JP2012208327A - Scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning microscope arranging first and second deflection elements nearby and reducing a total size.SOLUTION: A scanning microscope 10 has: a light source 20; an object lens 36; and a scanning unit 33. This scanning unit 33 has first to third deflection elements 33a to 33c, has the first and second deflection elements 33a, 33b arranged so that illumination light made incident from the light source 20 side is reflected by the first deflection element 33a and the second deflection element 33b and the illumination light is made to scan a sample surface 50 in a prescribed direction, in addition the first and second deflection elements 33a, 33b are rotated so that the rotational centers of the illumination light by the first and second deflection elements 33a, 33b are positioned more on the light source 20 side than the first deflection element 33a or positioned more on the sample surface 50 than the second deflection element 33b, furthermore, the third deflection element 33b is arranged to scan the sample surface 50 in a direction which is substantially orthogonal to the prescribed direction by having a reflection surface positioned in this rotational center of illumination light by the first and second deflection elements 33a, 33b.

Description

  The present invention relates to a scanning microscope.

  As is well known, a scanning confocal microscope (hereinafter referred to as a scanning microscope) irradiates an observation sample with a laser beam through an objective lens, thereby irradiating reflected light from the observation sample or an observation sample. As a result, the fluorescence generated from the sample is imaged in the form of a point again via the objective lens and the optical system, and this is detected by a detector to obtain a point image. At this time, a two-dimensional image can be obtained by scanning the entire two-dimensional XY plane of the observation sample using a galvanometer mirror that is a deflection element.

  FIG. 7 shows a specific path of the laser beam in such a scanning microscope. The laser beam incident from the A direction passes through the beam splitter 132 and passes through the two deflecting elements 133a and 133b to be two-dimensional. Scanned. Thereafter, the light enters the pupil projection lens 134 and passes through the pupil projection lens 134 to form a spot on the image plane of an objective lens (not shown). At this time, two-dimensional scanning is performed on the image surface of the objective lens by the rotation (swing angle) of the two deflecting elements 133a and 133b, so that the reflected light or fluorescence from the sample surface on the image surface is as described above. Is returned to the beam splitter 132, reflected by the beam splitter 132, further passes through the detection optical system, and is incident on a photodetector such as a photomultiplier (not shown). The converted two-dimensional image is displayed on each pixel of the monitor (not shown) in correspondence with the scanning positions in the X and Y directions of the two deflection elements 133a and 133b.

  By the way, in such a scanning microscope, in order to obtain a uniform two-dimensional image by two-dimensional scanning with a laser beam, the exit pupil of the objective lens is always used regardless of the rotation angle (swing angle) of the two deflection elements. And the center of rotation of the laser beam passing through the objective lens must coincide with each other. For this purpose, the rotational center of the deflecting element and the pupil position of the objective lens should be in a conjugate relationship. However, since two deflection elements are used for two-dimensional scanning of the laser beam, it is not always possible to set both of these two deflection elements so as to be conjugate with the pupil position of the objective lens. . Therefore, in Patent Document 1, a first deflecting element that deflects a light beam along one direction, a second deflecting element that further deflects the light beam deflected by the first deflecting element along the direction, And a driving unit that rotates the second deflecting element in synchronization with each other, and the center position of the deflection caused by the rotation of the light beam deflected by the second deflecting element is between the first and second deflecting elements. The first and second deflecting elements are arranged so as to coincide with the position conjugate with the pupil position of the objective lens.

JP 2001-091848 A

  However, in the above-described scanning microscope, since the center position (rotation center) of deflection is between the first and second deflection elements, the third deflection deflects along a direction perpendicular to the one direction. The element needs to be placed between the first and second deflection elements. For this reason, the distance between the first and second deflecting elements is widened, and the entire scanning microscope is increased in size.

  The present invention has been made in view of such a problem, and an object of the present invention is to provide a scanning microscope in which the first and second deflecting elements can be arranged close to each other and the whole is downsized.

  In order to solve the above-described problems, a scanning microscope according to the present invention is arranged between an objective lens that collects illumination light emitted from a light source and irradiates a sample surface, and the light source and the objective lens. A scanning unit that scans the sample surface with light, and each of the scanning units includes a first deflecting element and a second deflecting element each having a reflecting surface that is rotated about a rotation axis that faces the first direction. And a third deflecting element having a reflecting surface rotated about a rotation axis facing a second direction different from the first direction, and the illumination light incident from the light source side is the first The illumination light reflected by the deflection element and further reflected by the first deflection element is reflected by the second deflection element, and the illumination light is scanned in a direction substantially orthogonal to the first direction on the sample surface. The first deflecting element and the second deflecting element are arranged so that the first deflecting element The first deflection element and the second deflection element are arranged so that the rotation center of the illumination light by the deflection element and the second deflection element is located on the light source side with respect to the first deflection element or on the sample surface side with respect to the second deflection element. The second deflecting element rotates, and the third deflecting element is arranged so that the reflecting surface is located at the center of rotation and the sample surface is scanned in a direction substantially perpendicular to the second direction. Features.

  In such a scanning microscope, it is preferable that the first deflecting element and the second deflecting element are arranged so that the incident angle of the illumination light with respect to the reflecting surface is smaller than 45 °.

  In such a scanning microscope, the first deflecting element and the second deflecting element reflect the illumination light incident on the reflecting surface of the first deflecting element on the reflecting surface of the second deflecting element. It is preferable that the illumination lights emitted in this way are arranged so as to be substantially orthogonal and intersect.

  At this time, in the first deflecting element and the second deflecting element, the illumination light that is reflected by the reflecting surface of the second deflecting element and emitted is emitted with respect to the illumination light incident on the reflecting surface of the first deflecting element. It is preferable that they are arranged so as to be substantially orthogonal.

  Also, in such a scanning microscope, the first deflection element, the second deflection element, and the third deflection element can move along the optical axis direction of the objective lens without changing the positional relationship with each other. preferable.

  When the scanning microscope according to the present invention is configured as described above, the entire scanning microscope can be reduced in size by arranging the first and second deflecting elements close to each other.

It is explanatory drawing which shows the structure of the scanning microscope which concerns on this embodiment. It is explanatory drawing which shows the structure of the scanning unit which concerns on 1st Embodiment. It is explanatory drawing which shows the light beam when arrange | positioning the rotation center by the 1st and 2nd deflection | deviation element in the sample surface side, and scanning on a sample surface, Comprising: (a) shows the locus | trajectory of the light beam at the time of scanning, b) shows the upper ray in (a), (c) shows the ray on the optical axis in (a), and (d) shows the lower ray in (a). It is explanatory drawing which shows the light ray when arrange | positioning the rotation center by the 1st and 2nd deflection | deviation element in the light source side, and scanning on a sample surface, Comprising: (a) shows the locus | trajectory of the light ray at the time of scanning, (b ) Shows the upper light beam in (a), (c) shows the light beam on the optical axis in (a), and (d) shows the lower light beam in (a). It is explanatory drawing which compares the structure of the scanning unit which concerns on this embodiment, Comprising: (a) shows the scanning unit which concerns on 1st Embodiment, (b) shows the structure of the scanning unit which concerns on 2nd Embodiment. Show. It is explanatory drawing for demonstrating arrangement | positioning of the deflection | deviation element in a scanning unit, Comprising: (a) shows the conventional scanning microscope, (b) shows the scanning microscope concerning 2nd Embodiment. It is explanatory drawing for demonstrating arrangement | positioning of the deflection | deviation element in the conventional scanning microscope.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the scanning microscope according to the present embodiment will be described with reference to FIG. The scanning microscope 10 detects a laser beam (illumination light) emitted from the light source 20 by irradiating the sample surface 50 of the observation sample to scan, and reflected light or fluorescence from the sample surface 50. And a detection optical system 40.

  The scanning optical system 30 includes a beam expander 31, a beam splitter 32, a scanning unit 33, a pupil projection lens 34, an imaging lens 35, and an objective lens 36 in order from the light source 20 side. The detection optical system 40 is disposed on the side of the scanning optical system 30 and includes an imaging lens 41, a light shielding plate 42, and a photodetector 43 in this order from the beam splitter 32 side. In addition, the scanning microscope 10 is provided with a processing unit 70 that processes the position (coordinates on the sample surface 50) scanned by the scanning unit 33 and the value detected by the photodetector 43.

  In the scanning microscope 10, the laser beam emitted from the light source 20 becomes a substantially parallel light beam having a beam diameter required by the beam expander 31, passes through the beam splitter 32, and enters the scanning unit 33. The scanning unit 33 scans the laser beam two-dimensionally in a direction orthogonal to the optical axis as will be described later. For example, the scanning unit 33 deflects the laser beam by reflecting the laser beam as shown in FIG. The first deflecting element 33a, the second deflecting element 33b, and the third deflecting element 33c are made up of three deflecting elements. The first to third deflection elements 33 a to 33 c are configured to be rotated (swinged) by the motor of the drive unit 60. The laser beam (substantially parallel light beam) emitted from the scanning unit 33 is imaged on the primary image plane I by the pupil projection lens 34, and then passes through the image forming lens 35 to become a substantially parallel light beam again. 36 forms an image on the sample surface (focal plane of the objective lens 36) 50.

  The laser beam image formed on the sample surface 50 is a point image, and the diameter of the point image is determined by the numerical aperture (NA) of the objective lens 36. The reflected light from the point image (irradiation region) on the sample surface 50 is condensed again by the objective lens 36 to become a substantially parallel light beam, which is imaged on the primary image surface I by the imaging lens 35, and further pupil projected. It is made into a substantially parallel light beam by the lens 34 and enters the scanning unit 33. The reflected light (substantially parallel light beam) emitted from the scanning unit 33 is reflected by the beam splitter 32 and enters the detection optical system 40, and is condensed on the opening 42 a of the light shielding plate 42 by the imaging lens 41. . Only the light that has passed through the opening 42a of the light shielding plate 42 reaches the photodetector 43 and is detected.

  As described above, the opening 42 a of the light shielding plate 42 is conjugate with the point image of the laser beam imaged on the sample surface 50, and light (reflected light or fluorescence) emitted from the irradiation region on the sample surface 50 is It can pass through this opening 42a. On the other hand, most of the light emitted from other regions on the sample surface 50 is not imaged on the opening 42a and cannot pass therethrough. As described above, the processing unit 70 processes the optical signal detected by the photodetector 43 in synchronization with the scanning of the scanning unit 33, whereby a two-dimensional image of the sample surface 50 can be obtained. As a result, the scanning microscope 10 can obtain an image of the sample surface 50 with high resolution.

  Incidentally, as described above, in such a scanning microscope 10, in order to obtain a uniform two-dimensional image by two-dimensional scanning of a laser beam, the first to third deflection elements 33a constituting the scanning unit 33 are obtained. It is necessary to have a conjugate relationship between the rotation center (center of the swing angle) of the laser beam by ~ 33c and the position of the exit pupil P of the objective lens 36. The pupil projection lens 34 is configured to form an image of the exit pupil P of the objective lens 36 at the rotation center of the scanning unit 33. The configuration of the scanning unit 33 will be described below.

[First Embodiment]
FIG. 2 described above shows the configuration of the scanning unit 33 according to the first embodiment. As shown in FIG. 2, a predetermined direction (in FIG. 2, the vertical direction in the sample surface 50 substantially perpendicular to the optical axis) is shown. (Direction) is the x-axis direction, and the direction perpendicular to the x-axis direction (the left-right direction in FIG. 2) is the y-axis direction, the two images that scan the point image of the laser beam in the x-axis direction on the sample surface 50 The first deflection element (first and second deflection elements 33a and 33b) and one deflection element (third deflection element 33c) that scans in the y-axis direction. In the configuration of FIG. 2, the first and second deflection elements 33a and 33b have a scanning rotation axis extending in the y-axis direction, and the third deflection element 33c has a scanning rotation axis extending in the x-axis direction. These rotating shafts are rotated by the motor of the driving unit 60 described above, whereby the reflecting surfaces of the first and second deflecting elements 33a and 33b rotate (swing) in the x-axis direction. The reflecting surface of the third deflection element 33c rotates (swings) in the y-axis direction. Also, as shown in FIG. 3C, when the scanning unit 33 is emitted and a laser beam is irradiated onto a region on the optical axis in the x-axis direction of the sample surface 50, the first and second deflections are performed. The reflecting surfaces of the elements 33a and 33b are substantially parallel and are inclined by 45 ° with respect to the optical axis (the incident angle of the laser beam is 45 °).

  FIG. 3 shows a case where the laser light incident on the optical axis is scanned in the x-axis direction on the sample surface 50 by the first and second deflecting elements 33a and 33b. The first and second deflection elements 33a and 33b have scanning rotation axes extending substantially parallel to each other in the y-axis direction, and the second deflection element 33b is adjusted by adjusting the respective rotation angles (oscillation angles). This laser beam can be scanned with the point P ′ closer to the sample surface 50 as the center of rotation. Therefore, the rotation center of the third deflection element 33c that scans the laser beam in the y-axis direction is arranged at this point P ′, and the image of the exit pupil of the objective lens 36 formed by the pupil projection lens 34 is positioned. By doing so, the rotation center in the x-axis direction, the rotation center in the y-axis direction, and the image of the exit pupil of the objective lens 36 can be substantially matched, so that a two-dimensional image without unevenness is obtained by two-dimensional scanning of the laser beam. be able to.

  As shown in FIG. 4, these rotation angles (oscillations) are such that the rotation center P ′ by the first and second deflecting elements 33a and 33b is located closer to the light source 20 than the first deflecting element 33a. The rotation center of the third deflection element 33c is arranged at the rotation center P ', and the image of the exit pupil of the objective lens 36 formed by the pupil projection lens 34 is positioned. Is also possible. Further, the first and second deflecting elements 33a and 33b can be configured to scan the laser beam in the y-axis direction on the sample surface 50 and to scan in the x-axis direction using the third deflecting element 33c. is there.

  As described above, by disposing the third deflection element 33c on the sample surface 50 side of the second deflection element 33b or on the light source 20 side of the first deflection element 33a, the first and second deflection elements are arranged. Since 33a and 33b can be arranged close to each other, the scanning optical system 30 can be downsized, and as a result, the entire scanning microscope 10 can be downsized.

[Second Embodiment]
As shown in the scanning unit 33 according to the first embodiment, when the laser beam is emitted from the scanning unit 33 onto the optical axis, it is incident on the reflecting surfaces of the first and second deflecting elements 33a and 33b. When the incident angles of the laser beams are respectively set to 45 °, as shown in FIG. 5A, the light beam incident on the second deflection element 33b is rotated in the x-axis direction by the rotation (swing) of the first deflection element 33a. Therefore, the reflection surface of the second deflection element 33b becomes large. Therefore, as shown in FIG. 5B, the light beam incident on the first deflecting element 33a and the light beam emitted from the second deflecting element 33b are crossed, so that the reflecting surface of the second deflecting element 33b is made. It can be downsized. This is because the incident angle of the laser beam with respect to the first and second deflecting elements 33a and 33b (incident angle when emitted from the scanning unit 33 onto the optical axis) can be made smaller than 45 °. This is because the amount of movement of the light beam incident on the second deflection element 33b in the x-axis direction is reduced by the rotation of the first deflection element 33a. Also, with this arrangement, the first deflection element 33a can be reduced in size as compared with the case of FIG.

  Further, as shown in FIG. 5A, in a state where the scanning optical system 30 scans the center (on the optical axis) of the image plane, the light beam incident on the first deflection element 33a and the second deflection element 33b are emitted. When the luminous flux to be emitted is a substantially parallel luminous flux, the first deflection element 33a and the second deflection element 33b are also parallel. In this state, if the first deflecting element 33a and the second deflecting element 33b are brought closer than a certain distance, the second deflecting element 33b blocks the incident light flux. On the other hand, as shown in FIG. 5B, the configuration in which the light beam incident on the first deflecting element 33a and the light beam exiting from the second deflecting element 33b can be folded, the scanning optical system 30 can be folded. It can be made smaller.

  Table 1 below shows that the incident beam diameter φ is 5.5 mm, the maximum scanning angle θ is 6.89 °, and the surface interval L1 on the optical axis of the reflection surfaces of the first and second deflecting elements 33a and 33b is 10. When the distance L2 on the optical axis from 0 mm and the reflecting surface of the second deflecting element 33b to the plane P ′ conjugate with the entrance pupil of the objective lens 36 is 15.0 mm, the input shown in FIG. The effective diameters φ1 and φ2 of the reflection surfaces of the first and second deflecting elements 33a and 33b when the outgoing light beams are parallel and when the incoming and outgoing light beams shown in FIG.

  Further, as shown in FIG. 6B, when the intersection angle of the light beam incident on the first deflection element 33a and the light beam emitted from the second deflection element 33b is 90 °, It is possible to introduce the scanning optical system 30 according to the second embodiment without changing any other elements of the optical system because the incident light beam direction and the outgoing light beam direction of the normal two-dimensional scanning optical system shown in FIG. it can.

  The purpose of the first and second embodiments is to align the rotation center of the light beam with the pupil conjugate position of the observation optical system (which refers to the optical system closer to the sample surface 50 than the imaging lens 35) including the objective lens 36. is there. The pupil position of the observation optical system depends on the type of the objective lens 36 or when the observation optical system includes a variable magnification optical system (arranged between the objective lens 36 and the imaging lens 35), the magnification of the variable magnification optical system The pupil position moves. By moving the whole of the first to third deflecting elements 33a to 33c in the optical axis direction of the observation optical system (objective lens 36) without changing the relative positional relationship at the time of the movement, it is always possible to observe the observation optics. The pupil conjugate position of the system and the rotation center of the light beam can be matched.

DESCRIPTION OF SYMBOLS 10 Scanning microscope 20 Light source 33 Scanning unit 33a First deflection element 33b Second deflection element 33c Third deflection element 36 Objective lens

Claims (5)

An objective lens that collects the illumination light emitted from the light source and irradiates the sample surface;
A scanning unit that is arranged between the light source and the objective lens and scans the sample surface with the illumination light;
The scanning unit is
It rotates in a second direction different from the first direction, with the first deflection element and the second deflection element having reflective surfaces rotated about a rotation axis that faces the first direction. A third deflecting element having a reflecting surface rotated about an axis,
The illumination light incident from the light source side is reflected by the first deflection element, and further, the illumination light reflected by the first deflection element is reflected by the second deflection element, and the illumination light is reflected. The first deflection element and the second deflection element are arranged so as to scan in the direction substantially perpendicular to the first direction on the sample surface, and the first deflection element and the second deflection element The first deflection element is arranged such that the center of rotation of the illumination light by the deflection element is positioned closer to the light source than the first deflection element or closer to the sample surface than the second deflection element. And the second deflection element rotates,
The scanning microscope characterized in that the reflection surface is located at the rotation center, and the third deflection element is arranged so as to scan the sample surface in a direction substantially perpendicular to the second direction.   2. The scanning type according to claim 1, wherein the first deflection element and the second deflection element are arranged such that an incident angle of the illumination light with respect to the reflection surface is smaller than 45 °. microscope.   The first deflecting element and the second deflecting element are reflected by the reflecting surface of the second deflecting element and emitted with respect to the illumination light incident on the reflecting surface of the first deflecting element. The scanning microscope according to claim 1, wherein the illumination lights are arranged so as to intersect with each other.   The first deflecting element and the second deflecting element are reflected by the reflecting surface of the second deflecting element and emitted with respect to the illumination light incident on the reflecting surface of the first deflecting element. The scanning microscope according to claim 3, wherein the illumination lights are arranged so as to be substantially orthogonal to each other.   2. The first deflecting element, the second deflecting element, and the third deflecting element move along the optical axis direction of the objective lens without changing the positional relationship with each other. The scanning microscope as described in any one of -4.

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