram showing the trajectory of the beam of the excitation light beam incident on the scanning mirror 4 in the confocal microscope unit 1B.

Fig. 7 is a diagram showing a distribution of the airy disk range of the light spot of the excitation light on the sample M formed by the confocal microscope unit 1B.

Fig. 8 is a schematic configuration diagram of a confocal microscope unit 1C according to embodiment 3.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the description, the same elements or elements having the same function are denoted by the same reference numerals, and redundant description thereof will be omitted.

[ embodiment 1 ]

Fig. 1 is a schematic configuration diagram of a confocal microscope 101 according to embodiment 1. The confocal microscope 101 shown in fig. 1 is configured as a confocal microscope for acquiring a possible image of a structure of an optical tomographic image of a sample M to be observed, and the confocal microscope unit 1A is configured to be connected to a connection port P1 for connecting to an external unit of the microscope 50. The confocal microscope unit 1A according to embodiment 1 is a device that irradiates excitation light onto a sample M placed on a stage or the like of a microscope 50 via a microscope optical system such as an imaging lens 51 and an objective lens 52 in the microscope 50, and generates and outputs an optical tomographic image by imaging and receiving (detecting) fluorescence generated from the sample M by the excitation light via the microscope optical system of the microscope 50.

Specifically, the confocal microscope unit 1A includes a main housing 2, a lens barrel 3 constituting a part of the main housing 2 and detachably connected to a connection port P1 of the microscope 50, a scanning mirror 4 fixed in the main housing 2, a fixed mirror 5, 1 st to 2 nd sub-units 6a to 6b, and a scanning lens 7 fixed in the lens barrel 3. Hereinafter, each constituent element of the confocal microscope unit 1A will be described in detail.

The scanning lens 7 in the lens barrel 3 is an optical element having a function of forming a light spot on 1 st image plane of the microscope optical system of the microscope 50 while relaying the reflection surface of the scanning mirror 4 to the pupil position of the objective lens 52. Thereby, the exit pupils of the scanning mirror 4 and the objective lens 52 are in a conjugate relationship, i.e., an imaging relationship. The scanning lens 7 irradiates the sample M with the excitation light scanned by the scanning mirror 4 by guiding the excitation light to the microscope optical system, and guides the fluorescence generated from the sample M according to the irradiation to the scanning mirror 4.

The scanning mirror 4 in the main casing 2 is an optical scanning element such as a Micro Electro Mechanical System (MEMS) mirror having a reflection plate configured to be tiltable in two axes, for example. The scanning mirror 4 has a function of scanning the excitation light output from the 1 st to 2 nd subunits 6a to 6b on the sample M by continuously changing the reflection angle, and guiding the fluorescence generated by the excitation light to the 1 st to 2 nd subunits 6a to 6 b.

The fixed mirror 5 is a light reflecting element fixed in the main casing 2, and reflects the excitation light output from the 1 st to 2 nd sub-units 6a to 6b toward the scanning mirror 4, and reflects the fluorescence reflected by the scanning mirror 4 toward the 1 st to 2 nd sub-units 6a to 6b coaxially with the excitation light.

The 1 st subunit 6a has a substrate 8a, a dichroic mirror (1 st optical mirror) 9a disposed on the substrate 8a, a light source 10a, a collimator lens 15a, a dichroic mirror 11a, a blocking filter 16a, a condenser lens 17a, a pinhole plate (1 st aperture member) 12a, and a photodetector (1 st photodetector) 13 a.

The dichroic mirror 9a is fixed on the mirror 5 in the direction of reflection of the fluorescence and has a wavelength λ reflecting the light emitted from the 1 st subunit 6a₁And the wavelength range Δ λ of the 1 st excitation light and generated from the sample M based thereon₁And a beam splitter having a property of transmitting light having a longer wavelength than the 1 st excitation light and the 1 st fluorescence. The dichroic mirror 11a is disposed in the reflection direction of the 1 st fluorescence of the dichroic mirror 9a, and has a wavelength range of Δ λ₁The 1 st fluorescence of (2) is transmitted and the specific wavelength region is made to be DeltaLambda₁Short wavelength lambda₁1 excitation light reflecting property of the beam splitter. The light source 10a has an output wavelength λ₁The 1 st excitation light (e.g., laser light) emitting element (e.g., laser diode) of (1) is arranged such that the 1 st excitation light is reflected by the dichroic mirror 11a toward the dichroic mirror 9a coaxially with the 1 st fluorescence. The collimator lens 15a converts the 1 st excitation light output from the light source 10a into parallel light.

The blocking filter 16a is a filter member that is provided adjacent to the dichroic mirror 11a and cuts noise light other than the 1 st fluorescent light transmitted by the dichroic mirror 11 a. The condenser lens 17a condenses the 1 st fluorescence transmitted through the blocking filter 16a to the pinhole of the pinhole plate 12 a. The pinhole plate 12a is an aperture member that is disposed so that the position of the pinhole coincides with the conjugate position of the light spot of the 1 st excitation light of the sample M and limits the light flux of the 1 st fluorescence, and constitutes a confocal optical system together with the light source 10a and the like. The pinhole plate 12a can adjust the diameter of the pinhole from the outside, and can change the resolution of the image detected by the photodetector 13a and the signal intensity of the image.

The photodetector 13a is disposed so that its detection surface faces the pinhole plate 12a, and receives and detects the 1 st fluorescence passing through the pinhole plate 12 a. The photodetector 13a is a photomultiplier, a photodiode, an avalanche photodiode, an MPPC (Multi-Pixel Photon Counter), an HPD (Hybrid Photo Detector), an area image sensor, or the like.

The 2 nd subunit 6b has the same structural elements as the 1 st subunit 6 a.

That is, the 2 nd subunit 6b has a substrate 8b, a dichroic mirror (2 nd optical mirror) 9b, a light source 10b, a collimator lens 15b, a dichroic mirror 11b, a blocking filter 16b, a condenser lens 17b, a pinhole plate (2 nd aperture member) 12b, and a photodetector (2 nd photodetector) 13 b.

Color separationThe mirror 9b has a wavelength λ reflecting the radiation of the sub-unit 2b₂(＞λ₁) And the wavelength range Δ λ of the 2 nd excitation light and generated from the sample M based thereon₂And (3) the 2 nd fluorescence of (2), and a property of transmitting light having a longer wavelength than the 2 nd excitation light and the 2 nd fluorescence. The dichroic mirror 9b may be replaced with a simple mirror having no wavelength selectivity. The dichroic mirror 11b has a wavelength range of Δ λ₂The 2 nd fluorescence of (2) is transmitted and reflected within a specific wavelength range DeltaLambda₂Short wavelength lambda₂Property of the 2 nd excitation light. The light source 10b has an output wavelength λ₂The light-emitting element of the 2 nd excitation light of (1). The collimator lens 15b converts the 2 nd excitation light output from the light source 10b into parallel light.

The blocking filter 16b is a filter member that is provided adjacent to the dichroic mirror 11b and cuts noise light other than the 2 nd fluorescence. The condenser lens 17b condenses the 2 nd fluorescent light transmitted through the blocking filter 16b to the pinhole of the pinhole plate 12 b. The pinhole plate 12b is an aperture member that restricts the luminous flux of the 2 nd fluorescence, and the position of the pinhole coincides with the conjugate position of the spot of the 2 nd excitation light of the sample M.

The photodetector 13b is disposed so that its detection surface faces the pinhole plate 12b, and receives and detects the 2 nd fluorescence passing through the pinhole plate 12 b. The photodetector 13b is a photomultiplier, a photodiode, an avalanche photodiode, an MPPC (Multi-Pixel Photon Counter), an HPD (Hybrid Photo Detector), an area image sensor, or the like.

The arrangement of the 1 st to 2 nd subunits 6a to 6b having the above-described configuration will be described. In the following description, a direction along the optical axis of the scanning lens 7 is taken as a Z-axis, a direction perpendicular to the Z-axis and along the substrates 8a, 8b of the subunits 6a, 6b is taken as an X-axis, and a direction perpendicular to the Z-axis and the X-axis is taken as a Y-axis.

The 1 st and 2 nd sub-units 6a, 6b are fixed to the substrate 14 constituting the main body casing 2 so as to be arranged in this order in a direction away from the fixed mirror 5 along the light guiding direction (Z-axis direction) of the 1 st and 2 nd fluorescent lights by the scanning mirror 4 and the fixed mirror 5 and so as to have the dichroic mirrors 9a, 9b positioned on the optical paths of the 1 st and 2 nd fluorescent lights. In detail, the 2 nd subunit 6b is configured as a phaseThe 1 st subunit 6a is moved (shift) in a direction (X-axis direction) substantially perpendicular to the light guiding direction of the 1 st fluorescent light with reference to the center positions of the dichroic mirrors 9a, 9 b. The dichroic mirror 9a is set at an installation angle such that the reflected 1 st excitation light enters the center of the reflection surface of the scanning mirror 4 via the fixed mirror 5. Similarly, the dichroic mirror 9b is set at a setting angle such that the reflected 2 nd excitation light enters the center of the reflecting surface of the scanning mirror 4 via the dichroic mirror 9a and the fixed mirror 5. With this arrangement, the incident angle of the 1 st excitation light to the scanning mirror 4 is set to be shifted by a predetermined angle θ with respect to the incident angle of the 2 nd excitation light to the scanning mirror 4_S。

The amount of movement in the X-axis direction of the 1 st and 2 nd subunits 6a and 6b and the installation angles of the 2 dichroic mirrors 9a and 9b described above are set so as to satisfy the following conditions. Namely, the above-mentioned predetermined angle θ_SThe angle is set so that the airy disk of the light spot of the 1 st excitation light incident on the sample M does not overlap with the airy disk of the light spot of the 2 nd excitation light incident on the sample M. In general, the diameter Φ of the airy disk is calculated by the following equation (1) assuming that the wavelength of the incident excitation light is λ and the numerical aperture of the objective lens 52 is NA.

φ＝1.22×λ/NA…(1)

Therefore, the minimum value Δ d of the shift Δ d between the light spots of the 1 st and 2 nd excitation lights on the sample M for making the light spots of the 2 excitation lights not overlap_minCan be calculated by the following formula (2).

Δd_min＝φ₁/2+φ₂/2，

φ₁＝1.22×λ₁/NA，

φ₂＝1.22×λ₂/NA…(2)。

Here, since the scanning mirror 4 and the exit pupil of the objective lens 52 are in a conjugate relationship, the light beams of the 1 st and 2 nd excitation lights incident on the center of the reflecting surface of the scanning mirror 4 are incident on the center of the exit pupil of the objective lens 52. At this time, the incident angle to the objective lens 52 changes according to the imaging magnification of the scanning lens 7. Since the objective lens 52 is a telecentric lens, the principal rays of the 1 st and 2 nd excitation lights are aligned with the optical axis of the objective lens 52 on the sample MIn the parallel direction (Z-axis direction), the positions of the light spots of the 2 excitation lights are shifted on the sample M according to the incident angle to the exit pupil. In order to prevent the light spots of the 2 excitation lights from overlapping on the sample M, the angle θ of the shift of the incident angle to the scanning mirror 4 is set to be different_SThe angle theta calculated by the following formula (3) is set_SminThe above.

θ_Smin＝arctan(Δd_min/f)/mags…(3)

Here, mags is the magnification of the scanning lens 7, and f is the focal length of the objective lens 52.

The diameters of the pinholes of the pinhole plates 12a and 12b of the subunits 6a and 6b are set to be larger than the Airy Unit (AU) calculated by the following expressions (4) and (5)₁、AU₂) The value of (c) is small. By setting in this way, crosstalk between wavelengths in a fluorescence image can be effectively prevented.

AU₁＝mag_t×φ₁…(4)

AU₂＝mag_t×φ₂…(5)

Wherein mag_tThe total magnification of the optical system for observing fluorescence is shown.

FIG. 2 shows a range S of Airy spots of a light spot of the 1 st excitation light on the specimen M formed by the confocal microscope 101₁And the range S of Airy spots of the spot of the 2 nd excitation light₂Fig. 3 shows the light intensity distribution of each of the 1 st excitation light and the 2 nd excitation light irradiated onto the sample M by the confocal microscope 101 in one dimension along the X-axis direction. Thus, the range S of the light spot of the 1 st and 2 nd excitation lights formed on the sample M is adjusted₁、S₂The intensity distributions of the 2 excitation lights on the sample M are separated without overlapping each other, and as a result, the interference between the 2 wavelength bands of fluorescence images generated by the 2 excitation lights can be reduced.

According to the confocal microscope unit 1A described above, the 1 st excitation light output from the 1 st subunit 6a is scanned on the sample M via the scanning mirror 4, the 1 st fluorescence generated from the sample M according to the scanning mirror 4 enters the 1 st subunit 6a via the scanning mirror 4, and the image thereof is formed on the pinhole plate 12a in the 1 st subunit 6a, and the image is displayed on the pinhole plate 12aDetected by the photodetector 13 a. In addition, the 2 nd excitation light output from the 2 nd subunit 6b is scanned on the sample M via the scanning mirror 4, the 2 nd fluorescence generated from the sample M according to the scanning is incident into the 2 nd subunit 6b via the scanning mirror 4, and the pinhole plate 12b in the 2 nd subunit 6b forms an image thereof and is detected by the photodetector 13 b. Here, the scanning mirror 4 and the 1 st and 2 nd sub-units 6a and 6b are fixed to the main casing 2, and the incident angles of the 1 st and 2 nd excitation lights to the scanning mirror 4 from the 1 st and 2 nd sub-units 6a and 6b are shifted from each other by a predetermined angle θ_STherefore, the light spots of the 1 st and 2 nd excitation lights scanned on the sample M can be separated, and as a result, the light beams of the 1 st and 2 nd fluorescence guided to the 1 st and 2 nd sub-units 6a, 6b can be separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation. In addition, by adopting a configuration in which the sample M is scanned by the scanning mirror 4, the overall apparatus configuration can be simplified.

Alternatively, the confocal microscope 101 is a confocal microscope including the confocal microscope unit 1A and the microscope 50 having the microscope optical system and the connection port P1 to which the confocal microscope unit 1A is attached. According to such a confocal microscope 101, confocal imaging can be easily performed using the microscope 50 which is a general optical microscope.

In the confocal microscope unit 1A according to the above-described embodiment, the dichroic mirrors 9a and 9b are shifted by a predetermined angle θ from the incident angles of the 1 st and 2 nd excitation lights with respect to the scanning mirror 4_SIs disposed in the main casing 2. With such a configuration, the light spots of the 1 st and 2 nd excitation lights scanned on the sample M can be separated, and as a result, the 1 st and 2 nd fluorescence light beams guided to the 1 st and 2 nd sub-units 6a, 6b can be separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation.

In addition, in the confocal microscope unit 1A, a predetermined angle θ_SThe angle is set so that the 1 st excitation light airy disk and the 2 nd excitation light airy disk are separated from each other on the sample M. By setting such an angle, the light spots of the 1 st and 2 nd excitation lights scanned on the sample M can be completely separated. As a result, the 1 st and 2 nd fluorescent light beams guided to the 1 st and 2 nd subunits 6a, 6b can be completely separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation.

In the above embodiment, the scanning mirror 4 employs a MEMS mirror. With such a configuration, the cell can be easily miniaturized.

In the confocal microscope unit 1A, the 1 st subunit 6a and the 2 nd subunit 6b are fixed to the main casing 2 in a state of being arranged in order along the light guiding direction based on the fluorescence of the scanning mirror 4. With this configuration, the 2 nd excitation light emitted from the 2 nd subunit 6b can be emitted toward the sample M on the microscope 50 side via the 1 st subunit 6a, and the 2 nd fluorescence generated from the sample M according to the emission can be introduced into the 2 nd subunit 6b via the 1 st subunit 6 a. As a result, the unit that can realize confocal imaging in a plurality of wavelength regions can be miniaturized.

[ 2 nd embodiment ]

Fig. 4 is a schematic configuration diagram of a confocal microscope unit 1B according to embodiment 2. The confocal microscope unit 1B shown in fig. 4 is different from the confocal microscope unit 1A of embodiment 1 in that it has 4 subunits. Hereinafter, the structure of the confocal microscope unit 1B will be described focusing on differences from embodiment 1.

That is, the confocal microscope unit 1B includes 1 st to 4 th sub-units 6a to 6d in the main casing 2.

The 3 rd subunit 6c has a substrate 8c, a dichroic mirror (3 rd optical mirror) 9c, a light source 10c, a collimator lens 15c, a dichroic mirror 11c, a blocking filter 16c, a condenser lens 17c, a pinhole plate (3 rd aperture member) 12c, and a photodetector (3 rd photodetector) 13 c.

The dichroic mirror 9c has a wavelength λ reflecting the illumination of the 3 rd subunit 6c₃(＞λ₂) And the 3 rd excitation light and the wavelength range Δ λ generated from the sample M based thereon₃And (3) and transmitting light having a longer wavelength than the 3 rd excitation light and the 3 rd fluorescence. The dichroic mirror 11c has a wavelength range of Δ λ₃The 3 rd fluorescence of (2) is transmitted and reflected within a specific wavelength range DeltaLambda₃Short wavelength lambda₃Property of the 3 rd excitation light. The light source 10c has an output wavelength λ₃The light-emitting element of the 3 rd excitation light. The collimator lens 15c converts the 3 rd excitation light output from the light source 10c into parallel light.

The blocking filter 16c is a filter member that is provided adjacent to the dichroic mirror 11c and blocks noise light other than the 3 rd fluorescent light. The condenser lens 17c condenses the 3 rd fluorescent light transmitted through the blocking filter 16c to the pinhole of the pinhole plate 12 c. The pinhole plate 12c is an aperture member that restricts the 3 rd fluorescence beam, with the pinhole position thereof being aligned with the conjugate position of the 3 rd excitation light spot of the sample M.

The photodetector 13c is disposed so that its detection surface faces the pinhole plate 12c, and receives and detects the 3 rd fluorescence that has passed through the pinhole plate 12 c. The photodetector 13c is a photomultiplier, a photodiode, an avalanche photodiode, an MPPC (Multi-Pixel Photon Counter), an HPD (Hybrid Photo Detector), an area image sensor, or the like.

The 4 th subunit 6d has a substrate 8d, a total reflection mirror (4 th optical mirror) 9d, a light source 10d, a collimator lens 15d, a dichroic mirror 11d, a blocking filter 16d, a condenser lens 17d, a pinhole plate (4 th aperture member) 12d, and a photodetector (4 th photodetector) 13 d.

The total reflection mirror 9d reflects the wavelength λ irradiated by the 4 th subunit 6d₄(＞λ₃) And the wavelength range Δ λ generated from the sample M according to the 4 th excitation light of (1)₄Fluorescence of (4). The dichroic mirror 11d has a wavelength range DeltaLambda₄The 4 th fluorescence of (2) is transmitted and reflected within a specific wavelength range DeltaLambda₄Short wavelength lambda₄The property of the 4 th excitation light. Light source 10d is of output wavelength λ₄The light-emitting element of the 4 th excitation light of (1). The collimator lens 15d converts the 4 th excitation light output from the light source 10d into parallel light.

The blocking filter 16d is a filter member that is provided adjacent to the dichroic mirror 11d and cuts noise light other than the 4 th fluorescence. The condenser lens 17d condenses the 4 th fluorescent light transmitted through the blocking filter 16d to the pinhole of the pinhole plate 12 d. The pinhole plate 12d is an aperture member that restricts the luminous flux of the 4 th fluorescence, and the position of the pinhole coincides with the conjugate position of the luminous spot of the 4 th excitation light of the sample M.

The photodetector 13d is disposed so that its detection surface faces the pinhole plate 12d, and receives and detects the 4 th fluorescence that has passed through the pinhole plate 12 d. The photodetector 13d is a photomultiplier, a photodiode, an avalanche photodiode, an MPPC (Multi-Pixel Photon Counter), an HPD (Hybrid Photo Detector), an area image sensor, or the like.

The 1 st to 4 th subunits 6a to 6d are fixed in the main casing 2 so as to be arranged in the direction away from the fixed mirror 5 in this order along the light guiding direction (Z-axis direction) of the 1 st to 4 th fluorescent lights by the scanning mirror 4 and the fixed mirror 5, and so as to have the dichroic mirrors 9a to 9c and the total reflection mirror 9d positioned on the optical paths of the 1 st to 4 th fluorescent lights. Specifically, the 4 th subunit 6d is disposed to move in the X-axis direction with respect to the 3 rd subunit 6c with reference to the center positions of the dichroic mirror 9c and the total reflection mirror 9 d. The 3 rd and 4 th sub-units 6c and 6d are also arranged to move in the Y-axis direction with respect to the 1 st and 2 nd sub-units 6a and 6b with reference to the center positions of the dichroic mirrors 9a to 9c and the total reflection mirror 9 d. The dichroic mirrors 9a to 9c and the total reflection mirror 9d are set at respective installation angles so that the 1 st to 4 th excitation lights reflected by the dichroic mirrors enter the center of the reflection surface of the scanning mirror 4. With this arrangement, the incident angles of 2 fluorescent lights incident on the scanning mirror 4 from the adjacent 2 sub-units are set to be shifted from each other by a predetermined angle θ_SThe above.

Fig. 5 is a perspective view of a structure for moving the arrangement of the 3 rd and 4 th subunits 6c, 6d in the Y axis direction as viewed from the lens barrel 3 side. In this way, in the present embodiment, the flat plate-like spacer 21 is disposed on the substrate 14 of the main casing 2, and the 3 rd and 4 th sub-units 6c and 6d are mounted on the spacer 21. With this configuration, the 3 rd and 4 th sub-units 6c and 6d can be arranged to be moved in the Y-axis direction by a desired width with respect to the 1 st and 2 nd sub-units 6a and 6 b.

FIG. 6 shows a light beam B of the 1 st to 4 th excitation light beams incident on the scanning mirror 4 from the 1 st to 4 th sub-units 6a to 6d in the confocal microscope unit 1B₁～B₄FIG. 7 shows the range S of Airy spots of the light spots of the 1 st to 4 th excitation lights on the sample M formed by the confocal microscope unit 1B₁～S₄Distribution of (2). In FIG. 6, a solid line represents a light flux B of each excitation light flux₁～B₄The outer edge of (A) indicates the beam B of each excitation light beam by a chain line₁～B₄Center BC of₁～BC₄. According to the confocal microscope unit 1B, the 1 st to 4 th excitation light fluxes are set to enter the center of the incident surface RF of the scanning mirror 4, and the 1 st to 4 th excitation light fluxes are set such that the incident angles are displaced from each other in the two-dimensional direction. Further, the incident angles are shifted by a predetermined angle θ_SThus, the range S of the Airy spots of the light spots of the 1 st to 4 th excitation lights formed on the sample M is set₁～S₄Do not overlap each other.

According to the confocal microscope unit 1B of embodiment 2 described above, the light spots of the 1 st to 4 th excitation lights scanned on the sample M can be separated, and as a result, the light fluxes of the 1 st to 4 th fluorescence guided to the 1 st to 4 th sub-units 6a to 6d can be separated from each other. Thus, even if 4 kinds of excitation wavelengths are used, the fluorescence image can be observed with reduced penetration.

[ embodiment 3 ]

Fig. 8 is a schematic configuration diagram of a confocal microscope unit 1C according to embodiment 3. The confocal microscope unit 1C shown in fig. 8 is different from the confocal microscope unit 1A of embodiment 1 in that the dichroic mirrors 9a and 9b are arranged in the same manner on the substrates 8a and 8b of the 2 sub-units 6a and 6 b. Hereinafter, the structure of the confocal microscope unit 1C will be described focusing on differences from embodiment 1.

The arrangement of the elements other than the dichroic mirror 9a on the substrate 8a in the 1 st subunit 6a, that is, the elements including the light source 10a, the collimating lens 15a, the dichroic mirror 11a, the blocking filter 16a, the condensing lens 17a, the pinhole plate 12a, and the photodetector 13a with reference to the dichroic mirror 9a is adjusted to be different from the arrangement of the elements other than the dichroic mirror 9b on the substrate 8b in the 2 nd subunit 6b, that is, the elements including the light source 10b, the collimating lens 15b, the dichroic mirror 11b, the blocking filter 16b, the condensing lens 17b, the pinhole plate 12b, and the photodetector 13b with reference to the dichroic mirror 9 b. This adjustment is achieved, for example, by allowing elements other than the dichroic mirror 9a in the 1 st sub-unit 6a or elements other than the dichroic mirror 9b in the 2 nd sub-unit 6b to change their postures and positions integrally on the substrates 8a and 8 b.

More specifically, with the above-described configuration of the 1 st and 2 nd subunits 6a and 6b, the incident angle/incident position of the 1 st excitation light with respect to the dichroic mirror 9a in the 1 st subunit 6a and the incident angle/incident position of the 2 nd excitation light with respect to the dichroic mirror 9b in the 2 nd subunit 6b are set so that the incident angle of the 1 st excitation light to the scanning mirror 4 and the incident angle of the 2 nd excitation light to the scanning mirror 4 are shifted by a predetermined angle θ_S。

According to the confocal microscope unit 1C according to embodiment 2 described above, by setting the incident angle of the excitation light with respect to the 1 st and 2 nd dichroic mirrors 9a and 9b in the 1 st and 2 nd sub-units 6a and 6b, the light spots of the 1 st and 2 nd excitation lights scanned on the sample M can be separated, and as a result, the 1 st and 2 nd fluorescence light beams guided to the 1 st and 2 nd sub-units 6a and 6b can be separated from each other. This enables observation of a fluorescence image of 2 fluorescence wavelengths with reduced penetration.

While various embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and may be modified or applied to other embodiments within a scope not changing the gist described in the claims.

In the above-described embodiments, the pinhole plate is used as the aperture member to constitute the confocal optical system, but the aperture member may be any optical element as long as it restricts a light flux, and may be, for example, a color aperture or an optical fiber core. When a fiber output type light source is used, the position of the end face of the optical fiber core may be set as the aperture position (the position where the light flux is restricted).

In the above embodiment, a laser light source such as a solid laser or a diode laser can be used. In this case, the position of the beam waist of the laser light source may be set as a diaphragm position (a position where the beam is limited), and the light source itself functions as a diaphragm member.

In the above-described embodiments 1 to 3, the plurality of subunits are arranged in the direction away from the scanning mirror 4 side in the order of short wavelength ranges of the excitation light and the fluorescence to be processed, but may be arranged in the order of long wavelength ranges. However, in this case, the dichroic mirrors 9a to 9c have characteristics of reflecting the excitation light and fluorescence of longer wavelengths processed by the respective subunits 6a to 6c and transmitting the excitation light and fluorescence of shorter wavelengths processed by the other subunits.

In the above-described embodiment 2, the light spots of the excitation light formed on the sample M do not overlap between 2 of all the sub-cells 6a to 6d, but the light spots may not overlap between adjacent sub-cells. For example, the light spots of the excitation light may not overlap between the 1 st subunit 6a and the 2 nd subunit 6b, between the 2 nd subunit 6b and the 3 rd subunit 6c, and between the 3 rd subunit 6c and the 4 th subunit 6 d. In contrast, the spot of excitation light may overlap between the 1 st subunit 6a and the 3 rd subunit 6c or between the 2 nd subunit 6b and the 4 th subunit 6 d. In this case, the fluorescence of the observation target of one subunit and the fluorescence of the observation target of the other subunit can be easily separated by the dichroic mirror. As a specific configuration, in the confocal microscope unit 1B according to embodiment 2, the 3 rd and 4 th subunits 6c and 6d may be arranged so as not to be shifted in the Y axis direction with respect to the 1 st and 2 nd subunits 6a and 6B. With this configuration, the light spots of the 2 excitation lights close in wavelength range can be separated on the sample M, and therefore, the detected fluorescent light beams in the plurality of wavelength ranges can be prevented from affecting each other, and a plurality of fluorescent images can be observed with reduced penetration.

In the above embodiment, the 1 st subunit has a 1 st optical mirror that reflects the 1 st excitation light and the 1 st fluorescence and transmits the 2 nd excitation light and the 2 nd fluorescence, the 2 nd subunit has a 2 nd optical mirror that reflects the 2 nd excitation light and the 2 nd fluorescence, and the 1 st optical mirror and the 2 nd optical mirror are disposed in the main casing so that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror. Thus, by setting the arrangement of the 1 st and 2 nd optical mirrors in the main casing, the light spots of the 1 st and 2 nd excitation lights scanned on the sample can be separated, and as a result, the light beams of the 1 st and 2 nd fluorescence guided to the 1 st and 2 nd sub-units can be separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation.

In addition, the 1 st subunit may include a 1 st optical mirror that reflects the 1 st excitation light and the 1 st fluorescence and transmits the 2 nd excitation light and the 2 nd fluorescence, the 2 nd subunit may include a 2 nd optical mirror that reflects the 2 nd excitation light and the 2 nd fluorescence, and an incident angle of the 1 st excitation light to the 1 st optical mirror and an incident angle of the 2 nd excitation light to the 2 nd optical mirror in the 1 st subunit and the 2 nd subunit may be set so that an incident angle of the 1 st excitation light to the scanning mirror is shifted by a predetermined angle from an incident angle of the 2 nd excitation light to the scanning mirror. In this case, by setting the incident angle of the excitation light with respect to the 1 st and 2 nd optical mirrors in the 1 st and 2 nd sub-units, the light spots of the 1 st and 2 nd excitation lights scanned on the sample can be separated, and as a result, the light beams of the 1 st and 2 nd fluorescence guided to the 1 st and 2 nd sub-units can be separated from each other. This enables observation of a plurality of fluorescence images with reduced permeation.

The predetermined angle is preferably an angle at which the 1 st excitation light airy disk and the 2 nd excitation light airy disk are separated from each other on the sample. By setting such an angle, the light spots of the 1 st and 2 nd excitation lights scanned on the sample can be completely separated, and as a result, the light beams of the 1 st and 2 nd fluorescence guided to the 1 st and 2 nd sub-units can be completely separated from each other. This enables observation of a plurality of fluorescence images without permeation.

In the above embodiments, the scanning mirror may also be a MEMS mirror. In this case, the device can be easily reduced in size.

Further, the 1 st and 2 nd sub-units may be fixed to the main casing in a state of being arranged in the order of the 1 st and 2 nd sub-units along the light guiding direction based on the fluorescence of the scanning mirror. According to this configuration, the 2 nd excitation light emitted from the 2 nd subunit can be emitted toward the sample on the microscope side via the 1 st subunit, and the 2 nd fluorescence generated by this can be introduced into the 2 nd subunit via the 1 st subunit. As a result, the device can be miniaturized in which images are formed in a plurality of wavelength regions.

Industrial applicability of the invention

The embodiment uses a confocal microscope unit and a confocal microscope constituting a confocal microscope as usage purposes, and can realize fluorescence imaging using a plurality of fluorescence in a state of reduced penetration with a simple configuration.

Description of the symbols

M … test specimen, P1 … connection Port, θ_S…, 10 a-10 d … light source, 12 a-12 d … pinhole plate (diaphragm component), 13 a-13 d … photodetector, 6 a-6 d … 1 st-4 th subunit, 9 a-9C … dichroic mirror (1 st-3 rd optical mirror), 9d … total reflection mirror (4 th optical mirror), 1A, 1B, 1C … confocal microscope unit, 2 … main casing, 3 … lens cone, 4 … scanning mirror, 7 … scanning lens, 50 … microscope, 101 … confocal microscope.