JP6202979B2 - Microscope system - Google Patents

Description

  The present invention relates to a microscope system and a disk unit that acquire super-resolution images and confocal images that exceed the resolution limit of an optical system.

  Conventionally, in a microscope system, a sample is irradiated with illumination light through an objective lens, and reflected light reflected by the sample or fluorescence generated by excitation of a fluorescent dye labeled on the sample by irradiation of the illumination light is generated by the objective lens. Is acquired as observation light, and the specimen image is formed by forming an image of the observation light.

  As a technique for generating a sample image, a technique for generating an image having a resolution exceeding the resolution limit of an optical system (hereinafter referred to as super-resolution) is known. Structured illumination method (Structured Illumination Microscopy; SIM) is disclosed as a method for obtaining a super-resolution image (for example, refer to Patent Documents 1 and 2).

  In general, in wide field observation, illumination light is irradiated as uniformly as possible on a specimen. In SIM, a striped illumination light is irradiated on a specimen. In SIM, a plurality of sample images are acquired while sequentially shifting the irradiation position of the striped illumination light irradiated on the sample, and Fourier analysis is performed on these sample images to generate a super-resolution image. can do.

  As another technique for generating a super-resolution sample image, a generation method using a disc (Disc Scan Super Resolution Microscope; DSSRM) has been disclosed (see, for example, Patent Document 3). In DSRM, the spatial intensity distribution of observation light from a specimen is modulated by rotating a disk on which an optimal scan pattern is formed in order to obtain super-resolution observation light, and an image is obtained with respect to the modulated observation light. By performing processing, a super-resolution image is generated.

  By the way, as a technique for generating a specimen image, a technique for three-dimensionally displaying an object by extracting a confocal point from a plurality of images has been developed. In this technique, for example, only the observation light from the specimen surface, that is, the in-focus state is focused by rotating a disk in which a confocal aperture such as a slit or a pinhole is arranged at a position conjugate with the specimen surface (focus position of the objective lens). Only the observed observation light is acquired, and a confocal image is generated based on the obtained observation light.

International Publication No. 2007/043314 International Publication No. 2007/043382 JP 2012-78408 A

  By the way, in the conventional configuration disclosed in Patent Documents 1 to 3, it is necessary to provide a unit for acquiring super-resolution observation light and a unit for acquiring confocal image observation light, respectively. It has been difficult to switch between the super-resolution image and the confocal image during the observation of the specimen.

  The present invention has been made in view of the above, and an object of the present invention is to provide a microscope system and a disk unit that can easily switch between a super-resolution image and a confocal image.

In order to solve the above-described problems and achieve the object, a microscope system according to the present invention forms an illumination light generating unit that generates illumination light for irradiating a specimen to be observed, and images the observation light from the specimen. An imaging optical system that forms an image of the specimen, and a super-resolution modulation unit that modulates the spatial intensity distribution of the illumination light and the observation light into a spatial intensity distribution for generating a super-resolution image It said optical modulation means comprising a second disk having a confocal modulator modulating the spatial intensity distribution for generating a first disk, and confocal images, the axis passing through the center of the disk as the center axis rotates the disk, said one of the disks of the first and second disk, and disk drive means for the main surface of the one of the disks to move in a direction crossing the axis of the imaging optical system, wherein This Receiving the observation light having passed through the, to an image generating means for generating a super-resolution image data or confocal image data based on the observation light receiving light, comprising the.

  In the microscope system according to the present invention, in the above invention, the disk has a disk shape and is detachably provided at an image forming position by the imaging optical system. The focus modulation section is characterized by comprising a plurality of holes that penetrate in the thickness direction and are arranged at equal intervals along the circumferential direction.

  In the microscope system according to the present invention as set forth in the invention described above, the image generation means includes an image sensor that receives the observation light and photoelectrically converts the observation light, and the disk driving means corresponds to an exposure time of the image sensor. Accordingly, the rotational speed of the disk is changed accordingly.

  According to the present invention, illumination light generating means for generating illumination light for irradiating a specimen to be observed, an imaging optical system for forming an image of the specimen by imaging observation light from the specimen, illumination light and observation A super-resolution modulation unit that modulates the spatial intensity distribution of light into a spatial intensity distribution for generating a super-resolution image, and a confocal modulation unit that modulates the spatial intensity distribution to generate a confocal image Optical modulation means including at least a first disk or a second disk having a super-resolution modulation section and a third disk having a confocal modulation section, and the disk is rotated about an axis passing through the center of the disk. Disk drive means for moving the disk in a direction in which the main surface of the disk intersects the axis of the imaging optical system, and super-resolution image data based on the received observation light. Ma Since it was set to and an image generating means for generating a confocal image data, an effect that can be obtained by switching the image of the super-resolution, and a confocal image easily.

FIG. 1 is a schematic diagram illustrating a schematic configuration of the microscope system according to the first embodiment of the present invention. FIG. 2 is a schematic diagram illustrating a configuration of a main part of the microscope system according to the first embodiment of the present invention. FIG. 3 is a diagram for explaining holes formed in the disk of the microscope system according to the first embodiment of the present invention. FIG. 4 is a schematic diagram illustrating a schematic configuration of the microscope system according to the second embodiment of the present invention. FIG. 5 is a schematic diagram illustrating a configuration of a main part of the microscope system according to the second embodiment of the present invention.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment. The drawings referred to in the following description only schematically show the shape, size, and positional relationship so that the contents of the present invention can be understood. That is, the present invention is not limited only to the shape, size, and positional relationship illustrated in each drawing.

(Embodiment 1)
FIG. 1 is a schematic diagram illustrating a schematic configuration of the microscope system according to the first embodiment of the present invention. The microscope system 1 shown in FIG. 1 forms a specimen image by capturing fluorescence from the specimen S. The specimen S is held in a container such as a dish, a slide glass, or a beaker. Examples of the specimen S include biological samples such as biological tissue sections, cells separated from biological samples, cultured cells such as cell lines, cultures of cells separated from biological samples, and cultured cultures of cells. . The specimen S is fluorescently labeled with a fluorescent dye. When the specimen S is irradiated with excitation light, the labeled fluorescent dye is excited and emits fluorescence.

  The microscope system 1 includes at least an objective lens 2 that captures observation light from the specimen S, an imaging lens 3 that forms an intermediate image of the specimen S from observation light captured by the objective lens 2, and a position at which the intermediate image is formed. At least the spatial intensity distribution of the observation light imaged by the imaging lens 3 is removably provided at (image forming position), and the resolution exceeds the resolution limit of the optical system including the imaging lens 3 (hereinafter referred to as super-resolution). An optical modulation unit 4 (disk unit) having a composite pattern disk 4a that modulates a spatial intensity distribution for generating a confocal image and an optical path of illumination light in the optical axis N direction of the objective lens 2 The fluorescent cube 5 that allows the observation light from the sample S in the direction of the optical axis N to pass through, the relay lens 6 that relays the observation light that has passed through the fluorescent cube 5, and the relay lens 6 relays The received observation light is received, the photoelectric conversion process is performed on the received observation light, the imaging unit 7 that captures an intermediate image of the sample S, and the electrical signal generated by the imaging unit 7 are acquired, and the electrical An image processing unit 8 that performs predetermined image processing on an image according to a signal, a light source 9 that emits illumination light that illuminates the specimen S, and an illumination lens 10 that guides the illumination light emitted from the light source 9. . The objective lens 2 and the imaging lens 3 form an imaging optical system. The light source 9 and the illumination lens 10 constitute illumination light generation means.

  The optical modulation unit 4 includes a composite pattern disk 4a having a disk shape in which a plurality of holes for modulating the spatial intensity distribution of illumination light or observation light are formed, and a shaft 41 that supports the center of the composite pattern disk 4a. The rotary motor 42 that supports the shaft 41 and rotates the shaft 41 around the central axis of the shaft 41, the support portion 43 that supports the rotary motor 42, and the support portion 43 in a direction orthogonal to the optical axis N. And a holding portion 44 that holds it movably. The rotation motor 42 and the holding unit 44 rotate the shaft 41 and move the support unit 43 under the control of a control unit (not shown). The shaft 41, the rotation motor 42, the support part 43, and the holding part 44 constitute a disk drive stage.

  The fluorescent cube 5 has an excitation filter 51 that transmits only light having a predetermined wavelength as illumination light (excitation light) from the light source 9, and reflects light having a wavelength corresponding to the excitation light toward the sample S (optical axis). A dichroic mirror 52 that bends (in the N direction) and transmits light having a wavelength corresponding to the observation light (fluorescence) from the specimen S, and an absorption filter that transmits only a predetermined fluorescent component of the observation light transmitted through the dichroic mirror 52 53.

  The relay lens 6 relays the observation light (intermediate image) that has passed through the fluorescent cube 5 to the observation light receiving surface of the imaging unit 7. The cutoff frequency of the relay lens 6 is higher than the cutoff frequency of the imaging optical system (the objective lens 2 and the imaging lens 3). The cutoff frequency refers to a limit value of frequency components that can be transmitted. For example, if the cutoff frequency is fc, the relay lens 6 transmits a frequency component in the range of frequency ± fc.

  The imaging unit 7 is realized using a CCD image sensor or a CMOS image sensor. The Nyquist frequency of the imaging unit 7 is higher than the cutoff frequency of the imaging optical system.

  The image processing unit 8 performs image processing such as A / D conversion processing, white balance (WB) adjustment processing, gain adjustment processing, and γ correction processing based on the electrical signal (image signal) generated by the imaging unit 7. Then, image data to be displayed on a monitor (not shown) is generated. Specifically, the image processing unit 8 generates super-resolution image data and confocal image data based on the observation light that is optically modulated by the optical modulation unit 4.

  When generating super-resolution image data, the image processing unit 8 performs image processing that emphasizes high-frequency components from the obtained electrical signal. Thereby, super-resolution image data in which a super-resolution component that has not been reflected as image data in normal fluorescence observation is visualized is generated.

  Further, when generating the confocal image data, the image processing unit 8 extracts the confocal from a plurality of images (electrical signals corresponding to a plurality of frames), so that only the observation light from the specimen surface, that is, the in-focus state is obtained. Only the image that is being acquired is acquired, and confocal image data is generated based on the obtained image.

The light source 9 is realized using a mercury lamp, an LED, or the like, and emits incident illumination light.
Further, before and after the illumination lens 10, a lens that collects light from the light source 9, a light control filter that adjusts the amount of illumination light, and the like may be provided. In addition, when an arc light source such as a mercury lamp is used, a field stop may be provided that allows a part of the illumination light to pass through the aperture and narrows the irradiation range on the irradiation surface.

  In the microscope system 1, for example, the illumination light from the light source 9 is wavelength-selected by the excitation filter 51, and the illumination light is bent toward the objective lens 2 by the dichroic mirror 52. When the illumination light bent by the dichroic mirror 52 is irradiated onto the specimen S through the imaging lens 3 and the objective lens 2, the fluorescent dye labeled on the specimen S is excited and emits fluorescence. The objective lens 2 captures the fluorescence emitted from the sample S, and the captured fluorescence is imaged by the imaging lens 3, passes through the dichroic mirror 52 and the absorption filter 53, and enters the imaging unit 7 as observation light. To do. The observation light incident on the imaging unit 7 is photoelectrically converted and output to the image processing unit 8, and image data (super-resolution image data or confocal image data) is generated in the image processing unit 8.

  At this time, by changing the position of the composite pattern disk 4a disposed on the optical axis N by the movement of the support portion 43, a super-resolution image is generated for the spatial intensity distribution of the illumination light and the observation light. Switching to a spatial intensity distribution for generating a confocal image or a spatial intensity distribution for generating a confocal image.

  FIG. 2 is a schematic diagram showing a configuration of a main part of the microscope system according to the first embodiment, and shows a case where the composite pattern disk 4a is viewed from above. The composite pattern disk 4a has an annular shape in which an opening for inserting and fixing the shaft 41 is formed at the center position of the main surface.

  The composite pattern disk 4a is provided on the outer peripheral side, and is formed with a plurality of holes 401a that are formed along the circumferential direction to modulate the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a super-resolution image. A resolution modulation unit 401 and a common hole formed on the inner circumference side and formed with a plurality of holes 402a along the circumferential direction for modulating the spatial intensity distribution of observation light into a spatial intensity distribution for generating a confocal image. A focus modulation unit 402. The super-resolution modulation unit 401 and the confocal modulation unit 402 are provided on concentric circles, and holes (holes 401a and 402a) corresponding to the type of modulation are formed in a predetermined pattern in each region. The opening diameter of the hole 401a is smaller than the opening diameter of the hole 402a, for example, as shown in FIG.

  The confocal modulation unit 402 is provided so as to include a region that intersects the optical axis N due to the movement of the composite pattern disk 4a.

  FIG. 3 is a diagram for explaining holes formed in the disk of the microscope system according to the first embodiment. FIG. 3 is a cross-sectional view in which a plane perpendicular to the radial direction of the main surface of the composite pattern disk 4a is cut. As shown in FIG. 3, the hole H is formed so as to penetrate in the plate thickness direction (direction orthogonal to the main surface) of the composite pattern disk 4a. The composite pattern disk 4a is provided such that the plate thickness direction is substantially parallel to the optical axis N. Hereinafter, the holes 401a and 402a are collectively referred to as a hole H.

When the width of the opening of the hole H is W, and the formation interval (pitch) between adjacent holes H is P, the hole H formed in the super-resolution modulation unit 401 has a width based on the following equation (1). W is set. The formation interval P refers to the distance between the holes H along the circumferential direction of the composite pattern disk 4a.
W = (1 / fc) × (1/2) (1)
Here, fc is a cutoff frequency of the imaging optical system including the objective lens 2 and the imaging lens 3. Note that fc is a spatial frequency determined by the following expression (2) and determined by the numerical aperture NA of the objective lens 2.
fc = 2 × NA · (1 / λ · M · β) (2)
Where λ is the wavelength of light
M: Projection magnification of the imaging optical system
β: Projection magnification of relay lens 6 Note that β needs to be considered when considering the light receiving surface of the image sensor, but may be 1 when considering the disk surface.

The formation interval P of the holes H formed in the super-resolution modulation unit 401 is set to a value obtained based on the following equation (3).
P = 2 × (1 / fc)
= 4W (3)

  Note that when the formation interval P is P <4 W, the contrast is lowered, and when P> 4 W, the luminance (brightness) is lowered. Therefore, P = 4 W is preferable.

On the other hand, the width W of the hole H formed in the confocal modulation unit 402 is set based on the following expression (4).
W = k · λ · R / NA (4)
Here, k: constant (0.5 ≦ k ≦ 1.0)
R: Projection magnification on the composite pattern disk 4a

  The formation interval P of the holes H formed in the super-resolution modulation unit 401 is set to about 10 times the width W, for example.

  Further, when the super-resolution modulation unit 401 is located on the optical axis N, the rotational speed of the composite pattern disk 4a is required for imaging one frame (one image) of the imaging unit 7 (image sensor). The exposure time is set to a speed that is equal to or longer than the time required for half rotation of the composite pattern disk 4a. In other words, the time required for half rotation of the composite pattern disk 4 a is set to a speed that is shorter than the exposure time of the imaging unit 7.

  On the other hand, when the confocal modulation unit 402 is positioned on the optical axis N, the rotational speed of the composite pattern disk 4 a is set to a sufficiently large speed as compared with the exposure time of the imaging unit 7. Specifically, when the exposure time of the imaging unit 7 is, for example, 1/60 or 1/30, the composite pattern disk 4a is set to 1800 rpm, which makes a substantially half rotation during the exposure time.

  Here, in the composite pattern disk 4a, the linear velocities that pass through arbitrary points of the super-resolution modulation unit 401 and the confocal modulation unit 402 are different while the angular velocities are the same. If the rotational speed is set so as to be half or more, the super-resolution modulation unit 401 is positioned on the optical axis N and the confocal modulation unit 402 is positioned on the optical axis N. It is possible to modulate the spatial intensity distribution according to each image without changing the speed. That is, the modulation for generating a super-resolution image or the modulation for generating a confocal image can be switched only by moving the composite pattern disk 4a with respect to the optical axis N.

  According to the first embodiment described above, in the composite pattern disk 4a that optically modulates the observation light, a super solution in which a plurality of holes that modulate the spatial intensity distribution for generating a super resolution image is formed. An image modulation unit 401 and a confocal modulation unit 402 in which a plurality of holes for modulating a spatial intensity distribution for generating a confocal image are provided, and one of them intersects the optical axis N Since the spatial intensity distribution is selectively modulated by moving to, the super-resolution image and the confocal image can be easily switched and acquired.

  In addition, according to the first embodiment described above, the modulation of the spatial intensity distribution for generating a super-resolution image by one composite pattern disk 4a, and the modulation of the spatial intensity distribution for generating a confocal image, Therefore, the space occupied by the microscope system can be reduced and the cost of the microscope system can be reduced as compared with the case where units for performing respective optical modulations are attached.

  Further, according to the first embodiment described above, the super-resolution modulation unit 401 and the confocal modulation unit 402 are provided concentrically, and observation is performed by inserting a desired modulation unit with respect to the optical axis N. Therefore, for example, when observing a cell, first, the confocal modulation unit 402 is arranged on the optical axis N and the confocal observation is performed, so that the entire image of the cell is macroscopically observed in three dimensions, and then the super solution is obtained. By arranging the image modulation unit 401 on the optical axis N and performing super-resolution observation, it is possible to observe the molecular behavior of a cell, a minute region, and the like. That is, according to the first embodiment, the detailed observation of the sample S can be efficiently performed by simply switching between the macro three-dimensional observation by the confocal observation and the super-resolution observation of the portion to be noticed. Can do.

  In the first embodiment described above, it has been described that optical modulation can be performed at a constant speed regardless of the modulation unit positioned on the optical axis N. However, the modulation unit positioned on the optical axis N is described. Control to change the rotational speed of the disk may be performed by

  In the first embodiment described above, the super-resolution modulation unit 401 is provided on the outer peripheral side and the confocal modulation unit 402 is provided on the inner peripheral side. However, the super-resolution modulation unit 402 is provided on the inner peripheral side. In addition, a confocal modulation unit may be provided on the outer peripheral side.

(Embodiment 2)
FIG. 4 is a schematic diagram illustrating a schematic configuration of the microscope system according to the second embodiment of the present invention. FIG. 5 is a schematic diagram illustrating a configuration of a main part of the microscope system according to the second embodiment, and illustrates a case where the super-resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a are viewed from above. It is. In addition, the same code | symbol is attached | subjected to the component same as the structure demonstrated in FIG. The microscope system 1a according to the second embodiment includes an optical modulation unit 11 (disk unit) instead of the optical modulation unit 4 of the microscope system 1 according to the first embodiment described above.

  The optical modulation unit 11 includes a super-resolution modulation pattern disk 100a having a super-resolution modulation unit 1001 that modulates the observation light into a spatial intensity distribution for generating a super-resolution image. A super-resolution modulation switching unit 100 for moving the super-resolution modulation pattern disk 100a in a direction in which the main surface of the modulation pattern disk 100a intersects the axis (optical axis N) of the imaging optical system; A confocal modulation pattern disk 110a having a confocal modulation section 1101 for modulating the spatial intensity distribution for generating a confocal image, and the main surface of the confocal modulation pattern disk 110a is the axis of the imaging optical system. A confocal modulation switching unit 110 that moves the confocal modulation pattern disk 100a in a direction crossing the (optical axis N) is provided at a position facing the optical axis N.

  The super-resolution modulation switching unit 100 is a super-resolution modulation pattern having a disk shape in which a plurality of holes for modulating the spatial intensity distribution of the observation light into a spatial intensity distribution for generating a super-resolution image is formed. A disc 100a, a shaft 101 that supports the center of the super-resolution modulation pattern disc 100a, a rotation motor 102 that supports the shaft 101 and rotates the shaft 101 around the central axis of the shaft 101, and a rotation motor 102 And a holding portion 104 that holds the support portion 103 movably in a direction orthogonal to the optical axis N.

  The confocal modulation switching unit 110 includes a confocal modulation pattern disk 110a having a disk shape in which a plurality of holes for modulating the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a confocal image is formed. A shaft 111 that supports the center of the confocal modulation pattern disk 110a, a rotation motor 112 that supports the shaft 111 and rotates the shaft 111 around the central axis of the shaft 111, and a support that supports the rotation motor 112 Part 113 and holding part 114 that holds support part 113 movably in a direction orthogonal to optical axis N.

  The rotation motors 102 and 112 and the holding units 104 and 114 rotate the shafts 101 and 111 and move the support units 103 and 113 under the control of a control unit (not shown).

  The super-resolution modulation pattern disk 100a has an annular shape in which an opening for inserting and fixing the shaft 101 is formed at the center position of the main surface. The confocal modulation pattern disk 110a has an annular shape in which an opening for inserting and fixing the shaft 111 is formed at the center position of the main surface.

  The super-resolution modulation unit 1001 of the super-resolution modulation pattern disk 100a penetrates in a direction perpendicular to the main surface, and the spatial intensity distribution of the observation light is changed to a spatial intensity distribution for generating a super-resolution image. A plurality of holes 1001a to be modulated are formed. Further, the confocal modulation section 1101 of the confocal modulation pattern disk 110a penetrates in a direction orthogonal to the main surface, and modulates the spatial intensity distribution of the observation light into a spatial intensity distribution for generating a confocal image. A plurality of holes 1101a are formed. The plurality of holes 1001a and 1101a formed in the super resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a are arranged at equal intervals along the circumferential direction as shown in FIG. The diameter of the hole formed in the image modulation pattern disk 100a is smaller than the diameter of the hole formed in the confocal modulation pattern disk 110a.

  The holes formed in the super-resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a have the width W and the formation interval based on the equations (1) to (4), respectively, as in the first embodiment. (Pitch) P is set.

  In the microscope system 1a, as in the microscope system 1 described above, for example, the illumination light from the light source 9 is wavelength-selected by the excitation filter 51, the illumination light is reflected by the dichroic mirror 52, and the illumination light is directed toward the objective lens 2. It can be bent. When the illumination light bent by the dichroic mirror 52 is irradiated onto the specimen S through the imaging lens 3 and the objective lens 2, the fluorescent dye labeled on the specimen S is excited and emits fluorescence. The objective lens 2 captures the fluorescence emitted from the sample S, and the captured fluorescence is imaged by the imaging lens 3, passes through the dichroic mirror 52 and the absorption filter 53, and enters the imaging unit 7 as observation light. To do. The observation light incident on the imaging unit 7 is photoelectrically converted and output to the image processing unit 8, and image data (super-resolution image data or confocal image data) is generated in the image processing unit 8.

  At this time, a super-resolution image is generated by switching the disk arranged on the optical axis N to the super-resolution modulation pattern disk 100a or the confocal modulation pattern disk 110a by the movement of the support portions 103 and 113. Modulation for generating a confocal image can be switched.

  According to the second embodiment described above, the super-resolution modulation pattern disk 100a in which a plurality of holes for modulating the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a super-resolution image is formed, The optical axis N intersects one of the confocal modulation pattern disks 110a formed with a plurality of holes for modulating the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a confocal image. Since the optical modulation is selectively performed by moving the image, the super-resolution image and the confocal image can be easily switched and acquired.

  Also, according to the second embodiment described above, a super-resolution modulation pattern disk in which a plurality of holes for modulating the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a super-resolution image is formed. 100a and a confocal modulation pattern disk 110a in which a plurality of holes for modulating the spatial intensity distribution of the observation light into a spatial intensity distribution for generating a confocal image are individually provided, and illumination light and Since the spatial intensity distribution of the observation light is modulated, the degree of freedom of each modulation process can be improved, for example, when the thickness of the disk or the rotation speed of the disk is varied when performing each optical modulation.

  Furthermore, according to the second embodiment described above, a super-resolution modulation pattern disk in which a plurality of holes for modulating the spatial intensity distribution of the observation light into the spatial intensity distribution for generating a super-resolution image is formed. 100a and a confocal modulation pattern disk 110a in which a plurality of holes for modulating the spatial intensity distribution of the observation light into a spatial intensity distribution for generating a confocal image are formed. Can be formed. For example, in the confocal modulation pattern disk 110a, by forming holes having a plurality of different patterns for performing optical modulation for generating a confocal image with respect to the observation light, the degree of freedom of each modulation process Can be further improved.

  Further, according to the second embodiment described above, since observation is performed by inserting one of the super-resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a with respect to the optical axis N, for example, When observing cells, first, the support 113 is moved to place the confocal modulation pattern disk 110a on the optical axis N, and confocal observation is performed, so that the entire image of the cells is macroscopically three-dimensionally. Observation, and then withdrawing the support portion 113 from the optical axis N and moving the support portion 103 to place the super resolution modulation pattern disk 100a on the optical axis N to perform super resolution observation. It is possible to observe the molecular behavior of cells and microregions. That is, according to the second embodiment, the detailed observation of the sample S can be performed efficiently by simply switching between the macro three-dimensional observation by confocal observation and the super-resolution observation of the portion to be noted. Can do.

  In the second embodiment described above, the modulation mode is switched using two disks. However, the modulation mode may be switched using three or more disks. For example, in the case of using four disks, the modulation mode can be switched by moving in the direction orthogonal to the adjacent disks and arranging any of the disks on the optical axis.

  In the first and second embodiments described above, the hole is not only a through hole having a circular or elliptical shape, but also a slit (long hole) having an opening extending in the radial direction, or a rectangular hole. Also good. Further, the main surface of the disk may have a pinhole-like opening. In addition, the composite pattern disk 4a, the super resolution modulation pattern disk 100a, and the confocal modulation pattern disk 110a have been described as having a disk shape, but the main surface may have an elliptical shape. In addition, it may have a square shape. Furthermore, as long as light can pass therethrough, the hole may be filled with a transparent resin or the like.

  In the first and second embodiments described above, the optical modulation units 4 and 11 are provided as disk units so as to be detachable from the microscope systems 1 and 1a, and can be attached and detached as necessary. It is possible to mount discs having different hole shapes and patterns. In the second embodiment, the super-resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a may have a plurality of patterns like the composite pattern disk 4a of the first embodiment.

  In the first and second embodiments described above, the holding portions 44, 104, and 114 have been described as sliding the support portions 43, 103, and 113 in the direction perpendicular to the optical axis N. 4a, the super resolution modulation pattern disk 100a, and the confocal modulation pattern disk 110a can be applied as long as they can be moved forward and backward with respect to the optical axis N. That is, the insertion / removal direction of the composite pattern disk 4a, the super-resolution modulation pattern disk 100a, and the confocal modulation pattern disk 110a is not limited to the direction orthogonal to the optical axis N.

  Furthermore, in the first and second embodiments described above, the holding units 44, 104, and 114 automatically move the support units 43, 103, and 113 using a motor under the control of the control unit. Alternatively, it may be moved manually by a lever or dial.

  The support portions 103 and 113 are preferably moved in conjunction with each other in order to prevent interference due to movement. The super resolution modulation pattern disc 100a and the confocal modulation pattern disc 110a are respectively moved by one support portion. The super resolution modulation pattern disk 100a and the confocal modulation pattern disk 110a may be moved in conjunction with each other by being supported rotatably around the axis.

  Moreover, Embodiment 1 and 2 mentioned above are only the examples for implementing this invention, and this invention is not limited to these. Further, the present invention can form various inventions by appropriately combining a plurality of constituent elements disclosed in the respective embodiments and modifications. It is obvious from the above description that the present invention can be variously modified according to specifications and the like, and that various other embodiments are possible within the scope of the present invention.

  As described above, the microscope system and the disk unit according to the present invention are useful for easily switching and acquiring a super-resolution image and a confocal image.

DESCRIPTION OF SYMBOLS 1,1a Microscope system 2 Objective lens 3 Imaging lens 4,11 Optical modulation part 4a Composite pattern disk 5 Fluorescence cube 6 Relay lens 7 Imaging part 8 Image processing part 9 Light source 10 Illumination lens 41,101,111 Axis 42,102, DESCRIPTION OF SYMBOLS 112 Rotation motor 43,103,113 Support part 44,104,114 Holding part 51 Excitation filter 52 Dichroic mirror 53 Absorption filter 100 Super-resolution modulation switching part 100a Super-resolution modulation pattern disk 110 Confocal modulation switching part 110a Confocal Modulation pattern disc 401, 1001 Super-resolution modulation unit 402, 1101 Confocal modulation unit

Claims (3)

Illumination light generating means for generating illumination light for illuminating the specimen to be observed;
An imaging optical system that forms an image of the specimen by imaging the observation light from the specimen;
A first disk having a super-resolution modulation unit that modulates the spatial intensity distribution of the illumination light and the observation light into a spatial intensity distribution for generating a super-resolution image, and for generating a confocal image an optical modulation means comprising a second disk having a confocal modulator modulating the spatial intensity distribution,
The disk is rotated with an axis passing through the center of the disk as a central axis, and one of the first and second disks is rotated with a main surface of the one disk being an axis of the imaging optical system. a disk driving means for moving in a direction intersecting,
Image generation means for receiving the observation light that has passed through the disk and generating super-resolution image data or confocal image data based on the received observation light;
A microscope system comprising: The disk has a disk shape, and is provided so as to be detachable at an image forming position by the imaging optical system.
2. The microscope system according to claim 1, wherein the super-resolution modulation unit and the confocal modulation unit include a plurality of holes penetrating in the plate thickness direction and arranged at equal intervals along the circumferential direction. . The image generation means includes an image sensor that receives the observation light and performs photoelectric conversion,
The microscope system according to claim 1 , wherein the disk driving unit changes a rotation speed of the disk according to an exposure time of the image sensor.

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