JP7134727B2 - disk scanning microscope - Google Patents

Description

The present invention relates to disk scanning microscopes for obtaining super-resolution of observed objects.

A disk scanning microscope is known as one form of confocal microscope. A disk scanning microscope has a disk which is a confocal plate with a plurality of apertures on its surface at a position conjugate to the focal position of the objective lens. According to this microscope, it is possible to obtain only information on the focal position, and by rotating the disk, it is possible to sequentially obtain optical images at different positions in the observation object via the disk, thereby achieving high-speed observation. enable

2. Description of the Related Art In recent years, ISM (Image Scanning Microscopy) is known as one of techniques for realizing super-resolution observation (observation with resolution exceeding the resolution limit of a normal optical system) in a confocal microscope. In ISM, an array detector detects a light spot image from an observed object. Since each pixel in the array functions like a confocal aperture, multiple confocal images can be obtained simultaneously, but the confocal images are centroidally shifted.
FIG. 7 is a diagram for explaining deviation of the center-of-gravity position. For a detector that is d displaced from the center of the illumination PSF (PSF _ill ), the detection PSF (PSF _det ) is also d displaced. Therefore, the position of the center of gravity of the PSF as a confocal microscope also shifts, and if the shapes of the illumination PSF and the detection PSF are the same, the amount of shift is d/2.
High resolution and high detection efficiency can be achieved at the same time by correcting the deviation of the position of the center of gravity for observation. Techniques related to ISM are described in Non-Patent Document 1, for example.

Patent Literature 1 describes a technique for realizing super-resolution observation similar to ISM in a disk scanning microscope. In the technique of Patent Document 1, a lens array having a plurality of lens elements covering a plurality of apertures of the confocal plate is additionally provided on the confocal plate. This lens array functions to correct the position of the center of gravity, enabling super-resolution observation similar to that of ISM. The appropriate amount of center-of-gravity position correction varies depending on the optical system, but if the shapes of the illumination PSF and the detection PSF can be considered the same, the appropriate amount of correction is 1/2 of the amount of deviation from the center of the detection position. In other words, by projecting each spot onto the aperture of the confocal plate with a magnification of 1/2 using the lens array, it is possible to appropriately correct the position of the center of gravity.
The projection magnification of each spot onto the aperture by the lens array can be designed according to the optical system. FIG. 8 shows an example in which the illumination PSF is larger than the detection PSF. In this case, the center-of-gravity position shift amount d′ in the case of the shift amount d from the detection center position is larger than d/2. Therefore, the optimum projection magnification of each spot by the lens array is greater than 1/2.

JP 2016-212154 A

C. Muller and J. Enderlein, “Image Scanning Microscopy,” Physical Review Letters, Vol. 104, 198101, 2010.

When performing normal confocal observation and super-resolution observation, an example of exchanging a disc without a lens array with a disc with a lens array disclosed in Patent Document 1 can be considered. On the other hand, the appropriate projection magnification from the disc to the specimen surface may differ depending on the disc used. If the projection magnification deviates from the appropriate one during super-resolution observation, the illumination PSF will be changed, and the projection magnification of the lens array for obtaining super-resolution will change, making it impossible to obtain super-resolution. Therefore, in a configuration in which disks are exchanged depending on the application, a situation may arise in which it is difficult to bring out the full performance inherent in the disks.

In view of the above circumstances, it is an object of the present invention to provide a disk scanning microscope capable of performing excellent super-resolution observation and switching between super-resolution observation and normal observation.

A disk scanning microscope according to one aspect of the present invention includes an objective lens that irradiates an illumination spot on an observation object with illumination light and captures light generated from the observation object, a scanning optical system that scans the illumination light, forming a spot image that is an optical image of the light generation position generated by irradiating the illumination spot with the illumination light, and projecting the entire optical image formed by a set of the spot images of the plurality of different generation positions. An intermediate variable power optical system capable of changing magnification, an imaging device, and a relay optical system that relays the optical image and forms an image on the imaging device, wherein the scanning optical system is a light shielding portion on the surface One or more first disks for super-resolution observation having a plurality of apertures formed in the first disk and having microlenses covering the plurality of apertures; a second disk for confocal observation, a disk switching mechanism for switching and arranging the first disk and the second disk at a position where the spot image is formed, and a control device , wherein the control device controls the disc switching mechanism to switch and arrange the first disc or the second disc to a position where the spot image is formed, based on an input from the user, and controls the first disc or the When the second disc is placed at each spot image forming position, the intermediate variable magnification optical system is arranged so that the projection magnification of the entire optical image is suitable for the disc placed at the spot image forming position. is controlled, and the microlens reduces the spot image when the first disk is placed at a position where the spot image is formed.

According to the present invention, it is possible to provide a disk scanning microscope capable of performing excellent super-resolution observation and switching between super-resolution observation and normal observation.

It is a figure which shows the structure of the disk scanning microscope in 1st Embodiment. 3 is a diagram showing the configuration of a disk unit; FIG. FIG. 10 is a diagram showing a state in which discs are switched and arranged with respect to an optical system that does not have an intermediate variable magnification optical system; FIG. 2 is a diagram showing a state in which a disc unit for confocal observation is inserted into the optical path in the disc scanning microscope according to the first embodiment; It is a figure which shows the functional structure of a control apparatus. FIG. 10 is a diagram showing the configuration of an intermediate variable power optical system in a modified example; FIG. 10 is a diagram for explaining deviation of the center-of-gravity position when detected by an array detector; FIG. 10 is a diagram showing a deviation amount of the center-of-gravity position when the illumination PSF is larger than the detection PSF;

A disk scanning microscope system 100 according to the first embodiment will be described below. FIG. 1 is a diagram showing the configuration of a disk scanning microscope system 100. As shown in FIG.

A disk scanning microscope 100 includes a light source unit 1, an optical fiber 2, a beam expander 3, a scanning optical system 60, an intermediate variable power optical system 30, a lens 11, an objective lens 12, a relay optical system 35, an imaging device 15, and an image processing board. 16, an external storage device 17, a monitor 18, and a control device 20.

A light source unit 1 outputs illumination light for illuminating a sample S, which is an observation object. The optical fiber 2 guides the illumination light from the light source unit 1, and the beam expander 3 adjusts the illumination light flux to an appropriate size.

The scanning optical system 60 has disc units 40 and 50 , a turret 65 and a motor 19 . The scanning optical system 60 scans the illumination light. The disk unit 40 and the disk unit 50 are put into and removed from the optical path of the disk scanning microscope 100 by the turret 65 . In the example of FIG. 1, the disk unit 40 is shown arranged on the optical path of the disk scanning microscope 100 .

FIG. 2 is a diagram showing the configuration of the disk units 40 and 50. As shown in FIG. The disc unit 40 has an illumination disc 41 and a confocal disc 42 . The illumination disk 41 and the confocal disk 42 are rotated integrally about the axis 43 by the motor 19 while being placed on the optical path. The configuration of the disc unit 40 will be described below, taking as an example the state in which the disc unit 40 is arranged on the optical path as shown in FIG.

The illumination disc 41 has a plurality of microlenses 41a arranged on the disc surface. The confocal disc 42 has a plurality of openings 42a on a disc surface 42c, which is a light shielding portion, at positions directly below the microlenses 41a of the illumination disc 41. As shown in FIG. That is, the illumination disc 41 is arranged so that the illumination light passing through the microlens 41a passes through the aperture 42a of the confocal disc 42. FIG. Since the illumination disk 41 has the microlenses 41a, it is possible to prevent the illumination light from being blocked by the disk surface 42c other than the opening of the confocal disk 42, thereby improving the illumination efficiency.

The confocal disc 42 has a plurality of apertures 42a formed on the disc surface 42c. The confocal disk 42 is, for example, a Nipkow disk in which apertures are arranged at regular intervals. By rotating the confocal disk 42 , it functions as scanning means for sequentially irradiating different positions on the sample S with the illumination light through the objective lens 12 . Each small area on the specimen S irradiated with the illumination light is referred to as an illumination spot. The confocal disk 42 is arranged at a position conjugate with the front focal position of the objective lens 12, that is, at a position where the intermediate variable magnification optical system 30 forms an optical image of the sample S, which is an observation object. The optical image referred to here includes a spot image, which is an optical image of the position of the light generated by irradiating one illumination spot with the illumination light. More specifically, as the scanning optical system 60 scans, an entire optical image is formed by a set of spot images from a plurality of different light generation positions that are generated as each illumination spot is illuminated. The confocal disk 42 relays each spot image of the specimen S to the imaging device 15 in the subsequent stage by way of the dichroic mirror 6 . That is, when each aperture 42a of the confocal disk 42 is arranged on the optical path, the position of each aperture 42a and the front focal position of the objective lens 12 are conjugate positions. Therefore, only light generated from the in-focus position can be used for imaging.

Further, the confocal disk 42 has microlenses 42b covering the plurality of apertures 42a on the surface on the specimen S side. Since the confocal disk 42 has the microlenses 42b, the condensing angle of the light incident from the sample S side is increased. Each spot image is thereby reduced. Specifically, the distribution in each spot image is displaced toward the center of the aperture 42a by the action of the microlens 42b compared to when the confocal disc 42 does not have the microlens 42b. That is, the microlens 42b acts to reduce each spot image when the confocal disk 42 is placed at a position where each spot image is formed by a turret 65, which will be described later. Note that the microlens 42b does not reduce the entire optical image obtained by scanning the sample S with the illumination light by the scanning optical system 60. FIG.

In general, when acquiring a spot image with a microscope, light outside the central axis in the Airy disk is divided into an illumination PSF on the illumination system side that illuminates the sample S and an imaging PSF on the imaging side that forms the spot image of the sample S. There is a shift in the center of gravity position between them. Therefore, when an array-type detector such as the imaging device 15 is used, such deviation is reflected in the acquired image. It is known that the deviation of the center of gravity position at each position in the Airy disk is about 1/2 of the distance between the peak positions of the illumination PSF and the imaging PSF.

For this reason, by reducing the size of each spot image generated from the viewing plane by a factor of 1/2, the shift in the position of the center of gravity can be corrected. In other words, it is possible to remove the blur caused by the deviation of the center of gravity position and obtain super-resolution, which is a resolution exceeding the resolution limit of the confocal optical system. In the disk scanning microscope 100, the size of each spot image generated from the observation surface is reduced to 1 by increasing the divergence angle of the light from the specimen S when it reaches the opening 42a. It is possible to reduce the size to /2 times.

The disc unit 50 has an illumination disc 51 and a confocal disc 52 . Illumination disk 51 is equal to illumination disk 41 . Also, the confocal disk 52 differs from the confocal disk 42 in that it does not have the microlens 42b, but otherwise has the same configuration. That is, the disk unit 50 is a disk unit for performing normal confocal observation without performing super-resolution observation by centroid position correction.

The turret 65 switches and disposes the disc units 40 and 50 at positions where spot images are formed by the intermediate variable magnification optical system 30 . The turret 65 is also referred to as a disk switching mechanism because the disk units 40 and 50 are alternately arranged.

The intermediate variable power optical system 30 is provided between the lens 11 and the scanning optical system 60 . The intermediate variable power optical system 30 guides the light from the irradiation position on the sample S captured by the objective lens 12 and forms each spot image to the scanning optical system 60 . Also, the intermediate variable power optical system 30 changes the projection magnification of the intermediate variable power optical system 30 . The projection magnification changed by the intermediate variable-magnification optical system 30 refers to the projection magnification of the entire optical image formed by a set of spot images from a plurality of different light generation positions that are generated with the illumination of each illumination spot. . The intermediate variable power optical system 30 includes a base 8, a lens group 7, a lever 9, a motor 10, and mirrors 7a, 7b, 8a, 8b. The lens group 7 is a lens group having an enlargement magnification for projecting the light from the sample S in an enlarged manner.

A pedestal 8 supports mirrors 8 a and 8 b and is connected to a lever 9 . The position of the pedestal 8 is changed by moving the lever 9 by the motor 10 . The position of the pedestal 8 is changed so that the mirrors 8 a and 8 b are arranged on the optical axis of the objective lens 12 , and the light passes through the lens group 7 . On the other hand, by changing the position of the pedestal 8 so that the mirrors 8 a and 8 b are excluded from the optical axis, the light does not pass through the lens group 7 . That is, the intermediate variable magnification optical system 30 switches between two optical systems with different projection magnifications.

FIG. 3 is a diagram for explaining a problem that may occur when a disc is switched and placed in the optical system 200 that does not have an intermediate variable magnification optical system unlike the disc scanning microscope 100. FIG. On the left side of FIG. 3, a disk 210 corresponding to the confocal disk 52 of the disk unit 50 is arranged in the optical system, and on the right side of FIG. 3, a disk 220 corresponding to the confocal disk 42 of the disk unit 40 is arranged in the optical system. For example, when the state shown in FIG. 3 left to FIG. It is reduced by the action of the lens 221 . Therefore, the size of the luminous flux diameter at the pupil position of the objective lens may become smaller than the size that should be originally set, and the illumination PSF may be changed.

If the projection magnification of the entire optical image, which should be set for each disc, differs by simply switching discs, the size of the illumination spot may change, resulting in degradation of resolution.

In addition, in the configuration of the disk unit 40 that displaces the spot image by the action of the microlens to obtain super-resolution, if the illumination PSF is changed, the amount of displacement of the spot image required to obtain super-resolution will change. If there is a large difference between the amount of variation due to the microlens and the amount of variation required to obtain super-resolution, the displacement due to the microlens may actually increase the deviation of the center of gravity position. In this case, not only the original super-resolution performance cannot be obtained, but also the resolution is inferior to that of normal confocal observation. In other words, it is particularly important in the configuration of the disk unit 40 to appropriately match the projection magnification of the entire optical image. However, in the configuration of the disk scanning microscope 200, the projection magnification of the entire optical image cannot be adjusted appropriately, and performance in the original observation application may not be obtained.

On the other hand, in the disc scanning microscope 100, the intermediate variable magnification optical system 30 appropriately changes the projection magnification for each disc used, thereby preventing the above problem. FIG. 4 shows a state in which a disk unit 50 for normal confocal observation is inserted into the optical path. When the disk unit 40 for super-resolution observation shown in FIG. That is, when the turret 65 places the disc unit 40 at the position where each spot image is formed, the intermediate variable magnification optical system 30 makes the projection magnification higher than that of the disc unit 50 without the microlenses 42b.

In this example, by passing through the lens group 7 during super-resolution observation, the luminous flux diameter sufficiently fills the pupil of the objective lens 12, ensuring an appropriate illumination PSF.

According to the disk scanning microscope 100 described above, by changing the projection magnification of the intermediate variable magnification optical system 30 to a projection magnification suitable for each disk, the diameter of the light beam is adjusted to sufficiently satisfy the pupil diameter of the objective lens 12. It can be adjusted and observations can be made without compromising the inherent performance of the disc. Especially in the configuration for switching the disk unit, it is possible to prevent the illumination PSF from being changed, so that it is possible to perform normal confocal observation while performing excellent super-resolution observation.

It is conceivable that the disc unit 40 and the disc unit 50 may have different sizes of apertures formed in the confocal discs. Even in such a case, the intermediate variable magnification optical system 30 should be adjusted so that the projection magnification is suitable for each disc unit when the disc units 40 and 50 are arranged on the optical path. In particular, in order to perform super-resolution observation satisfactorily, the intermediate variable power optical system 30 is arranged in the optical path when the disk unit 40 for super-resolution observation does not have the microlens 42b. It is preferable that the projection magnification is higher than that. In addition, two or more disk units may be arranged so as to be switchable, and the intermediate variable-magnification optical system 30 can switch not only the lens group 7 but also a plurality of optical systems with different projection magnifications. may have in

A relay optical system 35 is installed after the dichroic mirror 6 and includes a fluorescence filter 13 and a lens 14 . The relay optical system 35 relays each spot image formed by the scanning optical system 60 to the imaging device 15 and forms an image.

The control device 20 is a computer for controlling each component of the disk scanning microscope 100 . The control device 20 may use the connected image processing board 16 and record data in the external storage device 17 during image processing, for example.
A monitor 18 displays an image output from the control device 20 .

FIG. 5 is a diagram showing the functional configuration of the control device 20. As shown in FIG. The control device 20 has an intermediate magnification control section 21 , a disc switching control section 22 , a light source control section 23 , an exposure control section 24 , a scanning control section 25 and an image control section 26 .

The intermediate variable power control section 21 controls the intermediate variable power optical system 30 . More specifically, the projection magnification is changed by driving the motor 19 based on the user's input from an input device such as a keyboard connected to the control device 20 . The intermediate variable power control section 21 may perform control so that the intermediate variable power optical system 30 changes the projection magnification when the disc unit 40 or the disc unit 50 is arranged at the formation position of each spot image. Alternatively, when an input to the effect of super-resolution observation is detected from the user, the corresponding disc is automatically arranged by the disc switching control unit 22, which will be described later, and the intermediate magnification control unit 21 selects the corresponding disc. Control may be performed so that the projection magnification is the same as above.

The disc switching control unit 22 controls the switching of the disc units 40 and 50 arranged on the optical path by controlling the rotation of the turret 65 based on the user's input from the input device.

The light source control unit 23 controls ON/OFF of the light source unit 1 and changes in intensity of output illumination light.

The exposure control section 24 controls the exposure time of the imaging device 15 .

The scanning control unit 25 rotates the installed disk unit by controlling the motor 19 to scan the sample S with the illumination light.

The image control unit 26 is an arithmetic device that performs image processing on the image captured by the imaging device 15 . The image control unit 26 may, for example, perform digital processing for emphasizing the super-resolution component on the super-resolution observation data from the disk unit 40 to enhance the resolution of the image. Note that the super-resolution component is a high-frequency component exceeding the cutoff frequency of the imaging optical system. Also, the confocal observation data from the disk unit 50 may be digitally processed to emphasize the super-resolution component contained in the confocal image data. Techniques such as Fourier or convolution filters, or deconvolution can be used as examples of digital processing.

As a result, the switching of the disc unit and the control of the intermediate variable magnification optical system 30 corresponding to the arranged disc unit are automatically performed by the controller 20, thereby reducing the burden on the user.

Further, the intermediate variable magnification optical system 30 may change the projection magnification according to the type of objective lens used (objective lens with different NA, etc.) in addition to switching the disc unit. That is, the intermediate magnification control unit 21 may control the change of the projection magnification by the intermediate magnification variable optical system 30 when the objective lens is switched.

Moreover, the scanning optical system 60 may include a plurality of types of disk units 40 for performing super-resolution observation and disk units 50 for performing normal confocal observation. For example, a plurality of disk units 40 and 50 having different opening diameters and opening intervals may be prepared. Also, the scanning optical system 60 may include a plurality of disk units 50 having microlenses with different magnifications. In other words, the scanning optical system 60 may have one or more disk units 40 and 50 .

Also, an intermediate variable power optical system 150, which is a modification of the intermediate variable power optical system 30, will be described. FIG. 6 shows the configuration of an intermediate variable magnification optical system 150 in a modified example. The intermediate variable magnification optical system 150 includes lenses 151 , 152 and 153 . At least one of lens 151, lens 152, and lens 153 is changed in position on the optical path by a motor. Since the lenses 151 to 153 function as a zoom lens as a whole, any projection magnification can be set within a specific range, increasing the flexibility of the system.

When the disk scanning microscope 100 has the intermediate variable magnification optical system 150, the projection magnification is switched by moving the lens. According to the intermediate variable power optical system 150 , it is possible to switch the projection magnification with less energy for driving than for moving the base 8 . Also, instead of the zoom lens, the lens system may be simplified by including a varifocal lens that maintains the image forming position only at a specific projection magnification.

The above-described embodiments are specific examples for easy understanding of the invention, and the invention is not limited to these embodiments. Various modifications and alterations can be made to the disk scanning microscope described above without departing from the scope of the present invention as defined in the claims.

1 light source unit 2 optical fiber 3 beam expander 6 dichroic mirror 7 lens group 7a, 7b, 8a, 8b mirror 8 pedestal 9 lever 10 motor 11, 14 lens 12 objective lens 13 fluorescence filter 15 imaging device 16 image processing board 17 external storage device 18 monitor 19 motor 20 control device 21 intermediate magnification control section 22 disc switching control section 23 light source control section 24 exposure control section 25 scanning control section 26 image control section 35 relay optical system 40, 50 disc units 41, 51 illumination disc 42, 52 confocal disk 60 scanning optical system 65 turrets 41a, 51a, 42b microlenses 42a, 52a apertures 42c, 52b light shielding plate

Claims (6)

an objective lens that irradiates an illumination spot on an observation object with illumination light and captures the light emitted from the observation object;
a scanning optical system that scans the illumination light;
forming a spot image that is an optical image of the light generation position generated by irradiating the illumination spot with the illumination light, and projecting the entire optical image formed by a set of the spot images of the plurality of different generation positions. an intermediate variable magnification optical system capable of changing magnification;
an imaging device;
a relay optical system that relays the optical image and forms an image on the imaging device;
The scanning optical system is
one or more first discs for super-resolution observation having a plurality of openings formed on a surface that is a light shielding portion and having microlenses covering the plurality of openings;
one or more second discs for confocal observation in which a plurality of apertures are formed on a surface that is a light shielding portion;
a disc switching mechanism for switching and arranging the first disc and the second disc at a position where the spot image is formed;
a controller ;
The control device controls the disc switching mechanism to switch and arrange the first disc or the second disc to a position where the spot image is formed, based on an input from a user, and When the disc or the second disc is placed at each spot image forming position, the intermediate variable is provided so that the projection magnification of the entire optical image is suitable for the disc placed at the spot image forming position. controls the magnification optics,
The disk scanning microscope, wherein the microlens reduces the spot image when the first disk is placed at a position where the spot image is formed. The disk scanning microscope of claim 1, wherein
The intermediate variable-magnification optical system is such that when the disc switching mechanism places the first disc at a position where the spot image is formed, the light captured by the objective lens fills the pupil diameter of the objective lens. A disk scanning microscope characterized by changing the projection magnification. In the disk scanning microscope according to claim 1 or claim 2,
The disk scanning microscope, wherein the intermediate variable magnification optical system switches between two or more optical systems having different projection magnifications. In the disk scanning microscope according to claim 1 or claim 2,
A disk scanning microscope, wherein the intermediate variable magnification optical system includes a zoom lens. In the disk scanning microscope according to any one of claims 1 to 4,
A disk scanning microscope, comprising image processing means for performing high-frequency emphasis processing for emphasizing frequency components above a specific frequency on an image captured by the imaging device. In the disk scanning microscope according to any one of claims 1 to 5,
A disc characterized by comprising control means for controlling the intermediate variable power optical system to change the projection magnification when the first disc or the second disc is placed at the spot image forming position. scanning microscope.

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