JP2006267432A - Scanning type viewing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To quickly perform alignment of an objective lens for a specimen in a preparatory stage by allowing a simple and accurate confirmation of the optical axis position of the objective lens relative to a part desired to be viewed on the specimen. <P>SOLUTION: This scanning type viewing apparatus 1 comprises: a first light source 2; an optical scanning section 3 that scans light L<SB>1</SB>from the first light source 2 over a specimen A; an objective lens 6 that forms an image on the specimen A with the light L<SB>1</SB>scanned in the optical scanning section 3; an optical detecting section 9 that detects the return light L<SB>2</SB>emitted from the specimen A; a second light source 12 for emitting visible light L<SB>3</SB>; a deflective optical element 16 that is arranged between the optical scanning section 3 and the objective lens 6 and that makes the visible light L<SB>3</SB>emitted from the second light source 12 enter the objective lens 6 along the optical axis C of the objective lens 6; and a beam shaping section 14 in which the visible light L<SB>3</SB>from the second light source 12 to be emitted on the surface of the specimen A through the objective lens 6 is formed, by the deflective optical element 16, into patterns P<SB>1</SB>-P<SB>3</SB>that indicate the optical axis C of the objective lens 6. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a scanning observation apparatus.

Conventionally, for example, a scanning laser microscope is known as a scanning observation apparatus (see, for example, Patent Document 1).
In this scanning laser microscope, a laser beam emitted from a laser light source is imaged on a sample by an objective lens via a proximity galvanometer mirror to scan the sample two-dimensionally, while being generated in the sample and The fluorescence returning through the lens and the proximity galvanometer mirror is detected by being branched from the laser beam.
JP 2000-275529 A

However, such a scanning observation apparatus has a drawback in that it is difficult to align the optical axis position of the objective lens with the portion of the sample to be observed.
That is, when observing a sample using a scanning observation apparatus, in the preparation stage, the position of the objective lens is roughly aligned with the position where the sample is to be observed, and the objective distance is set at a rough interval in consideration of the working distance. A lens is placed opposite the sample. However, in the preparatory stage where light is not emitted, it is necessary for the operator to perform alignment work with the intuition as a clue using the shape of the sample and the shape of the objective lens.

  Even if the light is emitted in the preparation stage, the light irradiated from the light source is scanned two-dimensionally on the sample by the proximity galvanometer mirror, so that the optical axis of the objective lens is changed from the light spot that changes every moment. It is difficult to identify. In particular, as in Patent Document 1, when the light from the light source is laser light, when the wavelength of the laser light is not in the visible range, the irradiation range cannot be confirmed in the same manner as described above, and the light is in the visible range. Even if it exists, alignment work is difficult by the glare peculiar to a laser beam. Furthermore, it is not preferable for the operator to directly observe the laser beam irradiated on the sample.

  The present invention has been made in view of the above-described circumstances, and makes it possible to easily and accurately confirm the position of the optical axis of the objective lens with respect to the portion of the sample to be observed. An object of the present invention is to provide a scanning observation apparatus that can perform alignment work quickly and can quickly obtain a desired image in an observation stage.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a first light source, an optical scanning unit that scans light from the first light source on the sample, an objective lens that forms an image of the light scanned by the optical scanning unit on the sample, and a sample. A light detector that detects the emitted return light, a second light source that emits visible light, and a visible light that is disposed between the optical scanning unit and the objective lens and that is emitted from the second light source. A deflecting optical element that is incident on the objective lens along the optical axis of the objective lens, and visible light from the second light source that is irradiated onto the surface of the sample via the objective lens by the deflecting optical element is converted into light from the objective lens. There is provided a scanning observation apparatus including a beam shaping unit that forms an axis in a pattern that can be designated.

  According to the present invention, the light emitted from the first light source is scanned by the optical scanning unit, and is irradiated onto the sample via the objective lens. Thereby, the light from the first light source is sequentially irradiated over a predetermined range in the sample, and the return light returning from the irradiation range is detected by the light detection unit via the objective lens and the optical scanning unit. A two-dimensional image can be acquired.

  In this case, in the preparatory stage prior to observation, when the second light source is activated without activating the first light source, visible light emitted from the second light source is incident on the objective lens by the deflecting optical element, The sample is irradiated. Since the visible light from the second light source is formed in a pattern that can indicate the optical axis of the objective lens by the beam shaping unit, the operator can see the visible light pattern irradiated on the surface of the sample, The optical axis position of the objective lens can be easily specified.

  Then, the relative position of the sample with respect to the objective lens is moved in the direction intersecting the optical axis of the objective lens, and the optical axis position of the objective lens is adjusted by matching the pattern of visible light on the sample surface with the portion to be observed on the sample. It can be properly aligned. In addition, by adjusting the distance between the objective lens and the sample so as to correct the sharpness of the visible light pattern on the surface of the sample, the objective lens can be arranged at an appropriate working distance with respect to the sample. Become.

  Then, after the preparation stage is completed in this way, the operation of the second light source is stopped and the first light source is operated, and the deflection optical element is removed from between the optical scanning unit and the objective lens, or By passing through the deflecting optical element and irradiating the sample with the light from the first light source via the objective lens, it is possible to quickly observe the site to be observed in the sample.

In the above invention, it is preferable that the deflecting optical element is a mirror and is detachably provided between the optical scanning unit and the objective lens.
In this way, in the preparation stage in which the visible light from the second light source is incident on the sample, the deflecting optical element made of a mirror is inserted between the optical scanning unit and the objective lens, and the second light source The object can be easily aligned by irradiating the sample with non-scanned fixed light from the object. Further, in the observation stage in which the light from the first light source is incident on the sample, the scanning light from the first light source is applied to the sample by extracting the deflection optical element from between the optical scanning unit and the objective lens. The return light that is incident and returned from the sample can be detected by the light detection unit without being lost by the deflecting optical element.

In the above invention, the deflection optical element may be a half mirror.
By doing so, the light from the first light source and the light from the second light source are incident on the sample while the deflecting optical element is inserted and disposed between the optical scanning unit and the objective lens. Can do. In this case, in the preparation stage in which the light from the second light source is incident on the sample, the sample surface of the light from the second light source is operated by stopping the operation of the first light source and operating the optical scanning unit. Can be detected by the light detection unit. As a result, for example, it is possible to adjust the scanning range by the optical scanning unit while viewing the image constructed based on the detection signal from the optical detection unit.

Moreover, in the said invention, the said beam shaping part is good also as changing the pattern of the visible light from a 2nd light source alternatively.
By doing so, it is possible to selectively switch to a pattern that matches the form of the sample surface and irradiate visible light. This makes it easier to align the objective lens and the sample and allows more rapid observation.

Furthermore, in the above invention, the second light source may be wavelength-switchable.
By doing in this way, visible light with a wavelength suitable for the color of the sample surface can be irradiated. When the surface colors of various samples are different, the visibility of the image of the second light source on the sample surface is different for visible light having the same wavelength. Therefore, by irradiating visible light with a wavelength that makes the image of the light source stand out according to the color of the sample surface, it is possible to more easily align the objective lens and the sample and to start observation more quickly. Become.

  According to the present invention, by irradiating the surface of the sample with the fixed visible light from the second light source without passing through the optical scanning unit, the objective lens is obtained using the image of the second light source formed on the sample as a clue. It is possible to easily and accurately align the optical axis of the sample and the portion of the sample to be observed. As a result, the time required for observation preparation is reduced, and the desired observation image can be obtained quickly as a whole.

Hereinafter, the scanning observation apparatus 1 according to the first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, the scanning observation apparatus 1 according to the present embodiment is a laser scanning fluorescence microscope apparatus, which includes a laser light source (first light source) 2 that emits laser light L ₁ , and a laser light source. A scanner (light scanning unit) 3 that two-dimensionally scans the laser beam L ₁ emitted from 2 and a pupil projection lens 4 that focuses the laser beam L ₁ scanned by the scanner 3 to form an intermediate image. When re-imaging the laser beam L ₁ forming the intermediate image and the imaging lens 5 to be converted to substantially condenses parallel light, laser light L _1, which is focused by said imaging lens 5 to the sample a Objective lens 6 to be operated.

  The scanner 3 is constituted by, for example, a so-called proximity galvanometer mirror in which galvanometer mirrors 3a and 3b provided so as to be able to swing around two axes arranged in directions orthogonal to each other are opposed to each other (see FIG. FIG. 1 schematically shows the galvanometer mirrors 3a and 3b. The objective lens 6 is, for example, a low-magnification objective lens 6 that requires a relatively large working distance from the sample A.

Further, scanning observation apparatus 1 according to this embodiment, occurs by a fluorescent substance contained in the specimen A is excited by the laser beam L _1, the objective lens 6, an imaging lens 5, the pupil projection lens 4 the light detecting a dichroic mirror 7 for splitting the fluorescence L ₂ back through the scanner 3 from the laser light L _1, a condenser lens 8 for converging the branched fluorescence L _2, the collected fluorescence L ₂ A detector (light detection unit) 9, a signal processing device 10 that processes an electric signal S ₁ generated by photoelectric conversion in the light detector 9 to form an image signal S ₂ , and a configured image signal S ₂ And a monitor 11 for display.

The photodetector 9 is, for example, a zero-dimensional photomultiplier tube (PMT). Accordingly, the photodetector 9 outputs a electrical signals S ₁ proportional to the amount of fluorescence L ₂ to be detected at each instant. The signal processing device 10 specifies the light quantity of the fluorescence L ₂ from the electrical signal S ₁ output from the photodetector 9, and based on the angle information of the galvanometer mirrors 3 a and 3 b sent from the scanner 3, The position of the fluorescence L _{2 on} the sample A is specified, and the fluorescence image signal S ₂ is constructed by combining these pieces of information.

Furthermore, scanning observation apparatus 1 according to this embodiment includes a visible light source 12 for generating visible light L _3, visible light L ₃ generated in a desired pattern visible light beam L ₄ of by passing the slit 13 In a state of being inserted on the optical axis C between the imaging lens 5 and the objective lens 6, the beam shaping means 14 for shaping, the collimating lens 15 for converting the shaped visible light beam L ₄ into parallel light, And a mirror 16 that deflects the visible light beam L ₄ made of parallel light and makes it incident on the objective lens 6 along the optical axis C of the objective lens 6.

The beam shaping means 14 includes, for example, a turret 18 including a plurality of slit plates 17 having slits 13 having a plurality of patterns P _{1 to} P ₃ as shown in FIGS. The motor 19 is made to rotate. By the fact that rotating the turret 18 by the operation of the motor 19 alternatively selects the pattern P ₁ to P ₃ in order to be able to shape the visible light beam L ₄ with a desired pattern P ₁ to P ₃ It has become.

For example, the pattern P ₁ in FIG. 2A is a cross-shaped pattern P ₁ composed of two intersecting straight lines, and is set so that the optical axis C of the objective lens 6 coincides with the intersection. , the worker performing the preparatory work, the intersection of the imaged visual beam L ₄ of the image on the sample a, so that it can be confirmed well easily and accurately to the optical axis C position of the objective lens 6 Yes. The pattern P ₂ of FIG. 2 (b) is a circular pattern P _2, by setting so that the optical axis C of the objective lens 6 to the center of the pattern P ₂ is matched, the worker , the center position of the formed image of the visible light beam L ₄ on the specimen a, so that the user can confirm that the optical axis C of the objective lens 6 is disposed.

Also, the pattern P ₃ in FIG. 2C is an arrow-shaped pattern P ₃ , and the arrow-shaped pattern P ₃ is set by setting the optical axis C of the objective lens 6 to coincide with the tip of the arrow. ₃ can indicate the position of the optical axis C of the objective lens 6.
Therefore, the operator can move the objective lens 6 and the sample A relative to each other so that the portion to be observed coincides with the position of the optical axis C of the objective lens 6 indicated by the visible light beam L ₄ of each pattern P _{1 to} P _3. By adjusting the positional relationship, the objective lens 6 can be aligned with the sample A simply and accurately.

  The mirror 16 is detachably provided on the optical axis C between the imaging lens 5 and the objective lens 6 as indicated by an arrow in FIG. When the visible light source 12 is operated to cause the visible light beam L4 to enter the sample A, it is inserted on the optical axis C between the imaging lens 5 and the objective lens 6 as shown by the solid line in FIG. When the light source 2 is operated to cause the laser light L1 to enter the sample A, it is removed from the optical axis C between the imaging lens 5 and the objective lens 6 as indicated by a chain line.

The operation of the scanning observation apparatus 1 according to the present embodiment configured as described above will be described below.
In order to observe the sample A using the scanning observation apparatus 1 according to the present embodiment, first, the objective lens 6 is aligned with the sample A in the preparation stage.

In this case, the visible light source 12 is operated with the laser light source 2 stopped, and the mirror 16 is inserted on the optical axis C between the imaging lens 5 and the objective lens 6. Further, the turret 18 is rotated by the operation of the motor 19 to select the slit plate 17 having the slits 13 of the desired patterns P _{1 to} P ₃ . Thereby, the visible light L ₃ emitted from the visible light source 12 is shaped into a visible light beam L ₄ having desired patterns P _{1 to} P ₃ by passing through the slit plate 17, and is collimated by the collimating lens 15. And enters the mirror 16. Since the mirror 16 is inserted on the optical axis C between the imaging lens 5 and the objective lens 6, the visible light beam L ₄ is deflected and incident on the objective lens 6. Is imaged on the surface.

For example, when the slit plate 17 having the slit 13 having the form shown in FIG. 2A is selected, a cross-shaped image B is formed on the surface of the sample A as shown in FIG. Imaged. The operator while watching the image B of the cross-shaped visible light beam L ₄ imaged on the surface of the specimen A, to move the entire scanning observation apparatus 1 including an objective lens 6 to the sample A, or, by moving the mounted stage of the sample a in the horizontal direction, part D to be observed on the sample a shown by the solid line, modulate to match the image B of the visible light beam L _4, as shown by the chain line be able to.

Since the intersection of two straight lines in the image B of the visible light beam L ₄ is adjusted so that the optical axis C of the objective lens 6 coincides, matches the site D to be observed the image B of the visible light beam L ₄ By doing so, the site D to be observed can be easily installed at the center of the observation range. Further, when the image B of the visible light beam L ₄ on the surface of the specimen A is unclear, since distance between the objective lens 6. and the sample A does not match the working distance, the objective lens to the sample A 6 Is moved in the direction of the optical axis C and set to a position where the image B of the visible light beam L ₄ is the clearest, the imaging position of the visible light beam L ₄ emitted from the objective lens 6 is set to the surface of the sample A. Can match.
Thereby, the optical axis C of the objective lens 6 can be made to coincide with the part D of the sample A to be observed, and the distance between the objective lens 6 and the sample A can be made to coincide with the working distance, and the preparation stage is completed.

Next, in order to observe the sample A, the operation of the visible light source 12 is stopped, and the mirror 16 is removed from between the imaging lens 5 and the objective lens 6. In this state, the laser light source 2, the scanner 3, and the photodetector 9 are operated to scan the sample A with the laser light L ₁ emitted from the laser light source 2. Thus, the fluorescence L ₂ is generated fluorescent substance is excited in the specimen A, fluorescence detected L ₂ generated is by the photodetector 9, the fluorescence image signal S ₂ is formed monitor 11 in the signal processing device 10 Will be displayed.

  According to the scanning observation apparatus 2 according to the present embodiment, the optical axis C of the objective lens 6 is accurately matched with the portion D to be observed of the sample A in the preparation stage. A fluorescent image to be observed can be acquired. Therefore, the time required for the entire observation process including from the preparation stage can be shortened. When the scanning observation apparatus 1 is a confocal microscope, the imaging position of the laser light L1 by the objective lens 6 is set to coincide with the surface of the sample A at the end of the preparation stage. By moving the objective lens 6 in the direction of the optical axis C with the position as a reference, a fluorescent image at a desired depth position can be acquired.

Further, according to the scanning observation apparatus 1 according to the present embodiment, the slit plate 17 having a plurality of patterns P _{1 to} P ₃ is attached to the turret 18, so that an image is formed on the surface of the sample A in the preparation stage. If the image B of the visible light beam L4 is not suitable for the sample A, the turret 18 can be rotated by the operation of the motor 19 to select the most suitable patterns P _{1 to} P _3. . For example, as a cross-shaped pattern P ₁ shown in FIG. 2 (a), the pattern P ₁ indicating the direct optical axis C position of the objective lens 6 by the intersection, the surface shape of the sample A, the intersection because if the location has a complex shape is disturbed statue B, as shown in FIG. 2 (b), a circular surrounding the observation position by the pattern P _2, indirectly the objective lens 6 It may be better to indicate the position of the optical axis C.

In the present embodiment, the sample A is irradiated as it is simply by shaping the visible light L ₃ from the visible light source 12, but instead, visible light L ₃ having a different wavelength can be emitted. A plurality of visible light sources 12 may be provided so as to be switched, and the visible light sources 12 may be switched according to the color of the surface of the sample A. By doing so, it highlights the image B of the visible light beam L _4, which is formed on the surface of the specimen A, it is possible to facilitate the positioning operation of the optical axis C of the objective lens 6. For example, when the surface of the sample A is red, if the red visible light beam L ₄ is irradiated, the image B of the visible light beam L ₄ formed on the surface of the sample A cannot be identified. It is preferable to switch to the visible light source 12 that emits visible light having a prominent wavelength.

Next, a scanning observation apparatus 20 according to a second embodiment of the present invention will be described below with reference to FIGS. 4 and 5.
In the description of the present embodiment, portions having the same configuration as those of the scanning observation apparatus 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.
The scanning observation apparatus 20 according to the present embodiment forms an image of a half mirror 21 as a deflection optical element, as shown in FIG. 4, instead of the mirror 16 of the scanning observation apparatus 1 according to the first embodiment. This is different from the first embodiment in that it is fixedly disposed between the lens 5 and the objective lens 6.

The operation of the scanning observation apparatus 20 according to the present embodiment configured as described above will be described below.
According to the scanning observation apparatus 20 according to the present embodiment, in the preparation stage, the laser light source 2 is stopped, the visible light source 12 is operated, and the visible light beam L4 shaped into the predetermined patterns P _{1 to} P ₃ is generated. When emitted, the visible light beam L 4 deflected in the direction of the optical axis C of the objective lens 6 by the half mirror 21 is imaged on the surface of the sample A by the objective lens 6. As in the first embodiment, the operator can adjust the optical axis C of the objective lens 6 so as to coincide with the portion D to be observed of the sample A while viewing the image B of the visible light beam L4 on the sample A.

According to the scanning observation apparatus 20 according to the present embodiment, an image is formed on the surface of the sample A by operating the scanner 3, the photodetector 9, the signal processing apparatus 10, and the monitor 11 in the preparation stage. The visible light beam L ₄ is reflected, returns to the objective lens 6, passes through the half mirror 21, and passes through the imaging lens 5, the pupil projection lens 4, the scanner 3, the dichroic mirror 7, and the condenser lens 8. The image of the visible light L ₃ detected by the signal processing device 10 and generated by the signal processing device 10 is displayed on the monitor 11.

That is, as shown by a solid line in FIG. 5, the image B on the surface of the sample A of the visible light beam L ₄ can be enlarged and observed on the monitor 11. At this time, when the image B on the surface of the sample A is deviated on the monitor 11 as indicated by a solid line in FIG. 5, the optical axis C of the objective lens 6 with respect to the scanning range by the scanner 3 It will be shifted. Therefore, by adjusting the swing angle range of each of the galvanometer mirrors 3a and 3b constituting the scanner 3, the position of the optical axis C of the objective lens 6 can be made substantially coincident with the center of the scanning range as shown by the chain line. it can. This completes the preparation stage.

Thereafter, the visible light source 12 is stopped and the laser light source 2 is operated, whereby the laser light L ₁ is scanned on the sample A, and a fluorescent image can be obtained. The obtained fluorescent image is a fluorescent image of the surface of the sample A corresponding to the scanning range with the optical axis C of the objective lens 6 as the center, and a desired fluorescent image arranged around the site D to be observed of the sample A Become.

Thus, according to the scanning examination apparatus 20 according to the present embodiment, matching in the preparation stage, the image B of the visible light beam L _4, the optical axis C position of the objective lens 6 in the region D to be observed in sample A In addition, the scanning range by the scanner 3 can be adjusted with respect to the position of the optical axis C of the objective lens 6, and a desired image including the part D to be observed in the observation stage can be quickly acquired.

  In each of the above embodiments, the laser light source 2 is employed as the first light source, but other arbitrary light sources may be used instead. That is, it may be an excitation light source that emits excitation light, or a light source that is a combination of a halogen lamp and an excitation light filter. Further, the present invention is not limited to a fluorescence observation apparatus that obtains fluorescence by irradiating excitation light.

Further, as the slit plate 17 of the beam shaping means 14, it has been illustrated as having a slit 13 of the pattern P ₁ to P ₃ shown in FIG. 2, instead of this, those having a slit 13 of any other pattern It may be adopted. In this case, any configuration may be used as long as it directly or indirectly indicates the position of the optical axis C of the objective lens 6. Further, the visible light beam L ₄ having predetermined patterns P _{1 to} P ₃ may be generated by a digital micromirror device (DMD) without being limited to the slit 13.

1 is an overall configuration diagram showing a scanning observation apparatus according to a first embodiment of the present invention. It is a figure which shows the example of a pattern of the visible light beam produced | generated by the beam shaping means of the scanning observation apparatus of FIG. FIG. 2 is a perspective view illustrating an example of an objective lens, a sample, and a visible light beam image formed on the sample of the scanning observation apparatus in FIG. 1. It is a whole block diagram which shows the scanning observation apparatus which concerns on the 2nd Embodiment of this invention. It is a figure which shows typically the image of the visible light beam displayed on the monitor of the scanning observation apparatus of FIG.

Explanation of symbols

A Sample C Optical axis L ₁ Laser light (light)
L ₂ fluorescence (return light)
L ₃ visible light P _{1 to} P ₃ patterns 1,20 Scanning observation apparatus 2 Laser light source (first light source)
3 Scanner (light scanning part)
6 Objective lens 9 Light detector (light detector)
12 Visible light source (second light source)
14 Beam shaping means (beam shaping part)
16 mirror (deflection optical element)
21 half mirror (deflection optical element)

Claims (5)

A first light source;
An optical scanning unit for scanning the sample with light from the first light source;
An objective lens that forms an image on the sample of the light scanned in the optical scanning unit;
A light detection unit for detecting return light emitted from the sample;
A second light source that emits visible light;
A deflecting optical element that is disposed between the optical scanning unit and the objective lens and that makes visible light emitted from the second light source incident on the objective lens along the optical axis of the objective lens;
Scanning observation provided with a beam shaping unit that forms visible light from a second light source irradiated onto the surface of the sample through the objective lens by the deflection optical element into a pattern capable of indicating the optical axis of the objective lens. apparatus.   The scanning observation apparatus according to claim 1, wherein the deflecting optical element includes a mirror and is detachably provided between the optical scanning unit and the objective lens.   The scanning observation apparatus according to claim 1, wherein the deflecting optical element is a half mirror.   The scanning observation apparatus according to any one of claims 1 to 3, wherein the beam shaping unit can selectively switch a pattern of visible light from the second light source.   The scanning observation apparatus according to any one of claims 1 to 4, wherein the second light source is wavelength-switchable.

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