Patent ID: 12216263

DESCRIPTION OF EMBODIMENTS

Hereinafter, an embodiment of the present disclosure will be described in detail with reference to the accompanying drawings. Additionally, in the description, the same reference numerals will be used for the same elements or elements having the same function and redundant description will be omitted.

FIG.1is a schematic configuration diagram of a confocal microscope A which is a kind of scanning microscope according to an embodiment. The confocal microscope A shown inFIG.1constitutes a confocal microscope that acquires an image enabling the construction of an optical sectioning image of a sample M to be observed and is configured such that a confocal microscope unit1which is a scanning microscope unit according to the embodiment is connected to a connection port P1used for connection to an external unit of a microscope50. This confocal microscope unit1is a device that irradiates the sample M disposed on a stage or the like of the microscope50via a microscope optical system such as an imaging lens51and an objective lens52inside the microscope50with excitation light, receives (detects) fluorescence generated from the sample M in response to the excitation light via the microscope optical system of the microscope50, generates an optical sectioning image, and outputs the image.

Specifically, the confocal microscope unit1includes a main housing2, a lens barrel (housing)3which constitutes a part of the main housing2and is removably connected to the connection port P1of the microscope50, a scan mirror4, a fixed mirror5, and first to fourth subunits6ato6dwhich are fixed into the main housing2, and a scan lens7which is fixed into the lens barrel3. Hereinafter, each component of the confocal microscope unit1will be described in detail.

The scan lens7in the lens barrel3is an optical element for relaying a reflection surface of the scan mirror4to a pupil position of the objective lens52and collecting the excitation light (irradiation light) on a primary image plane of the microscope optical system of the microscope50. The scan lens7irradiates the sample M by guiding the excitation light (irradiation light) scanned by the scan mirror4to the microscope optical system and guides the fluorescence (observation light) generated from the sample M in response to the excitation light to the scan mirror4. Specifically, the scan lens7is configured to focus the pupil of the objective lens52on the scan mirror4and guides the fluorescence focused by the objective lens52and the imaging lens51of the microscope50to the scan mirror4.

The scan mirror4in the main housing2is, for example, an optical scanning element such as a micro electro mechanical system (MEMS) mirror configured so that a reflector can be tilted in two axes. The scan mirror4has a function of scanning the excitation light (irradiation light) output from the first to fourth subunits6ato6don the sample M by continuously changing a reflection angle and guiding the fluorescence (observation light) generated in response to the excitation light to the first to fourth subunits6ato6d.

The fixed mirror5is an optical reflecting element fixed into the main housing2, reflects the excitation light output from the first to fourth subunits6ato6dto the scan mirror4, and reflects the fluorescence reflected by the scan mirror4to the first to fourth subunits6ato6dcoaxially with the excitation light.

The first subunit6aincludes a base plate8aand a dichroic mirror (first beam splitter)9a, a light source10a, a dichroic mirror11a, a pinhole plate (first aperture)12a, and a photodetector (first photodetector)13awhich are disposed on the base plate8a. The dichroic mirror9ais a beam splitter which is fixed in the reflection direction of the fluorescence of the fixed mirror5and has a property of reflecting first excitation light of a wavelength λ1irradiated by the first subunit6aand first fluorescence of a wavelength range Δλ1generated from the sample M in response to the first excitation light and transmitting light of a wavelength longer than those of the first excitation light and the first fluorescence. The dichroic mirror11ais a beam splitter which is provided in the reflection direction of the first fluorescence of the dichroic mirror9aand has a property of transmitting the first fluorescence of the wavelength range Δλ1and reflecting the first excitation light of the wavelength λ1shorter than the wavelength range Δλ1. The light source10ais a light emitting element (for example, laser diode) outputting the first excitation light (for example, laser beam) of the wavelength λ1and is disposed so that the first excitation light is reflected toward the dichroic mirror9acoaxially with the first fluorescence by the dichroic mirror11a. The pinhole plate12ais an aperture which is disposed so that a pinhole position coincides with a conjugate position of a spot of the first excitation light of the sample M and limits the luminous flux of the first fluorescence and constitutes a confocal optical system along with the light source10aand the like. This pinhole plate12ahas a pinhole diameter that can be adjusted from the outside and the resolution of the image detected by the photodetector13aand the signal intensity of the image can be changed. The photodetector13ais disposed so that a detection surface is opposed to the pinhole plate12aand receives and detects the first fluorescence having passed through the pinhole plate12a. Additionally, the photodetector13ais a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photo detector (HPD), an area image sensor, or the like.

The second to fourth subunits6bto6dalso have the same configuration as that of the first subunit6a.

That is, the second subunit6bincludes a base plate8b, a dichroic mirror (second beam splitter)9b, a light source10b, a dichroic mirror11b, a pinhole plate (second aperture)12b, and a photodetector (second photodetector)13b. The dichroic mirror9bhas a property of reflecting second excitation light of a wavelength λ2(>λ1) irradiated by the second subunit6band second fluorescence of a wavelength range Δλ2generated from the sample M in response to the second excitation light and transmitting light of a wavelength longer than those of the second excitation light and the second fluorescence. The dichroic mirror11bhas a property of transmitting the second fluorescence of the wavelength range Δλ2and reflecting the second excitation light of the wavelength λ2shorter than the wavelength range Δλ2. The light source10bis a light emitting element which outputs the second excitation light of the wavelength λ2. The pinhole plate12bis an aperture which is disposed so that a pinhole position coincides with a conjugate position of a spot of the second excitation light of the sample M and limits the luminous flux of the second fluorescence. The photodetector13bis disposed so that a detection surface is opposed to the pinhole plate12band receives and detects the second fluorescence having passed through the pinhole plate12b. Additionally, the photodetector13bis a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photo detector (HPD), an area image sensor, or the like.

The third subunit6cincludes a base plate8c, a dichroic mirror (third beam splitter)9c, a light source10c, a dichroic mirror11c, a pinhole plate (third aperture)12c, and a photodetector (third photodetector)13c. The dichroic mirror9chas a property of reflecting third excitation light of a wavelength λ3(>λ2) irradiated by the third subunit6cand third fluorescence of a wavelength range Δλ3generated from the sample M in response to the third excitation light and transmitting light of a wavelength longer than those of the third excitation light and the third fluorescence. The dichroic mirror11chas a property of transmitting the third fluorescence of the wavelength range Δλ3and reflecting the third excitation light of the wavelength λ3shorter than the wavelength range Δλ3. The light source10cis a light emitting element which outputs the third excitation light of the wavelength λ3. The pinhole plate12cis an aperture which is disposed so that a pinhole position coincides with a conjugate position of a spot of the third excitation light of the sample M and limits the luminous flux of the third fluorescence. The photodetector13cis disposed so that a detection surface is opposed to the pinhole plate12cand receives and detects the third fluorescence having passed through the pinhole plate12c. Additionally, the photodetector13cis a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photo detector (HPD), an area image sensor, or the like.

The fourth subunit6dincludes a base plate8d, a total reflection mirror9d, a light source10d, a dichroic mirror11d, a pinhole plate (fourth aperture)12d, and a photodetector (fourth photodetector)13d. The total reflection mirror9creflects fourth excitation light of a wavelength λ4(>λ3) irradiated by the fourth subunit6dand fourth fluorescence of a wavelength range Δλ4generated from the sample M in response to the fourth excitation light. The dichroic mirror11dhas a property of transmitting the fourth fluorescence of the wavelength range Δλ4and reflecting the fourth excitation light of the wavelength λ4shorter than the wavelength range Δλ4. The light source10dis a light emitting element which outputs the fourth excitation light of the wavelength λ4. The pinhole plate12dis an aperture which is disposed so that a pinhole position coincides with a conjugate position of a spot of the fourth excitation light of the sample M and limits the luminous flux of the fourth fluorescence. The photodetector13dis disposed so that a detection surface is opposed to the pinhole plate12dand receives and detects the fourth fluorescence having passed through the pinhole plate12d. Additionally, the photodetector13dis a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photo detector (HPD), an area image sensor, or the like.

A positional relationship of the first to fourth subunits6ato6dwith the above-described configuration will be described.

The first to fourth subunits6ato6dare fixed into the main housing2so that the first to fourth subunits are arranged in this order along the light guiding direction of the first to fourth fluorescences formed by the scan mirror4and the fixed mirror5to be away from the fixed mirror5and the dichroic mirrors9ato9cand the total reflection mirror9dare located on the optical paths of the first to fourth fluorescences. Specifically, the second to fourth subunits6bto6dare respectively disposed to be shifted from the first to third subunits6ato6cby a predetermined distance d in a direction perpendicular to the light guiding direction of the second to fourth fluorescences based on the center positions of the dichroic mirrors9ato9cand the total reflection mirror9d.

This predetermined distance d is set to be substantially the same as a shift amount δ generated due to the refraction of the fluorescence of each of the dichroic mirrors9ato9cin a direction perpendicular to the optical path of the fluorescence transmitted in the dichroic mirrors9ato9c. In this embodiment, since the thickness of the mirror members constituting the dichroic mirrors9ato9cis set to be the same, the shift amount generated in the dichroic mirrors9ato9cis substantially the same and hence the shift distance d between two subunits adjacent to each other among the first to fourth subunits6ato6dis also set to be the same. This shift distance d is appropriately set in response to the refractive index and the thickness of the mirror member constituting the dichroic mirrors9ato9c. Specifically, if the mirror member has a thickness t and a refractive index n, an incident angle θ of the fluorescence incident to the mirror member, and a refracting angle ϕ in the mirror member, the shift amount δ of the fluorescence due to the mirror member is obtained as inFIG.2. At this time, since the shift amount δ can be obtained as in the following formula (1), the shift distance (predetermined distance) d may be set in accordance with this shift amount δ. Additionally, the incident angle θ and the refracting angle ϕ have a relationship of the following formula (2).
δ=t·sin(θ−ϕ))/cos ϕ  (1)
ϕ=arcsin(sin θ/n)  (2)

Additionally, when the incident angle θ is set to 45°, d=δ=0.33 t if the refractive index n of the mirror member is 1.5 and d=δ=0.29 t when the refractive index n of the mirror member is 1.4.

Next, an attachment structure of the confocal microscope unit1to the microscope50will be described in detail.FIG.3is a cross-sectional view showing an attachment structure of the confocal microscope unit1to the microscope50.

As shown inFIG.3, the scan lens7including a plurality of lenses is fixed into the lens barrel3and a tilt adjustment mechanism23in which an attachment portion21and a movable portion22are integrated with each other is provided inside the front end of the lens barrel3. The attachment portion21has an annular shape which protrudes from the front end of the lens barrel3and the front end side thereof is provided with a structure (for example, a structure corresponding to a C mount) attachable to the connection port P1for camera connection of the microscope50. The movable portion22is formed to be continuous to the base end side of the attachment portion21and has a substantially annular shape and the outer surface thereof is formed as a spherical sliding surface. Further, the inner surface of the front end of the lens barrel3is provided with a spherical sliding surface24corresponding to the outer surface shape of the movable portion22. Here, the outer surface of the movable portion22and the inner surface of the lens barrel3are formed in a shape in which a center C1of a spherical surface including these shapes is located on the image plane S1of the microscope optical system of the microscope50in a state in which the movable portion22is fitted into the lens barrel3and the attachment portion21is connected to the connection port P1of the microscope50.

According to an attachment structure in which the tilt adjustment mechanism23with the above-described structure is fitted into the front end side of the lens barrel3, it is possible to support the lens barrel3so that an angle of the lens barrel3with respect to the attachment portion21is changeable by sliding the movable portion22with respect to the sliding surface24of the lens barrel3while the microscope unit is attached to the microscope50. At this time, since the outer surface of the movable portion22and the inner surface of the lens barrel3are formed as spherical surfaces, the lens barrel3is rotatable with respect to the attachment portion21and the angle of the center axis of the lens barrel3with respect to the center axis of the attachment portion21is adjustable two-dimensionally. That is, the tilt adjustment mechanism23is configured to change the angle of the lens barrel3with respect to the attachment portion21so that the optical axis of the microscope optical system of the microscope50is parallel to the optical axis of the scan lens7.

According to the above-described confocal microscope unit1, the excitation light (irradiation light) irradiated from the light sources10ato10dof the subunits6ato6dis scanned on the sample M via the scan mirror4, the scan lens7, and the external microscope50and the fluorescence (observation light) generated from the sample M in response to the excitation light is detected by the photodetectors13ato13dof the subunits6ato6dvia the external microscope50, the scan lens7, and the scan mirror4. In this confocal microscope unit1, the lens barrel3to which the scan lens7is fixed is attached to the connection port P1of the microscope50by the attachment portion21and the angle of the lens barrel3with respect to the attachment portion21is changeable. With such a configuration, the optical axis of the scan lens7can be aligned to the direction of the optical axis of the microscope optical system of the microscope50according to the microscope50at the attachment destination. As a result, it is possible to realize imaging in which signal intensity and resolution are maintained.

FIG.4is a graph showing wavelength distribution characteristics of the excitation light and the fluorescence handled by the first to fourth subunits6ato6d. The wavelength range Δλ1of the fluorescence generated in response to the excitation light of the wavelength λ1irradiated from the first subunit6ais generally in the vicinity of the wavelength λ1and in the range of the wavelength longer than the wavelength λ1. On the contrary, the wavelength λ2of the excitation light irradiated from the second subunit6band the wavelength range Δλ2of the fluorescence generated in response to the excitation light are in the range of the wavelength longer than the wavelength λ1and the wavelength range Δλ1. Here, the boundary wavelength λd1of the optical division of the dichroic mirror9aof the first subunit6ais set to a value which is longer than the wavelength λ1and the wavelength range Δλ1and is shorter than the wavelength λ2and the wavelength range Δλ2. Accordingly, it is possible to perform the confocal measurement in the range of the wavelength λ1and the wavelength range Δλ1using the first subunit6aand to perform the confocal measurement in the range of the wavelength λ2and the wavelength range Δλ2using the second subunit6bof the same device. Similarly, the boundary wavelength λd2of the optical division of the dichroic mirror9bof the second subunit6bis set to a value which is longer than the wavelength λ2and the wavelength range Δλ2and is shorter than the wavelength λ3and the wavelength range Δλ3and the boundary wavelength λd3of the optical division of the dichroic mirror9cof the third subunit6cis set to a value which is longer than the wavelength λ3and the wavelength range Δλ3and is shorter than the wavelength λ4and the wavelength range Δλ4. Accordingly, it is possible to perform the confocal measurement in the range of the wavelength λ3and the wavelength range Δλ3using the third subunit6cof the same device and to perform the confocal measurement in the range of the wavelength λ4and the wavelength range Δλ4using the fourth subunit6dof the same device.

Here, the movable portion22of the tilt adjustment mechanism23is configured to change the angle of the lens barrel3so that the optical axis of the scan lens7is parallel to the optical axis of the microscope optical system of the microscope50. In this case, since the direction of the optical axis of the scan lens7is aligned to the optical axis of the microscope optical system, the scan mirror4and the pupil positions of the objective lens52can be arranged at the conjugate position, the NA of the objective lens52can be used to the maximum, and the signal intensity and resolution of the signal detected by the photodetectors13ato13dcan be reliably improved.

In particular, the movable portion22is rotatable with respect to the attachment portion21. With such a configuration, the direction of the scan lens7and the optical axis of the microscope optical system can be aligned two-dimensionally and the signal intensity and resolution of the signal detected by the photodetectors13ato13dcan be reliably improved. Further, the rotation center C1of the movable portion22is formed to be included in the image plane S1of the microscope optical system. In this way, since the adjustment can be performed without affecting the field of view, it is not necessary to alternately repeat the field of view adjustment and the angle adjustment.

FIG.5shows an image of a light guide of the excitation light or fluorescence when the direction of the optical axis of the scan lens7in the confocal microscope unit1does not coincide with the direction of the optical axis of the microscope optical system andFIG.6shows an image of a light guide of the excitation light or fluorescence when the direction of the optical axis of the scan lens7in the confocal microscope unit1coincides with the direction of the optical axis of the microscope optical system.

In the confocal microscope unit1, the microscope optical system and the scan lens7are adjusted in advance so that the objective lens pupil of the microscope50is focused on the scan mirror4. Accordingly, since the changing of the angle by driving the scan mirror4is equivalent to the swinging the angle of the beam of the excitation light or fluorescence at the pupil of the objective lens52, it is possible to reduce the loss of the beam and to sufficiently exhibit the performance of the objective lens52. Here, the size of the objective lens pupil differs depending on the objective lens52and the beam diameter of the excitation light or fluorescence needs to be equal to or larger than the pupil size. However, since the scan mirror4configured as the MEMS mirror or the like generally has a small diameter and a large swing angle, the objective lens pupil may not be included in the scan mirror4when using the objective lens52with a large pupil having a low magnification and a low NA.

Further, in the microscope50, the optical axis of the microscope may be inclined with respect to the connection port P1(FIG.1) due to the assembling error. This inclination becomes a problem when photographing and detecting the observation light and particularly becomes a problem in the application to the scanning microscope. As a result, as shown inFIG.4, an optical axis A2of the scan lens7may be inclined with respect to an optical axis A1of the microscope optical system by an angle θ (>0) when the confocal microscope unit1is attached to the microscope50. In such a case, when the scan mirror4is driven to change the focus position of the excitation light and irradiates the sample M as the beams B1and B2, the positions of the beams B1and B2may be displaced from the aperture of the objective lens and a part of the excitation light may be cut off. As a result, the amount of signal and resolution in the image generated by using the subunits6ato6ddecrease.

In contrast, as shown inFIG.6, the positions of the beams B1and B2can be aligned to the aperture position of the objective lens when the focus position of the excitation light is changed and the sample M is irradiated with the beams B1and B2by adjusting the direction of the optical axis A2of the scan lens7to be aligned to the optical axis A1of the microscope optical system by the tilt adjustment mechanism23when the confocal microscope unit1is attached to the microscope50. As a result, it is possible to prevent the excitation light from being cut off and to increase the amount of signal and resolution in the image generated by using the subunits6ato6d.

The tilt adjustment using the tilt adjustment mechanism23of the embodiment can be realized by using a jig shown inFIG.7.FIG.7is a diagram showing a configuration of a jig used for adjusting the tilt of the confocal microscope unit1. As the jig, a jig including a disk-shaped target member31and a mount portion32for attaching the target member31to the microscope50instead of the objective lens52can be adopted. A circular marking33is provided at the center of the surface of the target member31and the jig is configured so that the position of the marking33may coincide with the pupil position of the objective lens52when it is attached to the microscope50by the mount portion32.

By using such a configuration, a spot SP1of the excitation light irradiated to the target member31while the excitation light is output using the confocal microscope unit1is observed using an external camera or the like. Then, it is possible to adjust the direction of the optical axis of the scan lens7to the optical axis of the microscope optical system by adjusting the tilt so that the spot SP1of the excitation light coincides with the marking33on the target member31.

Further, the scan mirror4may be a MEMS mirror. In this case, it is possible to easily realize the miniaturization of the device.

Further, the subunits6ato6dare respectively provided with the pinhole plates12ato12dwhich limit the luminous flux of the observation light returned via the scan mirror4and the photodetectors13ato13dare configured to detect the observation light having passed through the pinhole plates12ato12d. In this case, it is possible to realize imaging in which signal intensity and resolution are maintained in the confocal observation.

Although various embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiment and may be modified without departing from the spirit of each claim or may be applied to other embodiments.

The tilt adjustment mechanism23of the above-described embodiment is not limited to the configuration of the movable portion22having a spherical sliding surface and the movable portion22may be configured by using a rubber member or a bellows-shaped member.

Further, in the above-described embodiment, the excitation light is irradiated by each of the subunits6ato6dand the fluorescence generated in response to the excitation light is detected. However, the reflection light generated from the sample M in response to the irradiation of the irradiation light may be detected.

Further, the above-described embodiment is not limited to a case used as the confocal microscope and may be used as a general fluorescence microscope, reflection microscope, or the like if it is a scanning microscope using a scan mirror.

Further, the above-described embodiment is not limited to the application of the scanning microscope using the scan mirror and is also effective when inclining the housing of the optical unit so that the optical axis of the microscope optical system of the microscope50is parallel to the optical axis of the lens of the optical unit when the optical unit with the lens is attached to the connection port P1of the optical microscope. In this case, it is easy to adjust the optical axis between the optical microscope and the optical unit.

In the above-described embodiment, the pinhole plate is used as an aperture to form a confocal optical system, but may be, for example, an iris diaphragm, a fiber core, or the like if the aperture is any optical element that limits the luminous flux. When a fiber output type light source is used, the position of the end surface of the fiber core may be the aperture position (the position where the luminous flux is limited).

Further, in the above-described embodiment, a laser light source such as a solid-state laser or a diode laser can also be used. In this case, the position of the beam waist of these laser light sources may be set to the aperture position (the position where the luminous flux is limited) and the light source itself plays the role of the aperture.

In the above-described embodiment, the movable portion may change the angle of the housing so that the optical axis of the scan lens is parallel to the optical axis of the microscope optical system. In this case, it is possible to reliably improve the signal intensity and resolution of the signal detected by the photodetector by aligning the direction of the scan lens to the optical axis of the microscope optical system.

Further, the movable portion may be rotatable with respect to the attachment portion. By adopting such a configuration, it is possible to two-dimensionally align the direction of the scan lens to the optical axis of the microscope optical system and to reliably improve the signal intensity and resolution of the signal detected by the photodetector.

Further, the rotation center of the movable portion may be included in the image plane of the microscope optical system. In this way, it is possible to obtain an effect of increasing the signal amount detected by the photodetector and improving the resolution of the signal without affecting the field of view.

Further, the scan mirror may be a MEMS mirror. In this case, the miniaturization of the device can be easily realized.

Further, the photodetector may detect the fluorescence generated in response to the irradiation of the irradiation light as the observation light and detect the reflection light generated in response to the irradiation of the irradiation light as the observation light. According to such a configuration, it is possible to realize imaging in which signal intensity and resolution are maintained in the imaging of various kinds of observation light.

Further, the aperture configured to limit the luminous flux of the observation light returned via the scan mirror may be further provided and the photodetector may detect the observation light having passed through the aperture. In this case, it is possible to acquire an optical sectioning image by a confocal observation. Further, this aperture may be the pinhole plate.

INDUSTRIAL APPLICABILITY

In the embodiment, it is possible to realize imaging in which signal intensity and resolution are maintained in the scanning microscope unit constituting the scanning microscope.

REFERENCE SIGNS LIST

A1, A2: optical axis, C1: rotation center, M: sample, P1: connection port, S1: image plane, d: predetermined distance,10ato10d: light sources,12ato12d: pinhole plates (apertures),13ato13d: photodetectors,6ato6b: first to fourth subunits,9ato9c: dichroic mirrors,1: confocal microscope unit,2: main housing,3: lens barrel (housing),4: scan mirror,7: scan lens,21: attachment portion,22: movable portion,23: tilt adjustment mechanism,24: sliding surface,50: microscope,52: objective lens.