JP2020197633A - Confocal scanner and confocal microscope - Google Patents

Abstract

To provide a confocal scanner and a confocal microscope which can be flexibly installed without incurring a significant increase in compositional complexity and costs, and with which it is possible to obtain a homogeneous confocal image free of unevenness in brightness.SOLUTION: A confocal scanner 20 comprises: a fly-eye integrator 22 for uniformizing the intensity distribution of light output from a light source unit 10; a collimator lens 23 for converting light having intensity distribution uniformized by the fly-eye integrator 22 into parallel rays of light; and a disk unit 24 for generating illumination light for scanning a sample SP from the parallel rays of light converted by the collimator lens 23, the disk unit including a first disk 24a having a plurality of microlenses and a second disk 24b having a plurality of pin holes formed in correspondence to the microlenses and constituted so as to be rotatable together with the first disk.SELECTED DRAWING: Figure 1

Description

The present invention relates to a confocal scanner and a confocal microscope.

Conventionally, a confocal microscope using a disc scanning type confocal scanner has been developed. This confocal microscope has a microlens array disk having a plurality of microlenses formed in a predetermined pattern, and a plurality of pinholes associated with each of the microlenses and formed in the same pattern as the microlens. However, a confocal scanner equipped with a pinhole array disk configured to be rotatable together with the microlens array disk is used.

In such a confocal microscope, the disks (microlens array disk and pinhole array disk) provided in the confocal scanner are rotated to change the irradiation position of the laser beam on the sample (the sample is scanned by the laser beam). Therefore, a confocal image of the sample can be obtained. The following Patent Documents 1 to 4 disclose a conventional confocal microscope using a disk scanning confocal scanner.

Japanese Unexamined Patent Publication No. 5-60980 U.S. Pat. No. 8,922,887 JP-A-2017-207625 JP-A-2019-56734

By the way, in a conventional confocal microscope, if the intensity distribution of the light emitted from the confocal scanner (illumination light applied to the sample) is not uniform, the captured confocal image has uneven brightness. There was a problem that it would end up. For example, when the intensity distribution of the laser light emitted from the light source has a Gaussian distribution, the intensity distribution of the illumination light emitted from the confocal scanner is lower in the peripheral portion than in the central portion. Then, the confocal image to be photographed has a bright central portion and a dark peripheral portion with respect to the central portion, so that a homogeneous confocal image cannot be obtained. It should be noted that the confocal microscope is required to be capable of flexible installation without complicating the configuration and significantly increasing the cost, and to be able to capture a homogeneous confocal image.

The present invention has been made in view of the above circumstances, and it is possible to obtain a uniform confocal image without uneven brightness by enabling flexible installation without incurring complicated configuration and a significant increase in cost. It is an object of the present invention to provide a confocal scanner and a confocal microscope capable of providing the same.

In order to solve the above problems, the confocal scanner (20, 20A, 20B) according to one aspect of the present invention is a fly-eye integrator (22) that equalizes the intensity distribution of the light output from the light source unit (10, 10B). ), A collimating lens (23) that converts light whose intensity distribution is uniformed by the fly-eye integrator into parallel light, a first disk (24a) having a plurality of microlenses, and the microlens. A second disk (24b) having a plurality of pinholes formed in the lens and rotatably configured together with the first disk is provided, and a sample (SP) is provided from parallel light converted by the collimating lens. A disk unit (24) for generating illumination light for scanning the lens is provided.

Further, the confocal scanner according to one aspect of the present invention includes a speckle reduction unit (21, 21B) in which the light output from the light source unit is laser light and the speckle of the laser light is reduced.

Further, in the confocal scanner according to one aspect of the present invention, the speckle reducing unit has a speckle reducer (21b) that reduces the speckle of the laser light and a speckle reducer that collects the laser light on the speckle reducer. It includes a 1-condensing lens (21a) and a second condensing lens (21c) that condenses the laser light through the speckle reducer on the fly-eye integrator.

Further, the confocal scanner according to one aspect of the present invention includes a first bending mirror (M1) that bends the optical axis between the speckle reduction unit and the flyeye integrator, and the flyeye integrator and the collimating lens. A second bending mirror (M2) that bends the optical axis between them is provided.

Further, in the confocal scanner according to one aspect of the present invention, the fly-eye integrator preliminarily refers to the first fly-eye lens (22a) into which the light output from the light source unit is incident and the first fly-eye lens. It includes a second fly-eye lens (22b) arranged at a predetermined interval, and a Fourier lens (22c) into which light is incident through the second fly-eye lens.

Further, in the confocal scanner according to one aspect of the present invention, the collimating lens is arranged in the vicinity of the ejection side focal plane of the Fourier lens or the ejection side focal plane.

Further, in the confocal scanner according to one aspect of the present invention, the first fly-eye lens is arranged so as to be optically conjugated with the ejection side focal plane of the Fourier lens.

Further, the cofocal scanner according to one aspect of the present invention includes the lens pitch p _{LA of} the first fly-eye lens and the second fly-eye lens, the focal length f _LA1 of the first fly-eye lens, and the focal point of the Fourier lens. The distance f _FL is set so as to satisfy the following equation (1), where the width in the radial direction of the region where the microlens is formed on the first disk is D _LA3 .

Further, in the cofocal scanner according to one aspect of the present invention, the focal length f _{FL of the} Fourier lens and the lens pitch p _LA of the first fly-eye lens and the second fly-eye lens are the microlenses in the first disk. the focal length f _LA3, the diameter of the pin hole in the second disk when the d _PH, is set so as to satisfy the following equation (2).

The confocal microscope (1 to 5) according to one aspect of the present invention generates illumination light for scanning the sample (SP) from the light source unit (10) and the light output from the light source unit. The confocal scanner according to the above and a photographing device (40) for photographing a confocal image of the sample irradiated with the illumination light.

According to the present invention, it is possible to perform flexible installation without complicating the configuration and significantly increasing the cost, and it is possible to obtain a homogeneous confocal image with no uneven brightness.

It is a figure which shows the main part structure of the confocal microscope by 1st Embodiment of this invention. It is a figure which shows the structural example of the light source part of the confocal microscope according to 1st Embodiment of this invention. It is a figure which shows the structural example of the speckle reducer of the confocal microscope by 1st Embodiment of this invention. It is a figure for demonstrating the design requirement of the fly eye integrator in 1st Embodiment of this invention. It is a figure which shows the main part structure of the confocal microscope by the 2nd Embodiment of this invention. It is a figure which shows the main part structure of the confocal microscope according to the 3rd Embodiment of this invention. It is a block diagram which shows the schematic structure of the confocal microscope according to 4th Embodiment of this invention. It is a block diagram which shows the schematic structure of the confocal microscope according to the 5th Embodiment of this invention.

Hereinafter, the confocal scanner and the confocal microscope according to the embodiment of the present invention will be described in detail with reference to the drawings. Hereinafter, an outline of an embodiment of the present invention will be described first, and then details of each embodiment of the present invention will be described.

〔Overview〕
An embodiment of the present invention is a confocal scanner and a confocal microscope that can be flexibly installed without complicating the configuration and significantly increasing the cost, and can obtain a homogeneous confocal image without uneven brightness. It provides a microscope. Specifically, by sufficiently uniformizing the intensity distribution of the illumination light emitted from the confocal scanner used in the confocal microscope, it is possible to obtain a uniform confocal image without uneven brightness. It is a thing.

Here, Patent Document 2 described above discloses a technique for making the intensity distribution of illumination light uniform by providing a multimode fiber and an optical coupling unit between a light source and a confocal scanner. Specifically, the laser light emitted from the light source is guided by a multimode fiber to make the intensity distribution of the laser light uniform, and the laser light having the uniform intensity distribution is irradiated to the microlens array disk of the confocal scanner. This makes it possible to obtain illumination light with a uniform intensity distribution.

Further, in Patent Document 3 described above, the intensity distribution of the illumination light is made uniform by providing a magnifying optical system, an illumination homogenizing member using a glass rod or the like, and a relay optical system between the light source and the confocal scanner. The technology to be used is disclosed. Specifically, the laser beam emitted from the ejection end face of the optical fiber is irradiated to the incident end face of the illumination homogeneous member by the magnifying optical system, and the intensity distribution of the irradiated laser light is made uniform by the illumination homogeneous member to obtain the intensity distribution. By irradiating the microlens array disk of the confocal scanner with the uniformed laser light by the relay optical system, the illumination light having a uniform intensity distribution is obtained.

Further, Patent Document 4 described above discloses a technique for equalizing the intensity distribution of illumination light by providing an optical equalizing means using a microlens array, a fiber bundle, or the like between a light source and a confocal scanner. doing. Specifically, the laser beam emitted from the light source is irradiated to the incident end face of the light equalizing means, the intensity distribution of the irradiated laser light is made uniform by the light equalizing means, and the intensity distribution is made uniform. Is applied to the microlens array disk of the confocal scanner to obtain illumination light with a uniform intensity distribution.

However, in the technique disclosed in Patent Document 2 described above, in order to sufficiently make the intensity distribution of the laser light uniform, a multimode fiber having a sufficient length corresponding to the uniformity of the intensity distribution is required. .. Then, the cost increases significantly, and flexible installation becomes difficult due to the bending radius. Further, Patent Document 2 described above also discloses a technique of attaching a vibration exciter to a multimode fiber to randomize the phase of the laser beam guided by the multimode fiber, but the configuration becomes complicated and vibration occurs. Deterioration of the confocal image due to this is considered.

Further, in the technique disclosed in Patent Document 3 described above, in order to sufficiently homogenize the intensity distribution of the laser beam, an illumination homogeneous member having a sufficient length corresponding to the uniformity of the intensity distribution is required. .. Then, the cost is significantly increased, and the flexible installation becomes difficult due to the shape (columnar) of the illumination homogeneous member. Further, Patent Document 3 described above also discloses a technique for attaching a speckle removal mechanism to a magnifying optical system or a relay optical system, but it is attached to the magnifying optical system or the relay optical system as the cost configuration becomes complicated. Since the illumination light is diffused by the speckle removal mechanism, the loss of the illumination light becomes large.

Further, in the technique disclosed in Patent Document 4 described above, since the illumination light emitted from the light homogenizing means is not collimated, the illumination light is a microlens array of the confocal scanner depending on the location in the illumination region. Depending on the microlens of the disc, it may not be focused in the center of the pinhole. Then, the ratio of the illumination light passing through the pinhole differs depending on the location in the illumination region, and there is a problem that the intensity distribution of the illumination light emitted from the confocal scanner cannot be sufficiently made uniform.

Further, in the technique disclosed in Patent Document 4 described above, if the distance between the light equalizing means and the confocal scanner is set long, the illumination light can be regarded as parallel light generated from a position close to infinity. However, with such a setting, since only a small part of the illumination light can pass through the confocal scanner, the utilization efficiency of the illumination light is lowered, and it becomes difficult to capture a bright confocal image. In addition, the equipment becomes enormous, resulting in a significant increase in cost and difficulty in flexible installation.

In the embodiment of the present invention, the intensity distribution of the light output from the light source unit is made uniform by the flyeye integrator, the light having the uniformed intensity distribution is converted into parallel light by the collimating lens, and the light having a plurality of microlenses is provided. It is converted by a collimating lens by a disc unit provided with one disc and a second disc having a plurality of pinholes formed in association with the microlens and configured to be rotatable together with the first disc. Illumination light for scanning the sample is generated from the parallel light. As a result, the sample is irradiated with illumination light having a uniform intensity distribution, so that a uniform confocal image with no uneven brightness can be obtained. Further, in the embodiment of the present invention, since the intensity distribution of the light output from the light source unit is made uniform by the fly-eye integrator, flexible installation can be performed without complicating the configuration and significantly increasing the cost. It is possible.

[First Embodiment]
<Main components of confocal microscope>
FIG. 1 is a diagram showing a main configuration of a confocal microscope according to the first embodiment of the present invention. As shown in FIG. 1, the confocal microscope 1 of the present embodiment includes a light source unit 10, a confocal scanner 20, a microscope 30, and a camera 40 (imaging device). In such a confocal microscope 1, the confocal scanner 20 generates illumination light for scanning the sample SP from the light output from the light source unit 10, and the confocal image of the sample SP irradiated with the illumination light is captured by the camera. You get at 40.

The light source unit 10 outputs the light required to illuminate the sample SP. The light output from the light source unit 10 may be coherent light (laser light) or incoherent light. In the present embodiment, in order to facilitate understanding, the light output from the light source unit 10 is assumed to be laser light. The light source unit 10 outputs light in a wavelength range of, for example, 400 to 800 [nm]. The wavelength range of the light output from the light source unit 10 is not limited to the above wavelength range (400 to 800 [nm]), but may be an arbitrary wavelength range according to the optical characteristics of the sample SP. can do.

FIG. 2 is a diagram showing a configuration example of a light source portion of a confocal microscope according to the first embodiment of the present invention. The light source unit 10 shown in FIG. 2A has a configuration including a light source device 11 and an optical fiber 12. The light source device 11 outputs light in the above wavelength range (400 to 800 [nm]), for example. The light source device 11 may include a plurality of laser elements having different oscillation wavelengths, and may include one or a plurality of laser elements having variable oscillation wavelengths.

The optical fiber 12 guides the laser beam output from the light source device 11 to the confocal scanner 20. The optical fiber 12 may be a single mode fiber (SMF) or a multimode fiber (MMF). In the light source unit 10 shown in FIG. 2A, the output end T1 of the optical fiber 12 is the output end of the light source unit 10.

The light source unit 10 shown in FIG. 2B has a configuration including only the light source device 11. That is, the light source unit 10 shown in FIG. 2B has a configuration in which the optical fiber 12 of the light source unit 10 shown in FIG. 2A is omitted. The light source device 11 is the same as that shown in FIG. 2A. In the light source unit 10 shown in FIG. 2B, the output end T2 of the light source device 11 is the output end of the light source unit 10. The light source unit 10 shown in FIG. 2B is simpler and more compact than the light source unit 10 shown in FIG. 2A, and the loss of light due to the optical fiber 12 can be reduced. It can improve utilization efficiency.

The cofocal scanner 20 generates illumination light for scanning the sample SP from the light output from the light source unit 10, and also irradiates the sample SP with the illumination light to obtain reflected light, fluorescence, and the like (hereinafter, these are used). When collectively referred to, it simply guides the "return light") to the camera 40. The confocal scanner 20 includes a speckle reduction unit 21, a fly-eye integrator 22, a collimating lens 23, a disc unit 24, a beam splitter 25, a relay optical system 26, and an optical filter 27.

The speckle reduction unit 21 is provided to reduce the speckle of the light (laser light) output from the light source unit 10. If it is not necessary to reduce the speckle of the light output from the light source unit 10, the speckle reduction unit 21 can be omitted. The speckle reduction unit 21 includes a condenser lens 21a (first condenser lens), a speckle reducer 21b, and a condenser lens 21c (second condenser lens).

The condenser lens 21a collects the light output from the light source unit 10 on the speckle reducer 21b. The speckle reducer 21b reduces the speckle of the light condensed by the condenser lens 21a. The condenser lens 21c collects the light passing through the speckle reducer 21b on the fly-eye integrator 22. The condensing lens 21c does not condense the light passing through the speckle reducer 21b to one point of the flyeye integrator 22, but condenses the light (light having a spreading angle) passing through the speckle reducer 21b. Note that the light is parallel or generally parallel.

FIG. 3 is a diagram showing a configuration example of a speckle reducer of a confocal microscope according to the first embodiment of the present invention. The speckle reducer 21b shown in FIG. 3A is a rotary diffuser type including a diffuser DP and a motor MT. The diffuser DP is, for example, frosted glass in which fine and random irregularities are formed on the surface of a glass plate, a lens array in which microlenses are randomly formed, and the like. The motor MT rotates the diffuser DP in its plane. The speckle can be reduced by incidenting the light collected by the condenser lens 21a on the rotating diffuser plate DP.

The speckle reducer 21b shown in FIG. 3B is of a vibration diffuser type including a diffuser DP and an actuator AC. The diffuser plate DP is the same as the diffuser plate DP shown in FIG. 3 (a). However, the outer shape of the diffuser DP may be different from that of the diffuser DP shown in FIG. 3 (a). The actuator AC is, for example, a piezo element or an electroactive polymer, and vibrates (for example, reciprocates) the diffuser plate DP. The speckle can be reduced by incidenting the light collected by the condenser lens 21a on the vibrating diffuser DP.

The fly-eye integrator 22 outputs light from the light source unit 10 and makes the light intensity distribution uniform through the speckle reduction unit 21. The fly-eye integrator 22 includes a first fly-eye lens 22a, a second fly-eye lens 22b, and a Fourier lens 22c, and makes the intensity distribution of the Fourier lens 22c on the ejection side focal plane uniform.

The first fly-eye lens 22a and the second fly-eye lens 22b are lens bodies in which a plurality of lenses are arranged in a matrix with a predetermined lens pitch. The shape of the plurality of lenses (shapes viewed in the optical axis direction) arranged and formed on the first fly-eye lens 22a may be any shape, but is preferably rectangular. This is due to the following reasons.

That is, the shape (cross-sectional shape) of the light on the focal plane on the injection side of the Fourier lens 22c is similar to the shape of the lens formed on the first fly-eye lens 22a. Generally, since the shape of the image sensor provided in the camera 40 is rectangular, the loss of light can be reduced by making the outer shape of the light on the focal plane on the emission side of the Fourier lens 22c rectangular. This is because a bright confocal image can be taken.

The first fly-eye lens 22a is arranged at a position where the light collected by the condenser lens 21c of the speckle reduction unit 21 is incident. Here, the first fly-eye lens 22a is arranged so as to be optically conjugated with the focal plane on the injection side of the Fourier lens 22c. In such an arrangement, the injection aperture surface of each lens of the first fly-eye lens 22a is focused on the injection side of the Fourier lens 22c via the corresponding lenses of the second fly-eye lens 22b and the Fourier lens 22c. This is for image projection on a surface.

As a result, the light irradiation shape on the ejection side focal plane of the Fourier lens 22c becomes a shape similar to the ejection aperture of each lens of the first fly-eye lens 22a. Further, the light intensity distribution on the emission side focal plane of the Fourier lens 22c has few side lobes (transition region of the boundary between the light irradiation region and the non-irradiation region) and a steep flat top (light in the light irradiation region). The irradiation distribution is uniform).

The second fly-eye lens 22b is arranged with respect to the first fly-eye lens 22a at a predetermined interval in the optical axis direction. Here, the second fly-eye lens 22b is arranged so as to be optically conjugated with the output ends (output ends T1 and T2 shown in FIG. 2) of the light source unit 10 and the diffuser plate DP (see FIG. 3). The reason for such an arrangement is that a plurality of point light sources similar to the output end of the light source unit 10 are formed on the second fly-eye lens 22b.

The Fourier lens 22c is a lens into which light is incident through the second fly-eye lens 22b. Here, as described above, the second fly-eye lens 22b is formed with a plurality of point light sources similar to the output ends of the light source unit 10. The Fourier lens 22c is provided to collect the light emitted from each of the plurality of point light sources formed on the second fly-eye lens 22b and spatially superimpose the light.

The collimating lens 23 converts light whose intensity distribution is uniformed by the fly-eye integrator 22 into parallel light. The parallel light here refers to a state in which the light emitted from each of the plurality of point light sources formed on the second fly-eye lens 22b becomes parallel light. That is, it is a collection of a plurality of parallel lights having slightly different traveling directions. Here, the collimating lens 23 is arranged on the injection side focal plane (or in the vicinity of the injection side focal plane) of the Fourier lens 22c provided on the flyeye integrator 22. Such an arrangement maximizes the size of the parallel light (the size of the cross-sectional shape) emitted to the disk unit 24 while making the light intensity distribution on the focal plane on the injection side of the Fourier lens 22c uniform. Because.

The disk unit 24 generates illumination light for scanning the sample SP from the parallel light converted by the collimated lens 23. The disc unit 24 includes a microlens array disc 24a (first disc), a pinhole array disc 24b (second disc), a rotating shaft 24c, and the like. In FIG. 1, in order to simplify the illustration, the motor that rotationally drives the microlens array disk 24a and the pinhole array disk 24b around the rotation shaft 24c is omitted.

The microlens array disc 24a is a disc-shaped disc having a plurality of microlenses formed in a predetermined pattern. The pinhole array disk 24b is a disk-shaped disk having a plurality of pinholes formed in association with the microlens of the microlens array disk 24a. A microlens array disk 24a and a pinhole array disk 24b are attached to both ends of the rotating shaft 24c, respectively. Therefore, the microlens array disk 24a and the pinhole array disk 24b are both (integrally) rotatable around the rotation shaft 24c.

The beam splitter 25 transmits the light (illumination light) emitted to the sample SP and reflects the return light obtained by irradiating the sample SP with the illumination light toward the relay optical system 26. Specifically, the beam splitter 25 is arranged on the optical axis of the collimating lens 23, between the microlens array disk 24a provided in the disk unit 24 and the pinhole array disk 24b. As the beam splitter 25, a polarizing beam splitter, a half mirror, a dichroic mirror, or the like can be used.

The beam splitter 25 transmits a plurality of luminous fluxes divided and converged by a microlens provided on the microlens array disk 24a. The light flux transmitted through the beam splitter 25 is focused on a pinhole provided in the pinhole array disk 24b, passes through the pinhole, and is emitted to the outside of the disc unit 24. At this time, since the illumination light emitted to the microlens array disk 24a has a uniform intensity distribution and parallel light, the illumination light emitted to the outside of the disk unit 24 through the pinhole also has a uniform intensity distribution. It becomes.

This is because when the illumination light applied to the microlens array disk 24a has a uniform intensity distribution and parallel light, the amount of light incident on each of the microlenses of the microlens array disk 24a is the same. is there. Further, since the illumination light is focused near the center of the pinhole corresponding to each of the microlenses, the ratio of the illumination light passing through each of the pinholes is the same.

On the contrary, when the illumination light emitted to the microlens array disk 24a is not parallel light but divergent light or convergent light, the illumination light is near the center of the pinhole corresponding to the microlens depending on the location of the microlens. Does not collect light. As a result, the proportion of the illumination light passing through the corresponding pinhole changes depending on the location of the microlens, resulting in uneven brightness.

From the above, in order to emit the illumination light having a uniform intensity distribution from the disk unit 24, it is necessary that the illumination light emitted to the microlens array disk 24a is parallel light. Further, the beam splitter 25 reflects the return light transmitted through the pinhole provided in the pinhole array disk 24b toward the relay optical system 26 among the return light obtained by irradiating the sample SP with the illumination light.

The relay optical system 26 includes relay lenses 26a and 26b, and is an optical system that guides the return light reflected by the beam splitter 25 to the camera 40. The optical filter 27 is arranged between the relay lens 26a and the relay lens 26b of the relay optical system 26, and filters the return light guided to the camera 40 by the relay optical system 26. As the optical filter 27, a polarizing filter, an absorption filter (emission filter), a dichroic mirror, or the like can be used.

The microscope 30 irradiates the sample SP with the illumination light generated by the disk unit 24, and guides the return light obtained by irradiating the sample SP with the illumination light to the disk unit 24. The microscope 30 is an infinity correction optical system including an imaging lens 31 and an objective lens 32. Note that, in FIG. 1, the sample SP is shown inside the microscope 30 for convenience, but it should be noted that the sample SP does not constitute the microscope 30 and that the sample SP is replaceable. ..

The camera 40 obtains a confocal image of the sample SP. The camera 40 is equipped with a solid-state image sensor such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor), and captures a two-dimensional still image or moving image. It is a possible camera. The confocal image of the sample SP obtained by the camera 40 may be displayed on a display device (not shown), for example.

<Design requirements for fly-eye integrator>
FIG. 4 is a diagram for explaining the design requirements of the fly-eye integrator according to the first embodiment of the present invention. 4 (a) is, _(more precisely, a microlens array size D _FT in the radial direction of the disk _24a) size D _FT of parallel light is irradiated onto the disk unit 24 for the design requirements of the fly-eye integrator 22 (first It is a figure explaining 1 design requirement). FIG. 4B is a diagram illustrating a design requirement (second design requirement) of the fly-eye integrator 22 for emitting uniform illumination light from the disc unit 24.

<< First design requirements >>
As shown in FIG. 4A, the following parameters are defined for the flyeye integrator 22.
f _LA1 : Focal length of the first fly-eye lens 22a f _LA2 : Focal length of the second fly-eye lens 22b a ₁₂ : Spacing between the first fly-eye lens 22a and the second fly-eye lens 22b p _LA : First fly-eye Lens pitch of lens 22a and second fly-eye lens 22b f _FL : Focal length of Fourier lens 22c

Size D _FT of parallel light irradiated to the disk unit 24 is maximized when as described above, the collimator lens 23 is disposed on the exit-side focal plane of the Fourier lens 22c (or, near the exit-side focal plane) Become. In the case of such an arrangement, the magnitude _DFT of the parallel light applied to the disk unit 24 is represented by the following equation (3).

When the first fly-eye lens 22a is arranged so as to be optically coupled to the emission-side focal plane of the Fourier lens 22c, as described above, the light on the emission-side focal plane of the Fourier lens 22c The intensity distribution becomes uniform (specifically, the intensity distribution of a steep flat top with few side lobes). In order to make such an arrangement, f _LA1 = f _LA2 = a ₁₂ may be set. Then, the above equation (3) is represented by the following equation (4).

Here, in order to obtain a homogeneous confocal image without uneven brightness, the sample SP is irradiated with illumination light without loss, and the entire sample SP (the portion where the confocal image is obtained by the camera 40) is obtained. It is necessary to ensure that uniform illumination light is emitted (over the entire area). For this purpose, the magnitude _DFT of the parallel light applied to the disc unit 24 is the width D _LA3 in the radial direction of the region where the microlens is formed in the microlens array disc 24a (see FIG. 4A). It is desirable that the width is close to 1/4 of the width D _LA3 or more.

Therefore, the fly-eye integrator 22 is designed so as to satisfy the following equation (5).

<< Second design requirements >>
As shown in FIG. 4B, the following parameters are defined for the fly-eye integrator 22 and the disk unit 24.
d _IN : Diameter of the luminous flux incident on the first fly-eye lens 22a f _LA3 : Focal length of the microlens on the microlens array disk 24a d _PH : Diameter of the pinhole on the pinhole array disk 24b

In the first fly-eye lens 22a, the position of the center of the outermost lens on which the luminous flux is incident is at a distance indicated by (d _IN −p _LA ) / 2 from the optical axis. As shown in FIG. 4B, the light that has passed through this lens is obliquely incident on the focal plane on the emission side of the Fourier lens 22c at an angle φ (an angle to be the optical axis). This angle φ is expressed by the following equation (6).

Further, a plurality of luminous fluxes divided by the microlens provided on the microlens array disk 24a also obliquely enter the pinhole array disk 24b at the above angle φ (angle to be the optical axis). In order for the plurality of luminous fluxes divided by the microlens to pass through the pinholes formed in the pinhole array disk 24b without loss, the plurality of luminous fluxes divided by the microlenses pass through the inside of the inner wall of the pinholes. Must. For this purpose, it is necessary to satisfy the following equation (7).

From the above equation (6) and the above equation (7), the following equation (8) can be obtained.

By modifying the above equation (8), the following equation (9) can be obtained.

Here, in order to obtain light having a uniform intensity distribution in the fly-eye integrator 22, at least four (2 × 2) or more lenses among the lenses formed on the first fly-eye lens 22a have a luminous flux. Need to be incident. This is because when the luminous flux is incident on only one lens, the incident luminous flux is not divided and the divided luminous flux is not superposed, and the intensity distribution is not uniform.

Therefore, the diameter d _IN of the luminous flux incident on the first fly-eye lens 22a needs to satisfy the following equation (10).

By substituting the above equation (10) into the above equation (9), the following equation (11) is obtained. The fly-eye integrator 22 is designed so as to satisfy the following equation (11).

<Operation of confocal microscope>
When the operation of the confocal microscope 1 is started, light is output from the light source unit 10 and the rotation of the disc unit 24 (rotation around the rotation axis 24c) is started. The light output from the light source unit 10 is input to the confocal scanner 20. It is assumed that the light output from the light source unit 10 is light having an intensity distribution (for example, light having a Gaussian distribution).

The light input to the confocal scanner 20 first enters the speckle reduction unit 21, and is condensed by the condenser lens 21a to the speckle reducer 21b. Specifically, the light is collected on the diffuser DP (rotating or vibrating diffuser DP shown in FIG. 3) provided in the speckle reducer 21b. The speckle is reduced by the light incident on the speckle reduction unit 21 passing through the diffuser DP. The light passing through the speckle reducer 21b (light with reduced speckle) is condensed by the condenser lens 21c and irradiated to the first flyeye lens 22a of the flyeye integrator 22.

The light irradiated to the first fly-eye lens 22a is divided into a plurality of luminous fluxes by a plurality of lenses provided on the first fly-eye lens 22a. The plurality of divided light fluxes are imaged on the plurality of lenses provided on the second fly-eye lens 22b, respectively. As a result, a plurality of point light sources similar to the output ends (output ends T1 and T2 shown in FIG. 2) of the light source unit 10 are formed on the second fly-eye lens 22b.

The light emitted from each of the plurality of point light sources formed on the second fly-eye lens 22b is collected by the Fourier lens 22c and spatially superimposed. The superimposed light is converted into parallel light by the collimated lens 23 arranged on the emission side focal plane (or near the emission side focal plane) of the Fourier lens 22c, and is converted into parallel light on the microlens array disk 24a of the disk unit 24. Be irradiated. The parallel light emitted to the microlens array disk 24a has a uniform intensity distribution by the fly-eye integrator 22 (specifically, a steep flat top intensity distribution with few side lobes). ).

The parallel light irradiated on the microlens array disc 24a is divided into a plurality of luminous fluxes by the microlens provided on the microlens array disc 24a. The plurality of luminous fluxes divided by the microlens pass through the beam splitter 25 and are emitted to the outside of the disc unit 24 as illumination light through the pinholes formed in the pinhole array disk 24b corresponding to the microlens. The lens.

The illumination light emitted to the outside of the disk unit 24 irradiates the sample SP through the imaging lens 31 and the objective lens 32 provided in the microscope 30 in this order. Here, the illumination light emitted to the outside of the disc unit 24 is obtained by dividing the parallel light having a uniform intensity distribution. Therefore, the sample SP is irradiated with illumination light having a uniform intensity distribution.

The return light from the sample SP (the return light obtained by irradiating the sample SP with the illumination light) enters the disk unit 24 through the objective lens 32 and the imaging lens 31 provided in the microscope 30 in this order. Then, after passing through the pinhole formed in the pinhole array disk 24b of the disk unit 24, the beam splitter 25 reflects the pinhole toward the relay optical system 26.

The return light reflected by the beam splitter 25 enters the camera 40 in order through the relay lens 26a of the relay optical system 26, the optical filter 27, and the relay lens 26b of the relay optical system 26, and forms an image. Here, since the disc unit 24 rotates around the rotation shaft 24c, the illumination light applied to the sample SP is scanned according to the rotation of the disc unit 24. As a result, the return light corresponding to the scanning position of the illumination light is sequentially input to the camera 40. In this way, a confocal image of the sample SP is obtained by the camera 40.

As described above, in the present embodiment, the confocal scanner 20 provided with the fly-eye integrator 22, the collimating lens 23, and the disk unit 24 is used to homogenize the intensity distribution of the light output from the light source unit 10 and convert it into parallel light. , The illumination light for scanning the sample SP is generated from the parallel light. As a result, the sample SP can be irradiated with illumination light having a uniform intensity distribution, so that a uniform confocal image with no uneven brightness can be obtained.

Further, in the present embodiment, since the intensity distribution of the light output from the light source unit 10 is made uniform by the fly-eye integrator 22, flexible installation can be performed without complicating the configuration and significantly increasing the cost. It is possible. In addition, in the present embodiment, the shapes of the plurality of lenses (shapes viewed in the optical axis direction) arranged in the first fly-eye lens 22a of the fly-eye integrator 22 are changed to the shape of the image sensor provided in the camera 40 (shapes seen in the optical axis direction). Since it has a rectangular shape), a bright confocal image can be obtained with little light loss.

[Second Embodiment]
FIG. 5 is a diagram showing a main configuration of a confocal microscope according to the second embodiment of the present invention. In FIG. 5, the same reference numerals are given to the same configurations as those shown in FIG. 1. As shown in FIG. 5, the confocal microscope 2 of the present embodiment has a configuration in which the confocal scanner 20 of the confocal microscope 1 shown in FIG. 1 is replaced with the confocal scanner 20A.

The confocal scanner 20A has a configuration in which a folding mirror M1 (first folding mirror) and a folding mirror M2 (second folding mirror) are added to the confocal scanner 20 shown in FIG. The folding mirror M1 is a mirror that bends the optical axis between the speckle reduction unit 21 and the fly-eye integrator 22. The folding mirror M2 is a mirror that bends the optical axis between the fly-eye integrator 22 and the collimating lens 23.

By providing these folding mirrors M1 and M2, the optical path from the speckle reduction unit 21 to the confocal scanner 20A can be made into a Z shape, and the confocal scanner 20A can be made compact. The confocal scanner 20A of the present embodiment is different from the confocal scanner 20 of the first embodiment in that it includes the folding mirrors M1 and M2. Therefore, also in the present embodiment, flexible installation is possible without complicating the configuration and significantly increasing the cost, and a homogeneous confocal image without uneven brightness can be obtained.

[Third Embodiment]
FIG. 6 is a diagram showing a main configuration of a confocal microscope according to a third embodiment of the present invention. In FIG. 6, the same reference numerals are given to the same configurations as those shown in FIG. 1. As shown in FIG. 6, the confocal microscope 3 of the present embodiment has a configuration in which the light source unit 10 and the confocal scanner 20 of the confocal microscope 1 shown in FIG. 1 are replaced with the light source unit 10B and the confocal scanner 20B, respectively.

The light source unit 10B is different from the light source unit 10 shown in FIG. 1 in that it outputs collimated light (for example, laser light). The confocal scanner 20B has a configuration in which the speckle reduction unit 21 of the confocal scanner 20 shown in FIG. 1 is replaced with the speckle reduction unit 21B. The speckle reduction unit 21B has a configuration in which the condenser lens 21a of the speckle reduction unit 21 shown in FIG. 1 is omitted, and a speckle reducer 21b and a condenser lens 21c are provided.

In the confocal microscope 3 of the present embodiment, the light output from the light source unit 10B is directly incident on the speckle reducer 21b provided in the speckle reduction unit 21B. As described above, in the present embodiment, the condenser lens 21a shown in FIG. 1 can be omitted, so that the condenser lens 21a can be made compact. Further, in the present embodiment, the light output from the light source unit 10B is directly incident on the speckle reducer 21b to reduce the light loss, so that the light utilization efficiency can be improved. The confocal scanner 20B in this embodiment is different from the confocal scanner 20 in the first embodiment in that the condenser lens 21a is omitted. Therefore, also in the present embodiment, flexible installation is possible without complicating the configuration and significantly increasing the cost, and a homogeneous confocal image without uneven brightness can be obtained.

[Fourth Embodiment]
FIG. 7 is a block diagram showing a schematic configuration of a confocal microscope according to a fourth embodiment of the present invention. In the confocal microscope of the present embodiment, the confocal microscopes 1 to 3 according to the first to third embodiments are divided into a plurality of units, and the combination of each unit can be changed as needed. In the following, an example in which the confocal microscope 1 according to the first embodiment shown in FIG. 1 is divided into a plurality of units will be described.

As shown in FIG. 7A, the confocal microscope 4 of the present embodiment includes a light source unit U1, a homogenization unit U2, a scanner unit U3, a microscope unit U4, and a camera unit U5. The light source unit U1 is a unit in which the light source unit 10 shown in FIG. 1 is housed in a housing. The light source unit U1 can output the light output from the light source unit 10 from the output end T11 formed in the housing.

The homogenization unit U2 is a unit in which the speckle reduction unit 21, the fly-eye integrator 22, and the collimating lens 23 of the confocal scanner 20 shown in FIG. 1 are housed in a housing. The homogenization unit U2 equalizes the intensity distribution of the light input from the input end T21 formed in the housing and converts it into parallel light, and the parallel light having the uniform intensity distribution is formed in the housing. Output is possible from the output end T22.

The scanner unit U3 is a unit in which the disk unit 24, the beam splitter 25, the relay optical system 26, the optical filter 27, and the mirrors m1 and m2 of the confocal scanner 20 shown in FIG. 1 are housed in a housing. In FIG. 7, only the disk unit 24, the beam splitter 25, and the mirrors m1 and m2 are shown, and the relay optical system 26 and the optical filter 27 are not shown.

The scanner unit U3 can reflect the light input from the input end T31 formed in the housing by the mirror m1 and output the light from the output end T32 formed in the housing. Further, the scanner unit U3 reflects the light input from the input end T33 formed in the housing toward the disk unit 24 by the mirror m2, and the illumination light generated by the disk unit 24 (to scan the sample SP). It is possible to output the illumination light) from the input / output end T34 formed in the housing. Further, the scanner unit U3 reflects the return light (return light obtained by irradiating the sample SP with illumination light) input from the input / output terminal T34 by the beam splitter 25 from the output terminal T35 formed in the housing. It is possible to output.

Here, the mirrors m1 and m2 provided in the scanner unit U3 are configured to be movable. By moving the mirrors m1 and m2, the optical path of light in the scanner unit U3 can be changed. For example, in the example shown in FIG. 7A, when the mirrors m1 and m2 are retracted from the optical path between the input end T31 and the disk unit 24, the mirrors m1 and m2 are input from the input end T31 as shown in FIG. 7B. It is possible to direct the emitted light directly to the disc unit 24 without going through the homogenizing unit U2.

When it is necessary to equalize the light intensity of the light input from the input end T31, as shown in FIG. 7A, the equalization unit U2 is attached to the scanner unit U3, and the input end T31 and the disk unit 24 are used. The mirrors m1 and m2 may be arranged on the optical path between the two. On the other hand, when it is not necessary to equalize the light intensity of the light input from the input end T31, the equalization unit U2 is not attached to the scanner unit U3 as shown in FIG. 7B, and the input end is not equalized. The mirrors m1 and m2 may be retracted from the optical path between the T31 and the disk unit 24.

The microscope unit U4 is a unit in which the microscope 30 shown in FIG. 1 is housed in a housing. The microscope unit U4 can irradiate the sample SP (not shown) with the light input from the input / output end T41 formed in the housing, and output the return light from the sample SP from the input / output end T41. .. The camera unit U5 is a unit in which the camera 40 shown in FIG. 1 is housed in a housing. The camera unit U5 can obtain a confocal image from the light input from the input end T51 stored in the housing.

As described above, in the confocal microscope 4 of the present embodiment, the configurations of the confocal microscopes 1 to 3 according to the first to third embodiments are the light source unit U1, the homogenization unit U2, the scanner unit U3, and the microscope unit U4. And the camera unit U5. Thereby, for example, the light source unit U1 to be combined with the scanner unit U3 can be changed, or whether or not the uniformization unit U2 can be combined with the scanner unit U3 can be selected according to the optical properties of the sample SP. , Practicality can be improved.

[Fifth Embodiment]
FIG. 8 is a block diagram showing a schematic configuration of a confocal microscope according to a fifth embodiment of the present invention. In FIG. 8, the same reference numerals are given to the same configurations as those shown in FIG. 7. The confocal microscope of the present embodiment is the same as the confocal microscope 4 of the fourth embodiment, in which the confocal microscopes 1 to 3 according to the first to third embodiments are divided into a plurality of units. However, the confocal microscope of the present embodiment is different from the confocal microscope 4 of the fourth embodiment in that a light source unit and a homogenization unit can be added as needed.

As shown in FIG. 8, the confocal microscope 5 of the present embodiment includes a light source unit U1, a scanner unit U3A, a microscope unit U4, and a camera unit U5. In this confocal microscope 5, the light source unit U1A and the homogenization unit U2A can be added to the outside of the scanner unit U3A as needed. The scanner unit U3A has a configuration in which the mirror m1 of the scanner unit U3 shown in FIG. 7A is omitted, and the uniformization unit U2 is built in the optical path between the input terminal T31 and the mirror m2.

The light source unit U1A is the same as the light source unit U1 shown in FIG. 7A, and can output light from the output end T12 formed in the housing. The light output from the output end T12 of the light source unit U1A and the light output from the output end T11 of the light source unit U1 may be the same or different. For example, light having the same wavelength may be used, or light having different wavelengths may be used.

The homogenization unit U2A has the same configuration as the homogenization unit U2, and homogenizes the intensity distribution of the light input from the input end T23 formed in the housing and converts it into parallel light to make the intensity distribution uniform. The parallel light can be output from the output end T24 formed in the housing. The light output from the output end T24 of the homogenizing unit U2A is input into the scanner unit U3A from the input end T33 formed in the housing of the scanner unit U3A.

In such a confocal microscope 5, for example, a light source unit U1 provided with a laser light source and a light source unit U1A provided with an LED (Light Emitting Diode) light source can be used. Then, by arranging the mirror m2 on the optical path between the input end T31 and the disk unit 24 or retracting the mirror m2 on the optical path, it is possible to select the optimum type of light source according to the purpose of the experiment. it can.

Further, in the confocal microscope 5, for example, a light source unit U1 including a visible light light source and a light source unit U1A including an infrared light source can be used. Then, by using a dichroic mirror as the mirror m2 and arranging the mirror m2 fixedly on the optical path between the input end T31 and the disk unit 24, visible light and infrared light can be selected or infrared light is selected according to the purpose of the experiment. Visible light and infrared light can be used together.

Further, in the confocal microscope 5, for example, the light source unit U1 and U1A whose polarization directions of the output laser light are orthogonal to each other can be used. Then, by using a polarizing beam splitter as the mirror m2 and arranging the mirror m2 fixedly on the optical path between the input end T31 and the disk unit 24, either one of the polarizations is selected according to the purpose of the experiment, or both. Can be used in combination.

When the light source unit U1 and the light source unit U1A are used, it is not always necessary to include the homogenization unit U2 and the homogenization unit U2A together. For example, when it is not necessary to equalize the intensity of the light output from the light source unit U1, the equalization unit U2 can be omitted. Further, when it is not necessary to equalize the intensity of the light output from the light source unit U1A, the uniformization unit U2A can be omitted.

Although the confocal scanner and the confocal microscope according to the embodiment of the present invention have been described above, the present invention is not limited to the above embodiment and can be freely changed within the scope of the present invention. For example, in the fourth embodiment, the example in which the confocal scanner 20 is divided into the homogenization unit U2 and the scanner unit U3 has been described, but the confocal scanner 20 may be one unit. Further, in the fifth embodiment, the example in which the homogenization unit U2 is built in the scanner unit U3A has been described, but the configuration provided in the homogenization unit U2 (the speckle reduction unit of the confocal scanner 20 shown in FIG. 1). 21, the fly-eye integrator 22 and the collimating lens 23) may be built in the scanner unit U3A without being a unit.

1-5 Confocal microscope 10,10B Light source 20, 20A, 20B Confocal scanner 21,21B Speckle reduction 21a, 21c Condensing lens 22 Fly-eye integrator 22a First fly-eye lens 22b Second fly-eye lens 22c Fourier Lens 23 Confocal Lens 24 Disc Unit 24a Microlens Array Disc 24b Pinhole Array Disc 21b Speckle Reducer 40 Camera M1, M2 Folding Mirror SP Sample

Claims (10)

A fly-eye integrator that equalizes the intensity distribution of light output from the light source,
A collimated lens that converts light whose intensity distribution is uniformed by the fly-eye integrator into parallel light, and
A first disk having a plurality of microlenses and a second disk having a plurality of pinholes formed in association with the microlenses and configured to be rotatable together with the first disk are provided. , A disk unit that generates illumination light for scanning a sample from parallel light converted by the collimating lens, and
Confocal scanner with. The light output from the light source unit is laser light.
A speckle reduction unit for reducing the speckle of the laser beam is provided.
The confocal scanner according to claim 1. The speckle reduction unit includes a speckle reducer that reduces the speckle of the laser beam and a speckle reducer.
A first condensing lens that condenses the laser light on the speckle reducer,
A second condensing lens that condenses the laser light through the speckle reducer on the fly-eye integrator, and
2. The confocal scanner according to claim 2. A first bending mirror that bends the optical axis between the speckle reduction unit and the flyeye integrator,
A second folding mirror that bends the optical axis between the fly-eye integrator and the collimating lens,
2. The confocal scanner according to claim 2 or 3. The fly-eye integrator includes a first fly-eye lens into which the light output from the light source unit is incident.
A second fly-eye lens arranged at a predetermined interval with respect to the first fly-eye lens, and
A Fourier lens in which light is incident through the second fly-eye lens and
The confocal scanner according to any one of claims 1 to 4. The confocal scanner according to claim 5, wherein the collimating lens is arranged in the vicinity of the ejection side focal plane of the Fourier lens or the ejection side focal plane. The confocal scanner according to claim 5 or 6, wherein the first fly-eye lens is arranged so as to be optically conjugated with the ejection side focal plane of the Fourier lens. The lens pitch p _{LA of} the first fly-eye lens and the second fly-eye lens, the focal length f _LA1 of the first fly-eye lens, and the focal length f _FL of the Fourier lens are the microlenses in the first disk. The _cofocal length scanner according to any one of claims 5 to 7, wherein the width of the region in which the lens is formed is D _LA3, and the lens is set so as to satisfy the following equation (1).

The focal length f _{FL of the} Fourier lens and the lens pitch p _LA of the first fly eye lens and the second fly eye lens are such that the focal length of the microlens on the first disc is f _LA3 and the focal length on the second disc is f _LA3 . The cofocal length scanner according to any one of claims 5 to 8, which is set so as to satisfy the following equation (2), where _dPH is the diameter of the pinhole.

Light source and
The confocal scanner according to any one of claims 1 to 9, which generates illumination light for scanning a sample from the light output from the light source unit.
An imaging device that captures a confocal image of the sample irradiated with the illumination light, and
Confocal microscope equipped with.

Images