JP7415674B2 - Spinning disks, confocal scanners, and microscopy systems - Google Patents

Description

The present invention relates to spinning disks, confocal scanners, and microscopy systems.

BACKGROUND ART In recent years, a technique of imaging samples such as biological tissues, organs, cells, etc. into two-dimensional or three-dimensional images using a disk scanning confocal microscope has been attracting attention. This technique obtains a confocal image of a sample by rotating a spinning disk to change the irradiation position of illumination light (excitation light) on the sample (scanning the sample with the illumination light). This technique has the advantage of providing images with excellent resolution and contrast because only information in the focal plane is obtained.

Patent Documents 1 and 2 below disclose disk scanning confocal microscopes that can simultaneously obtain multicolor confocal images at the same location. Specifically, in the disk scanning confocal microscope disclosed in Patent Documents 1 and 2 below, a sample is irradiated with illumination light that includes a plurality of excitation lights with different wavelengths, and the fluorescence obtained from the sample is By distinguishing for each wavelength band, multicolor confocal images at the same location can be obtained simultaneously.

JP2009-288087A Patent No. 6484234

By the way, fluorescence obtained from a sample generally has a wide wavelength band (broad spectral width). For this reason, the fluorescence spectrum obtained when a sample is irradiated with illumination light containing multiple excitation lights with similar wavelengths is the overlap of the fluorescence spectra obtained when the sample is irradiated with each excitation light individually. It can become a thing. Since it is difficult to distinguish fluorescence whose spectra overlap each other for each observation wavelength band, a phenomenon occurs in which each fluorescence becomes noise (hereinafter referred to as "fluorescence crosstalk"). When such fluorescence crosstalk occurs, there is a problem in that the resolution and contrast of the confocal image are reduced.

The present invention has been made in view of the above circumstances, and provides a spinning disk, a confocal scanner, and a microscope system that can obtain confocal images with excellent resolution and contrast with almost no influence of fluorescence crosstalk. The purpose is to

In order to solve the above problems, a spinning disk according to one aspect of the present invention is a spinning disk (21) that generates illumination light for scanning a sample (SP) from light including a plurality of wavelengths, and includes a plurality of a first disk (32) having a pinhole (32a) and configured to be rotatable; It includes an optical filter (33) that transmits light in a specific wavelength band that is different for each defined region (R1 to R8, R11 to R14, R21, R22).

Further, in the spinning disk according to one aspect of the present invention, a second disk (31) has a plurality of microlenses (31a) formed in correspondence with the pinhole, and is configured to be rotatable together with the first disk. It further includes:

Further, the spinning disk according to one aspect of the present invention further includes a third disk (35) on which the optical filter is formed and configured to be rotatable together with the first disk.

Alternatively, in the spinning disk according to one aspect of the present invention, the optical filter is formed on the first disk.

Alternatively, in the spinning disk according to one aspect of the present invention, the optical filter is formed on the second disk.

Further, in the spinning disk according to one aspect of the present invention, the third disk is arranged close to the first disk or the second disk.

A confocal scanner (20) according to one aspect of the present invention includes the spinning disk described above and fluorescence obtained by irradiating the sample with the illumination light, which is set to correspond to the specific wavelength band. The fluorescence discriminator unit (24, 25a to 25c, 26a to 26d, 27a to 27d) that discriminates each observation wavelength band.

Further, in the confocal scanner according to one aspect of the present invention, the optical filter may include light in a first specific wavelength band (λ1), light in a second specific wavelength band (λ2), and light in a third specific wavelength band (λ3). , and a region (R1 to R8, R11 to R14) that transmits light in a fourth specific wavelength band (λ4), and the fluorescence discriminating unit distinguishes the fluorescence in a manner corresponding to the first specific wavelength band. A set first observation wavelength band (WB1), a second observation wavelength band (WB2) set corresponding to the second specific wavelength band, and a third observation set corresponding to the third specific wavelength band. Discrimination is made for each wavelength band (WB3) and a fourth observation wavelength band (WB4) set corresponding to the fourth specific wavelength band.

Alternatively, in the confocal scanner according to one aspect of the present invention, the optical filter has a first specific wavelength band (λ1), a second specific wavelength band (λ2), and a third specific wavelength band (λ3) arranged in order of wavelength length. ), and a first region (R21) that transmits light in the first specific wavelength band and third specific wavelength band among the fourth specific wavelength band (λ4), and the second specific wavelength band and the fourth specific wavelength. a second region (R22) that transmits light in a band, and the fluorescence discriminator unit converts the fluorescence into a first observation wavelength band (WB1) set corresponding to the first specific wavelength band. , a second observation wavelength band (WB2) set corresponding to the second specific wavelength band, a third observation wavelength band (WB3) set corresponding to the third specific wavelength band, and the fourth specific wavelength band. Discrimination is performed for each fourth observation wavelength band (WB4) set corresponding to the wavelength band.

A microscope system (1) according to one aspect of the present invention includes a light source unit (10) that outputs light including a plurality of wavelengths, and generates illumination light for scanning a sample from the light output from the light source unit. , a confocal scanner (20) according to any one of the above, which discriminates fluorescence obtained by irradiating the sample with the illumination light for each of the observation wavelength bands, and a confocal image of the sample for each of the observation wavelength bands. The image processing apparatus includes a plurality of photographing devices (30a to 30d) that photograph images, and an image processing device (40) that performs image processing of the confocal images photographed by the photographing devices.

According to the present invention, there is an effect that a confocal image with excellent resolution and contrast can be obtained with almost no influence of fluorescence crosstalk.

1 is a diagram showing a main part configuration of a microscope system according to an embodiment of the present invention. 1 is a diagram illustrating a spinning disk according to an embodiment of the invention. FIG. FIG. 3 is a diagram illustrating an example of the relationship between the wavelength of illumination light irradiated onto a sample and the wavelength band to be observed in an embodiment of the present invention. It is a side view which shows the modification of the spinning disk by embodiment of this invention. FIG. 7 is a plan perspective view showing another modification of the spinning disk according to the embodiment of the present invention.

Hereinafter, a spinning disk, a confocal scanner, and a microscope system according to embodiments of the present invention will be described in detail with reference to the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Below, an overview of an embodiment of the present invention will first be described, and then details of the embodiment of the present invention will be described.

〔overview〕
Embodiments of the present invention provide a spinning disk, a confocal scanner, and a microscope system that can obtain confocal images with excellent resolution and contrast with almost no influence of fluorescence crosstalk. Specifically, by irradiating the sample with illumination light that includes only light in a specific wavelength band that differs for each predefined region from light that includes multiple wavelengths, using the first disk and optical filter, the fluorescent cross It is possible to obtain a confocal image with almost no influence of talk and excellent resolution and contrast.

Here, in the invention disclosed in Patent Document 1 mentioned above, by irradiating a sample with light including a plurality of excitation lights having different wavelengths and discriminating the fluorescence obtained from the sample for each observation wavelength band, Multicolor confocal images are obtained simultaneously. In addition, in the technology disclosed in Patent Document 2 mentioned above, fluorescence obtained from the sample is obtained by irradiating the sample with illumination light that includes two excitation lights whose wavelengths are separated by a certain degree to obtain fluorescence with less spectral overlap. By distinguishing the images for each observation wavelength band, multicolor confocal images of the same location can be obtained simultaneously.

The invention disclosed in Patent Document 1 mentioned above irradiates the sample with light containing a plurality of excitation lights with different wavelengths, so if the wavelengths of the excitation lights included in the illumination light are close, the amount of light obtained from the sample is The emitted fluorescence may have overlapping spectra. Fluorescence with such overlapping spectra is difficult to distinguish for each observed wavelength band, resulting in fluorescence crosstalk. As a result, the resolution and contrast of the confocal image may be reduced.

The technology disclosed in Patent Document 2 mentioned above obtains fluorescence with less spectral overlap and reduces fluorescence crosstalk by irradiating a sample with illumination light that includes two excitation lights whose wavelengths are separated to some extent. That's what I do. However, in the technique disclosed in Patent Document 2 mentioned above, as mentioned above, it is necessary to irradiate the sample with illumination light that includes two excitation lights whose wavelengths are separated to some extent, so that the same spot can be obtained at the same time. It is conceivable that the wavelength band of the confocal image is limited.

In an embodiment of the present invention, a first disk having a plurality of pinholes and configured to be rotatable; By using a spinning disk equipped with an optical filter that transmits light in a specific wavelength band that differs in each predefined area, illumination light that includes only light in a specific wavelength band that differs in each predefined area from light that includes multiple wavelengths is produced. The sample is irradiated with As a result, from the point where the illumination light is irradiated, the fluorescence that is obtained when the sample is individually irradiated with light in a specific wavelength band is obtained, so there is almost no influence of fluorescence crosstalk and a common image with excellent resolution and contrast is obtained. A focused image can be obtained.

Furthermore, in the embodiment of the present invention, since the optical filter rotates as the first disk rotates, the wavelength band (specific wavelength band) of the illumination light irradiated onto the sample is sequentially switched. Thereby, confocal images can be sequentially obtained when a sample is sequentially irradiated with light in different specific wavelength bands as illumination light.

[Embodiment]
<Main configuration of microscope system>
FIG. 1 is a diagram showing the main configuration of a microscope system according to an embodiment of the present invention. As shown in FIG. 1, the microscope system 1 of this embodiment includes a light source section 10, a confocal scanner 20, cameras 30a to 30d (imaging devices), an image processing device 40, and a display device 50.

In such a microscope system 1, the confocal scanner 20 generates illumination light for scanning the sample SP from light output from the light source section 10, and the camera 30a captures a confocal image of the sample SP irradiated with the illumination light. Shoot at ~30d. The microscope system 1 processes the confocal images taken by the cameras 30a to 30d using the image processing device 40 and displays the processed images on the display device 50.

The light source unit 10 outputs light necessary to illuminate the sample SP. The light output from the light source section 10 may be coherent light (laser light) or incoherent light. In this embodiment, in order to facilitate understanding, it is assumed that the light output from the light source section 10 is a laser beam.

The light source section 10 outputs light including a plurality of wavelengths. For example, the light source unit 10 can produce a laser beam with wavelength λ1 (first specific wavelength band), a laser beam with wavelength λ2 (second specific wavelength band), a laser beam with wavelength λ3 (third specific wavelength band), a laser beam with wavelength λ4 (third specific wavelength band), and a laser beam with wavelength λ4 (third specific wavelength band). Outputs light including laser light of 4 specific wavelength bands). For example, the wavelength λ1 is 405 [nm], the wavelength λ2 is 488 [nm], the wavelength λ3 is 561 [nm], and the wavelength λ4 is 640 [nm]. Note that the light source section 10 may output white light including these wavelengths. Furthermore, the wavelength range of the light output from the light source section 10 is not limited to the above wavelength range (405 to 640 [nm]), but may be any wavelength range depending on the optical characteristics of the sample SP. can do.

The confocal scanner 20 generates illumination light for scanning the sample SP from the light output from the light source unit 10, and also generates reflected light, fluorescence, etc. (hereinafter referred to as these) obtained by irradiating the sample SP with the illumination light. When referred to collectively, it is simply referred to as "return light") that is guided to the cameras 30a to 30d. The confocal scanner 20 includes a spinning disk 21, an objective optical system 22, a dichroic mirror 23, a relay optical system 24 (fluorescence discrimination section), dichroic mirrors 25a to 25c (fluorescence discrimination section), and relay optical systems 26a to 26d (fluorescence discrimination section). ), and bandpass filters 27a to 27d (fluorescence discriminator).

The spinning disk 21 generates illumination light for scanning the sample SP from the light output from the light source section 10. FIG. 2 is a diagram illustrating a spinning disk according to an embodiment of the invention. Note that FIG. 2(a) is a side view of the spinning disk 21, and FIG. 2(b) is a plan perspective view of the spinning disk 21. As shown in FIG. 2, the spinning disk 21 includes a microlens array disk 31 (second disk), a pinhole array disk 32 (first disk), a bandpass coating 33 (optical filter), and a rotating shaft 34. In addition, in FIG. 1, in order to simplify the illustration, illustration of a motor that rotates the microlens array disk 31, pinhole array disk 32, etc. around the rotation axis 34 is omitted.

The microlens array disk 31 is a disc-shaped disk having a plurality of microlenses 31a formed in a predetermined pattern. An irradiation area A1 (see FIG. 2(b)) set on the first surface P11 of the microlens array disk 31 is irradiated with light output from the light source section 10. The pinhole array disk 32 is a disc-shaped disk having a plurality of pinholes 32a formed in correspondence with the microlenses 31a of the microlens array disk 31. The pinholes 32a of the pinhole array disk 32 are arranged in a spiral radial pattern, as shown in FIG. 2(b). Note that the microlenses 31a of the microlens array disk 31 are also formed in a spiral radial shape, similar to the pinholes 32a shown in FIG. 2(b).

The bandpass coating 33 is formed on the second surface P12 of the microlens array disk 31, and is used to identify different areas from the light output from the light source unit 10 in a predefined area in a plane perpendicular to the rotation axis 34. It allows light in a wavelength range to pass through. This bandpass coating 33 is provided to ensure that only light in a specific wavelength band is irradiated to one location on the sample SP (light that includes a plurality of wavelengths is not irradiated). Note that although one location on the sample SP is not irradiated with light in a plurality of specific wavelength bands, different locations on the sample SP may be irradiated with light in different specific wavelength bands at the same time.

For example, as shown in FIG. 2(b), the bandpass coating 33 is evenly divided into eight spiral radial regions R1 to R8 in a plane perpendicular to the rotation axis . For example, in FIG. 2(b), regions R1 and R5 are regions that transmit light of wavelength λ1, regions R2 and R6 are regions that transmit light of wavelength λ2, and regions R3 and R7 are regions that transmit light of wavelength λ3. The regions R4 and R8 are regions that transmit light of wavelength λ4. Note that the regions R1 to R8 may be set so that any two of the regions R1 to R8 transmit light of the same wavelength, and are arranged around the rotation axis 34 in the order of the length of the wavelength to be transmitted. It is not necessarily necessary.

A microlens array disk 31 and a pinhole array disk 32 are attached to both ends of the rotating shaft 34, respectively. Therefore, the microlens array disk 31 and the pinhole array disk 32 are configured to be rotatable together (integrally) around the rotation axis 34. Note that since the bandpass coating 33 is formed on the second surface P12 of the microlens array disk 31, it is rotatable around the rotation axis 34 together with the microlens array disk 31 and the pinhole array disk 32.

The relationship between the microlens 31a formed on the microlens array disk 31, the pinhole 32a formed on the pinhole array disk 32, and the regions R1 to R8 of the bandpass coating 33 does not change even if the spinning disk 21 rotates. . Therefore, the illumination light passing through each of the pinholes 32a provided in the pinhole array disk 32 is one of a laser beam with a wavelength λ1, a laser beam with a wavelength λ2, a laser beam with a wavelength λ3, and a laser beam with a wavelength λ4. Become.

The objective optical system 22 irradiates the sample SP with the illumination light generated by the spinning disk 21 and guides the returned light obtained by irradiating the sample SP with the illumination light to the spinning disk 21. Although the objective optical system 22 and the sample SP are shown inside the confocal scanner 20 in FIG. 1, the objective optical system 22 and the sample SP may be provided outside the confocal scanner 20.

The dichroic mirror 23 transmits the light (illumination light) irradiated onto the sample SP, and reflects the return light obtained by irradiating the sample SP with the illumination light toward the relay optical system 24 . Specifically, the dichroic mirror 23 is disposed on the optical axis of the objective optical system 22 and between a microlens array disk 31 and a pinhole array disk 32 provided on the spinning disk 21. Note that a half mirror can be used instead of the dichroic mirror 23.

Note that the dichroic mirror 23 transmits a plurality of light beams divided and converged by the microlens 31a provided on the microlens array disk 31. The light beam transmitted through the dichroic mirror 23 is focused on a pinhole 32a provided in a pinhole array disk 32, passes through the pinhole 32a, and is emitted to the outside of the spinning disk 21.

The relay optical system 24 and the relay optical systems 26a to 26d constitute an optical system that guides the return light reflected by the dichroic mirror 23 to the cameras 30a to 30d. The optical system constituted by the relay optical system 24 and the relay optical systems 26a to 26d is preferably a telecentric optical system in order to eliminate image shading (unevenness in brightness). Here, the telecentric optical system refers to an optical system in which the chief ray is parallel to the optical axis.

When a telecentric optical system is configured by the relay optical system 24 and the relay optical systems 26a to 26d, the object-side focal plane of the relay optical system 24 is set at the position of the pinhole 32a of the pinhole array disk 32. It is set to coincide with the image-side focal plane of the objective optical system 22. Further, the image-side focal planes of the relay optical systems 26a to 26d are set to coincide with the imaging planes of the cameras 30a to 30d, respectively.

The dichroic mirrors 25a to 25c are arranged in order on the optical axis between the relay optical system 24 and the relay optical system 26d, and reflect or transmit the return light reflected by the dichroic mirror 23 depending on the wavelength. The dichroic mirrors 25a to 25c generally reflect light in a short wavelength band and transmit light in a long wavelength band.

Specifically, the dichroic mirror 25a reflects the light in the wavelength band WB1 (first wavelength observation band) to be observed of the fluorescence obtained by irradiating the sample SP with the illumination light with the wavelength λ1, and Light in a wavelength band longer than the band WB1 is transmitted. The dichroic mirror 25b reflects the light in the wavelength band WB2 (second wavelength observation band) that is the object of observation of the fluorescence obtained by irradiating the sample SP with the illumination light with the wavelength λ2. Transmits light in the long wavelength band.

The dichroic mirror 25c reflects the light in the wavelength band WB3 (third wavelength observation band) that is the object of observation of the fluorescence obtained by irradiating the sample SP with the illumination light with the wavelength λ3. Transmits light in the long wavelength band. The light transmitted through the dichroic mirror 25c includes light in the observed wavelength band WB4 (fourth wavelength observation band) of fluorescence obtained by irradiating the sample SP with the illumination light having the wavelength λ4.

FIG. 3 is a diagram showing an example of the relationship between the wavelength of illumination light irradiated onto a sample and the wavelength band to be observed in the embodiment of the present invention. As shown in FIG. 3, when the sample SP is irradiated with illumination light of wavelength λ1, fluorescence having spectrum SP1 is obtained, and when the sample SP is irradiated with illumination light of wavelength λ2, fluorescence having spectrum SP2 is obtained. is obtained. Further, when the sample SP is irradiated with illumination light of wavelength λ3, fluorescence having a spectrum SP3 is obtained, and when the sample SP is irradiated with illumination light of wavelength λ4, fluorescence having a spectrum SP4 is obtained.

When the wavelength λ1 of the illumination light irradiated to the sample SP is 405 [nm], the wavelength band WB1 is set to, for example, 422.5 to 476.5 [nm], and the wavelength band WB1 of the illumination light irradiated to the sample SP is When the wavelength λ2 is 488 [nm], the wavelength band WB2 is set to, for example, 500.5 to 550 [nm]. Furthermore, when the wavelength λ3 of the illumination light irradiated onto the sample SP is 561 [nm], the wavelength band WB3 is, for example, 581.5 to 618.5 [nm], and the illumination light irradiated onto the sample SP is When the wavelength λ4 is 640 [nm], the wavelength band WB4 is, for example, 661.5 to 690.5 [nm]. Note that the wavelength bands WB1 to WB4 described above are just examples, and any wavelength band can be set depending on the wavelength of the laser light irradiated onto the sample SP.

The bandpass filters 27a to 27d are filters that have a characteristic of transmitting light in a certain wavelength band and blocking light in other wavelength bands. Specifically, the bandpass filter 27a has a property of transmitting light in the wavelength band WB1, the bandpass filter 27b has a property of transmitting light in the wavelength band WB2, and the bandpass filter 27c has a property of transmitting light in the wavelength band WB2. The bandpass filter 27d has a characteristic of transmitting light of wavelength band WB3, and the bandpass filter 27d has a characteristic of transmitting light of wavelength band WB4.

Note that the relay optical system 26a and the bandpass filter 27a are arranged on the optical path of the light reflected by the dichroic mirror 25a, and the relay optical system 26b and the bandpass filter 27b are arranged on the optical path of the light reflected by the dichroic mirror 25b. The relay optical system 26c and the bandpass filter 27c are arranged on the optical path of the light reflected by the dichroic mirror 25c. The relay optical system 26d and the bandpass filter 27d are arranged on the optical path of the light transmitted through the dichroic mirror 25c.

The cameras 30a to 30d are for obtaining confocal images of the sample SP. The cameras 30a to 30d are equipped with a solid-state imaging device such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor), and can capture two-dimensional still images or moving images. It is a camera that can take pictures. The cameras 30a to 30d are positioned with respect to the confocal scanner 20 so that the light passing through the bandpass filters 27a to 27d is incident on the imaging plane.

The image processing device 40 sequentially stores image data of confocal images taken by the cameras 30a to 30d, reads out the stored image data, and performs image processing. The image processing device 40 is realized by, for example, a desktop personal computer or a workstation. The display device 50 includes, for example, a liquid crystal display device, and displays a confocal image subjected to image processing by the image processing device 40.

<Microscope system operation>
When the operation of the microscope system 1 is started, light including laser light with wavelengths λ1 to λ4 is output from the light source section 10, and rotation of the spinning disk 21 (rotation around the rotation axis 34) is started. Light output from the light source section 10 is input to a confocal scanner 20. The light input to the confocal scanner 20 is irradiated onto an irradiation area A1 (see FIG. 2(b)) set on the first surface P11 of the microlens array disk 31 of the spinning disk 21.

The light irradiated onto the microlens array disk 31 is divided into a plurality of light beams by a microlens 31a provided on the microlens array disk 31. The plurality of light beams divided by the microlens 31a enter the bandpass coating 33 formed on the second surface P12 of the microlens array disk 31. Then, among the plurality of wavelength components included in each luminous flux, only light having different wavelengths for each region R1 to R8 shown in FIG. 2(b) passes through the bandpass coating 33.

For example, for the light beams incident on regions R1 and R5 of the bandpass coating 33, only the light of wavelength λ1 passes through, and for the light fluxes incident on the regions R2 and R6 of the bandpass coating 33, only the light of wavelength λ2 passes. do. Also, for the light beams incident on the regions R3 and R7 of the bandpass coating 33, only the light with the wavelength λ3 passes, and for the light fluxes incident on the regions R4 and R8 of the bandpass coating 33, only the light of the wavelength λ4 passes. do.

The light that has passed through the bandpass coating 33 is transmitted through the dichroic mirror 23 and is emitted to the outside of the spinning disk 21 as illumination light through pinholes 32a formed in the pinhole array disk 32 in correspondence with the microlenses 31a. Ru. At this time, the light transmitted through the dichroic mirror 23 is focused on the pinhole 32a and then emitted to the outside of the spinning disk 21.

The illumination light emitted to the outside of the spinning disk 21 is irradiated onto the sample SP via the objective optical system 22. Here, although different parts of the sample SP may be irradiated with light of different wavelengths at the same time, one part of the sample SP is irradiated with only light of a specific wavelength, and light containing multiple wavelengths is irradiated. Never. That is, when viewed locally, the sample SP is so-called monochromatically excited.

Return light from the sample SP (return light obtained by irradiating the sample SP with illumination light) enters the spinning disk 21 via the objective optical system 22 . After passing through the pinhole 32a formed in the pinhole array disk 32 of the spinning disk 21, the light is reflected by the dichroic mirror 23 toward the relay optical system 24.

The return light reflected by the dichroic mirror 23 passes through the relay optical system 24, the dichroic mirrors 25a to 25c, the relay optical systems 26a to 26d, and the bandpass filters 27a to 27d in this order and enters the cameras 30a to 30d to form an image. .

Specifically, among the returned light reflected by the dichroic mirror 23, the light in the wavelength band WB1 enters the camera 30a through the relay optical system 24, the dichroic mirror 25a, the relay optical system 26a, and the bandpass filter 27a in this order. and forms an image. Further, among the return light reflected by the dichroic mirror 23, the light in the wavelength band WB2 enters the camera 30b via the relay optical system 24, the dichroic mirrors 25a and 25b, the relay optical system 26b, and the bandpass filter 27b in this order. and forms an image.

Further, among the return light reflected by the dichroic mirror 23, the light in the wavelength band WB3 enters the camera 30c via the relay optical system 24, the dichroic mirrors 25a to 25c, the relay optical system 26c, and the bandpass filter 27c in this order. and forms an image. Further, among the return light reflected by the dichroic mirror 23, the light in the wavelength band WB4 enters the camera 30d via the relay optical system 24, the dichroic mirrors 25a to 25c, the relay optical system 26d, and the bandpass filter 27d in this order. and forms an image.

Here, since the spinning disk 21 is rotating around the rotation axis 34, the illumination light irradiated onto the sample SP is scanned in accordance with the rotation of the spinning disk 21. As a result, returned light corresponding to the scanning position of the illumination light is sequentially input to the cameras 30a to 30d. In this way, confocal images of the sample SP are obtained by the cameras 30a to 30d.

Note that, as shown in FIG. 2(b), the bandpass coating 33 is equally divided into eight spiral radial regions R1 to R8 in a plane perpendicular to the rotation axis . Therefore, the sample SP is sequentially scanned with illumination light of different wavelengths, or different positions are simultaneously scanned with illumination light of different wavelengths. Therefore, the confocal images obtained by the cameras 30a to 30d may be of a part of the region (observation target region) where the sample SP is irradiated with illumination light.

Image data of confocal images taken by the cameras 30a to 30d are sequentially output to the image processing device 40. The image processing device 40 sequentially stores image data sequentially output from the cameras 30a to 30d, reads out the stored image data, and performs image processing to create a confocal image of the sample SP. This image processing is, for example, a process of synthesizing confocal images of a part of the observation target area to obtain a confocal image of the entire observation target area. The confocal image of the sample SP thus obtained is displayed on the display device 50.

As described above, in this embodiment, the spinning disk 21 including the microlens array disk 31, the pinhole array disk 32, and the bandpass coating 33 is used, and only illumination light of a specific wavelength is irradiated to one location of the sample SP. I'm trying to make it happen. As a result, from the point irradiated with the illumination light, the fluorescence obtained when the sample is individually irradiated with light of a specific wavelength is obtained, so there is almost no influence of fluorescence crosstalk, and a common image with excellent resolution and contrast is obtained. A focused image can be obtained.

In addition, in this embodiment, although illumination light including multiple wavelengths is not irradiated to one part of the observation target area of the sample SP, the observation target area of the sample SP is scanned with light of different wavelengths λ1 to λ4. will be done. Therefore, it is possible to obtain confocal images for each of the different wavelengths λ1 to λ4 for the observation target area, and as in Patent Document 2 mentioned above, the wavelength band of the confocal images at the same location that can be obtained simultaneously is There are no restrictions.

Further, in this embodiment, the return light from the sample SP (the return light obtained by irradiating the sample SP with illumination light) is reflected by the dichroic mirror 23 toward the relay optical system 24, and the microlens array disk 31 It does not pass through the bandpass coating 33 formed on the substrate. Therefore, the degree of freedom in the type of fluorescent dye to be used can be increased.

<Modified example>
FIG. 4 is a side view showing a modification of the spinning disk according to the embodiment of the present invention. In FIG. 4, the same components as those shown in FIG. 2(a) are designated by the same reference numerals. The spinning disk 21 shown in FIG. 4A has a configuration in which the bandpass coating 33 formed on the microlens array disk 31 of the spinning disk 21 shown in FIG. 2 is omitted, and an auxiliary disk 35 (third disk) is added. It is.

The auxiliary disk 35 is a disc-shaped disk that is transparent to at least the light output from the light source section 10. For example, the auxiliary disk 35 is a disc-shaped disk made of transparent glass. The auxiliary disk 35 is configured to be rotatable around the rotation axis 34 together with the microlens array disk 31 and the pinhole array disk 32 (integrally). A bandpass coating 33 is formed on the first surface P31 of this auxiliary disk 35. As shown in FIG. 4A, the auxiliary disk 35 is placed close to the microlens array disk 31 with the second surface P32 facing the first surface P11 of the microlens array disk 31.

Note that the auxiliary disk 35 may be arranged close to the microlens array disk 31 with the first surface P31 on which the bandpass coating 33 is formed facing the first surface P11 side of the microlens array disk 31. . Further, the auxiliary disk 35 may be arranged close to the microlens array disk 31 between the microlens array disk 31 and the pinhole array disk 32.

In this way, the spinning disk 21 shown in FIG. 4A is provided with an auxiliary disk 35 on which a bandpass coating 33 is formed, in addition to the microlens array disk 31. In the spinning disk 21 shown in FIG. 4A, there is no need to form the bandpass coating 33 on the microlens array disk 31, so the spinning disk 21 can be easily produced.

The spinning disk 21 shown in FIG. 4(b) omits the bandpass coating 33 formed on the microlens array disk 31 of the spinning disk 21 shown in FIG. 2, and forms the bandpass coating 33 on the pinhole array disk 32. This is the configuration. The bandpass coating 33 is formed on the first surface P21 of the pinhole array disk 32, as shown in FIG. 4(b). Note that the pinhole array disk 32 may be formed on the second surface P22 of the pinhole array disk 32.

The spinning disk 21 shown in FIG. 4(c), like the spinning disk 21 shown in FIG. 4(a), omits the bandpass coating 33 formed on the microlens array disk 31 of the spinning disk 21 shown in FIG. However, it has a configuration in which an auxiliary disk 35 (third disk) is added. However, in the spinning disk 21 shown in FIG. 4(c), the auxiliary disk 35 is placed close to the pinhole array disk 32.

Specifically, the auxiliary disk 35 is arranged close to the pinhole array disk 32 with the first surface P31 on which the bandpass coating 33 is formed facing toward the second surface P22 of the pinhole array disk 32. . Note that the auxiliary disk 35 may be placed close to the pinhole array disk 32 with the second surface P32 facing the second surface P22 side of the pinhole array disk 32. Further, the auxiliary disk 35 may be arranged close to the pinhole array disk 32 between the microlens array disk 31 and the pinhole array disk 32.

In this way, the spinning disk 21 shown in FIG. 4(c), like the spinning disk 21 shown in FIG. 35. Therefore, in the spinning disk 21 shown in FIG. 4(c), it is not necessary to form the bandpass coating 33 on the pinhole array disk 32, so that the spinning disk 21 can be easily produced.

The spinning disk 21 shown in FIG. 4(d) has a configuration in which an auxiliary disk 35 is provided in place of the microlens array disk 31 of the spinning disk 21 shown in FIG. The auxiliary disk 35 is attached to the rotating shaft 34 with the second surface P32 facing the first surface P21 of the pinhole array disk 32. Note that the auxiliary disk 35 may be attached to the rotating shaft 34 with the first surface P31 on which the bandpass coating 33 is formed facing the first surface P21 of the pinhole array disk 32.

FIG. 5 is a plan perspective view showing another modification of the spinning disk according to the embodiment of the present invention. In FIG. 5, the same components as those shown in FIG. 2(b) are designated by the same reference numerals. The spinning disk 21 shown in FIGS. 5(a) and 5(b) is obtained by changing the method of dividing the bandpass coating 33 formed on the microlens array disk 31 of the spinning disk 21 shown in FIG. 2.

In the spinning disk 21 shown in FIG. 5(a), the bandpass coating 33 is equally divided into four regions R11 to R14 by straight lines extending radially from the center in a plane perpendicular to the rotation axis 34. For example, region R11 is a region that transmits light of wavelength λ1, region R12 is a region that transmits light of wavelength λ2, region R13 is a region that transmits light of wavelength λ3, and region R14 is a region that transmits light of wavelength λ4. This is an area that allows light to pass through. Note that the regions R11 to R14 do not necessarily have to be arranged around the rotation axis 34 in the order of the length of the wavelength to be transmitted.

In the spinning disk 21 shown in FIG. 5(b), the bandpass coating 33 is formed into two regions R21 (first region) and R22 (first region) in a straight line extending radially from the center in a plane perpendicular to the rotation axis 34. 2 areas). For example, region R21 is a region that transmits light of wavelength λ1 and light of wavelength λ3, and region R22 is a region that transmits light of wavelength λ2 and light of wavelength λ4.

In the spinning disk 21 shown in FIG. 5(b), the sample SP is irradiated with illumination light including a plurality of wavelengths. Specifically, illumination light including light with a wavelength λ1 and light with a wavelength λ3, or illumination light including light with a wavelength λ2 and light with a wavelength λ4 is irradiated onto one location of the sample SP. However, the wavelengths of the lights of wavelengths λ1 and λ3 included in the illumination light irradiated onto the sample SP are separated to some extent. Further, the wavelengths of the lights of wavelengths λ2 and λ4 included in the illumination light irradiated onto the sample SP are also separated to some extent. Therefore, although some fluorescence crosstalk may occur, a confocal image with excellent resolution and contrast can be obtained.

Although the spinning disk, confocal scanner, and microscope system according to the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be freely modified within the scope of the present invention. For example, in the embodiment described above, the light source unit 10 outputs light including four wavelengths λ1 to λ4, and the confocal scanner 20 discriminates the returned light obtained from the sample SP into four wavelength bands WB1 to WB4. explained. However, the number of wavelengths included in the light output from the light source section 10 may be other than four (for example, two, three, or five or more), and the wavelength bands discriminated by the confocal scanner 20 are four. The number may be other than one (for example, two, three, or five or more).

1 Microscope system 10 Light source 20 Confocal scanner 21 Spinning disk 24 Relay optical system 25a-25c Dichroic mirror 26a-26d Relay optical system 27a-27d Bandpass filter 30a-30d Camera 31 Microlens array disk 31a Microlens 32 Pinhole array Disk 32a Pinhole 33 Bandpass coating 34 Rotating shaft 35 Auxiliary disk 40 Image processing device R1 to R8 area R11 to R14 area R21, R22 area SP Sample WB1 to WB4 Wavelength band λ1 to λ4 Wavelength

Claims (5)

A spinning disk that generates illumination light for scanning a sample from light containing multiple wavelengths, the spinning disk comprising:
a first disk having a plurality of pinholes and configured to be rotatable;
a second disk having a plurality of microlenses formed in correspondence with the pinholes and configured to be rotatable together with the first disk;
an optical filter that is rotatable together with the first disk and that transmits light in a specific wavelength band that is different for each predefined region in a plane perpendicular to the rotation axis of the first disk;
Equipped with
the optical filter is formed on the second disk;
spinning disc. A spinning disk according to claim 1 ;
a fluorescence discrimination unit that discriminates fluorescence obtained by irradiating the sample with the illumination light for each observation wavelength band set corresponding to the specific wavelength band;
Confocal scanner with The optical filter includes a region that transmits light in a first specific wavelength band, light in a second specific wavelength band, light in a third specific wavelength band, and light in a fourth specific wavelength band,
The fluorescence discrimination unit separates the fluorescence into a first observation wavelength band set corresponding to the first specific wavelength band, a second observation wavelength band set corresponding to the second specific wavelength band, and a second observation wavelength band set corresponding to the second specific wavelength band. 3 discrimination for each of a third observation wavelength band set corresponding to the specific wavelength band and a fourth observation wavelength band set corresponding to the fourth specific wavelength band,
The confocal scanner according to claim 2 . The optical filter is configured to select the first specific wavelength band and the third specific wavelength band among a first specific wavelength band, a second specific wavelength band, a third specific wavelength band, and a fourth specific wavelength band arranged in order of wavelength length. a first region that transmits light in the band; and a second region that transmits light in the second specific wavelength band and the fourth specific wavelength band;
The fluorescence discrimination unit separates the fluorescence into a first observation wavelength band set corresponding to the first specific wavelength band, a second observation wavelength band set corresponding to the second specific wavelength band, and a second observation wavelength band set corresponding to the second specific wavelength band. 3 discrimination for each of a third observation wavelength band set corresponding to the specific wavelength band and a fourth observation wavelength band set corresponding to the fourth specific wavelength band,
The confocal scanner according to claim 2 . a light source unit that outputs light including multiple wavelengths;
5. The illumination light for scanning the sample is generated from the light output from the light source section, and the fluorescence obtained by irradiating the sample with the illumination light is discriminated for each of the observation wavelength bands. a confocal scanner according to any one of the items;
a plurality of imaging devices that take confocal images of each of the observation wavelength bands of the sample;
an image processing device that performs image processing of the confocal image taken by the imaging device;
A microscope system equipped with

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