JP6090607B2 - Confocal scanner, confocal microscope - Google Patents

Description

  The present invention relates to a confocal scanner that obtains super-resolution by optical processing, and more particularly to a confocal scanner capable of reducing the complexity of position adjustment of optical components and a confocal using such a confocal scanner. It relates to a microscope.

  A confocal microscope that irradiates a target object with light at a pin point, selectively detects only the focused light from the irradiation point, and scans that point to obtain an image. It is an optical microscope that can be reconstructed. Confocal microscopes are widely used in the life science field, and various related techniques have been proposed.

  For example, in Patent Document 1, regarding a confocal scanner that is a scanning unit of a confocal microscope, a microlens disk provided with a plurality of microlenses, and a pinhole disk in which pinholes are formed in the same pattern as the microlens There is disclosed a confocal scanner that performs multi-beam scanning by rotating these while applying illumination light.

  Conventionally, optical microscopes including confocal microscopes have Abbe's diffraction limit, that is, the resolution limit based on the theory that an object smaller than half of the wavelength of light applied to the object cannot be seen. In recent years, a super-resolution technique for obtaining an image with a resolution exceeding the resolution limit has been developed and put into practical use.

  For example, Patent Document 2 captures an image having a high-frequency component exceeding the resolution limit using a scan mask that modulates the spatial intensity distribution of excitation light from a light source and return light from an object, and performs high-frequency enhancement processing. It is described that a super-resolution image is obtained by applying. Further, Non-Patent Document 1 discloses that an image in which a large number of bright spots are recorded using a shutter that allows illumination light from a light source device to pass through in a strobe shape, with the positions of the bright spots being slightly different from each other. It is described that a super-resolution image is obtained by synthesizing each image after taking a picture and performing image processing so that each image has a luminescent spot size of ½.

  The super-resolution techniques described in Patent Document 2 and Non-Patent Document 1 are not suitable for real-time observation because they perform complicated or large-scale image processing, and require a large calculation load and processing time.

  On the other hand, Patent Document 3 describes a technique that can obtain a super-resolution image at high speed by optical processing. FIG. 19 is a diagram showing the configuration of the optical system of the microscope system described in Patent Document 3. As shown in FIG.

  As shown in the figure, the microscope system 400 is configured to irradiate the sample 430 with the laser light emitted from the light source 410 and photograph the return light with the camera 420. The collimated illumination light emitted from the light source 410 is divided into a number of illumination light beamlets by the microlens array 411. The illumination light beamlet is reflected by the galvanometer mirror 413 and condensed on the sample 430 by the objective lens 414.

  The sample 430 emits return light based on the illumination light. In particular, in the case of observation of a fluorescent sample, the sample 430 is obtained by staining a specific structure with a fluorescent dye or the like, and the fluorescent dye molecule is excited by the illumination light and emits fluorescence having a longer wavelength than the illumination light.

  Return light from the sample 430 is reflected by the galvanometer mirror 413, further reflected by the beam splitter 412, and passes through the lens 415. The return light that has passed through the lens 415 reaches the pinhole array 416 in which many pinholes are formed, but only the light from the focal plane of the sample 430 is focused on the pinhole array 416 and passes through the pinhole. To do.

  The return light that has passed through the pinhole passes through the microlens array 417 and microlens array 418 in which a large number of microlenses are arranged, is reflected by the galvano mirror 413, and is imaged by the camera 420. The return light is a part of the sample 430 irradiated with the illumination light beamlet, but the entire sample 430 can be scanned by turning the galvanometer mirror 413.

  Here, the pinhole array 416 is precisely arranged so that each pinhole is in a conjugate position with the focal spot of the objective lens 414. The individual pinholes of the pinhole array 416 are precisely arranged at the focal positions of the individual microlenses of the microlens array 417. Furthermore, each microlens of the microlens array 417 and each microlens of the microlens array 418 are precisely arranged so as to be coaxial.

  The focal length of each microlens in the microlens array 418 is shorter than the focal length of each microlens in the microlens array 417.

  In this configuration, the return light from the focused surface that has passed through the pinhole of the pinhole array 416 is converted into parallel light by the microlens array 417 and is incident on the microlens array 418. Since the focal length of each microlens of the microlens array 418 is shorter than the focal length of each microlens of the microlens array 417, the return light has a larger numerical aperture when passing through the microlens array 417. For example, if the focal length of each microlens of the microlens array 418 is ½ of the focal length of each microlens of the microlens array 417, the microlens array 417 is converted into a light beam having a numerical aperture of twice.

  A super-resolution image of the sample 430 can be obtained by capturing this light beam with the camera 420 while turning the galvanometer mirror 413. At this time, since it is not necessary to perform complicated image processing and a large number of times of imaging, a super-resolution image can be obtained easily at high speed.

Japanese Patent Laid-Open No. 10-062691 JP 2012-78408 A International Publication No. 2013/126762

Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.Proceedings of the National Academy of Sciences of United States of America, Vol.110, pp.21000-21005 (2013)

  As described above, a super-resolution image can be easily obtained at high speed by using a confocal scanner that obtains super-resolution by optical processing. However, a confocal scanner that obtains super-resolution by optical processing can precisely adjust the optical components of the microlens array for illumination light, the objective lens, the two microlens arrays for return light, and the pinhole array. is necessary. In the optical system described in Patent Document 3, these optical components are independently spaced apart from each other, and precise position adjustment is not easy.

  Precise position adjustment of optical components increases costs, and is susceptible to environmental changes and changes over time, making maintenance complicated. Therefore, it is desirable to reduce the burden of precise position adjustment as much as possible.

  Accordingly, an object of the present invention is to reduce the burden of precise position adjustment in a confocal scanner that obtains super-resolution by optical processing.

  In order to solve the above-described problem, the confocal scanner according to the first aspect of the present invention includes a first microlens disk on which a plurality of microlenses are arranged, and a plurality of the corresponding microfocus patterns corresponding to the arrangement pattern of the first microlens disks. The second microlens disk having a rotation axis common to the first microlens disk, the illumination light irradiating the object is guided to the first microlens disk, and the first microphone lens A beam splitter for guiding the return light from the object that has passed through each microlens of the disk to the corresponding microlens of the second microlens disk, and an opening of the microlens disposed on the second microlens disk The number is the numerical aperture of the microlens arranged on the first microlens disk. It is also big and said Ri.

  Here, a pinhole may be disposed at each focal position on the side opposite to the object of each microlens disposed on the second microlens disk.

  Alternatively, a pinhole may be arranged at each focal position on the object side of each microlens arranged on the first microlens disk.

  At this time, an image reversal microlens corresponding to each pinhole may be disposed on the object side.

  Alternatively, an image reversal microlens is disposed at a position farther than each focal position on the object side of each microlens disposed on the first microlens disk, and the illumination light of each image reversal microlens A pinhole may be arranged at each in-focus position.

  The microlens disposed on the second microlens may be a concave lens.

  In either case, the numerical aperture of the microlens disposed on the second microlens disk can be approximately twice the numerical aperture of the microlens disposed on the first microlens disk.

  The aperture of the microlens disposed on the first microlens disk is preferably smaller than the aperture of the microlens disposed on the second microlens disc.

  The illumination light guided to the first microlens disk by the beam splitter can travel obliquely with respect to the optical axis of the microlens of the first microphone lens disk.

  Alternatively, the illumination light guided to the first microlens disk by the beam splitter travels in parallel with the optical axis of the microlens of the first microphone lens disk, and the first microlens disk and the second microlens disk. An optical member for correcting an optical path shift generated by the beam splitter may be disposed between the optical members.

  In order to solve the above problems, a confocal microscope according to a second aspect of the present invention includes any one of the above-described confocal scanners, a light source unit that emits parallel illumination light to the beam splitter, and the first An objective lens disposed on the microlens disk side and an image pickup device disposed on the second microlens disk side are provided.

  A variable magnification optical system may be disposed between the first microlens disk and the objective lens, and between the second microlens disk and the imaging element.

  According to the present invention, it is possible to reduce the burden of precise position adjustment in a confocal scanner that obtains super-resolution by optical processing.

It is a figure which shows typically the structure of the confocal microscope using the confocal scanner of 1st Example. It is a figure which shows the detail of the positional relationship of a microlens disc, a pinhole disc with a microlens, and a beam splitter. It is a figure which shows typically the state of the scanning of a confocal microscope. It is the figure which represented typically the confocal optical system using a camera. It is a figure which shows the general form of received light quantity I (x). It is a figure which shows the structural example of the light source unit which radiate | emits parallel illumination light. It is a figure which shows the 1st modification in 1st Example. It is a figure which shows the 2nd modification in 1st Example. It is a figure which shows the 3rd modification in 1st Example. It is a figure which shows typically the structure of the confocal microscope using the confocal scanner of 2nd Example. It is a figure which shows the detail of the positional relationship of a pinhole disk, a micro lens disk, a 2nd micro lens disk, and a beam splitter. It is a figure which shows the 2nd modification in 2nd Example. It is a figure which shows the 3rd modification in 2nd Example. It is a figure explaining an inverted image. It is a figure explaining the adjacent inverted point image. It is a figure which shows the improved type corresponding to 2nd Example. It is a figure which shows another example of the improved type corresponding to 2nd Example. It is a figure which shows another example of the improved type corresponding to 2nd Example. It is a figure which shows the structure of the optical system of the conventional microscope system which obtains super-resolution by optical processing.

<First embodiment>
Embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a diagram schematically showing a configuration of a confocal microscope 10 using the confocal scanner 20 of the first embodiment.

  As shown in the figure, the confocal microscope 10 using the confocal scanner 20 includes a light source unit 110 that emits parallel light as illumination light, an objective lens 120, a photographing lens 130, a relay lens 140, and a camera 150. Assume that a super-resolution confocal image of the sample 30 is taken.

  The confocal scanner 20 includes a microlens disk 210 in which a large number of microlenses 211 are regularly arranged, a number of microlenses 221 on one surface, and a number of pinholes 223 formed on the other surface. A pinhole disk 220 with a microlens, a motor 230, and a beam splitter 240 are provided. The microlens disc 210 and the pinhole disc 220 with a microlens overlap with each other, and rotate integrally with the motor 230 around the center axis.

  By integrally forming the microlens disc 210 and the pinhole disc 220 with microlenses, mechanical adjustment of both discs becomes unnecessary, and stability against environmental changes and changes over time can be improved.

  The microlens 221 of the pinhole disk 220 with a microlens is disposed on the microlens disk 210 side, and a light shielding mask 222 is provided on the opposite surface. The individual microlenses 221 of the pinhole disk 220 with a microlens are arranged so as to be coaxial with the individual microlenses 211 of the microlens disk 210.

  The pinhole disk 220 with a microlens is formed of a material that transmits return light at least at a portion where the microlens 221 is disposed, and the thickness thereof is a focal length of the microlens 221.

  The pinhole 223 is configured by a minute opening formed in the light shielding mask 222, and each pinhole 223 is disposed so as to be a focal position of each microlens 221. The size of the pinhole 223 is preferably about the same as the diffraction limit of the light collected by the microlens 221.

  In this example, both the microlens 211 and the microlens 221 are convex lenses, but other optical elements may be used as long as they have a lens effect. For example, a Fresnel lens or a diffractive optical element may be used. Further, it may be a single lens or a compound lens.

  The beam splitter 240 is disposed between the microlens disk 210 and the pinhole disk 220 with a microlens. The beam splitter 240 has a characteristic of reflecting the wavelength of illumination light and transmitting fluorescence having a long wavelength emitted from the sample 30 by the illumination light.

  FIG. 2 shows details of the positional relationship among the microlens disc 210, the pinhole disc 220 with microlens, and the beam splitter 240. In this figure, a pair of microlenses 211 arranged on the same axis and the central axis of the microlenses 211 are indicated by a one-dot chain line.

  Since the beam splitter 240 has a certain thickness, the light path of obliquely incident light is shifted by refraction. In the present embodiment, in order to correct this optical path shift, the angle of the beam splitter 240 with respect to the microlens disk 210 is made slightly smaller than 45 degrees. For this reason, the light reflected by the beam splitter 240 is obliquely directed to the microlens disk 210 with a slight angle with the one-dot chain line as shown in the figure.

  The light from the microlens 211 is also directed to the beam splitter 240 through the reverse optical path of the same angle, and the angle and the arrangement are such that the light refracted by the beam splitter 240 is incident on the microlens 221.

  Here, the outer diameter of the micro lens 211 is configured to be slightly smaller than the outer diameter of the micro lens 221. As an optical path shift correction method, the angle of the beam splitter 240 may be set to 45 degrees, and the microlens 211 and the microlens 221 may be disposed slightly inclined with respect to the central axis. In this case, the light incident on the microlens 211 from the beam splitter 240 is parallel to the optical axis indicated by the alternate long and short dash line.

  In this embodiment, the numerical aperture (NA) of the light collected by the microlens 211 is configured to be twice the numerical aperture of the light collected by the microlens 221. That is, if the angle of the light collected by the microlens 211 is θ, the parameters of both microlenses are selected so that the angle of the light collected by the microlens 221 and diverging from the pinhole 223 is 2θ. In principle, double the resolution is the best, but other numerical values may be used as long as the numerical aperture of the light collected by the microlens 221 is larger.

  Here, if the angle with respect to the optical axis center is φ and the refractive index of the medium is n, NA = n · sin (φ). Further, FIG. 2 shows the diameter of the micro lens large for convenience of explanation, but the actual diameter of the micro lens is as small as 1 mm or less. That is, the angles of θ and 2θ are extremely small values. When the angle is very small, sin (φ) ≈φ can be set, so that the numerical aperture of the light collected by the microlens 221 is approximately twice the numerical aperture of the light collected by the microlens 211. It turns out that it is.

  Returning to the description of FIG. 1, the photographing lens 130 and the objective lens 120 are arranged in the downward direction of the confocal scanner 20, and the sample 30 is arranged at the focal position of the objective lens 120. A relay lens 140 is disposed above the confocal scanner 20 and projects a pinhole surface image onto the image sensor 151 surface of the camera 150. Note that a filter that allows only desired fluorescence to pass through may be disposed in front of the camera 150.

  In such a configuration, the illumination light emitted from the light source unit 110 is reflected by the beam splitter 240 and guided to the microlens disk 210. Then, it is divided into a number of illumination light beamlets by a number of microlenses 211 on the microlens disk 210. Each illumination light beamlet is focused once, converted into parallel light by the photographing lens 130, and condensed at each position on the sample 30 by the objective lens 120.

  The sample 30 emits return light based on the illumination light beamlet. In particular, in the case of fluorescent sample observation, the sample 30 is obtained by staining a specific structure with a fluorescent dye or the like, and the fluorescent dye molecule is excited by the illumination light and emits fluorescence having a longer wavelength than the illumination light.

  Each return light captured by the objective lens 120 returns on the same optical path as the illumination light. That is, it becomes parallel light by the objective lens 120, is once focused by the photographing lens 130, enters the microlens disk 210, and is converted back to parallel light by the action of the microlens 211. That is, the microlens disk 210 is used for both illumination light and return light. As a result, optical components that require position adjustment can be reduced.

  Each return light passes through the beam splitter 240 and enters the microlens 221 of the pinhole disk 220 with a microlens in a parallel light state. At this time, as shown in FIG. 2, the shift of the optical path due to refraction in the beam splitter 240 is corrected by adjusting the angle of the beam splitter 240 or the like.

  The parallel light that has passed through the beam splitter 240 is condensed on the pinhole 223 by the action of the microlens 221. As described above, since the numerical aperture of the microlens 221 is twice as large as the numerical aperture of the microlens 211, the focal point on the pinhole 223 is larger than the focal point where the beamlet is connected before the microlens 211. Is about ½ in size and passes through this pinhole 223.

  At this time, only the return light from the sample-side focal plane of the objective lens 120 passes through the pinhole 223. On the other hand, the return light from other than the in-focus plane is not focused on the pinhole 223 and is blocked by the light shielding mask 222 and cannot pass through the pinhole 223.

  The light that has passed through the pinhole 223 is imaged on the image sensor 151 of the camera 150 by the action of the relay lens 140. By rotating the microlens disk 210 and the pinhole disk 220 with microlens by the motor 230 and scanning the entire sample 30 with illumination light, a confocal image of the sample 30 can be captured by the camera 150. FIG. 3 schematically shows this scanning state. A three-dimensional image can be obtained by scanning the sample 30 in the z-axis direction.

  Here, the reason why a super-resolution image can be obtained with the confocal microscope 10 using the confocal scanner 20 of the present embodiment will be described. FIG. 4 is a diagram schematically showing a confocal optical system using a camera which is a two-dimensional image sensor. For simplicity, the illumination light side and the return light side are shown separately on the left and right sides, and the point light source to the sample surface is the illumination light side, and the sample surface to the image surface is the return light side. In addition, for the sake of simplicity, the magnification of the objective lens is set to 1, but this assumption does not impair the generality of the discussion. Assuming that the magnification is 1, the sample plane and the image plane can be handled with the coordinates of an equal scale upside down.

  Illumination light emitted from a point light source on the optical axis is focused on the sample surface by the objective lens. At this time, due to light diffraction, the intensity distribution of the illumination light on the sample surface has a certain spread around the coordinate 0 as shown in the figure. This spread of light is generally called an Airy disk.

  Next, let us consider return light emitted from three points of coordinates 0, d / 2, and d on the sample surface generated by irradiating the sample with illumination light. These three points are assumed to be in the airy disk of illumination light. The intensity distribution on the image plane of the return light emitted from the coordinates 0, d / 2, d on the sample surface has peaks at the coordinates 0, d / 2, d on the image plane, as shown in the figure. The height of the peak is proportional to the illumination light intensity at the sample plane coordinates 0, d / 2, and d.

  The return light is received by the image sensor of the camera on the image plane. Here, the amount of received light at the position of the coordinate d on the image plane (position corresponding to pixel 2 in the figure) is considered. When the intensity distributions of the return lights emitted from the coordinates 0, d / 2, and d on the sample plane are compared at the position of the coordinates d on the image plane, as shown in FIG. The return light is the largest. That is, the pixel 2 at the position of the coordinate d on the image plane receives the brightest return light from the coordinate d / 2 on the sample surface, not the return light from the coordinate d on the sample surface. This indicates that in the microscopic region in the Airy disc, the projection is magnified twice from the sample surface to the image surface.

The above is explained as follows using mathematical formulas. Assuming that the position on the sample surface where the return light is generated is x, the received light amount I (x) at the position d on the image plane is
It is expressed. Here, PSF _ill (x) and PSF _img (x) are point spread functions on the illumination side and the imaging side, respectively. In general, the point spread function PSF (x) by using the numerical aperture NA and the wavelength λ of the first kind Bessel function J ₁ and the optical system,
It is expressed.

  From [Equation 1], I (x) is the product of two point spread functions whose peak positions differ by a distance d, so that I (x) peaks at d / 2 as shown schematically in FIG. have. That is, it is also shown from [Equation 1] that the pixel at the position of the coordinate d on the image plane receives the brightest return light from the coordinate d / 2 on the sample plane.

  As described above, in a confocal optical system using a camera, an area in an Airy disk centered on each bright spot of a non-scanning confocal image is projected twice magnified from the sample surface to the image surface. Therefore, the high-frequency component exceeding the resolution limit of the optical system can be obtained by reducing the airy disk area by half and performing a correction process for matching the coordinates on the sample surface with the coordinates on the image surface. . This is because the process of reducing the Airy disk area by half corresponds to halving the width of the point spread function of the optical system. As a result, a super-resolution image having a resolution twice the resolution limit (diffraction limit) of the optical system can be obtained.

  In this embodiment, the width of the point spread function of the optical system is optically changed by doubling the numerical aperture of the return light from the objective lens 120 by the microlens disk 210 and the pinhole disk 220 with a microlens. 1/2 due to processing. That is, the airy disc area is projected onto the camera 150 in a form reduced to ½.

In this embodiment, the numerical aperture when the return light from the objective lens 120 is optically projected on the camera 150 is optically converted to double, so that the image is only in the plane of the captured image (in the XY plane). In addition, the resolution is improved in the optical axis direction (Z-axis direction) perpendicular to the image. This is because the point spread function in the optical axis direction is expressed by [Equation 3], and the width of the point spread function in the optical axis direction is inversely proportional to the square of the numerical aperture. Therefore, it is suitable for observing the three-dimensional structure of the sample in detail.

  Note that the light source unit 110 that emits parallel illumination light includes, for example, a collimator lens 112 disposed at an appropriate location in front of a laser light emitting element 111 such as a laser diode as illustrated in FIG. Can be configured.

  Alternatively, as shown in FIG. 6B, the laser light is introduced from the laser light emitting element 111 to one end of the single mode fiber 113, and the emitted light from the other end of the single mode fiber 113 is collimated by the collimator lens 112. It may be configured. In this case, the laser light emitting element 111 can be disposed far from the confocal scanner 20.

  As shown in FIG. 6C, light may be incident on the rod integrator 115 using the LED 114 as a light source. The rod integrator 115 emits uniform illumination from the end by internal reflection. Then, this light is enlarged and projected by the two enlargement projection lenses 116 and 117. At this time, the projection surface is configured to coincide with the microlens disk 210. By enlarging and projecting the light, near the microlens disk 210, the light is substantially equivalent to the collimated light, and can be regarded as substantially parallel light. In this example, an inexpensive LED can be used to provide uniform illumination.

  As shown in FIG. 6D, a multimode fiber 118 may be used instead of the rod integrator 115. Since the multimode fiber 118 is flexible, the LEDs 114 can be placed at any location.

  Next, a modification of the first embodiment will be described. FIG. 7 is a diagram illustrating another example of optical path shift correction that is the first modification. The first modification is also configured such that the numerical aperture of the light condensed by the microlens 221 and diverging from the pinhole 223 is double the numerical aperture of the light collected by the microlens 211. . That is, when the angle with respect to the axis of the light collected by the microlens 211 is θ, the parameters of both microlenses are selected so that the angle of the light diverging from the pinhole 223 becomes 2θ.

  In the first modification, the beam splitter 240 is disposed at an angle of 45 degrees with respect to the axis, and the illumination light is guided to the microlens disk 210 in parallel with the optical axes of the microlenses 211 and 221.

  Then, an optical path correction glass plate 241 having the same material and the same thickness as the beam splitter 240 is disposed above the beam splitter 240 (on the side of the pinhole disk 220 with a microlens) and tilted by 45 degrees in the opposite direction.

  The return light from the sample 30 passes through the same optical path as the illumination light and passes through the beam splitter 240. At this time, the optical path shifts, but the shift is canceled by passing through the optical path correction glass plate 241, and enters the microlens 221. Since it is not necessary to adjust the angle of the beam splitter 240, this can be realized easily.

  FIG. 8 is a diagram for explaining a second modification of the first embodiment. In the second modification, the beam splitter 240 is disposed at an angle of 45 degrees with respect to the axis, and the optical path correction filter 242 is used to correct the optical path shift. Since the optical path correction filter 242 is thicker than the beam splitter 240, it is arranged at an angle smaller than 45 degrees with respect to the microlens disk 210.

  In addition, a filter film 243 that blocks laser light and passes only fluorescence is provided on one surface of the optical path correction filter 242. Thereby, for example, when part of the laser light is reflected by the microlens disk 210 and passes through the beam splitter 240 and emits fluorescence at the surface of the pinhole disk 220 with microlenses, the light shielding mask 222, etc., there is no filter film 243. And these fluorescence becomes noise. On the other hand, by using the optical path correction filter 242 provided with the filter film 243, the wavelength of the laser light is blocked from proceeding to the pinhole disk 220 with a microlens, so that these noise lights can be eliminated. it can.

  In the second modification, the distance between the microlens disk 210 and the pinhole disk 220 with a microlens can be made smaller than in the first modification, so that the rigidity of the disk unit integrated by connecting both can be increased. .

  A third modification of the first embodiment will be described with reference to FIG. In the third modification, the objective side variable power optical system 131 is used instead of the photographic lens 130, and the camera side variable power optical system 141 is used instead of the relay lens 140. By using an objective-side variable optical system 131 such as a zoom lens instead of the taking lens 130, the objective-side magnification is changed so that an optimum focal length satisfying the pupil diameter can be obtained even when the objective lens 120 having a different pupil diameter is used. The optical system 131 can be adjusted.

  For example, in the high-magnification objective lens 120 with a small pupil diameter, the focal length of the objective-side variable magnification optical system 131 is shortened so that the light emitted from the microlens disk 210 satisfies the pupil diameter of the objective lens 120. In the low-magnification objective lens 120 having a large pupil diameter, the light emitted from the microlens disk 210 satisfies the pupil diameter of the objective lens 120 by increasing the focal length of the objective-side variable magnification optical system 131. Can be adjusted.

  In addition, the magnification of the microscope is originally determined by the ratio of the focal lengths of the objective lens 120 and the objective-side variable magnification optical system 131 (photographing lens 130). Therefore, when the focal length of the objective side variable magnification optical system 131 is changed, the magnification is also changed. This is corrected by the camera side variable magnification optical system 141.

  For example, assuming that the focal length of the objective-side variable magnification optical system 131 is lengthened and adjusted to a value twice the focal length of the normal photographing lens 130 in order to cope with the low-magnification objective lens 120, a microlens is attached. On the surface of the pinhole disk 220, an image twice as large as usual can be obtained. For example, when a 10 × objective lens is used, an image equivalent to 20 × is obtained. On the other hand, the camera side variable magnification optical system 141 projects the image on the surface of the pinhole disk 220 with a microlens to the image pickup device 151 of the camera 150 so as to be ½, so that it is the same size as usual. An image of the size is obtained.

In order to realize these configurations, it is difficult if the photographing range is not wider than in the past, but in this configuration, the light is parallel light between the microlens disc 210 and the pinhole disc 220 with a microlens. Therefore, since it is easy to widen these distances, it can be realized easily. In addition, since the optimum light can be guided to the pupil diameter of the objective lens 120, not only the resolution is high but also the light utilization efficiency is improved. In other words, in the case of an optical system adjusted to a low magnification, the objective lens 120 having a high magnification and a small pupil diameter does not use only a part of the light and is highly efficient.
<Second embodiment>

  Next, a second embodiment of the present invention will be described. FIG. 10 is a diagram schematically showing the configuration of the confocal microscope 11 using the confocal scanner 21 of the second embodiment. The same parts as those in the first embodiment are denoted by the same reference numerals. The light source unit 110, the objective lens 120, the photographing lens 130, the relay lens 140, and the camera 150 are the same as in the first embodiment.

  The confocal scanner 21 includes a microlens disk 210 in which a large number of microlenses 211 are regularly arranged, a second microlens disk 260 in which a large number of microlenses 261 are regularly arranged, and a microscopic aperture of the light shielding mask 251. , A pinhole disk 250 in which a large number of pinholes 252 are regularly formed, a motor 230 and a beam splitter 240 are provided. The pinhole disc 250, the microlens disc 210, and the second microlens disc 260 are overlapped with each other at the central axis, and rotate integrally with the motor 230 around the central axis.

  A pinhole disk 250 and a microlens disk 210 are disposed from the side close to the objective lens 120, and a second microlens disk 260 is disposed with the beam splitter 240 interposed therebetween. Each pinhole 252 of the pinhole disk 250, each microlens 211 of the microlens disk 210, and each microlens 261 of the second microlens disk 260 are arranged so as to be coaxial.

  That is, in the second embodiment, the pinhole 252 is disposed on the objective lens 120 side, and not only the return light but also the illumination light passes through the pinhole 252. Since the return light from the focal plane by the light that has passed through the pinhole 252 returns to the pinhole 252 again, the adjustment of the optical system is further facilitated.

  The distance between the microlens disc 210 and the pinhole disc 250 is the focal length of the microlens 211. Note that. The pinhole disk 250 and the microlens disk 210 may be integrated as in the first embodiment.

  FIG. 11 shows details of the positional relationship among the pinhole disk 250, the microlens disk 210, the second microlens disk 260, and the beam splitter 240 of the second embodiment. In this figure, the central axes of a pair of pinholes 252, microlenses 211, and microlenses 261 that are arranged on the same axis are indicated by a one-dot chain line.

  Since the beam splitter 240 has a certain thickness, the light path of obliquely incident light is shifted by refraction. Also in the second embodiment, in order to correct this optical path shift, the angle of the beam splitter 240 with respect to the microlens disk 210 is made slightly smaller than 45 degrees. For this reason, the light reflected by the beam splitter 240 travels toward the microlens disc 210 with a slight angle with the one-dot chain line as shown in the figure.

  The light from the microlens 211 is also directed to the beam splitter 240 through the reverse optical path of the same angle, and the angle and the arrangement are such that the light refracted by the beam splitter 240 is incident on the microlens 221.

  In the second embodiment, the numerical aperture (NA) of light collected by the microlens 211 is configured to be twice the numerical aperture of light collected by the microlens 261. That is, if the angle of the light collected by the microlens 211 is θ, the parameters of both microlenses are selected so that the angle of the microlens 261 is 2θ. In principle, 2 times gives the best resolution, but other numerical values may be used.

  In the second embodiment, the image sensor 151 may be directly arranged at the focal position of the micro lens 261. In this case, an optical system such as the relay lens 140 is not necessary, and the configuration is simplified.

  Next, a modification of the second embodiment will be described. In the second embodiment, similarly to the first modification of the first embodiment shown in FIG. 7, the beam splitter 240 is arranged at an angle of 45 degrees with respect to the axis, and the illumination light is transmitted through the microlenses 211 and 261. An optical path correction glass plate is guided parallel to the optical axis to the microlens disk 210, and the optical path correction glass plate having the same material and the same thickness as the beam splitter 240 is inclined at 45 degrees in the opposite direction above the beam splitter 240 to correct the optical path. Can be done.

  A second modification of the second embodiment will be described with reference to FIG. In the second modification of the second embodiment, the beam splitter 240 is disposed at an angle of 45 degrees with respect to the axis, and the optical path correction filter 242 is used to correct the optical path shift. Since the optical path correction filter 242 is thicker than the beam splitter 240, it is arranged at an angle smaller than 45 degrees with respect to the microlens disk 210. At this time, a filter film that blocks laser light and passes only fluorescence may be disposed on one surface of the optical path correction filter 242. In the example of this figure, the microlens 261 is disposed on the surface of the microlens disc 260 on the beam splitter 240 side, but may be disposed on the opposite surface.

A third modification of the second embodiment will be described with reference to FIG. In the third modification of the second embodiment, the optical path shift is corrected by making the angle of the beam splitter 240 shallower than 45 degrees with respect to the microlens disk 210. Further, the microlens disk 210, the pinhole disk 250, An optical path correction glass plate 244 is disposed between the two. In the second example, the distance between the microlens 211 and the pinhole 252 than the distance between the microlens 221 and the pinhole 223 in the first example, that is, the focal length of the microlens 221 (first example), That is, since the focal length of the microlens 221 (second embodiment) is generally longer, it is preferable to correct the inclination of the optical axis accompanying the correction of the optical path shift. For this reason, the optical path correction glass plate 244 is disposed obliquely between the microlens disk 210 and the pinhole disk 250 to correct the inclination of the optical axis.
<Improved type>

  By the way, the confocal microscope 10 shown in FIG. 1 and the confocal microscope 11 shown in FIG. 10 have two convex lenses and one pinhole arranged in the direction of the rotation axis of the disk rotating integrally. For this reason, the point image of the sample 30 passes through the convex lens one time and reaches the image sensor 151 as compared with the general Nipkow disk system with a microlens array in which one convex lens and one pinhole are arranged. .

  Therefore, when a point image of the sample 30 that emits fluorescence in the upper right region as shown in FIG. 14A is taken, in a general Nipo Disc confocal microscope with a microlens array, as shown in FIG. 14B. In contrast, the confocal microscope 11 of the second embodiment is a point image of an inverted image as shown in FIG. 14C.

  Here, the sample 30 that emits circular fluorescence as shown in FIG. 15A is scanned as four points as shown in FIG. 15B as point images (represented by rectangular images for convenience). Assuming that an image is formed. These point images are irradiated with different illumination light beamlets and are captured in the same frame. However, the example of this figure is simplified for the sake of simplicity.

  In the confocal microscope 11 of the second embodiment, since each point image is captured as an inverted image, an image in which the point image of each region is inverted is obtained as shown in FIG. Fine continuity will be impaired. For this reason, there is room for further improvement from the viewpoint of improving the resolution.

  Therefore, it is conceivable that the inverted image is returned to the upright image by passing the convex lens once more in the integrally rotating disc. Thereby, since fine continuity between adjacent point images is maintained, further improvement in resolution can be realized.

  FIG. 16 shows details of the positional relationship among the improved pinhole disk 250, microlens disk 210, second microlens disk 260, and beam splitter 240 corresponding to the second embodiment shown in FIG.

  In the improved type, microlenses 253 are arranged on the surface opposite to the pinhole forming surface of the pinhole disk 250 so as to correspond to each pinhole 252 in order to return to an upright image. In this figure, the central axes of a set of microlenses 253, pinholes 252, microlenses 211, and microlenses 261 arranged on the same axis are indicated by a one-dot chain line.

  The illumination light emitted from the light source 110 (FIG. 10) is reflected by the beam splitter 240, passes through the microlens 211, and is divided into a number of illumination light beamlets. Each illumination light beamlet is condensed at the pinhole 252 and is incident on the microlens 253 in a state of being slightly expanded in the thickness direction of the pinhole disk 250. Then, the light is condensed again by the microlens 253, spreads and enters the photographing lens (FIG. 10), and is condensed on the sample 30 by the objective lens 120 (FIG. 10).

  In the improved type, both the angle condensed by the pinhole 252 and the angle condensed by the microlens 253 (the angle that spreads after being condensed) are both designed to be θ.

  The return light from the sample 30 passes through the same optical path as the illumination light in the order of the objective lens 120 (FIG. 10), the photographic lens (FIG. 10), the microlens 253, the pinhole 252, and the microlens 211, and becomes parallel light. Then, the light passes through the beam splitter 240 and enters the microlens 261. Then, the light is collected at an angle 2θ.

  A region other than the microlens 253 on the microlens 253 side of the pinhole disk 250 may be subjected to light shielding processing. Thereby, it is possible to prevent the light returning at an angle larger than θ from entering the peripheral microlens 253 and degrading the image.

  Similar to the first modification of the second embodiment, the beam splitter 240 is disposed at an angle of 45 degrees with respect to the axis, and the optical path correction glass plate is disposed at an angle of 45 degrees in the opposite direction, thereby correcting the optical path. As in the second modification, the optical path correction filter 242 is used to correct the optical path shift, and the angle of the beam splitter 240 is 45 degrees with respect to the microlens disk 210 as in the third modification. In addition, the optical path correction glass plate 244 may be disposed.

  Further, as shown in FIG. 17, the pinhole surface of the pinhole disk 250 on which the microlens 253 is arranged may be directed to the sample 30 side. In this case, the distance between the microlens 211 and the microlens 253 is made longer than the focal length of the microlens 211, and each illumination light beamlet is condensed in front of the microlens 253 and slightly expanded. So that it is incident on. Then, the light travels through the pinhole disk 250 and is collected by the pinhole 252, spreads and enters the photographing lens (FIG. 10), and is focused on the sample 30 by the objective lens 120 (FIG. 10).

  In this example, both the angle incident on the microlens 253 and the angle to be appreciated from the pinhole 252 are designed to be θ.

  The return light from the sample 30 passes through the same optical path as the illumination light in the order of the objective lens 120 (FIG. 10), the photographing lens (FIG. 10), the pinhole 252, the microlens 253, and the microlens 211, and becomes parallel light. Then, the light passes through the beam splitter 240 and enters the microlens 261. Then, the light is collected at an angle 2θ.

  Alternatively, as shown in FIG. 18, a micro concave lens disk 270 may be used instead of the second micro lens disk 260 without arranging the micro lens on the pinhole disk 250. In this case, the inverted image is not returned to the erect image by passing the convex lens once more, but the point image does not become an inverted image by reducing the number of times the convex lens passes.

  The micro concave lens disk 270 is a concave lens formed by the convex lens formed on the second micro lens disk 260, and each concave lens is designed to diverge the parallel light return light by 2θ. A line 261 in the figure indicates the focal plane of the concave lens.

  In this example, the relay lens 140 (FIG. 10) projects an image of the focal plane 261 on the image sensor (FIG. 10) surface of the camera 150. The configuration of this example has an advantage that the configuration is further simplified in addition to the point images being erect images.

DESCRIPTION OF SYMBOLS 10 ... Confocal microscope, 11 ... Confocal microscope, 20 ... Confocal scanner, 21 ... Confocal scanner, 30 ... Sample, 110 ... Light source unit, 111 ... Laser light emitting element, 112 ... Collimating lens, 113 ... Single mode fiber, DESCRIPTION OF SYMBOLS 115 ... Rod integrator, 116 ... Magnification projection lens, 117 ... Magnification projection lens, 118 ... Multimode fiber, 120 ... Objective lens, 130 ... Shooting lens, 131 ... Objective side variable magnification optical system, 140 ... Relay lens, 141 ... Camera Side variable optical system, 150 ... camera, 151 ... imaging device, 210 ... microlens disk, 211 ... microlens, 220 ... pinhole disk with microlens, 221 ... microlens, 222 ... light shielding mask, 223 ... pinhole, 230 ... motor, 240 ... beam splitter, 2 DESCRIPTION OF SYMBOLS 1 ... Optical path correction glass plate, 242 ... Optical path correction filter, 243 ... Filter film, 244 ... Optical path correction glass plate, 250 ... Pinhole disk, 251 ... Shading mask, 252 ... Pinhole, 260 ... Microlens disk, 261 ... Micro Lens, 270 ... Micro concave lens disk

Claims (6)

A first microlens disk having a plurality of microlenses disposed thereon;
A plurality of microlenses arranged corresponding to the arrangement pattern of the first microlens disk, and a second microlens disk having a rotation axis in common with the first microlens disk;
Corresponding to the arrangement pattern of the first microlens disk, a plurality of image reversal microlenses are arranged on one surface and a plurality of pinholes are arranged on the other surface, and are common to the first microlens disk. A third microlens disc provided on the opposite side of the second microlens disc,
The illumination light irradiating the object is guided to the first microlens disk, and the return light from the object that has passed through each microlens of the first microlens disk is converted into the corresponding micro of the second microlens disk. With a beam splitter leading to the lens,
A confocal scanner, wherein a numerical aperture of a microlens disposed on the second microlens disk is larger than a numerical aperture of a microlens disposed on the first microlens disk.   2. The confocal point according to claim 1, wherein the numerical aperture of the image reversal microlens disposed on the third microlens disk is equal to the numerical aperture of the microlens disposed on the first microlens disk. Scanner. Wherein each focal point of the object side of the first microlenses disposed on the microlens disk confocal scanner according to claim 1 or 2, characterized in that the pin holes are arranged.   The image reversal microlens is disposed at a position farther from the focal position on the object side of each microlens disposed on the first microlens disk, and the illumination light of each image reversal microlens is The confocal scanner according to claim 1, wherein the pinhole is disposed at each in-focus position.   5. The numerical aperture of the microlens disposed on the second microlens disk is approximately twice the numerical aperture of the microlens disposed on the first microlens disk. A confocal scanner according to claim 1.   The confocal scanner according to any one of claims 1 to 5,
  A light source unit that emits parallel illumination light to the beam splitter;
  An objective lens disposed on the third microlens disk side;
  A confocal microscope comprising: an image pickup device disposed on the second microlens disk side.