Confocal scanner and confocal microscope

A confocal scanner includes a first micro lens disk having a plurality of micro lenses arranged thereon, a second micro lens disk having a plurality of micro lenses, which is arranged in correspondence to an arrangement pattern of the first micro lens disk, and having a common rotation axis to the first micro lens disk, and a beam splitter configured to guide an illumination light, which is to be irradiated to an object, to the first micro lens disk, and to guide a return light from the object having passed through each micro lens of the first micro lens disk to the corresponding micro lens of the second micro lens disk. A numerical aperture of each micro lens arranged on the second micro lens disk is greater than a numerical aperture of each micro lens arranged on the first micro lens disk.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priorities from Japanese Patent Applications No. 2014-247673 filed on Dec. 8, 2014 and No. 2015-103644 filed on May 21, 2015, the entire contents of which are incorporated herein by reference.

BACKGROUND

Technical Field

The present disclosure relates to a confocal scanner configured to obtain a super resolution by optical processing, and more particularly, to a confocal scanner capable of reducing complication of a positioning operation of an optical component and a confocal microscope using the confocal scanner.

Related Art

A confocal microscope configured to irradiate a light to an object with pinpoint, to selectively detect only a focusing light from an irradiation point, to scan the irradiation point and to obtain an image is an optical microscope capable of reconstructing an image of a high resolution and three-dimensional information. The confocal microscope is widely used in the biological science field and the like, and a variety of related technologies have been suggested.

For example, Patent Document 1 discloses a confocal scanner, which is a scan unit of a confocal microscope, includes a micro lens disk having a plurality of micro lenses and a pinhole disk having pinholes formed in the same pattern as the micro lenses, and is configured to perform a multi-beam scanning by rotating the disks while irradiating an illumination light.

In the related art, an optical microscope including the confocal microscope has an Abbe's diffraction limit, i.e., a resolution limit based on a theory that an object smaller than a half wavelength of the light to be irradiated to the object cannot be seen. In recent years, a super resolution technology of obtaining an image having a resolution beyond the resolution limit has been developed and put to practical use.

For example, Patent Document 2 discloses a technology of using a scan mask, which is configured to modulate spatial intensity distributions of an excitation light from a light source and a return light from an object, to capture an image having a high frequency component beyond a resolution limit, and performing high frequency enhancement processing to obtain a super resolution. Also, Non-Patent Document 1 discloses a technology of using a shutter, which is configured to pass an illumination light from a light source apparatus in a strobe shape, to capture hundreds of images having a plurality of bright spots recorded therein while slightly changing positions of the bright spots, performing image processing in which the bright spot becomes a half size for each image, and synthesizing the respective images to obtain a super resolution image.

According to the super resolution technologies disclosed in Patent Document 2 and Non-Patent Document 1, since the complicated or the large amount of image processing is performed, the calculation load is high and much time is consumed for the processing. That is, the corresponding super resolution technologies are inappropriate to the real-time observation.

In contrast, Patent Document 3 discloses a technology capable of obtaining a super resolution image at high speed by optical processing.FIG. 19shows a configuration of an optical system of a microscope system disclosed in Patent Document 3.

As shown inFIG. 19, a microscope system400is configured to irradiate a laser light, which is to be emitted from a light source410, to a sample430, and to capture a return light with a camera420. The collimated illumination light emitted from the light source410is divided into a plurality of illumination light beamlets by a micro lens array411. The illumination light beamlets are reflected on a galvanometer mirror413, and are concentrated on the sample430by an objective lens414.

The sample430is configured to radiate the return light based on the illumination light. In particular, in case of fluorescent sample observation, the sample430is a specific structure dyed by a fluorescent dye and the like, and is configured so that fluorescent dye molecules are excited by the illumination light and radiate the fluorescence having a longer wavelength than the illumination light.

The return light from the sample430is reflected on the galvanometer mirror413, is reflected on a beam splitter412and then passes through a lens415. The return light having passed through the lens415reaches a pinhole array416having a plurality of pinholes. However, only the light from a focal plane of the sample430is focused on the pinhole array416and passes through the pinholes.

The return light having passed through the pinhole passes through a micro lens array417and a micro lens array418each of which having a plurality of micro lenses, is reflected on the galvanometer mirror413, and is captured by the camera420. The return light is a part of the sample430on which the illumination light beamlets are reflected, but can scan the entire sample430by changing a direction of the galvanometer mirror413.

Here, the pinhole array416is precisely arranged so that each pinhole is disposed at a position conjugate with a focusing spot of the objective lens414. Also, each pinhole of the pinhole array416is precisely arranged at a focal position of each micro lens of the micro lens array417. Further, each micro lens of the micro lens array417and each micro lens of the micro lens array418are precisely arranged to be coaxial with each other.

A focal length of each micro lens of the micro lens array418is set to be shorter than a focal length of each micro lens of the micro lens array417.

In the above configuration, the return light of the focusing plane having passed through the pinholes of the pinhole array416is converted into a parallel light by the micro lens array417, which is then incident to the micro lens array418. Since the focal length of each micro lens of the micro lens array418is shorter than the focal length of each micro lens of the micro lens array417, a numerical aperture of the return light increases when passing through the micro lens array417. For example, when the focal length of each micro lens of the micro lens array418is a half of the focal length of each micro lens of the micro lens array417, the return light is converted into a light beam having a double numerical aperture.

When the light beam is captured by the camera420while changing a direction of the galvanometer mirror413, a super resolution image of the sample430can be obtained. At this time, since it is not necessary to perform the troublesome image processing and the plurality of capturing processing, it is possible to simply obtain the super resolution image at high speed.[Patent Document 1] Japanese Patent Application Publication No. Hei 10-062691A[Patent Document 2] Japanese Patent Application Publication No. 2012-78408A[Patent Document 3] International Patent Application Publication No. WO2013/126762[Non-Patent Document 1] 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, it is possible to simply obtain the super resolution image at high speed by using the confocal scanner configured to obtain a super resolution with the optical processing. However, in case of the confocal scanner configured to obtain a super resolution image with the optical processing, it is necessary to perform the precise positioning with respect to each optical component of the micro lens array for the illumination light, the objective lens, the two micro lens arrays for the return light, and the pinhole array. According to the optical system disclosed in Patent Document 3, the optical components are independently arranged with being spatially spaced, so that the precise positioning is not easy.

The precise positioning of the optical component causes the cost increase and is easily influenced by environment and temporal changes, and the maintenance thereof is complicated. Therefore, it is preferably to reduce a burden on the precise positioning as much as possible.

SUMMARY

Exemplary embodiments of the invention provide a confocal scanner and a confocal microscope using the confocal scanner which can reduce a burden on a precise positioning in the confocal scanner configured to obtain a super resolution image with optical processing.

A confocal scanner according to an exemplary embodiment of the invention, comprises:

a first micro lens disk having a plurality of micro lenses arranged thereon;

a second micro lens disk having a plurality of micro lenses, which is arranged in correspondence to an arrangement pattern of the first micro lens disk, and having a common rotation axis to the first micro lens disk; and

a beam splitter configured to guide an illumination light, which is to be irradiated to an object, to the first micro lens disk, and to guide a return light from the object having passed through each micro lens of the first micro lens disk to the corresponding micro lens of the second micro lens disk,

wherein a numerical aperture of each micro lens arranged on the second micro lens disk is greater than a numerical aperture of each micro lens arranged on the first micro lens disk.

Pinholes may be arranged at respective focal positions of the respective micro lenses, which are arranged on the second micro lens disk, on a side opposite to the object.

Pinholes may be arranged at respective focal positions of the respective micro lenses, which are arranged on the first micro lens disk, on a side facing the object.

Micro lenses for image reversal corresponding to the respective pinholes may be arranged on the side facing the object.

Micro lenses for image reversal may be arranged at more distant positions than respective focal positions of the respective micro lenses, which are arranged on the first micro lens disk, on a side of the first micro lens disk facing the object, and pinholes are arranged at each focusing position of the illumination lights of the micro lenses for image reversal.

The micro lenses arranged on the second micro lens disk may be concave lenses.

The numerical aperture of each micro lens arranged on the second micro lens disk may be substantially twice as large as the numerical aperture of each micro lens arranged on the first micro lens disk.

A diameter of each micro lens arranged on the first micro lens disk may be smaller than a diameter of each micro lens arranged on the second micro lens disk.

The illumination light that is to be guided to the first micro lens disk by the beam splitter may obliquely advance relative to an optical axis of each micro lens on the first micro lens disk.

The illumination light that is to be guided to the first micro lens disk by the beam splitter may advance in parallel with an optical axis of each micro lens on the first micro lens disk, and the confocal scanner may comprise an optical member configured to correct an optical path shift, which is caused due to the beam splitter, and disposed between the first micro lens disk and the second micro lens disk.

A confocal microscope according to an exemplary embodiment comprises:

the confocal scanner;

a light source unit configured to emit an illumination light of a parallel light to the beam splitter;

an objective lens disposed at a first micro lens disk-side; and

a capturing element arranged at a second micro lens disk-side.

The confocal microscope may comprise:

a first variable power optical system disposed between the first micro lens disk and the objective lens; and

a second variable power optical system disposed between the second micro lens disk and the capturing element.

According to the present invention, it is possible to reduce the burden on the precise positioning in the confocal scanner configured to obtain the super resolution image with the optical processing.

DETAILED DESCRIPTION

First Exemplary Embodiment

Exemplary embodiments of the present invention will be described with reference to the drawings.FIG. 1pictorially shows a configuration of a confocal microscope10using a confocal scanner20of a first exemplary embodiment.

As shown inFIG. 1, the confocal microscope10using the confocal scanner20includes a light source unit110configured to emit a parallel light as an illumination light, an objective lens120, a capturing lens130, a relay lens140, and a camera150, and is configured to capture a super resolution confocal image of a sample30.

The confocal scanner20includes a micro lens disk210having a plurality of micro lenses211regularly arranged thereon, a pinhole disk220having a plurality of micro lenses221arranged on one surface thereof and a plurality of pinholes223formed on the other surface, a motor230and a beam splitter240. The micro lens disk210and the pinhole disk220having the micro lenses are arranged so that central axes thereof overlap with each other, and are configured to integrally rotate about the central axes by the motor230.

The micro lens disk210and the pinhole disk220having the micro lenses are integrally formed, so that it is not necessary to mechanically adjust both the disks and it is possible to increase the stability with respect to the environmental and temporal changes.

The micro lenses221of the pinhole disk220are arranged on a side facing the micro lens disk210, and a light shield mask222is provided on an opposite side. The respective micro lenses221of the pinhole disk220are arranged to be coaxial with the respective micro lenses211of the micro lens disk210.

At least a part of the pinhole disk220, on which the micro lenses221are arranged, is made of a material through which a return light penetrates, and a thickness of the pinhole disk220is set to a focal length of the micro lens221.

The pinholes223are configured by fine apertures formed in the light shield mask222, and each pinhole223is arranged at a focal position of each micro lens221. A size of the pinhole223is preferably equivalent to a diffraction limit of the light to be concentrated at the micro lens221.

In the exemplary embodiment, both the micro lens211and the micro lens221are convex lenses. However, the other optical elements such as a Fresnel lens and a diffraction optical element may also be adopted inasmuch as a lens effect can be accomplished. Also, a single lens or a compound lens may be adopted.

The beam splitter240is arranged between the micro lens disk210and the pinhole disk220having the micro lenses. The beam splitter240has characteristics of reflecting a wavelength of an illumination light and enabling the fluorescence having a long wavelength, which is generated from the sample30by the illumination light, to penetrate therethrough.

FIG. 2shows in detail a positional relation among the micro lens disk210, the pinhole disk220having the micro lenses and the beam splitter240. InFIG. 2, the central axes of the micro lens211and the micro lens221of one set arranged on the same axis are denoted by the dashed-dotted line.

Since the beam splitter240has a thickness of a predetermined level, an optical path of the obliquely incident light is shifted by refraction. In this exemplary embodiment, in order to correct the optical path shift, an angle of the beam splitter240relative to the micro lens disk210is set to be slightly smaller than 45°. For this reason, the light reflected on the beam splitter240obliquely faces towards the micro lens disk210at a slight angle relative to the shown dashed-dotted line.

The light from the micro lens211also faces towards the beam splitter240along an optical path of a reverse direction of the same angle, and the beam splitter240is disposed at an angle at which the light refracted at the beam splitter240is correctly incident to the micro lens221.

Here, an outer diameter of the micro lens211is formed to be slightly smaller than an outer diameter of the micro lens221. In the meantime, as the method of correcting the optical path shift, the angle of the beam splitter240may be set to 45°, and the micro lens211and the micro lens221may be slightly inclined relative to the central axis. In this case, the light incident to the micro lens211from the beam splitter240is parallel with the optical axis shown with the dashed-dotted line.

In this exemplary embodiment, a numerical aperture (NA) of the light that is to be concentrated by the micro lens221is set to be twice as large as a numerical aperture of the light that is to be concentrated by the micro lens211. That is, parameters of both the micro lenses are selected so that when an angle of the light to be concentrated at the micro lens211is denoted as θ, an angle of the light to be concentrated at the micro lens221and to diverge from the pinhole223is 2θ. In the meantime, the resolution is highest at the double numerical aperture, in principle. However, the other multiples are also possible inasmuch as the numerical aperture of the light that is to be concentrated by the micro lens221is larger.

Here, when the angle relative to a center of the optical axis is denoted as φ and a refractive index of a medium is denoted as n, NA=n·sin (φ). Also, although a diameter of the micro lens is largely shown for convenience of explanations inFIG. 2, an actual diameter of the micro lens is small, for example 1 mm or less. That is, the angels of θ and 2θ are very small. When the angle is small, it can be assumed that sin (φ)≈φ. Therefore, it can be seen that the numerical aperture of the light that is to be concentrated by the micro lens221is substantially twice as large as the numerical aperture of the light that is to be concentrated by the micro lens211.

Returning toFIG. 1, the capturing lens130and the objective lens120are arranged below the confocal scanner20, and the sample30is arranged at a focal position of the objective lens120. Also, the relay lens140is arranged above the confocal scanner20, and is configured to project an image of a pinhole surface onto a surface of a capturing element151of the camera150. In the meantime, a filter configured to pass only the desired fluorescence may be arranged on a front surface of the camera150.

In the above configuration, the illumination light emitted from the light source unit110is reflected on the beam splitter240and is guided to the micro lens disk210. Then, the light is divided into a plurality of illumination light beamlets by the plurality of micro lenses211on the micro lens disk210. After each illumination light beamlet is once focused, it is converted into a parallel light at the capturing lens130, and is concentrated at each point on the sample30by the objective lens120.

The sample30is configured to radiate return lights based on the illumination light beamlets. In particular, in case of fluorescent sample observation, the sample30is a specific structure dyed by a fluorescent dye and the like, and is configured so that fluorescent dye molecules are excited by the illumination light and radiate the fluorescence having a longer wavelength than the illumination light.

Each return light captured by the objective lens120returns along the same optical path as the illumination light. That is, the return light is converted into the parallel light by the objective lens120, is once focused by the capturing lens130, is incident to the micro lens disk210, and is then again converted into the parallel light by the micro lens211. That is, the micro lens disk210serves as a micro lens disk for the illumination light and a micro lens disk for the return light. Thereby, it is possible to reduce the number of optical components for which the positioning is required.

Each return light passes through the beam splitter240, and is incident to the micro lens221of the pinhole disk220, as the parallel light. At this time, as shown inFIG. 2, the optical path shift due to the refraction at the beam splitter240is corrected by the angle adjustment of the beam splitter240, for example.

The parallel light having passed through the beam splitter240is concentrated on the pinhole223by the micro lens221. As described above, since the numerical aperture of the micro lens221is twice as large as the numerical aperture of the micro lens211, a focus on the pinhole223is about a half of a focus formed by the beamlet in front of the micro lens211, and the light passes through the pinhole223.

At this time, only the return light from the sample-side focusing plane of the objective lens120passes through the pinhole223. On the other hand, since the return light from a plane except for the focusing plane does not form a focus on the pinhole223, it is shielded by the light shield mask222and cannot thus pass through the pinhole223.

The light having passed through the pinhole223is imaged on the capturing element151of the camera150by the relay lens140. The micro lens disk210and the pinhole disk220having the micro lenses are rotated by the motor230to scan the entire sample30with the illumination light, so that a confocal image of the sample30can be captured by the camera150.FIG. 3pictorially shows a scanning state. Also, it is possible to obtain a three-dimensional image by scanning the sample30in a z-axis direction.

The reason to obtain a super resolution image by the confocal microscope10using the confocal scanner20of this exemplary embodiment is described.FIG. 4pictorially shows a confocal optical system using a camera, which is a two-dimensional image sensor. For simplification, the illumination light-side and the return light-side are shown at left and right sides, respectively, and the illumination light-side ranges from a point light source to a sample plane and the return light-side ranges from the sample plane to an image plane. Also, the magnification of the objective lens is one time for simplification. However, such assumption does not damage the generality of the discussion. By assuming the magnification as one time, it is possible to treat the sample plane and the image plane with upside-down coordinates of equal scales.

The illumination light generated from a point light source on the optical axis is focused on a sample plane by the objective lens. At this time, an intensity distribution of the illumination light on the sample plane has a predetermined area about the coordinate 0 due to the diffraction of the light, as shown inFIG. 4. The area of the intensity distribution of the light is generally referred to as an airy disk.

Then, the return lights, which are generated at three points of coordinates 0, d/2 and d on the sample plane by the irradiation of the illumination light to the sample, are considered. It is assumed that the three points are in the airy disk of the illumination light. The intensity distribution on the image plane of the return lights generated from the coordinates 0, d/2, d on the sample plane has peaks at the coordinates 0, d/2, d on the image plane, as shown inFIG. 4. Heights of the respective peaks are proportional to the illumination light intensity at the coordinates 0, d/2, d on the sample plane.

The return light is received by the capturing element of the camera on the image plane. Here, a light receiving amount at a position (position corresponding to a pixel2inFIG. 4) of the coordinate d on the image plane is considered. Comparing the intensity distributions of the respective return lights, generated from the coordinates 0, d/2, d on the sample plane, at the position of the coordinate d on the image plane, the return light from the coordinate d/2 on the sample plane is highest, as shown inFIG. 4. That is, the pixel2at the position of the coordinate d on the sample plane most brightly receives the return light from the coordinate d/2 on the sample plane, not the return light from the coordinate d on the sample plane. This means that the light is projected with being twice enlarged from the sample plane onto the image plane in the microscopic area referred to as the airy disk.

The above can be described by equations, as follows. When a position on the sample plane at which the return light is generated is denoted as x, a light receiving amount I(x) at a position d on the image plane is expressed as follows.
I(x)=PSFill(x)×PSFimg(x−d)  [equation 1]

Here, PSFill(x) and PSFimg(x) indicate point image distribution functions at the illumination-side and the capturing-side. In general, the point image distribution function PSF(x) is expressed using Bessel function of the first kind J1, a numerical aperture NA of the optical system and a wavelength λ, as follows.

From the equation 1, since the light receiving amount I(x) is a product of two point image distribution functions of which peak positions are different by a distance d, the light receiving amount I(x) has a peak at a position d/2, as shown in an outline ofFIG. 5. That is, it can also be seen from the equation 1 that the pixel2at the position of the coordinate d on the sample plane most brightly receives the return light from the coordinate d/2 on the sample plane.

Like this, according to the confocal optical system using the camera, in the airy disk about each bright spot of a non-scanning confocal image, since the light is projected with being twice enlarged from the sample plane onto the image plane, it is possible to obtain a high-frequency component beyond the resolution limit of the optical system by performing correction processing of reducing the area of the airy disk to a half and matching the coordinates on the sample plane and the coordinates on the image plane. The reason is that the processing of reducing the area of the airy disk to a half corresponds to processing of making a width of the point image distribution function of the optical system to a half. As a result, it is possible to obtain a super resolution image having a resolution twice as high as the resolution limit (diffraction limit) of the optical system.

In this exemplary embodiment, the numerical aperture of the return light from the objective lens120is converted into the double numerical aperture by the micro lens disk210and the pinhole disk220having the micro lenses, so that the width of the point image distribution function of the optical system is reduced to a half by the optical processing. That is, the light is projected to the camera150with the area of the airy disk being reduced to a half.

Also, in this exemplary embodiment, since the numerical aperture is optically converted into the double numerical aperture when the return light from the objective lens120is projected to the camera150, the resolution is enhanced not only in a plane (XY plane) of a captured image but also in an optical axis direction (Z-axis direction) perpendicular to the image. The reason is that the point image distribution function in the optical axis direction is expressed by an equation 3 and a width of the point image distribution function in the optical axis direction is inversely proportional to a square of the numerical aperture. Therefore, the present invention is also appropriate to the detailed observation of a three-dimensional structure of the sample.

In the meantime, the light source unit110configured to emit the illumination light of the parallel light may be configured by disposing a collimate lens112at an appropriate place in front of a laser light emitting element111such as a laser diode, as shown inFIG. 6A, for example.

Alternatively, as shown inFIG. 6B, the laser light may be introduced to one end of a single mode fiber113from the laser light emitting element111, and an emission light from the other end of the single mode fiber113may be collimated at the collimate lens112. In this case, the laser light emitting element111may be disposed at a place distant from the confocal scanner20.

Also, as shown inFIG. 6C, the light may be made to be incident to a rod integrator115by using an LED114as the light source. The rod integrator115is configured to generate a uniform illumination light from an end portion by internal reflection. Then, the light is enlarged and projected by two enlargement and projection lenses116,117. At this time, a projection plane is matched with the micro lens disk210. By enlarging and projecting the light, the light is substantially equivalent to the collimate light in the vicinity of the micro lens disk210, so that it can be regarded as the parallel light. In this example, it is possible to provide the uniform illumination light by using the low-priced LED.

Also, as shown inFIG. 6D, a multimode fiber118may be used, instead of the rod integrator115. Since the multimode fiber118is flexible, it is possible to dispose the LED114at any place.

In the below, modified examples of the first exemplary embodiment are described.FIG. 7shows another example of the optical path shift correction of a first modified example. Also in the first modified example, the numerical aperture of the light that is to be concentrated by the micro lens221and to diverge from the pinhole223is set to be twice as large as the numerical aperture of the light that is to be concentrated by the micro lens211. That is, the parameters of both the micro lenses are selected so that when an angle of the light to be concentrated at the micro lens211relative to the axis is denoted as θ, an angle of the light to diverge from the pinhole223is 2θ.

In the first modified example, the beam splitter240is disposed at the angle of 45° relative to the axis and the illumination light is guided to the micro lens disk210in parallel with the optical axes of the micro lenses211,221.

Above the beam splitter240(at a side facing the pinhole disk220having the micro lenses), an optical path correction glass plate241made of the same material as the beam splitter240and having the same thickness is obliquely disposed at an angle of 45° in an opposite direction to the beam splitter240.

The return light from the sample30passes the same optical path as the illumination light, and passes through the beam splitter240. At this time, although the optical path is shifted, the shifted optical path is cancelled because the light passes through the optical path correction glass plate241, and the light is incident to the micro lens221. Since it is not necessary to adjust the angle of the beam splitter240, the incidence of the light can be simply implemented.

FIG. 8shows a second modified example of the first exemplary embodiment. In the second modified example, the beam splitter240is disposed at the angle of 45° relative to the axis, and an optical path correction filter242is used so as to correct the optical path shift. Since the optical path correction filter242is thicker than the beam splitter240, the optical path correction filter242is disposed at an angle smaller than 45° relative to the micro lens disk210.

Also, one surface of the optical path correction filter242is provided with a filter film243configured to shield the laser light and to pass only the fluorescence. Thereby, for example, when a part of the laser light is reflected on the micro lens disk210and passes through the beam splitter240and the fluorescence is radiated from the surface of the pinhole disk220having the micro lenses, the light shield mask222and the like, such a fluorescence becomes a noise if the filter film243is not provided. In contrast, when the optical path correction filter242having the filter film243is used, the wavelength of the laser light is interrupted from advancing towards the pinhole disk220having the micro lenses, so that the noise light can be excluded.

In the second modified example, it is possible to reduce a distance between the micro lens disk210and the pinhole disk220having the micro lenses, as compared to the first modified example. Therefore, it is possible to increase the stiffness of the disk unit in which the micro lens disk and the pinhole disk are connected and integrated.

A third modified example of the first exemplary embodiment is described with reference toFIG. 9. In the third modified example, an object-side variable power optical system131is used instead of the capturing lens130, and a camera-side variable power optical system141is used instead of the relay lens140. The objective-side variable power optical system131such as a zoom lens is used instead of the capturing lens130. Thereby, even when the objective lens120having a different pupil diameter is used, it is possible to regulate the objective-side variable power optical system131so that an optimal focal length satisfying the pupil diameter is obtained.

For example, in case of the high-power objective lens120having a small pupil diameter, the focal length of the objective-side variable power optical system131is shortened so that the light emitted from the micro lens disk210satisfies the pupil diameter of the objective lens120. In case of the low-power objective lens120having a large pupil diameter, the focal length of the objective-side variable power optical system131is lengthened so that the light emitted from the micro lens disk210satisfies the pupil diameter of the objective lens120.

The magnification of the microscope is generally determined by a ratio of the focal lengths of the objective lens120and the objective-side variable power optical system131(capturing lens130). Therefore, when the focal length of the objective-side variable power optical system131is changed, the magnification is also changed. This is corrected by the camera-side variable power optical system141.

For example, when the focal length of the objective-side variable power optical system131is lengthened to a double value of the normal focal length of the capturing lens130so as to cope with the low-power objective lens120, an image having a double size of a usual size can be obtained on the surface of the pinhole disk220having the micro lenses. For example, when a 10× (ten times) objective lens is used, an image equivalent to a twenty times size is obtained. In contrast, the camera-side variable power optical system141is configured to project the image on the surface of the pinhole disk220having the micro lenses to the capturing element151of the camera150so that a size thereof becomes a half. Thereby, an image having the same size as usual is obtained.

In order to achieve the above configurations, a capturable range should be wider than the related art. However, in the exemplary embodiment, since the light between the micro lens disk210and the pinhole disk220having the micro lenses is the parallel light, it is possible to easily wide the distance therebetween, so that the above configurations can be simply implemented. Also, since it is possible to guide the optimal light to the pupil diameter of the objective lens120, the resolution increases and the using efficiency of the light is also improved. That is, in case of a low-power optical system, a situation where only a part of the light is used in the high-power objective lens120having a small pupil diameter does not occur. That is, the efficiency is high.

Second Exemplary Embodiment

In the below, a second exemplary embodiment of the present invention is described.FIG. 10pictorially shows a configuration of a confocal microscope11using a confocal scanner21of the second exemplary embodiment. The same parts as the first exemplary embodiment are denoted by the same reference numerals. The light source unit110, the objective lens120, the capturing lens130, the relay lens140and the camera150are the same as the first exemplary embodiment.

The confocal scanner21includes the micro lens disk210having the plurality of micro lenses211regularly arranged thereon, a second micro lens disk260having a plurality of micro lenses261regularly arranged thereon, a pinhole disk250having a plurality of pinholes252regularly formed by fine apertures of a light shield mask251, the motor230and the beam splitter240. The pinhole disk250, the micro lens disk210and the second micro lens disk260are arranged so that central axes thereof overlap with each other, and are configured to integrally rotate about the central axes by the motor230.

The pinhole disk250and the micro lens disk210are arranged from a side closer to the objective lens120, and the second micro lens disk260is arranged with the beam splitter240being interposed therebetween. The respective pinholes252of the pinhole disk250, the respective micro lenses211of the micro lens disk210and the respective micro lenses261of the second micro lens disk260are coaxially arranged.

That is, in the second exemplary embodiment, the pinhole252is arranged at the objective lens120-side, so that not only the return light but also the illumination light passes through the pinhole252. Since the return light from the focusing plane by the light having passed through the pinhole252returns to the pinhole252, it is possible to adjust the optical system more easily.

A distance between the micro lens disk210and the pinhole disk250is a focal length of the micro lens211. In the meantime, the pinhole disk250and the micro lens disk210may be integrated, like the first exemplary embodiment.

FIG. 11shows in detail a positional relation of the pinhole disk250, the micro lens disk210, the second micro lens disk260and the beam splitter240of the second exemplary embodiment. InFIG. 11, the central axes of the pinhole252, the micro lens211and the micro lens261of one set arranged on the same axis are denoted by the dashed-dotted line.

Since the beam splitter240has a thickness of a predetermined level, an optical path of the obliquely incident light is shifted by the refraction. Also in the second exemplary embodiment, in order to correct the optical path shift, an angle of the beam splitter240relative to the micro lens disk210is set to be slightly smaller than 45°. For this reason, the light reflected on the beam splitter240faces towards the micro lens disk210at a slight angle relative to the shown dashed-dotted line.

The light from the micro lens211also faces towards the beam splitter240along an optical path of a reverse direction of the same angle, and the beam splitter240is disposed at an angle at which the light refracted at the beam splitter240is correctly incident to the micro lens261.

In the second exemplary embodiment, a numerical aperture (NA) of the light that is to be concentrated by the micro lens261is set to be twice as large as a numerical aperture of the light that is to be concentrated by the micro lens211. That is, parameters of both the micro lenses are selected so that when an angle of the light to be concentrated at the micro lens211is denoted as θ, an angle of the light to be concentrated at the micro lens261is 2θ. In the meantime, the resolution is highest at the double numerical aperture, in principle. However, the other multiples are also possible.

In the second exemplary embodiment, the capturing element151may be directly arranged at a focal position of the micro lens261. In this case, since it is possible to omit the optical system such as the relay lens140, the configuration is simpler.

Subsequently, modified examples of the second exemplary embodiment are described. Also in the second exemplary embodiment, like the first modified example of the first exemplary embodiment shown inFIG. 7, the beam splitter240is disposed at an angle of 45° relative to the axis, the illumination light is guided to the micro lens disk210in parallel with the optical axes of the micro lenses211,261, and the optical path correction glass plate made of the same material as the beam splitter240and having the same thickness is obliquely disposed at an angle of 45° in an opposite direction to the beam splitter240above the beam splitter240so as to correct the optical path.

A second modified example of the second exemplary embodiment is described with reference toFIG. 12. In the second modified example of the second exemplary embodiment, the beam splitter240is disposed at an angle of 45° relative to the axis, and the optical path correction filter242is used so as to correct the optical path shift. Since the optical path correction filter242is thicker than the beam splitter240, the optical path correction filter242is disposed at an angle smaller than 45° relative to the micro lens disk210. At this time, a filter film configured to shield the laser light and to pass only the fluorescence may be disposed on one surface of the optical path correction filter242. In this embodiment, the micro lens261is disposed on the surface of the micro lens disk260facing the beam splitter240. However, the micro lens261may be disposed on the opposite surface.

A third modified example of the second exemplary embodiment is described with reference toFIG. 13. In the third modified example of the second exemplary embodiment, the optical path shift is corrected with the beam splitter240being disposed at an angle smaller than 45° relative to the micro lens disk210, and an optical path correction glass plate244is disposed between the micro lens disk210and the pinhole disk250. In the second exemplary embodiment, a distance between the micro lens211and the pinhole and252, i.e., a focal length of the micro lens221(the second exemplary embodiment) is generally longer than the distance between the micro lens221and the pinhole223in the first exemplary embodiment, i.e., the focal length of the micro lens221(the first exemplary embodiment). Therefore, it is preferably to correct the inclination of the optical axis accompanied by the correction of the optical path shift. To this end, the optical path correction glass plate244is obliquely disposed between the micro lens disk210and the pinhole disk250, thereby correcting the inclination of the optical axis.

According to the confocal microscope10shown inFIG. 1and the confocal microscope11shown inFIG. 10, two convex lenses and one pinhole are disposed in the direction of the rotation axis of the disks configured to integrally rotate. For this reason, the point image of the sample30passes through the convex lenses one more time and reaches the capturing element151, as compared to a Nipkow disk type having a general micro lens array in which one convex lens and one pinhole are disposed.

Therefore, when a point image of the sample30radiating the fluorescence at a right-upper area as shown inFIG. 14Ais captured, a point image of an upright image as shown inFIG. 14Bis obtained in a confocal microscope of a Nipkow disk type having a general micro lens array but a point image of an inverted image as shown inFIG. 14Cis obtained in the confocal microscope11of the second exemplary embodiment.

Here, a case where the sample30radiating the circular fluorescence as shown inFIG. 15Ais imaged by scanning four areas as shown inFIG. 15Bas point images (shown with rectangular images for descriptive purposes) is assumed. It is assumed that the point images are captured with the same frames by irradiating the separate illumination light beamlets. At this time, the shown example is simplified for simple descriptions.

According to the confocal microscope11of the second exemplary embodiment, since each point image is captured as an inverted image, an image in which the point images of the respective areas are inverted is obtained as shown inFIG. 15C, and the fine continuity between the adjacent point images is damaged. For this reason, there is room for further improvement from a standpoint of the resolution.

Therefore, it is considered to return the inverted image to the upright image by enabling the light to additionally pass through the convex lens one more time in the disks configured to integrally rotate. Thereby, since the fine continuity between the adjacent point images is maintained, it is possible to further improve the resolution.

FIG. 16shows in detail a positional relation among the pinhole disk250, the micro lens disk210, the second micro lens disk260and the beam splitter240in an improved version corresponding to the second exemplary embodiment shown inFIG. 10.

In the improved version, micro lenses253are disposed on an opposite surface of the pinhole disk250to the surface on which the pinholes are formed, in correspondence to the respective pinholes252, so as to return the inverted image to the upright image. InFIG. 16, the central axes of the micro lens253, the pinhole252, the micro lens211and the micro lens261of one set disposed on the same axis are denoted by the dashed-dotted line.

The illumination light emitted from the light source110(FIG. 10) is reflected on the beam splitter240, passes through the micro lens211and is divided into a plurality of illumination light beamlets. The respective illumination light beamlets are concentrated in the pinhole252, and are incident to the micro lens253with being slightly widened in the thickness direction of the pinhole disk250. Then, the beamlets are again concentrated at the micro lens253, are widened, are incident to the capturing lens (refer toFIG. 10), and are concentrated on the sample130by the objective lens120(refer toFIG. 10).

In the improved version, the concentration angle of light in the pinhole252and the concentration angle of light at the micro lens253(the widened angle after the concentration of light) are designed to be θ.

The return light from the sample30passes through the objective lens120(refer toFIG. 10), the capturing lens (refer toFIG. 10), the micro lens253, the pinhole252and the micro lens211in corresponding order along the same optical path as the illumination light, becomes the parallel light, passes through the beam splitter240and are incident to the micro lens261. Then, the return light is concentrated at the angle of 2θ.

An area of the micro lens253-side of the pinhole disk250except for the micro lenses253may be subject to the light shield processing. Thereby, it is possible to prevent the light, which returns at an angle greater than θ, from being incident to the surrounding micro lenses253to deteriorate an image.

Like the first modified example of the second exemplary embodiment, the beam splitter240may be disposed at the angle of 45° relative to the axis, and the optical path correction glass plate may be obliquely disposed at the angle of 45° in the opposite direction so as to correct the optical path correction. Like the second modified example, the optical path correction filter242may be used so as to correct the optical path shift. Like the third modified example, the beam splitter240may be disposed at an angle smaller than 45° relative to the micro lens disk210, and the optical path correction glass plate244may be disposed.

Also, as shown inFIG. 17, regarding the pinhole disk250having the micro lenses253arranged thereon, the pinhole surface may be made to face towards the sample30. In this case, a distance between the micro lens211and the micro lens253is set to be longer than the focal length of the micro lens211, and each illumination light beamlet is concentrated in front of the micro lens253and is incident to the micro lens253with being slightly widened. Each beamlet advances in the pinhole disk250, is concentrated in the pinhole252, is widened, is incident to the capturing lens (refer toFIG. 10), and is concentrated on the sample30by the objective lens120(refer toFIG. 10).

In this example, the incident angle of light to the micro lens253and the emission angle of light from the pinhole252are designed to be θ.

The return light from the sample30passes through the objective lens120(refer toFIG. 10), the capturing lens (refer toFIG. 10), the pinhole252, the micro lens253, and the micro lens211in corresponding order along the same optical path as the illumination light, becomes the parallel light, passes through the beam splitter240and are incident to the micro lens261. Then, the return light is concentrated at the angle of 2θ.

Alternatively, as shown inFIG. 18, the pinhole disk250may not be provided with the micro lenses, and a micro concave lens disk270may be used instead of the second micro lens disk260. In this case, instead of the configuration of returning the inverted image to the upright image by enabling the light to additionally pass through the convex lens one more time, the point image is prevented from being the inverted image by reducing the number of passing times of the light through the convex lens.

The micro concave lens disk270adopts the convex lenses formed on the second micro lens disk260, as concave lenses. Each concave lens is designed to radiate the return light of the parallel light at the angle of 2θ. A line261inFIG. 18indicates a focal plane of the concave lenses.

In this example, the relay lens140(refer toFIG. 10) is configured to project an image of the focal plane261onto the surface of the capturing element (refer toFIG. 10) of the camera150. The configuration of this example has merits that each point image is an upright image and the configuration is further simpler.