JP5523937B2 - Confocal microscope - Google Patents

Description

  The present invention relates to a confocal microscope capable of observing an observation object by irradiating and reflecting light on the observation object and selectively detecting reflected light focused on the observation object.

  Conventionally, a disk scanning method or a laser scanning method has been widely used as a scanning method for a confocal microscope (see Patent Documents 1 and 2).

  A disc scanning confocal microscope has a nipou disc with a number of pinholes formed in a line shape between the light source and the object to be observed. The image of the observation object can be obtained by detecting the above with a detector.

  Further, a laser scanner type confocal microscope irradiates an observation target object with light from a light source via a scanner mirror, and detects the reflected light with a detection element via the scanner mirror or pinhole. At this time, an image of the observation object can be obtained by moving the angle of the scanner mirror and scanning the irradiation position of the light on the observation object.

JP-A-5-88092 JP 2010-26344 A US Pat. No. 5,022,743

  However, in the laser scanning method, during scanning, reflected light is condensed at one fixed point by a pinhole, and the detection element detects the light at the fixed point. For this reason, in order to finally obtain an image of the entire observation object, a computer is used based on information such as the relationship between the light irradiation position of the observation object and the angle of the scanner mirror and the light intensity of the detection element. It is necessary to reconstruct the image of the observation object. For this reason, for example, if the position and angle of the actual scanner mirror deviate from the position and angle recognized on the computer, the image reconstructed by the computer is distorted.

  On the other hand, in the above-described disk scanning method, since it is not necessary to reconstruct an image by a computer, image distortion hardly occurs. However, since the light from the light source that does not pass through the pinhole is blocked by the Niipou disk, much of the light is lost and the light from the light source is not used efficiently.

  The present invention has been made in view of this point, and an object of the present invention is to provide a confocal microscope that can reduce the loss of the light amount of the light source while preventing distortion of the image of the observation object.

In order to achieve the above object, the present invention provides a light source and light that is disposed on an optical path between the light source and an observation object and that is incident on the optical path in the first incident direction from the light source side. A scanner mirror that reflects the object side and reflects the reflected light from the observation object side to the light source side, and can change an irradiation position of light on the observation object by changing an angle with respect to the optical path; and Of the reflected light reflected by the scanner mirror from the side of the observation object, the light selection unit through which the reflected light focused on the observation object passes, and the reflected light that has passed through the light selection unit are the first light an optical system for the second incidence direction different from the incident direction is incident again on the same side of the scanner mirror is reflected to form an image of the observation object from the light reflected again by the scanner mirror It possesses a forming section, wherein the optical system introduces light from the light source side injection to the first incident direction relative to the scanner mirror, yet at the scanner mirror from the observation object side A first optical fiber that introduces reflected reflected light, an optical branching unit that branches the reflected light introduced into the first optical fiber unit from the first optical fiber, and the optical branching unit. A second optical fiber that emits the reflected light to the scanner mirror in the second incident direction, and an introduction end portion of the reflected light of the first optical fiber includes the light selection unit. This is a confocal microscope.

  According to the present invention, since the reflected light that has passed through the light selection unit from the observation object side is reflected again by the same scanner mirror, the two-dimensional plane corresponding to the light irradiation position of the observation object in the image forming unit. An image can be formed. Since the irradiation position of light on the observation object can be scanned by the scanner mirror, a two-dimensional planar image of the observation object can be obtained. As a result, it is not necessary to reconstruct the image on the computer based on information such as the relationship between the irradiation position of the light of the observation object and the angle of the scanner mirror as in the past, and the image can be acquired directly. Distortion of the image of the observation object can be prevented. In addition, since light is not blocked by a disk such as a Nipkow disk, loss of the light amount of the light source can be reduced.

  The scanner mirror may include a first mirror that can rotate around a first axis, and a second mirror that can rotate around a second axis perpendicular to the first axis.

  The image forming unit may include a detection element capable of detecting the reflected light reflected again by the scanner mirror on a two-dimensional plane.

  ADVANTAGE OF THE INVENTION According to this invention, the loss of the light quantity of a light source can be reduced, preventing the distortion of the image of an observation target object. Therefore, high-accuracy observation can be performed with a low-output light source.

It is a schematic diagram which shows the outline of a structure of the optical system of the confocal microscope which has an optical fiber. It is a schematic diagram which shows the outline of a structure of the optical system of the confocal microscope which has a beam splitter. It is a schematic diagram which shows the outline of a structure of the optical system of a confocal microscope in case a scanner mirror has two mirrors.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram illustrating an example of a configuration of an optical system of a confocal microscope 1 according to the present embodiment.

  The confocal microscope 1 includes, for example, a light source 10, a scanner mirror 11, a light selection unit 12, an optical system 13, and an image forming unit 14.

  The light source 10 is, for example, a laser light source. The scanner mirror 11 is disposed on the optical path between the light source 10 and the observation target A, reflects light incident from the laser light source 10 side on the optical path to the observation target A side, and from the observation target A side. Can be reflected back to the light source 10 side. In addition, a MEMS (Micro Electro Mechanical Systems) mirror or the like is used as the scanner mirror 11 and can be rotated around two orthogonal axes to change the installation angle with respect to the optical path. Thereby, the scanner mirror 11 can scan the irradiation position of the light on the observation object A in a two-dimensional direction. The light after being reflected by the observation object A is referred to as “reflected light”.

  The optical system 13 includes, for example, a first optical fiber 20, an optical branching unit 21, and a second optical fiber 22.

  For example, the first optical fiber 20 can introduce light from the light source 10 side from the first end 20 a and emit the light from the second end 20 b to the scanner mirror 11 in the first incident direction B. Moreover, the 1st optical fiber 20 can introduce | transduce the reflected light reflected by the scanner mirror 11 from the observation object A side from the 2nd edge part 20b. The second end portion 20 b as the introduction end portion of the reflected light of the first optical fiber 20 is the light selection unit 12. The light selection unit 12 performs the same function as a pinhole, and selectively passes reflected light focused on the observation object A.

  The optical branching unit 21 can branch the reflected light introduced into the first optical fiber 20 from the first optical fiber 20. The optical branching unit 21 is, for example, a fiber coupler.

  The second optical fiber 22 can emit the reflected light branched by the light branching unit 21 toward the second incident direction C different from the first incident direction B with respect to the scanner mirror 11. For example, the incident angle in the second incident direction C is larger than the incident angle in the first incident direction B at the scanner mirror 11. This injection can be performed from the injection end 22a. The other end of the second optical fiber 22 is connected to the fiber coupler of the optical branching unit 21.

  The image forming unit 14 includes, for example, an imaging lens 30 and a detection element 31. The imaging lens 30 collects the reflected light reflected by the scanner mirror 11 and forms an image. The detection element 31 is, for example, a CCD camera as a two-dimensional imaging element, and can form an image on a two-dimensional plane and detect the image.

  In addition, the confocal microscope 1 is configured such that, for example, a condensing lens (light source unit lens) 40 that collects light emitted from the light source 10 and light emitted from the second end 20b of the first optical fiber 20 are collected. A collimating first collimator lens 41, a bending mirror 42 having a fixed angle that reflects light reflected by the scanner mirror 11 toward the observation object A, an objective lens 43, and the second optical fiber 22. A second collimator lens 44 that collimates the light emitted from the emission end 22a.

  Next, the operation of the confocal microscope 1 configured as described above will be described.

  First, laser light is emitted from the light source 10, the light is collected by the condenser lens 40, and introduced into the first optical fiber 20 from the first end 20 a. The light introduced into the first optical fiber 20 is emitted from the second end 20b through the inside thereof. This emission is performed toward the first incident direction B with respect to the scanner mirror 11.

  The light emitted from the second end 20 b is collimated by the first collimator lens 41 and then enters the scanner mirror 11. The light is reflected by the scanner mirror 11 and further reflected by the bending mirror 42 in the direction in which the object A is observed. Next, the light is condensed through the objective lens 43, irradiated onto the observation object A, and an image corresponding to the light spot (the second end 20b (the light selection unit 12) of the first optical fiber 20). ).

  The reflected light reflected by the observation object A passes through the objective lens 43 again, is reflected by the bending mirror 42, enters the scanner mirror 11, and is reflected. Thereafter, the reflected light is collected through the first collimator lens 41 and introduced into the first optical fiber 20 from the second end 20b. When introduced into the first optical fiber 20, the reflected light passes through the light selector 12 at the second end 20b. At this time, when an image (spot of light) corresponding to the light selection unit 12 of the first optical fiber 20 is formed on the observation object A, that is, when the light is in focus, the reflected light is light. In other cases, the reflected light does not pass through the light selector 12. Therefore, only the reflected light in focus is introduced into the first optical fiber 20, and an image is formed by the detection element 31 as described later.

  The reflected light introduced into the first optical fiber 20 is branched by the light branching unit 21 and introduced into the second optical fiber 22. The reflected light passes through the second optical fiber 22 and is emitted from the emission end 22 a toward the scanner mirror 11. At this time, the reflected light is emitted to the scanner mirror 11 in a second incident direction C different from the first incident direction B from the first optical fiber 20 to the scanner mirror 11.

  The reflected light emitted from the second optical fiber 22 is collimated by the second collimator lens 44 and then enters the scanner mirror 11 again. The reflected light is reflected by the scanner mirror 11 and imaged by the imaging lens 30, and then spotted on the two-dimensional plane by the detection element 31 (observation object corresponding to the exit end 22 a of the second optical fiber 22). An image of an irradiation position of the object A is formed, and the image is detected.

  The angle of the scanner mirror 11 with respect to the optical path is changed, and the light irradiation position (spot formation position) of the observation object A is scanned over the entire light irradiation region (field of view) of the observation object A. As a result, a two-dimensional planar image of the entire observation object A in the field of view can be acquired by the detection element 31.

  Next, the relationship between the light irradiation position on the observation object A and the image formation position on the detection element 31 will be described.

  For example, it is assumed that spots are formed at the center of the visual field on the observation object A and the center of the detection element 31 when the scanner mirror 11 is at the reference angular position.

As shown in FIG. 1, when the scanner mirror 11 is tilted by θ _{M in a} certain direction from the reference angular position, the angular displacements θ _Obj and θ _Tube of light incident on the objective lens 43 and the imaging lens 30 are both
θ _Obj = θ _Tube = 2θ _M
It becomes.

As a result, the movement amounts y _Obj and y _Img of the spot on the observation object A and the detection element 31 are respectively
y _Obj = f _Obj × tanθ _Obj = f _Obj × tan (2θ _M )
y _Img = f _Tube x tan θ _Tube = f _Tube x tan (2θ _M )
It becomes. F _Obj is the focal length of the objective lens 43, and f _Tube is the focal length of the imaging lens 30.

As a result, the imaging magnification m is
m = y _Img / y _Obj = f _Tube / f _Obj
It becomes. This relationship is equivalent to the infinity correction optical system.

  Accordingly, by changing the angle of the scanner mirror 11 around two orthogonal axes and scanning the irradiation position of the light of the observation object A over the entire field of view, an image similar to the observation object A in the entire field of view is obtained. be able to.

  According to the above embodiment, since the reflected light that has passed through the light selection unit 12 from the observation object A side is reflected again by the same scanner mirror 11, the detection element 31 irradiates the light of the observation object A. An image (spot) on a two-dimensional plane corresponding to the position can be formed. Then, by scanning the irradiation position on the observation object A with the scanner mirror 11, a two-dimensional planar image of the entire observation object A in the field of view can be finally obtained by the detection element 31. As a result, it is not necessary to reconstruct the image on the computer based on the relationship between the irradiation position of the light of the observation object A and the angle of the scanner mirror as in the prior art, and the image can be acquired directly. Distortion of the image of the object A can be prevented. In addition, since the image can be acquired in real time, the image acquisition time can be shortened. Further, since the light is not blocked unlike the Nipkow disk, the loss of the light amount of the light source 10 can be reduced, and the light of the light source 10 can be used efficiently.

  Further, since the optical fibers 20 and 22 are used in the optical system 13, the optical system 13 can be arranged flexibly.

  In the above embodiment, the optical system 13 uses the first optical fiber 20, the optical branching unit 21, and the second optical fiber 22, but may have other configurations.

  For example, as shown in FIG. 2, the optical system 13 may include a beam splitter 50 on the optical path between the light source 10 and the observation object A instead of the optical fibers 20 and 22. For example, a pinhole 51 and a fixed mirror 52 as a light selection unit are provided on the observation object A side of the beam splitter 50 on the optical path. The beam splitter 50 can reflect the reflected light that has passed through the pinhole 51 and emit the reflected light toward the scanner mirror 11 in the second incident direction C. The fixed mirror 52 can reflect the light from the light source 10 toward the scanner mirror 11 and can reflect the light reflected by the scanner mirror 11 from the observation object A side to the light source 10 side. Since other configurations are the same as those in the above embodiment, the same reference numerals are used and description thereof is omitted.

  In such a case, first, the light emitted from the light source 10 is collected by the condenser lens 40, passes through the beam splitter 50, and then passes through the pinhole 51. Thereafter, the light is reflected by the fixed mirror 52, passes through the first collimator lens 41, and then enters the scanner mirror 11. The light is reflected by the scanner mirror 11 and further reflected by the bending mirror 42 in the direction of the observation object A. Next, the light is condensed through the objective lens 43 and irradiated onto the observation object A to form a spot (an image corresponding to the pinhole 51) on the observation object A.

  The reflected light reflected by the observation object A passes through the objective lens 43 again, is reflected by the bending mirror 42, and is reflected by the scanner mirror 11. Thereafter, the reflected light is collected through the first collimator lens 41, reflected by the fixed mirror 52, and then incident on the pinhole 51. At this time, when an image (spot of light) corresponding to the pinhole 51 is formed on the observation object A, that is, when the focus is on, the reflected light passes through the pinhole 51, and otherwise In this case, the reflected light does not pass through the pinhole 51. Therefore, only the reflected light in focus forms an image with the detection element 31 as described later.

  The reflected light that has passed through the pinhole 51 is reflected by the beam splitter 50, passes through the second collimator lens 44, and enters the scanner mirror 11 again. The reflected light is reflected by the scanner mirror 11, is imaged by the imaging lens 30, and is spotted on the two-dimensional plane in the detection element 31 (an image of the irradiation position of the observation object A corresponding to the pinhole 51. ) And the image is detected.

The angle of the scanner mirror 11 with respect to the optical path is changed, and the irradiation position (spot formation position) of the observation object A is scanned over the entire visual field of the observation object A. As a result, a two-dimensional planar image of the entire observation object A in the field of view can be acquired by the detection element 31. Note that the relationship between the observation object A and the amount of movement y _Obj , y _Img of the spot on the detection element 31, the imaging magnification m, and the like are the same as in the above embodiment.

  Also in this example, the reflected light from the observation object A is reflected again by the scanner mirror 11 by the beam splitter 50, and an image (spot) is formed on the two-dimensional plane corresponding to the irradiation position of the observation object A by the detection element 31. Further, by scanning the irradiation position on the observation object A with the scanner mirror 11, finally, a two-dimensional planar image of the entire observation object A in the field of view is obtained by the detection element 31. Can do. As a result, it is not necessary to reconstruct an image by a computer as in the above embodiment, and distortion of the image of the observation object A can be prevented. Moreover, the loss of the light quantity of the light source 10 can be reduced. Furthermore, since the structure is simplified without using an optical fiber, the confocal microscope 1 can be downsized as compared with the above embodiment.

  The scanner mirror 11 described in the above embodiment can be rotated around a first mirror 60 rotatable around a first axis and a second axis orthogonal to the first axis, for example, as shown in FIG. The second mirror 61 may be included. For the first mirror 60 and the second mirror 61, for example, a galvanometer mirror that can handle a large-diameter light beam is used. The mirrors 60 and 61 are provided with a drive source 62 such as a motor for driving the respective shaft rotations.

  In such a case, first, the light emitted from the light source 10 is collected by the condenser lens 40, introduced into the first optical fiber 20 from the first end 20 a, and the second of the first optical fiber 20. Injected from the end 20b. Thereafter, the light passes through the first collimator lens 41, is reflected by the first mirror 60 of the scanner mirror 11, and then is reflected by the second mirror 61. Thereafter, the light is reflected by the bending mirror 42, collected through the objective lens 43, and irradiated on the observation object A to form a spot (an image corresponding to the light selection unit 12 of the first optical fiber 20). To do.

  The reflected light reflected by the observation object A passes through the objective lens 43 again, is reflected by the bending mirror 42, is reflected by the second mirror 61 of the scanner mirror 11, and is reflected by the first mirror 60. Thereafter, the reflected light is collected through the first collimator lens 41 and introduced into the first optical fiber 20 from the second end 20b. At this time, the reflected light passes through the light selector 12. At this time, when an image (spot of light) corresponding to the light selection unit 12 of the first optical fiber 20 is formed on the observation object A, that is, when the light is in focus, the reflected light is light. In other cases, the reflected light does not pass through the light selector 12. Therefore, only the focused light is introduced into the first optical fiber 20 and an image is formed in the detection element 31 as described later.

  The reflected light introduced into the first optical fiber 20 is branched by the light branching unit 21 and introduced into the second optical fiber 22. The reflected light passes through the second optical fiber 22 and is emitted from the emission end 22 a toward the scanner mirror 11.

  The reflected light emitted from the second optical fiber 22 passes through the second collimator lens 44, is reflected again by the first mirror 60 of the scanner mirror 11, and is reflected by the second mirror 61. The reflected light is then imaged through the imaging lens 30 and then spotted on the two-dimensional plane in the detection element 31 (the light of the observation object A corresponding to the exit end 20a of the second optical fiber 22). The image of the irradiation position is formed, and the image is detected.

The angle of the first mirror 60 and the second mirror 61 with respect to the optical path is varied, and the irradiation position (spot formation position) of the observation object A is scanned over the entire visual field of the observation object A. As a result, a two-dimensional planar image of the entire observation object A in the field of view can be acquired by the detection element 31. Note that the relationship between the observation object A and the amount of movement y _Obj , y _Img of the spot on the detection element 31, the imaging magnification m, and the like are the same as in the above embodiment.

  According to this example, since the irradiation position of the light on the observation object A can be scanned in a two-dimensional direction using the two mirrors 60 and 61 that rotate one axis, compared to a two-axis rotation mirror made of MEMS or the like. Can handle light with a large beam diameter. Therefore, for example, a larger observation object A can be observed. In the example in which the beam splitter 50 is used instead of the optical fibers 20 and 22 described above, the two mirrors 60 and 61 may be used as the scanner mirror 11.

  The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to such examples. It is obvious for those skilled in the art that various modifications or modifications can be conceived within the scope of the idea described in the claims, and these naturally belong to the technical scope of the present invention. It is understood.

  For example, in the above embodiment, the detection element 31 is provided in the image forming unit 14, but the present invention can also be applied to a case where an image formed in a plane is observed with the naked eye.

  The present invention is useful for realizing a confocal microscope that reduces the loss of light quantity of a light source while preventing distortion of an image of an observation object.

DESCRIPTION OF SYMBOLS 1 Confocal microscope 10 Light source 11 Scanner mirror 12 Light selection part 13 Optical system 14 Image formation part 20 1st optical fiber 21 Optical branch part 22 2nd optical fiber 30 Imaging lens 31 Detection element 43 Objective lens A Observation object B First incident direction C Second incident direction

Claims (3)

A light source;
It is arranged on the optical path between the light source and the observation object, and reflects the light incident in the first incident direction from the light source side on the optical path to the observation object side and the reflection from the observation object side. A scanner mirror that reflects light toward the light source and can change an irradiation position of light on the observation object by changing an angle with respect to the optical path;
A light selection unit through which reflected light focused on the observation object passes among reflected light reflected by the scanner mirror from the observation object side;
An optical system that reflects and reflects the reflected light that has passed through the light selection unit from a second incident direction different from the first incident direction to the same surface of the scanner mirror;
Have a, an image forming unit that forms an image of the observation object from the light reflected again by the scanner mirror,
The optical system is
A first optical fiber that introduces light from the light source side, exits the scanner mirror in the first incident direction, and introduces reflected light reflected from the scanner mirror from the observation object side; ,
An optical branching unit for branching the reflected light introduced into the first optical fiber unit from the first optical fiber;
A second optical fiber that emits the reflected light branched at the light branching portion in the second incident direction with respect to the scanner mirror;
The confocal microscope , wherein an introduction end of reflected light of the first optical fiber constitutes the light selection unit . The scanner mirror has first and one mirror rotatable first axis around the second mirror rotatable second shaft around which is orthogonal to the first axis, according to claim 1 Confocal microscope. The confocal microscope according to claim 1 , wherein the image forming unit includes a detection element capable of detecting reflected light reflected again by the scanner mirror on a two-dimensional plane.

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