Light sheet microscope and sample observation method
阅读说明:本技术 光片显微镜以及试样观察方法 (Light sheet microscope and sample observation method ) 是由 泷口优 田中瑠美花 高本尚宜 于 2018-12-26 设计创作,主要内容包括:光片显微镜包括照射光学系统、检测光学系统和光检测器。照射光学系统具有:作为激发光输出波长随时间变化的光的波长扫描光源;分光元件,其入射从波长扫描光源输出的激发光,并且以与激发光的波长对应的出射角出射激发光;包含柱面透镜的中继光学系统,其中,从分光元件出射的激发光以与出射角对应的入射角入射于柱面透镜;和第一物镜,其使由中继光学系统引导来的激发光会聚,并使片状的激发光照射于试样。(The light sheet microscope includes an illumination optical system, a detection optical system, and a photodetector. The irradiation optical system includes: a wavelength-scanning light source that outputs light with a wavelength varying with time as excitation light; a spectroscopic element that enters the excitation light output from the wavelength-scanning light source and emits the excitation light at an emission angle corresponding to the wavelength of the excitation light; a relay optical system including a cylindrical lens on which excitation light emitted from the light splitting element is incident at an incident angle corresponding to the emission angle; and a first objective lens that condenses the excitation light guided by the relay optical system and irradiates the sample with the sheet-like excitation light.)
1. A light sheet microscope, comprising:
an irradiation optical system that irradiates an excitation light having a wavelength capable of exciting a sample to the sample;
a detection optical system that guides detection light emitted from the sample in association with irradiation of the excitation light; and
a light detector that detects the detection light guided by the detection optical system,
the illumination optical system has:
a wavelength-scanning light source as light whose excitation light output wavelength varies with time;
a spectroscopic element that enters the excitation light output from the wavelength-scanning light source and emits the excitation light at an emission angle corresponding to a wavelength of the excitation light;
a relay optical system including a cylindrical lens on which the excitation light emitted from the light splitting element is incident at an incident angle corresponding to the emission angle; and
and a first objective lens that focuses the excitation light guided by the relay optical system and irradiates the sample with the excitation light in a sheet form.
2. The light sheet microscope of claim 1, wherein:
the light splitting element is a diffraction grating.
3. The light sheet microscope of claim 1, wherein:
the light splitting element is a prism.
4. A light sheet microscope as claimed in any one of claims 1 to 3 wherein:
the detection optical system has: a second objective lens that receives the detection light; and a focus position adjuster that changes a focus position of the second objective lens in synchronization with a change in wavelength of the excitation light output from the wavelength-scanning light source.
5. The light sheet microscope of claim 4, wherein:
the focus position adjuster is a liquid lens.
6. A light sheet microscope as claimed in any one of claims 1 to 5 wherein:
the wavelength-scanning light source is configured to be capable of switching the center wavelength of the excitation light between a plurality of center wavelengths,
the spectroscopic element is rotatable according to the center wavelength of the excitation light output from the wavelength scanning light source.
7. A light sheet microscope as claimed in any one of claims 1 to 5 wherein:
the illumination optical system has a plurality of the wavelength-scanning light sources,
the plurality of wavelength-scanning light sources output light having center wavelengths different from each other as the excitation light,
the excitation light output from the plurality of wavelength-scanning light sources is incident on the spectroscopic element at an incident angle corresponding to the center wavelength thereof.
8. A light sheet microscope as claimed in any one of claims 1 to 7 wherein:
further comprising a moving mechanism that maintains a positional relationship between the light splitting element and the cylindrical lens, and moves the wavelength scanning light source, the light splitting element, and the cylindrical lens along an optical axis of the cylindrical lens.
9. A light sheet microscope as claimed in any one of claims 1 to 8 wherein:
the optical device further includes a reflecting unit that reflects the excitation light emitted from the first objective lens toward the sample.
10. The light sheet microscope of claim 9, wherein:
further comprising a container capable of holding the sample,
the reflecting portion is provided to the container.
11. A light sheet microscope as claimed in any one of claims 1 to 10 wherein:
the detection optical system has a second objective lens that enters the detection light,
the optical axis of the second objective lens is parallel to the optical axis of the first objective lens.
12. A sample observation method characterized by comprising:
irradiating the sample with excitation light having a wavelength capable of exciting the sample;
guiding detection light emitted from the sample in association with irradiation of the excitation light; and
a step of detecting the detection light, wherein,
the step of irradiating the sample with the excitation light includes:
a step of outputting light whose wavelength changes with time from a wavelength-scanning light source as the excitation light;
a step of causing the excitation light output from the wavelength-scanning light source to enter a spectroscopic element and causing the excitation light to exit the spectroscopic element at an exit angle corresponding to a wavelength of the excitation light;
causing the excitation light emitted from the spectroscopic element to enter a cylindrical lens at an incident angle corresponding to the emission angle; and
and converging the excitation light guided by a relay optical system including the cylindrical lens, and irradiating the sample with the excitation light in a sheet form.
Technical Field
One aspect of the present disclosure relates to a light sheet microscope and a specimen observation method.
Background
There is known a light sheet microscope which irradiates a specimen with excitation light in a sheet form and detects detection light emitted from the specimen with the irradiation of the excitation light (for example, see patent document 1). In this light sheet microscope, a container containing a sample is held by a holder, and at the time of observation of the sample, scanning of the irradiation position of the sample by excitation light is performed by moving or rotating the holder.
Disclosure of Invention
Technical problem to be solved by the invention
However, in the above-described light sheet microscope, there is a risk that the specimen may be shaken during observation, and the observation may not be stably performed. In addition, since the scanning speed of the excitation light is determined by the moving speed of the holder, it is difficult to increase the speed of observation. Although it is conceivable to dispose the sample on the stage and move or rotate the stage, there is a similar technical problem to the case of moving or rotating the holder from the viewpoint of stability and high speed.
An object of one aspect of the present disclosure is to provide a light sheet microscope and a sample observation method that can realize high-speed and stable observation.
Means for solving the problems
A light sheet microscope according to one aspect of the present disclosure includes: an irradiation optical system that irradiates the sample with excitation light having a wavelength capable of exciting the sample; a detection optical system that guides detection light emitted from the sample in association with irradiation of the excitation light; and a photodetector that detects the detection light guided by the detection optical system, the irradiation optical system having: a wavelength-scanning light source that outputs light with a wavelength varying with time as excitation light; a spectroscopic element that enters the excitation light output from the wavelength-scanning light source and emits the excitation light at an emission angle corresponding to the wavelength of the excitation light; a relay optical system including a cylindrical lens on which excitation light emitted from the light splitting element is incident at an incident angle corresponding to the emission angle; and a first objective lens that condenses the excitation light guided by the relay optical system and irradiates the sample with the sheet-like excitation light.
In this optical sheet microscope, light whose wavelength changes with time is output as excitation light from a wavelength-scanning light source, the excitation light output from the wavelength-scanning light source is output from a spectroscopic element at an output angle corresponding to the wavelength, and the excitation light output from the spectroscopic element is incident on a cylindrical lens at an incident angle corresponding to the output angle. This enables scanning of the sample at the irradiation position of the sheet-like excitation light. As a result, since it is not necessary to move or rotate the sample during observation, the sample can be stably observed while suppressing shaking of the sample. Further, since the irradiation position of the excitation light on the sample is scanned using the wavelength scanning light source, the spectroscopic element, and the cylindrical lens, the scanning of the excitation light on the sample can be speeded up. Therefore, according to the light sheet microscope, high-speed and stable observation can be realized.
In the light sheet microscope according to one aspect of the present disclosure, the light splitting element may be a diffraction grating or a prism. According to these configurations, the above-described operational effects that enable high-speed and stable observation are remarkably exhibited.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the detection optical system includes: a second objective lens for incidence of the detection light; and a focus position adjuster that changes a focus position of the second objective lens in synchronization with a change in wavelength of the excitation light output from the wavelength scanning light source. With this configuration, even when the irradiation position of the sample with the excitation light is scanned at a high speed, the detection light can be detected with high accuracy.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the focus position adjuster is a liquid lens. According to this configuration, the above-described operational effect of being able to detect the detection light with high accuracy is remarkably exhibited even in the case where the irradiation position of the sample with the excitation light is scanned at high speed.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the wavelength-scanning light source is configured to be capable of switching the center wavelength of the excitation light between a plurality of center wavelengths, and the spectroscopic element is capable of rotating in accordance with the center wavelength of the excitation light output from the wavelength-scanning light source. According to this configuration, excitation light of a plurality of center wavelengths can be irradiated to the sample.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the irradiation optical system has a plurality of wavelength scanning light sources that output light having center wavelengths different from each other as excitation light, and the excitation light output from the plurality of wavelength scanning light sources is incident on the spectroscopic element at an incident angle corresponding to the center wavelength thereof. According to this configuration, excitation light of a plurality of center wavelengths can be irradiated to the sample.
A light sheet microscope to which one aspect of the present disclosure relates may be: the optical scanning device further includes a moving mechanism that maintains a positional relationship between the spectroscopic element and the cylindrical lens and moves the wavelength scanning light source, the spectroscopic element, and the cylindrical lens along an optical axis of the cylindrical lens. According to this configuration, the position of the beam waist of the excitation light emitted from the first objective lens, that is, the formation position of the sheet-like excitation light in the irradiation direction of the excitation light to the sample can be adjusted.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the optical element further includes a reflecting section for reflecting the excitation light emitted from the first objective lens toward the sample. In addition, in the light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the sample processing apparatus further comprises a container in which the sample can be placed, and the container is provided with a reflecting portion. According to this configuration, the sheet-like excitation light can be irradiated to the sample without being affected by the wall portion of the container.
In a light sheet microscope according to an aspect of the present disclosure, the light sheet microscope may be: the detection optical system has a second objective lens for incidence of the detection light, and an optical axis of the second objective lens is parallel to an optical axis of the first objective lens. With this configuration, it is possible to facilitate the installation of the microscope such as an inverted microscope or an upright microscope.
A specimen observation method according to an aspect of the present disclosure includes: irradiating a sample with excitation light having a wavelength capable of exciting the sample; a step of guiding detection light emitted from the sample in association with irradiation of the excitation light; and a step of detecting the detection light, the step of irradiating the sample with the excitation light including: a step of outputting light of a wavelength varying with time from a wavelength-scanning light source as excitation light; a step of causing excitation light output from the wavelength-scanning light source to enter the spectroscopic element and causing the excitation light to exit the spectroscopic element at an exit angle corresponding to the wavelength of the excitation light; a step of causing excitation light emitted from the spectroscopic element to enter the cylindrical lens at an incident angle corresponding to the emission angle; and a step of converging the excitation light guided by the relay optical system including the cylindrical lens and irradiating the specimen with the sheet-like excitation light.
In this sample observation method, light whose wavelength changes with time is output as excitation light from the wavelength-scanning light source, the excitation light output from the wavelength-scanning light source is output from the spectroscopic element at an output angle corresponding to the wavelength, and the excitation light output from the spectroscopic element is incident on the cylindrical lens at an incident angle corresponding to the output angle. This enables scanning of the sample at the irradiation position of the sheet-like excitation light. As a result, since it is not necessary to move or rotate the sample during observation, the sample can be stably observed while suppressing shaking of the sample. Further, since the irradiation position of the excitation light on the sample is scanned using the wavelength scanning light source, the spectroscopic element, and the cylindrical lens, the scanning of the excitation light on the sample can be speeded up. Therefore, according to the light sheet microscope, high-speed and stable observation can be realized
ADVANTAGEOUS EFFECTS OF INVENTION
According to an aspect of the present disclosure, it is possible to provide a light sheet microscope and a specimen observation method capable of realizing high-speed and stable observation.
Drawings
Fig. 1 is a diagram showing a structure of a light sheet microscope according to an embodiment.
Fig. 2 is a diagram for explaining the spectroscopic element.
Fig. 3 is a diagram showing the irradiation optical system when viewed from the X-axis direction.
Fig. 4 (a) and 4 (b) are diagrams illustrating the irradiation optical system when viewed from the Y-axis direction.
Fig. 5 (a) to 5 (c) are views for explaining the adjustment of the beam waist position.
Fig. 6 (a) and 6 (b) are diagrams showing the structure around the container in which the sample is placed.
Fig. 7 (a) to 7 (c) show modifications.
Fig. 8 is a diagram showing another modification.
Detailed Description
Hereinafter, one embodiment of the present disclosure will be described in detail with reference to the drawings. In the following description, the same reference numerals are used for the same elements or elements having the same function, and redundant description is omitted.
The light sheet microscope 1 shown in fig. 1 is a device that irradiates a sheet-like (planar) excitation light L1 on a sample S, and detects a detection light L2 emitted from the sample S in association with the irradiation of the excitation light L1, thereby acquiring an image of the sample S. In the light sheet microscope 1, the irradiation position of the excitation light L1 is scanned over the sample S, and an image of the sample S is acquired at each irradiation position. In the light sheet microscope 1, since the region in which the excitation light L1 is irradiated on the sample S is narrow, it is possible to suppress deterioration of the sample S such as photobleaching or phototoxicity, and to speed up image acquisition.
The sample S is, for example, a sample of a cell, a living body, or the like containing a fluorescent substance such as a fluorescent dye or a fluorescent gene. When light of a predetermined wavelength range is irradiated, the sample S emits detection light L2 such as fluorescence. The sample S is disposed in the container 5 having light transmittance with respect to at least the excitation light L1 and the detection light L2, for example. Details of the container 5 will be described later.
As shown in fig. 1, the optical sheet microscope 1 includes an irradiation optical system 10, a detection optical system 20, a photodetector 30, and a control section 40. The irradiation optical system 10 irradiates the sample S with the excitation light L1. The detection optical system 20 guides the detection light L2 emitted from the sample S as the excitation light L1 is irradiated to the photodetector 30. The light detector 30 detects the detection light L2 guided by the detection optical system 20. The control unit 40 controls operations of the irradiation optical system 10, the detection optical system 20, the photodetector 30, and the like.
The irradiation optical system 10 has a wavelength scanning light source 11, a
The wavelength-scanning light source 11 outputs excitation light L1 having a wavelength that can excite the sample S. The wavelength-scanning light source 11 outputs light whose wavelength changes with time as the excitation light L1. More specifically, the wavelength-scanning light source 11 is a light source that performs wavelength scanning in a predetermined wavelength range by periodically changing the wavelength of the output excitation light L1 at a high speed.
The wavelength-scanning light source 11 may be any light source, but is preferably a light source whose emission angle does not change during wavelength scanning. As such a wavelength-scanning light source 11, for example, a semiconductor laser light source in which the wavelength of output light is variable by a change in cavity length or a change in temperature or current value is cited. The wavelength scanning light source 11 may be a unit that combines a light source that outputs white laser light and an AOTF (Acousto-Optic Tunable Filter) that selectively transmits light in a specific wavelength range. Alternatively, the wavelength scanning light source 11 is a littrow-type external resonator semiconductor Laser light source, a Vertical resonator Surface Emitting Laser (VCSEL) light source, or an external resonator semiconductor Laser light source using a KTN crystal.
The wavelength-scanning light source 11 may emit coherent light. As the laser light source that outputs coherent light, a light source that oscillates Continuous Wave (Continuous Wave) may be used, or a light source that oscillates pulsed light such as ultra-short pulsed light or a light source that outputs intensity-modulated light may be used. Further, a unit in which these light sources are combined with an optical shutter or an AOM (Acousto-Optic Modulator) for pulse modulation may be used.
As shown in fig. 1 and 2, the excitation light L1 output from the wavelength-scanning light source 11 enters the
The excitation light L1 emitted from the
The shaping of the excitation light L1 into a sheet shape by the irradiation optical system 10 will be described with reference to fig. 3 and 4. The surface of the
As shown in fig. 3, the
As shown in fig. 4 (a) and 4 (b), the
As shown in fig. 4 (b), the emission angle θ is the emission angle of the excitation light L1 from the
The height H of the sheet-like excitation light L1 emitted from the first objective lens 15 (the distance from the optical axis C in the X-axis direction) corresponds to the incident angle of the excitation light L1 incident on the
With reference to fig. 5 a to 5 c, the adjustment of the position B of the beam waist (the portion where the thickness of the sheet-like excitation light is smallest when viewed from the Y-axis direction) of the excitation light L1 emitted from the first objective lens 15 will be described. The excitation light L1 at the beam waist B is irradiated to the sample S as the sheet-like excitation light L1. That is, the position of the beam waist B corresponds to the position of the formation of the sheet-like excitation light L1 in the irradiation direction of the excitation light L1 with respect to the sample S.
As a technical means for adjusting the position of the beam waist B, the optical sheet microscope 1 further includes a moving mechanism 3 that maintains the positional relationship (optical distance) between the
As shown in fig. 5 (a) and 5 (B), the wavelength scanning light source 11, the
As shown in fig. 6 (a) and 6 (b), the sample S is disposed in the container 5. The container 5 is made of, for example, glass, plastic, or the like, and has a bottom wall portion 51 and a side wall portion 52. The bottom wall 51 is, for example, a cover glass or a glass chassis. The side wall portion 52 extends, for example, from an edge portion of the bottom wall portion 51 in a direction orthogonal to the bottom wall portion 51, and assumes a cylindrical or square-cylindrical shape. The container 5 is disposed such that the bottom wall portion 51 is positioned vertically below the side wall portion 52, for example. The sample S is disposed in the accommodation space defined by the bottom wall 51 and the side wall 52, for example, together with the culture solution. The sample S is placed on the bottom wall 51. In the case where the sample is a dried product, the container 5 may not be used.
As shown in fig. 6 (a), the first objective lens 15 is, for example, a water immersion objective lens, and is disposed so as to be immersed in the culture medium. The bottom wall 51 of the container 5 is provided with a mirror (reflecting portion) 53 that reflects the excitation light L1 emitted from the first objective lens 15 toward the sample S at a predetermined angle (for example, vertically). The reflecting mirror 53 has a reflecting surface 53a inclined and extending at 45 degrees with respect to each of the optical axis of the first objective lens 15 and the bottom wall portion 51. The scanning direction of the sheet-like excitation light L1 with respect to the sample S is the direction indicated by the arrow a (the direction along the optical axis (optical axis C) of the first objective lens 15). Further, the reflecting mirror (reflecting portion) 53 may not be provided to the container 5. For example, the mirror 53 may be mounted to the first objective lens 15 via a connector.
As shown in fig. 6 (b), the first objective lens 15 may be a dry objective lens or may be disposed outside the culture medium. The Working distance (Working distance) of the dry objective lens is longer than that of the water immersion objective lens. In this case, a prism (reflection portion) 54 that reflects the excitation light L1 emitted from the first objective lens 15 toward the sample S at a predetermined angle (for example, perpendicularly) is provided on the bottom wall portion 51 of the container 5. The prism 54 has a reflection surface 54a inclined and extending at 45 degrees to each of the optical axis of the first objective lens 15 and the bottom wall portion 51. The scanning direction of the sheet-like excitation light L1 with respect to the sample S is the direction indicated by the arrow a. Further, a prism (reflection portion) 54 may be provided to the container 5. For example, the prism 54 may be mounted to the first objective lens 15 via a connector.
When the reflecting portion is the mirror 53, a water immersion objective lens having a short working distance can be used. In this case, since the same medium exists between the first objective lens 15 and the sample S and it is not necessary to correct aberration occurring when passing through an interface of different kinds of media, the Numerical Aperture (NA) of the first objective lens 15 can be increased. On the other hand, in the case where the reflecting portion is the prism 54, a dry objective lens having a long working distance may be used. In this case, it is possible to save labor for cleaning the lens, and to easily perform measurement in which the sample S is frequently exchanged, measurement using a liquid that has entered the first objective lens 15, measurement over a long period of time, and the like.
The detection optical system 20 has a second
The liquid lens 22 is a lens whose focal point distance is variable according to an input signal. In the detection optical system 20, the focal position of the second
The light detector 30 images the detection light L2 guided by the second
The control unit 40 is constituted by a computer including a processor, a memory, and the like, for example. The controller 40 controls the movement of the moving mechanism 3, the wavelength scanning light source 11, the liquid lens 22, the photodetector 30, and the like by a processor, and executes various controls. For example, the control unit 40 changes the wavelength of the excitation light L1 output from the wavelength-scanning light source 11 over time so that the emission angle θ of the excitation light L1 emitted from the
As explained above, in the optical sheet microscope 1, light whose wavelength varies with time is output from the wavelength-scanning light source 11 as the excitation light L1, and the excitation light L1 output from the wavelength-scanning light source 11 is output from the
In the light sheet microscope 1, the
In the light sheet microscope 1, the detection optical system 20 has a liquid lens 22 (focus position adjuster) that changes the focus position of the second
The optical sheet microscope 1 further includes a moving mechanism 3 that maintains a positional relationship between the
In the light sheet microscope 1, the container 5 is provided with a mirror 53 or a prism 54 (reflection unit) for reflecting the excitation light L1 emitted from the first objective lens 15 toward the sample S. This allows the excitation light L1 in the form of a sheet to be applied to the sample S without being affected by the side wall 52 of the container 5. Further, the reflecting portion for guiding the excitation light L1 can be integrated with the container 5, and the number of components and the size of the device can be reduced. In addition, the excitation light L1 can be irradiated to the sample S with reliability and accuracy, as compared with the case where the reflecting portion is separate from the container 5. Further, the first objective lens 15 and the second
In the light sheet microscope 1, the optical axis of the second
The specimen observation method according to the light sheet microscope 1 includes: irradiating the sample S with excitation light L1 having a wavelength capable of exciting the sample S; a step of guiding the detection light L2 emitted from the sample S in association with the irradiation of the excitation light L1; and a step of detecting the detection light L2. The step of irradiating the sample S with the excitation light L1 includes: a step of outputting light whose wavelength changes with time from the wavelength-scanning light source 11 as excitation light L1; a step of causing excitation light L1 output from the wavelength-scanning light source 11 to enter the
The above description has been made of an embodiment of the present disclosure, but the present disclosure is not limited to the above embodiment. For example, the material and shape of each structure are not limited to those described above, and various materials and shapes may be employed.
As shown in fig. 7 (a), the
In the above-described embodiment, the wavelength-scanning light source 11 may be configured to be able to switch the center wavelength of the excitation light L1 between a plurality of center wavelengths. In this case, the
As shown in fig. 8, the irradiation optical system 10 may have a plurality of wavelength
In the above embodiment, the liquid lens 22 may be omitted. In this case, the second
The
Description of the symbols
1 … … light sheet microscope; 3 … … moving mechanism; 5 … … container; 10 … … illumination optics; 11 … … wavelength scanning light source; 12 … … light-splitting element; 14 … … relay optical system; 15 … … first objective lens; 16 … … cylindrical lenses; 20 … … detection optics; 21 … … second objective lens; 22 … … liquid lens (focal position adjuster); 30 … … light detector; a 53 … … mirror (reflection section); 54 … … prism (reflective portion); b … … beam waist; a C … … optical axis; l1 … … excitation light; l2 … … detects light; sample S … …; theta … … exit angle.
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