JP2018028706A - Adjustment method for microscope, and microscope - Google Patents

Adjustment method for microscope, and microscope Download PDF

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JP2018028706A
JP2018028706A JP2017232184A JP2017232184A JP2018028706A JP 2018028706 A JP2018028706 A JP 2018028706A JP 2017232184 A JP2017232184 A JP 2017232184A JP 2017232184 A JP2017232184 A JP 2017232184A JP 2018028706 A JP2018028706 A JP 2018028706A
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light
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objective lens
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元夫 小山
Motoo Koyama
元夫 小山
大内　由美子
Yumiko Ouchi
由美子 大内
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Nikon Corp
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Abstract

PROBLEM TO BE SOLVED: To provide an adjustment method for a microscope capable of highly accurately collimating illumination light emitted to a specimen via an objective lens, and to provide a microscope capable of conducting the adjustment method.SOLUTION: In an adjustment method for a total reflection fluorescence microscope 10 having an illuminating optical system 11 that relays light from a light source 1 by a relay optical system 2 and forms a conjugate image of the light source 1 near an incidence pupil face P of an objective lens 5, thereby making the light substantially parallel light by the objective lens 5 to be emitted to a specimen, displacement of a position of the conjugate image of the light source 1 is detected with respect to a position of the incidence pupil face P of the objective lens 5, and this displacement is reduced, thereby making the light emitted to the specimen closer to parallel light.SELECTED DRAWING: Figure 2

Description

本発明は、顕微鏡の調整方法及び顕微鏡に関する。   The present invention relates to a method for adjusting a microscope and a microscope.

図５に示すように、顕微鏡の一種である全反射蛍光顕微鏡１０は、照明光学系の光軸から偏心した位置Ａに配置された光源１から放射された照明光ＬＩを、対物レンズ５を介して略平行光束にした状態で試料台（カバーガラス６）とこのカバーガラス６上に載置された試料（図示せず）との境界面に照射して全反射させ、この全反射面の裏側に染み出す照明光（エバネッセント光）により試料を励起し、この励起により発生する蛍光ＬＯを観察するように構成されている（例えば、特許文献１参照）。   As shown in FIG. 5, a total reflection fluorescent microscope 10 which is a kind of microscope uses illumination light LI emitted from a light source 1 disposed at a position A decentered from the optical axis of an illumination optical system via an objective lens 5. In a state where the light beam is substantially parallel, the boundary surface between the sample stage (cover glass 6) and the sample (not shown) placed on the cover glass 6 is irradiated and totally reflected, and the back side of the total reflection surface The sample is excited by illumination light (evanescent light) that oozes out, and fluorescence LO generated by this excitation is observed (for example, see Patent Document 1).

このような全反射蛍光顕微鏡１０において、光源１の光軸とこの全反射蛍光顕微鏡１０の照明光学系の光軸とが一致するように光源１を配置すると（図５の位置Ｂ）、対物レンズ５を通過した照明光ＬＩ′は光軸上を進み、図５に示すように、十分な遠方に配置されたスクリーン５０にスポット像として投射される。ここで、例えばリレー光学系２を構成するレンズの一部を光軸方向に移動させてスクリーン５０上のスポット像の径が所定の値以下になる（最も小さくなる）ように調整を行うと、結果としてリレー光学系２により対物レンズ５の入射瞳面Ｐの近傍に集光された光源１の共役像が光軸に沿って移動してこの入射瞳面Ｐと略一致することになり、これにより、試料とカバーガラス６の境界面に照射される照明光ＬＩ′をコリメートすることができる。   In such a total reflection fluorescence microscope 10, when the light source 1 is arranged so that the optical axis of the light source 1 and the optical axis of the illumination optical system of the total reflection fluorescence microscope 10 coincide (position B in FIG. 5), the objective lens The illumination light LI ′ that has passed through 5 travels on the optical axis, and is projected as a spot image on a screen 50 disposed sufficiently far away, as shown in FIG. Here, for example, when a part of the lenses constituting the relay optical system 2 is moved in the optical axis direction so that the diameter of the spot image on the screen 50 is adjusted to be a predetermined value or less (smallest), As a result, the conjugate image of the light source 1 condensed in the vicinity of the entrance pupil plane P of the objective lens 5 by the relay optical system 2 moves along the optical axis and substantially coincides with the entrance pupil plane P. Thus, the illumination light LI ′ irradiated on the interface between the sample and the cover glass 6 can be collimated.

特開２００６−１８９７４１号公報JP 2006-189741 A

しかしながら、上述したような調整方法では、スクリーン５０上のスポット像の径を目視して調整するため、照明光のコリメート精度に限界があるという課題があった。また、照明光として紫外光や赤外光を用いる場合には、目視で確認することができず、この調整方法を用いることはできない。   However, in the adjustment method as described above, since the diameter of the spot image on the screen 50 is visually adjusted, there is a problem that the collimation accuracy of the illumination light is limited. Further, when ultraviolet light or infrared light is used as illumination light, it cannot be visually confirmed, and this adjustment method cannot be used.

本発明はこのような課題に鑑みてなされたものであり、対物レンズを介して試料に照射される照明光を高精度にコリメートすることができる顕微鏡の調整方法、及び、この調整方法を実行可能な顕微鏡を提供することを目的とする。   The present invention has been made in view of such a problem, and a microscope adjustment method capable of collimating illumination light irradiated onto a sample through an objective lens with high accuracy, and the adjustment method can be executed. Is to provide a simple microscope.

前記課題を解決するために、本発明に係る顕微鏡の調整方法は、明細書に記載の発明である。   In order to solve the above-described problems, a microscope adjustment method according to the present invention is an invention described in the specification.

本発明によると、高精度にコリメートすることができる顕微鏡の調整方法、及び、この調整方法を実行可能な顕微鏡を提供することができる。   ADVANTAGE OF THE INVENTION According to this invention, the microscope adjustment method which can collimate with high precision and the microscope which can perform this adjustment method can be provided.

全反射蛍光顕微鏡の構成を示す説明図である。It is explanatory drawing which shows the structure of a total reflection fluorescence microscope. 第１の実施形態に係る調整装置が取り付けられた全反射蛍光顕微鏡の構成を示す説明図である。It is explanatory drawing which shows the structure of the total reflection fluorescence microscope to which the adjustment apparatus which concerns on 1st Embodiment was attached. 第１の実施形態の変形例の構成を示す説明図である。It is explanatory drawing which shows the structure of the modification of 1st Embodiment. 第２の実施形態に係る調整装置が取り付けられた全反射蛍光顕微鏡の構成を示す説明図である。It is explanatory drawing which shows the structure of the total reflection fluorescence microscope to which the adjustment apparatus which concerns on 2nd Embodiment was attached. 全反射蛍光顕微鏡の従来の調整方法を示す説明図である。It is explanatory drawing which shows the conventional adjustment method of a total reflection fluorescence microscope.

以下、本発明の好ましい実施形態について図面を参照して説明する。まず、図１を用いて顕微鏡の一種である全反射蛍光顕微鏡１０の構成について説明する。この全反射蛍光顕微鏡１０は、光源１から放射された照明光ＬＩを試料台（以下「カバーガラス６」と呼ぶ）上に載置された試料Ｏに照射する照明光学系１１と、この照明光ＬＩにより励起された試料Ｏから発生する蛍光ＬＯをＣＣＤ等からなる撮像素子８の撮像面上に集光する結像光学系１２と、から構成される。なお、この図１における光源１は、別の光源装置から放射された照明光をこの照明光学系１１に導く光ファイバーの端面でも良い。   Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of a total reflection fluorescence microscope 10 which is a kind of microscope will be described with reference to FIG. The total reflection fluorescence microscope 10 includes an illumination optical system 11 that irradiates a sample O placed on a sample stage (hereinafter referred to as “cover glass 6”) with illumination light LI emitted from the light source 1, and the illumination light. An imaging optical system 12 that condenses the fluorescence LO generated from the sample O excited by LI onto the imaging surface of the imaging device 8 made of a CCD or the like. The light source 1 in FIG. 1 may be an end face of an optical fiber that guides illumination light emitted from another light source device to the illumination optical system 11.

結像光学系１２は、カバーガラス６側から順に、対物レンズ５と、ダイクロイックミラー４と、第２対物レンズ７と、を有し、この第２対物レンズ７の像側の焦点面が撮像素子８の撮像面と略一致するように配置されている。なお、この全反射蛍光顕微鏡１０において、試料Ｏはカバーガラス６の対物レンズ５と反対側の面に載置される。なお、対物レンズ５の物体側の面とカバーガラス６の対物レンズ５側の面との間にはこのカバーガラス６とほぼ同じ屈折力を有するオイルＩＯが満たされている。   The imaging optical system 12 includes an objective lens 5, a dichroic mirror 4, and a second objective lens 7 in order from the cover glass 6 side, and the focal plane on the image side of the second objective lens 7 is an image sensor. 8 are arranged so as to substantially coincide with the image pickup surface 8. In this total reflection fluorescence microscope 10, the sample O is placed on the surface of the cover glass 6 opposite to the objective lens 5. Note that oil IO having substantially the same refractive power as that of the cover glass 6 is filled between the object side surface of the objective lens 5 and the surface of the cover glass 6 on the objective lens 5 side.

また、照明光学系１１は、ダイクロイックミラー４の側方に配置され、光源１側から順に、リレー光学系２と、遮光部材３とから構成されている。なお、照明光学系１１は、ダイクロイックミラー４及び対物レンズ５を結像光学系１２と共用している。このような照明光学系１１において、リレー光学系２は光源１の像を対物レンズ５の入射瞳面Ｐ若しくはその近傍に形成するために、その光源１側の焦点面はこの光源１（光ファイバーの場合はその射出端）と略一致するように配置されている。また、遮光部材３は、対物レンズ５の入射瞳面Ｐの近傍でこの照明光学系１１の光路の断面の略半分を遮光するように配置されている。さらに、ダイクロイックミラー４は、光源１からの照明光ＬＩを対物レンズ５側に反射して結像光学系１２の光路に導き、また、試料Ｏから射出する蛍光ＬＯを透過させて第２対物レンズ７に導くように構成されている。   The illumination optical system 11 is disposed on the side of the dichroic mirror 4 and includes a relay optical system 2 and a light shielding member 3 in order from the light source 1 side. The illumination optical system 11 shares the dichroic mirror 4 and the objective lens 5 with the imaging optical system 12. In such an illumination optical system 11, the relay optical system 2 forms an image of the light source 1 on or near the entrance pupil plane P of the objective lens 5, so that the focal plane on the light source 1 side is the light source 1 (optical fiber). In this case, it is arranged so as to substantially coincide with the injection end). The light shielding member 3 is disposed in the vicinity of the entrance pupil plane P of the objective lens 5 so as to shield substantially half of the cross section of the optical path of the illumination optical system 11. Further, the dichroic mirror 4 reflects the illumination light LI from the light source 1 toward the objective lens 5 and guides it to the optical path of the imaging optical system 12, and transmits the fluorescence LO emitted from the sample O to transmit the second objective lens. 7 to lead to 7.

この全反射蛍光顕微鏡１０において、光源１を、照明光学系１１の光軸と直交する面内で移動させて所望量光軸から離した（偏心させた）状態で照明光ＬＩを放射させると、リレー光学系２でリレーされて対物レンズ５の入射瞳面Ｐ若しくはその近傍の光軸から離れた位置に光源１の像を形成し、さらに、この照明光ＬＩはダイクロイックミラー４に入射して対物レンズ５側に反射する。そして、この照明光ＬＩは、対物レンズ５によりコリメートされて略平行光束となりカバーガラス６に照射される。このとき、光源１が偏心して配置されていることから、この照明光ＬＩはカバーガラス６に対して所定の入射角を有して斜めに照射されるが、この入射角が試料Ｏとカバーガラス６との境界面６ａの臨界角を超えているときは、この境界面６ａで全反射し、対物レンズ５で集光されてダイクロイックミラー４で反射され、対物レンズ５の入射瞳面Ｐに再び集光される。そして、この位置に上述した遮光部材３を配置することにより、全反射した照明光ＬＩが結像光学系１２に入射して迷光になることを防止することができる。   In the total reflection fluorescent microscope 10, when the light source 1 is moved in a plane orthogonal to the optical axis of the illumination optical system 11 and is separated from the optical axis by a desired amount (decentered), the illumination light LI is emitted. An image of the light source 1 is formed at a position separated from the entrance pupil plane P of the objective lens 5 or the optical axis in the vicinity thereof by being relayed by the relay optical system 2, and the illumination light LI is incident on the dichroic mirror 4 to enter the objective. Reflected to the lens 5 side. The illumination light LI is collimated by the objective lens 5 to become a substantially parallel light beam and is irradiated onto the cover glass 6. At this time, since the light source 1 is arranged eccentrically, the illumination light LI is irradiated obliquely with a predetermined incident angle with respect to the cover glass 6. 6 exceeds the critical angle of the boundary surface 6a, the light is totally reflected by the boundary surface 6a, collected by the objective lens 5, reflected by the dichroic mirror 4, and again on the entrance pupil plane P of the objective lens 5. Focused. By disposing the above-described light shielding member 3 at this position, it is possible to prevent the totally reflected illumination light LI from entering the imaging optical system 12 and becoming stray light.

照明光ＬＩが全反射するカバーガラス６と試料Ｏとの境界面６ａでは、試料Ｏ側に光（エバネッセント光）が染み出しエバネッセン場を形成し、試料Ｏ側の厚さ数百ナノメートルの範囲が照明される。このエバネッセント光により励起された試料Ｏからは蛍光ＬＯが発生する。この位置に対物レンズ５の物体側の焦点面が位置するように調整すると、この蛍光ＬＯは対物レンズ５で集光されて略平行光束となってダイクロイックミラー４を通過し、第２対物レンズ７により撮像素子８の撮像面に集光され、蛍光ＬＯによる試料Ｏの像が形成される。このように、この全反射蛍光顕微鏡１０によると、背景に光ノイズの少ない非常に暗い状態で試料Ｏを励起することができるので、コントラストの高い像を取得することができる。   At the boundary surface 6a between the cover glass 6 and the sample O where the illumination light LI is totally reflected, light (evanescent light) oozes out on the sample O side to form an evanescent field, and the thickness on the sample O side is in the range of several hundred nanometers. Is illuminated. Fluorescence LO is generated from the sample O excited by the evanescent light. When adjustment is made so that the object-side focal plane of the objective lens 5 is positioned at this position, the fluorescence LO is condensed by the objective lens 5 to pass through the dichroic mirror 4 as a substantially parallel light beam, and the second objective lens 7. As a result, the light is condensed on the image pickup surface of the image pickup device 8, and an image of the sample O is formed by the fluorescence LO. As described above, according to the total reflection fluorescence microscope 10, the sample O can be excited in a very dark state with little optical noise in the background, so that an image with high contrast can be acquired.

このような全反射蛍光顕微鏡１０において、上述した境界面６ａで染み出すエバネッセント光の量は、この境界面６ａに入射する照明光ＬＩの入射角に依存する。そのため、境界面６ａに入射する照明光ＬＩを平行光に近づける必要がある。この全反射蛍光顕微鏡１０には、光源１若しくはリレー光学系２を構成するレンズの少なくとも一部を光軸方向に移動させて境界面６ａに照射される照明光ＬＩを平行光に近づけるための調整を行う調整部１３が設けられている。それでは、上述した構成の全反射蛍光顕微鏡１０において、対物レンズ５で集光されて境界面６ａに照射される照明光ＬＩを略平行光とするための調整方法について説明する。   In such a total reflection fluorescent microscope 10, the amount of evanescent light that permeates at the boundary surface 6a described above depends on the incident angle of the illumination light LI incident on the boundary surface 6a. Therefore, it is necessary to make the illumination light LI incident on the boundary surface 6a close to parallel light. The total reflection fluorescent microscope 10 includes an adjustment for moving the illumination light LI irradiated to the boundary surface 6a closer to parallel light by moving at least a part of the lens constituting the light source 1 or the relay optical system 2 in the optical axis direction. An adjustment unit 13 for performing the above is provided. Now, an adjustment method for making the illumination light LI condensed by the objective lens 5 and applied to the boundary surface 6a in the total reflection fluorescent microscope 10 having the above-described configuration to be substantially parallel light will be described.

［第１の実施形態］
図２は、全反射蛍光顕微鏡１０に第１の実施形態に係る調整装置２０を取り付けて調整を行う構成を示している。この調整装置２０は、図２に示すように、カバーガラス６を挟んで対物レンズ５の反対側に、この全反射蛍光顕微鏡１０の筐体に設けられた取付部９を介して取り付けるように構成されており、カバーガラス６側から順に、正の屈折力を有する集光光学系２１と、この集光光学系２１の焦点面に位置するように配置された検出器２２と、を有している。 [First Embodiment]
FIG. 2 shows a configuration in which adjustment is performed by attaching the adjusting device 20 according to the first embodiment to the total reflection fluorescent microscope 10. As shown in FIG. 2, the adjustment device 20 is configured to be attached to the opposite side of the objective lens 5 with the cover glass 6 interposed therebetween via an attachment portion 9 provided in the casing of the total reflection fluorescence microscope 10. In order from the cover glass 6 side, a condensing optical system 21 having a positive refractive power and a detector 22 arranged so as to be positioned on the focal plane of the condensing optical system 21 are provided. Yes.

上述したように、光源１の光軸とこの全反射蛍光顕微鏡１０の照明光学系１１の光軸とが略一致するように光源１を配置すると（図２の位置Ｂ）、対物レンズ５を通過した照明光は光軸上を進む。このとき、リレー光学系２により形成された光源１の像が対物レンズ５の入射瞳面Ｐと略一致していると、対物レンズ５から出射した照明光ＬＩ′は略平行光束となる。調整装置２０の検出器２２は集光光学系２１の焦点面と略一致するように配置されているため、対物レンズ５を出射した照明光ＬＩ′が略平行光束であれば、検出器２２で検出される照明光ＬＩ′のスポット像の径は所定の値以下になる（最も小さくなる）。すなわち、調整装置２０の検出器２２で検出されるスポット像の径が所定の値以下になる（最も小さくなる）ように、調整部１３により、リレー光学系２を構成するレンズの少なくとも一部を光軸に沿って移動させることにより、境界面６ａに照射される照明光ＬＩ′を略平行光束とすることができる。あるいは、調整部１３により、光源１の位置を照明光学系１１の光軸方向に移動させても良い。   As described above, when the light source 1 is arranged so that the optical axis of the light source 1 and the optical axis of the illumination optical system 11 of the total reflection fluorescence microscope 10 substantially coincide with each other (position B in FIG. 2), it passes through the objective lens 5. The illuminated light travels on the optical axis. At this time, if the image of the light source 1 formed by the relay optical system 2 substantially coincides with the entrance pupil plane P of the objective lens 5, the illumination light LI 'emitted from the objective lens 5 becomes a substantially parallel light beam. Since the detector 22 of the adjustment device 20 is arranged so as to substantially coincide with the focal plane of the condensing optical system 21, if the illumination light LI 'emitted from the objective lens 5 is a substantially parallel light beam, the detector 22 The diameter of the spot image of the detected illumination light LI ′ is less than a predetermined value (smallest). That is, at least a part of the lenses constituting the relay optical system 2 is adjusted by the adjusting unit 13 so that the diameter of the spot image detected by the detector 22 of the adjusting device 20 is equal to or smaller than a predetermined value (smallest). By moving along the optical axis, the illumination light LI ′ irradiated on the boundary surface 6a can be made into a substantially parallel light beam. Alternatively, the position of the light source 1 may be moved in the optical axis direction of the illumination optical system 11 by the adjusting unit 13.

なお、光源１は不図示のレーザダイオード（ＬＤ）とファイバーから構成され、点光源を形成するためにはシングルモードファイバーを用いることが好ましい。また、レーザ光を出射する光源であれば、ＬＤ以外でも使用可能である。レーザ波長としては、例えば、可視域では、４８８ｎｍ、５６１ｎｍ等が用いられ、赤外域では、７３０ｎｍ、７８５ｎｍが用いられ、紫外域では、３７５ｎｍ、４０５ｎｍが用いられる。   The light source 1 is composed of a laser diode (LD) (not shown) and a fiber, and it is preferable to use a single mode fiber in order to form a point light source. Further, any light source that emits laser light can be used other than the LD. As the laser wavelength, for example, 488 nm and 561 nm are used in the visible region, 730 nm and 785 nm are used in the infrared region, and 375 nm and 405 nm are used in the ultraviolet region.

ここで、調整装置２０を構成する集光光学系２１は、図２に示すように、少なくとも１枚の正の屈折力を有するレンズと、少なくとも１枚の負の屈折力を有するレンズとを有することが好ましい。このような構成とすることで、この集光光学系２１を、その全長に対して焦点距離を長くすることが可能となり、集光光学系２１から検出器２２までの機械的な間隔が多少の誤差を有していたとしても、照明光ＬＩ′の光束の集光点を検出器２２上に作ることで、十分な精度でコリメート状態にすることができる。なお、この集光光学系２１の全系の焦点距離は対物レンズ５の焦点距離の２倍以上であれば良い。   Here, as shown in FIG. 2, the condensing optical system 21 constituting the adjusting device 20 includes at least one lens having a positive refractive power and at least one lens having a negative refractive power. It is preferable. With such a configuration, it becomes possible to increase the focal length of the condensing optical system 21 with respect to the entire length thereof, and the mechanical distance from the condensing optical system 21 to the detector 22 is slightly increased. Even if there is an error, a collimating state can be achieved with sufficient accuracy by forming a condensing point of the light beam of the illumination light LI ′ on the detector 22. Note that the focal length of the entire system of the condensing optical system 21 may be at least twice the focal length of the objective lens 5.

以上のように、この第１の実施形態に係る調整方法によると、集光光学系２１と検出器２２とを有するコンパクトで簡単な構成の調整装置２０を、全反射蛍光顕微鏡１０に取り付け、検出器２２で検出される照明光ＬＩ′のスポット像の径が所定の値以下になる（最も小さくなる）ように調整部１３によりリレー光学系２の位置（又は光源１の位置）を調整するという簡単な操作で精度良く、境界面６ａに照射される照明光ＬＩ，ＬＩ′を略平行光束とすることができる。この場合、光源１から放射される照明光ＬＩ，ＬＩ′が紫外光や赤外光のような不可視光であっても、検出器２２の検出範囲の波長であれば調整を行うことができる。なお、この調整装置２０は、上述したように全反射蛍光顕微鏡１０の筐体に対して着脱可能であることが望ましい。あるいは、この全反射蛍光顕微鏡１０の筐体内に取り付けられ、照明光学系１１の光路上から、少なくとも集光光学系２１を挿脱可能に構成しても良い。   As described above, according to the adjustment method according to the first embodiment, the adjustment device 20 having a compact and simple configuration including the condensing optical system 21 and the detector 22 is attached to the total reflection fluorescence microscope 10 and detected. The position of the relay optical system 2 (or the position of the light source 1) is adjusted by the adjusting unit 13 so that the diameter of the spot image of the illumination light LI ′ detected by the device 22 is not more than a predetermined value (smallest). Illumination light LI, LI 'irradiated to the boundary surface 6a can be made into a substantially parallel light beam with high accuracy by a simple operation. In this case, even if the illumination light LI, LI ′ emitted from the light source 1 is invisible light such as ultraviolet light or infrared light, adjustment can be performed as long as the wavelength is within the detection range of the detector 22. In addition, as for this adjustment apparatus 20, it is desirable to be detachable with respect to the housing | casing of the total reflection fluorescence microscope 10, as mentioned above. Alternatively, it may be configured to be mounted in the casing of the total reflection fluorescence microscope 10 so that at least the condensing optical system 21 can be inserted and removed from the optical path of the illumination optical system 11.

なお、この全反射蛍光顕微鏡１０においては、上述した調整が終了したときは、図１に示すように、光源１を全反射蛍光顕微鏡１０の光軸に直交する面内でこの光軸から離すように移動させることで（図２の位置Ａ）、境界面６ａに対して照明光ＬＩを斜めに（全反射する角度で）照射することができる。   In the total reflection fluorescent microscope 10, when the above-described adjustment is completed, the light source 1 is separated from the optical axis in a plane orthogonal to the optical axis of the total reflection fluorescent microscope 10, as shown in FIG. 2 (position A in FIG. 2), the illumination light LI can be irradiated obliquely (at an angle for total reflection) to the boundary surface 6a.

［第１の実施形態の変形例］
図３に示すように、カバーガラス６を挟んだ対物レンズ５の反対側に、カバーガラス６に載置された試料を透過照明するための照明装置６０が設けられている場合には、この照明装置６０に上述した検出器２２を装着することにより、同様の方法で照明光の調整を行うことができる。この図３に示す照明装置６０は、透過照明用の光源６１を有し、この光源６１側から順に、集光レンズ６２と、この集光レンズ６２の焦点面と略一致するように配置された開口絞り６３と、開口絞り６３を通過した照明光を略平行光にするコンデンサレンズ６４と、コンデンサレンズ６４を出射した略平行光をカバーガラス６上の試料に照射するミラー６５と、を有して構成されている。そこで、コンデンサレンズ６４の入射瞳の位置に相当する開口絞り６３の付近の筐体に取付部９′を設け、この取付部９′を介して上述した検出器２２と同じ検出器２２′を有する調整装置２０′を装着することにより、照明光ＬＩ′のスポット像を検出することができる。なお、検出器２２′を配置する代わりに、この位置に、照明光ＬＩ′の像を投影するスクリーンを配置し、このスクリーンに投影されたスポット像をＣＣＤ等を有する撮像装置で撮像して、そのスポット像の径を検出するように構成することも可能である。 [Modification of First Embodiment]
As shown in FIG. 3, when an illuminating device 60 for transmitting and illuminating a sample placed on the cover glass 6 is provided on the opposite side of the objective lens 5 sandwiching the cover glass 6, this illumination is provided. By mounting the above-described detector 22 on the device 60, the illumination light can be adjusted in the same manner. The illuminating device 60 shown in FIG. 3 has a light source 61 for transmitted illumination, and is arranged so as to substantially coincide with the condenser lens 62 and the focal plane of the condenser lens 62 in this order from the light source 61 side. An aperture stop 63; a condenser lens 64 that converts the illumination light that has passed through the aperture stop 63 into substantially parallel light; and a mirror 65 that irradiates the sample on the cover glass 6 with substantially parallel light emitted from the condenser lens 64. Configured. Therefore, a mounting portion 9 ′ is provided in the casing near the aperture stop 63 corresponding to the position of the entrance pupil of the condenser lens 64, and the same detector 22 ′ as the detector 22 described above is provided via this mounting portion 9 ′. A spot image of the illumination light LI ′ can be detected by mounting the adjusting device 20 ′. Instead of arranging the detector 22 ′, a screen for projecting the image of the illumination light LI ′ is arranged at this position, and the spot image projected on the screen is imaged by an imaging device having a CCD or the like, It is also possible to configure to detect the diameter of the spot image.

［第２の実施形態］
図４は、全反射蛍光顕微鏡１０に第２の実施形態に係る調整装置３０を取り付けて調整を行う構成を示している。この調整装置３０は、図４に示すように、リレー光学系２の中に配置されるビームスプリッタ３１と、このビームスプリッタ３１で反射された光を集光する集光レンズ３２と、集光レンズ３２で集光された光を検出する検出器３３と、から構成されている。ここで、検出器３３は、対物レンズ５の入射瞳面Ｐと共役な位置に配置されている。なお、第１の実施形態で用いた遮光部材３は、この第２の実施形態では設けなくても良い。 [Second Embodiment]
FIG. 4 shows a configuration in which adjustment is performed by attaching the adjustment device 30 according to the second embodiment to the total reflection fluorescent microscope 10. As shown in FIG. 4, the adjusting device 30 includes a beam splitter 31 arranged in the relay optical system 2, a condenser lens 32 that condenses the light reflected by the beam splitter 31, and a condenser lens. And a detector 33 for detecting the light condensed at 32. Here, the detector 33 is arranged at a position conjugate with the entrance pupil plane P of the objective lens 5. Note that the light shielding member 3 used in the first embodiment may not be provided in the second embodiment.

このような状態で光源１から照明光ＬＩが放射されると、この照明光ＬＩはリレー光学系２に入射し、一部の光がビームスプリッタ３１を透過し、このリレー光学系２でリレーされて対物レンズ５の瞳面Ｐ若しくはその近傍の光軸から離れた位置に光源１の像を形成する。さらに、この照明光ＬＩはダイクロイックミラー４に入射して対物レンズ５側に反射し、対物レンズ５を介してカバーガラス６に照射される。そして、カバーガラス６を透過した照明光ＬＩはカバーガラス６の対物レンズ５と反対側の面（すなわち、上述した境界面６ａ）で全反射し、対物レンズ５で集光されてダイクロイックミラー４で反射され、対物レンズ５の瞳面Ｐに再び集光される。この第２の実施形態においては、照明光学系１１に遮光部材３が設けられていないため、入射瞳面Ｐを透過した照明光ＬＩは、リレー光学系２に入射し、一部の光がビームスプリッタ３１で反射して集光レンズ３２により検出器３３に集光される。   When the illumination light LI is emitted from the light source 1 in such a state, the illumination light LI enters the relay optical system 2, and a part of the light passes through the beam splitter 31 and is relayed by the relay optical system 2. Thus, an image of the light source 1 is formed at a position away from the pupil plane P of the objective lens 5 or the optical axis in the vicinity thereof. Further, the illumination light LI is incident on the dichroic mirror 4, is reflected toward the objective lens 5, and is irradiated onto the cover glass 6 through the objective lens 5. The illumination light LI transmitted through the cover glass 6 is totally reflected by the surface of the cover glass 6 opposite to the objective lens 5 (that is, the boundary surface 6 a described above), and is condensed by the objective lens 5 and is collected by the dichroic mirror 4. It is reflected and condensed again on the pupil plane P of the objective lens 5. In the second embodiment, since the illumination optical system 11 is not provided with the light blocking member 3, the illumination light LI transmitted through the entrance pupil plane P is incident on the relay optical system 2 and a part of the light is beamed. The light is reflected by the splitter 31 and condensed on the detector 33 by the condenser lens 32.

上述したように対物レンズ５の入射瞳面Ｐと検出器３３とは共役な位置に配置されているため、対物レンズ５を出射した照明光ＬＩが略平行光束であれば、照明光ＬＩのスポット像は検出器３３で検出される。すなわち、調整装置３０の検出器３３でスポット像が検出されるように、調整部１３により、リレー光学系２を構成するレンズのうち、ビームスプリッタ３１よりも光源１側のレンズを光軸に沿って移動させることにより、境界面６ａ（この境界面６ａに配置されたミラー３４）に照射される照明光ＬＩを略平行光束とすることができる。調整部１３により、リレー光学系２を構成するレンズのうち、ビームスプリッタ３１よりも対物レンズ５側のレンズを光軸に沿って移動させて調整することも可能であるが、上述したように、検出器３３は対物レンズ５の入射瞳面ＰＩと共役な位置に配置されているため、この関係に影響を与えない、ビームスプリッタ３１よりも光源１側のレンズを移動させる方が望ましい。また、調整部１３により、光源１を光軸方向に移動させて調整しても良い。   As described above, since the entrance pupil plane P of the objective lens 5 and the detector 33 are arranged in a conjugate position, if the illumination light LI emitted from the objective lens 5 is a substantially parallel light beam, the spot of the illumination light LI. The image is detected by the detector 33. That is, among the lenses constituting the relay optical system 2, the lens closer to the light source 1 than the beam splitter 31 is aligned along the optical axis by the adjustment unit 13 so that the spot image is detected by the detector 33 of the adjustment device 30. The illumination light LI irradiated to the boundary surface 6a (the mirror 34 disposed on the boundary surface 6a) can be made into a substantially parallel light beam. The adjustment unit 13 can adjust the lens constituting the relay optical system 2 by moving the lens closer to the objective lens 5 than the beam splitter 31 along the optical axis. Since the detector 33 is arranged at a position conjugate with the entrance pupil plane PI of the objective lens 5, it is desirable to move the lens on the light source 1 side rather than the beam splitter 31 without affecting this relationship. The adjustment unit 13 may adjust the light source 1 by moving it in the optical axis direction.

このように、この第２の実施形態に係る調整方法によると、ビームスプリッタ３１、集光レンズ３２及び検出器３３を有するコンパクトで簡単な構成の調整装置３０を、全反射蛍光顕微鏡１０に設け、検出器３３により検出される照明光ＬＩのスポット像の径が所定の値以下になる（最も小さくなる）ように調整部１３によりリレー光学系２の位置を調整するという簡単な操作で精度良く、境界面６ａに照射される照明光ＬＩを略平行光束とすることができる。第１の実施形態で説明した調整方法は、光源１を一旦照明光学系１１の光軸上に移動して調整を行い（図１の位置Ｂ）、その後、境界面６ａで照明光ＬＩが全反射する位置（図１の位置Ａ）に移動させていたが、この第２の実施形態に示す調整方法では、実際に観察を行う位置に光源１を配置して調整を行っているので、調整の前後で光源１の移動を行う必要がなく、光源１の移動による誤差の発生を防ぐことができる。   Thus, according to the adjustment method according to the second embodiment, the adjustment apparatus 30 having a compact and simple configuration including the beam splitter 31, the condenser lens 32, and the detector 33 is provided in the total reflection fluorescence microscope 10, With the simple operation of adjusting the position of the relay optical system 2 by the adjusting unit 13 so that the diameter of the spot image of the illumination light LI detected by the detector 33 is less than or equal to a predetermined value (smallest), the operation is accurate. The illumination light LI irradiated on the boundary surface 6a can be a substantially parallel light beam. In the adjustment method described in the first embodiment, adjustment is performed by moving the light source 1 once onto the optical axis of the illumination optical system 11 (position B in FIG. 1), and thereafter, the illumination light LI is completely emitted from the boundary surface 6a. Although it has been moved to the reflecting position (position A in FIG. 1), in the adjustment method shown in the second embodiment, since the light source 1 is arranged at the position where observation is actually performed, adjustment is performed. Therefore, it is not necessary to move the light source 1 before and after the above, and it is possible to prevent an error due to the movement of the light source 1.

なお、カバーガラス６に対する照明光ＬＩの入射角がこのカバーガラス６上に試料を載置しないと全反射しない角度の場合は、図４に示すように、カバーガラス６の上面（境界面６ａ）にミラー３４を取り付けても良い。   In addition, when the incident angle of the illumination light LI with respect to the cover glass 6 is an angle that does not totally reflect unless a sample is placed on the cover glass 6, as shown in FIG. 4, the upper surface (boundary surface 6a) of the cover glass 6 A mirror 34 may be attached to the mirror.

また、この第２の実施形態に係る調整装置３０は、全反射蛍光顕微鏡３０の筐体に図示しない取付部を介して着脱可能に構成しても良いし、ビームスプリッタ３１のみを照明光学系１１の光路上に挿脱可能に構成しても良い（調整時は、ビームスプリッタ３１を光路に挿入し、調整後は、ビームスプリッタ３１を光路から脱却させる）。しかし、照明光ＬＩのコリメートの調整にために検出器３３で検出する光量は小さくて良いため、ビームスプリッタ３１の透過率を大きくする（反射率を小さくする）ことにより、調整装置３０は、この全反射蛍光顕微鏡３０に固定して配置することができる。このとき、ビームスプリッタ３１は、リレー光学系２内であればどこに配置しても良いが、このリレー光学系２内の光束が略平行になる部分に配置する方が、収差等が発生が少なくなり好ましい。   In addition, the adjusting device 30 according to the second embodiment may be configured to be detachable from the housing of the total reflection fluorescent microscope 30 via a mounting portion (not shown), or only the beam splitter 31 is provided in the illumination optical system 11. The beam splitter 31 may be inserted into the optical path during adjustment (the beam splitter 31 is inserted into the optical path during adjustment, and the beam splitter 31 is removed from the optical path after adjustment). However, since the amount of light detected by the detector 33 may be small in order to adjust the collimation of the illumination light LI, the adjustment device 30 can be adjusted by increasing the transmittance of the beam splitter 31 (decreasing the reflectance). The total reflection fluorescent microscope 30 can be fixedly disposed. At this time, the beam splitter 31 may be arranged anywhere in the relay optical system 2, but aberrations and the like are less generated when the beam splitter 31 in the relay optical system 2 is arranged in a substantially parallel portion. It is preferable.

また、光源１の照明光学系１１の光軸の偏心量により照明光ＬＩの境界面６ａに対する入射角が決定する。この光源１の位置は、対物レンズ５の入射瞳面Ｐにおける光源１の像の位置に対応する。そのため、検出器３３で検出されるスポット像の位置により、光源１の位置を調整することも可能である。   Further, the incident angle of the illumination light LI with respect to the boundary surface 6a is determined by the amount of eccentricity of the optical axis of the illumination optical system 11 of the light source 1. The position of the light source 1 corresponds to the position of the image of the light source 1 on the entrance pupil plane P of the objective lens 5. Therefore, it is possible to adjust the position of the light source 1 according to the position of the spot image detected by the detector 33.

本発明の実施形態は、手動による調整に限られず、例えば、検出器２２，２２′，３３（２次元撮像素子等）で検出されたビーム径の大きさから所望の大きさ以下になるように、不図示の制御部によって、リレー光学系２を構成するレンズの少なくとも一部を光軸に沿って移動させる(調整部１３を駆動させる）、光源１の位置を照明光学系１１の光軸方向に移動させる（調整部１３を駆動させる）ようにしても良い。また、レーザ波長の切り替えに連動して、検出器２２，２２′，３３（２次元撮像素子等）で検出されたビーム径の大きさから所望の大きさ以下になる（最も小さくなる）ように、不図示の制御部によって、前述の調整部１３で照明光の平行化を行ってもよい。   The embodiment of the present invention is not limited to manual adjustment. For example, the beam diameter detected by the detectors 22, 22 ′, and 33 (two-dimensional imaging device or the like) is set to a desired size or less. The control unit (not shown) moves at least a part of the lens constituting the relay optical system 2 along the optical axis (drives the adjustment unit 13), and the position of the light source 1 is the optical axis direction of the illumination optical system 11. (Adjustment unit 13 may be driven). Further, in conjunction with the switching of the laser wavelength, the beam diameter detected by the detectors 22, 22 ', 33 (two-dimensional imaging device or the like) is made smaller than the desired size (smallest). The adjusting unit 13 may make the illumination light parallel by a control unit (not shown).

前記課題を解決するために、本発明に係る顕微鏡の調整方法は、光源からの光をリレー光学系でリレーしてこの光源の共役像を対物レンズの入射瞳面の近傍に形成することにより、前記光を対物レンズで略平行光にして試料に照射する照明光学系を有する顕微鏡において、対物レンズの入射瞳面の位置に対する光源の共役像の位置のずれを検出し、このずれを少なくすることにより試料に照射される光を平行光に近づけることを特徴とする。
このような顕微鏡の調整方法は、照明光学系の光軸と光源の光軸とを略一致させた状態で、対物レンズから出射した光を集光して形成した光のスポット像を検出し、このスポット像の径が所定の値以下になるように調整することにより試料に照射される光を平行光に近づけることが好ましい。
また、このような顕微鏡の調整方法は、照明光学系の光軸に対して光源の光軸を偏心させた状態で、試料で反射した光を対物レンズで集光し、さらに、この対物レンズで集光された光を集光して対物レンズの入射瞳面と共役な位置に配置された検出器で検出されるように調整することにより試料に照射される光を平行光に近づけることが好ましい。
また、このような顕微鏡の調整方法は、リレー光学系を構成するレンズの少なくとも一部、又は、光源を、照明光学系の光軸方向に移動させて調整することが好ましい。
また、第１の本発明に係る顕微鏡は、光源から放射された光をリレーして、光源の共役像を形成するリレー光学系と、光源の共役像の近傍に入射瞳面が位置するように配置され、リレー光学系でリレーされた光を試料に照射する対物レンズと、を含む照明光学系を有する顕微鏡であって、対物レンズを透過した光を集光する集光レンズと、集光レンズの焦点面に配置され、集光レンズで集光された光のスポット像を検出する検出器と、を有する調整装置を着脱する取付部と、調整装置を取付部に装着して光源の光軸と照明光学系の光軸とが略一致している状態で、試料に照射される光を平行光に近づけるための調整を行う調整部と、を備えたことを特徴とする。
また、第２の本発明に係る顕微鏡は、光源から放射された光をリレーして、光源の共役像を形成するリレー光学系と、光源の共役像の近傍に入射瞳面が位置するように配置され、リレー光学系でリレーされた光を試料に照射する対物レンズと、を含む照明光学系と、試料を介して対物レンズと反対側に配置されたコンデンサレンズを含む透過照明光学系と、を有する顕微鏡であって、対物レンズを透過した光を集光するコンデンサレンズの瞳位置近傍に、光のスポット像を検出する検出器を有する調整装置を着脱する取付部と、調整装置を取付部に装着し光源の光軸と照明光学系の光軸とが略一致している状態で、試料に照射される光を平行光に近づけるための調整を行う調整部と、を備えたことを特徴とする。
このような顕微鏡において、調整部は、検出器で検出されるスポット像の径が所定の値以下になるように調整することが好ましい。
また、このような顕微鏡において、調整部は、リレー光学系を構成するレンズの少なくとも一部を照明光学系の光軸に沿って移動させることにより調整を行うことが好ましい。
また、このような顕微鏡において、調整部は、光源を照明光学系の光軸方向に移動させることにより調整を行うことが好ましい。
また、第３の本発明に係る顕微鏡は、光軸から偏心した位置に配置された光源から放射された光をリレーして、光源の共役像を形成するリレー光学系と、光源の共役像の近傍に入射瞳面が位置するように配置され、リレー光学系でリレーされた光を試料に照射する対物レンズと、を含む照明光学系を有する顕微鏡であって、リレー光学系を通過する光の一部を透過し、残りを反射するビームスプリッタと、試料で反射した光を対物レンズで集光してリレー光学系に導き、このリレー光学系に入射した光のうち、ビームスプリッタで反射した光を集光する集光レンズと、入射瞳面と略共役な位置に配置され、集光レンズで集光された光を検出する検出器と、試料に照射される光を平行光に近づける調整を行うために、試料で反射した光が検出器で検出されるように調整する調整部と、を備えたことを特徴する。
このような顕微鏡において、調整部は、リレー光学系を構成するレンズのうち、ビームスプリッタよりも光源側にあるレンズの少なくとも一部を照明光学系の光軸に沿って移動させることにより調整を行うことが好ましい。
また、このような顕微鏡において、調整部は、光源を照明光学系の光軸方向に移動させることにより調整を行うことが好ましい。
また、このような顕微鏡は、ビームスプリッタ、集光レンズ及び検出器のうち、少なくともビームスプリッタを挿脱可能又は着脱可能に構成することが好ましい。 In order to solve the above-described problem, the microscope adjusting method according to the present invention relays light from a light source with a relay optical system and forms a conjugate image of the light source in the vicinity of the entrance pupil plane of the objective lens. In a microscope having an illumination optical system that irradiates the sample with the light being made substantially parallel light by an objective lens, the deviation of the position of the conjugate image of the light source with respect to the position of the entrance pupil plane of the objective lens is detected and this deviation is reduced Thus, the light irradiated onto the sample is made to approach parallel light.
Such a microscope adjustment method detects a spot image of light formed by condensing the light emitted from the objective lens in a state in which the optical axis of the illumination optical system and the optical axis of the light source are substantially matched. It is preferable that the light irradiated to the sample is brought close to parallel light by adjusting the diameter of the spot image to be a predetermined value or less.
In addition, such a microscope adjustment method condenses the light reflected by the sample with the objective lens while the optical axis of the light source is decentered with respect to the optical axis of the illumination optical system. It is preferable that the light irradiated on the sample is brought close to parallel light by adjusting the light so as to be collected and detected by a detector disposed at a position conjugate with the entrance pupil plane of the objective lens. .
Moreover, it is preferable that the adjustment method of such a microscope adjusts by moving at least one part of the lens which comprises a relay optical system, or a light source to the optical axis direction of an illumination optical system.
The microscope according to the first aspect of the present invention relays light emitted from a light source to form a conjugate image of the light source and an entrance pupil plane in the vicinity of the conjugate image of the light source. An objective lens that irradiates a sample with light that is arranged and relayed by a relay optical system, and a microscope that has an illumination optical system, and that collects light that has passed through the objective lens, and a condenser lens And a detector for detecting a spot image of the light collected by the condenser lens, a mounting portion for attaching / detaching the adjusting device, and an optical axis of the light source by attaching the adjusting device to the mounting portion And an adjustment unit that performs adjustment to bring the light irradiated to the sample closer to parallel light in a state where the optical axis of the illumination optical system substantially coincides with the optical axis of the illumination optical system.
The microscope according to the second aspect of the present invention relays light emitted from a light source to form a conjugate image of the light source and an entrance pupil plane in the vicinity of the conjugate image of the light source. An objective lens that irradiates the sample with the light relayed by the relay optical system, and a transmission illumination optical system that includes a condenser lens disposed on the opposite side of the objective lens through the sample; A mounting portion for attaching / detaching an adjusting device having a detector for detecting a spot image of light in the vicinity of a pupil position of a condenser lens that collects light transmitted through the objective lens, and a mounting portion for mounting the adjusting device. And an adjustment unit that adjusts the light irradiated to the sample to be close to parallel light in a state where the optical axis of the light source and the optical axis of the illumination optical system substantially coincide with each other. And
In such a microscope, the adjustment unit preferably adjusts so that the diameter of the spot image detected by the detector is a predetermined value or less.
In such a microscope, the adjustment unit preferably performs adjustment by moving at least a part of the lens constituting the relay optical system along the optical axis of the illumination optical system.
In such a microscope, it is preferable that the adjustment unit adjusts the light source by moving the light source in the optical axis direction of the illumination optical system.
A microscope according to a third aspect of the present invention includes a relay optical system that relays light emitted from a light source arranged at a position decentered from the optical axis to form a conjugate image of the light source, and a conjugate image of the light source. An microscope having an illumination optical system that is arranged so that an entrance pupil plane is positioned in the vicinity and irradiates a sample with light relayed by a relay optical system, the light passing through the relay optical system A beam splitter that transmits part of the light and reflects the rest, and the light reflected by the sample is collected by the objective lens and guided to the relay optical system. Of the light incident on this relay optical system, the light reflected by the beam splitter A condensing lens that collects the light, a detector that is positioned substantially conjugate with the entrance pupil plane, detects the light collected by the condensing lens, and adjusts the light applied to the sample closer to parallel light To do so, the light reflected from the sample To further comprising a, an adjustment unit that adjusts as detected.
In such a microscope, the adjustment unit performs adjustment by moving at least a part of the lens constituting the relay optical system that is closer to the light source than the beam splitter along the optical axis of the illumination optical system. It is preferable.
In such a microscope, it is preferable that the adjustment unit adjusts the light source by moving the light source in the optical axis direction of the illumination optical system.
Moreover, it is preferable that such a microscope is configured such that at least the beam splitter among the beam splitter, the condenser lens, and the detector can be inserted and removed.

１ 光源 ２ リレー光学系 ５ 対物レンズ ９，９′ 取付部
１０ 全反射蛍光顕微鏡（顕微鏡） １１ 照明光学系 １３ 調整部
２０，２０′，３０ 調整装置 ２１ 集光レンズ ２２，２２′ 検出器
３１ ビームスプリッタ ３２ 集光レンズ ３３ 検出器
Ｐ 入射瞳 Ｏ 試料 DESCRIPTION OF SYMBOLS 1 Light source 2 Relay optical system 5 Objective lens 9, 9 'Mounting part 10 Total reflection fluorescence microscope (microscope) 11 Illumination optical system 13 Adjustment part 20, 20', 30 Adjustment apparatus 21 Condensing lens 22, 22 'Detector 31 Beam Splitter 32 Condenser lens 33 Detector P Entrance pupil O Sample

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明細書に記載の発明。   Invention described in the specification.
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