JP4842442B2

JP4842442B2 - Convergent light phase contrast microscope apparatus and converging light phase contrast microscope observation method

Info

Publication number: JP4842442B2
Application number: JP2001016103A
Authority: JP
Inventors: 正夫美濃部; 晋也内海
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2001-01-24
Filing date: 2001-01-24
Publication date: 2011-12-21
Anticipated expiration: 2021-01-24
Also published as: JP2002221667A

Description

【０００１】
【発明の属する技術分野】
本発明は、各種材料の組織構造の観察に適した収束光位相差顕微鏡装置および収束光位相差顕微鏡観察方法に関するものである。本発明の収束光位相差顕微鏡装置および収束光位相差顕微鏡観察方法の観察対象となり得る材料のとして、ポリエチレンフィルム、ポリプロピレンフィルム等の高分子材料や、植物、病理組織等の生体材料や、塗液、乳液等の懸濁液や、半導体材料等が挙げられる。
【０００２】
【従来の技術】
各種材料の組織構造は、その物性と密接に相関していることから、その組織構造を正確に評価・解析することが重要となる。そのために、多くの手法が開発され活用されているが、光学顕微鏡装置はその中でも、利用のしやすさや得られる情報の多様性等から、材料の組織構造観察法として最も一般的に利用される手法となっている。
【０００３】
そして、従来の光学顕微鏡装置では、被検査物に対する照明法として、被検査物を一様に照射し、かつ像の分解能を高めるために、平行光を入射するケーラー照明法が通常用いられている。また、対物レンズの後ろ側焦点面上に位相板（フェーズプレート）を配置し、入射した光のうち直接光もしくは回折光の位相を変化させ両者の干渉によりコントラストをつける位相差顕微鏡装置も従来からしばしば用いられている。以下、これらを合わせて従来の顕微鏡装置と略称する。
【０００４】
【発明が解決しようとする課題】
しかし、従来の光学顕微鏡装置による観察は、対物レンズによって被検査物の拡大された光学像を単に形成し、それを接眼レンズでさらに拡大して行うだけである。すなわち、被検査物で反射あるいは透過した光の強度（振幅）に起因する像を観察するだけなので、ごくわずかな屈折率差しか有さない被検査物（位相物体）には適用できなかった。これを解決する手段として上記の位相差顕微鏡装置が利用されるわけであるが、従来の位相差顕微鏡装置では、試料によっては必ずしもコントラストが十分ではなかった。もちろん従来の位相差顕微鏡装置では位相物体の回折像を観察することはできなかった。
【０００５】
【課題を解決するための手段】
本発明の収束光位相差顕微鏡装置および収束光位相差顕微鏡観察方法はこのような課題を解決するためになされたものである。
【０００６】
本発明の収束光位相差顕微鏡装置は、空間の一点に収束する収束光を照明光として照射する照明手段と、前記照明光を被検査物の手前で略単色化する単色化装置と、前記照明光の前記収束点の手前で前記被検査物を載置する被検査物載置台と、前記照明光が前記被検査物において透過もしくは反射した光をひとたび前記収束点に収束させた後に入射するように配置された対物レンズと、対物レンズの前方であって、前記収束点を含み前記照明光の光軸に直交する回折像面上に設置され、かつ前記被検査物によって回折または散乱されることなく前記回折像面上に形成される回折像の中心付近に入射する直接光もしくは前記被検査物によって回折または散乱され前記回折像の中心付近以外に入射する回折光または散乱光の位相を遅らせる位相板（フェーズプレート）と、前記回折像面と前記被検査物との相対的な位置を任意に変更できる調整機構とを備えることを特徴とする。
【０００７】
このように、平行光に代わって単色化した収束光を照明光として用いると、極めてコントラストの高い、焦点深度の深い位相差顕微鏡像を得ることができる。さらに、直接光もしくは回折像の中心付近以外に入射する回折光の位相を遅らせる位相板を備えるため、屈折率の小さい被検査物（位相物体）に対しても明瞭な位相差顕微鏡像を得ることができる。
【０００８】
ここで位相物体とは、物体各部分を透過または反射した光に位相の差（光波の進行段階の差）を生じさせるが強度の差を生じさせないもののことである。強度の差が生じないために、そのままでは物体各部分を明暗の違いとして観察できない。この場合、位相板を用いて直接光の位相を回折光に対して変化させた後に回折光と干渉させるか、もしくは回折光の位相を直接光に対して変化させた後に直接光と干渉させることにより、物体各部分を透過または反射した光に強度の差が生じて観察することができるようになる。これが位相差顕微鏡の原理である。
【０００９】
従来の光学顕微鏡装置では、回折像は対物レンズの後側焦点面、すなわち、鏡筒内部に形成されるので、接眼レンズをはずさない限り観察できないし、いわんや操作を加えたりすることは不可能である。また、従来の位相差光学顕微鏡装置では、回折像が鏡筒内部に形成されるのにともない位相板が対物レンズに取り付けられており、位相差用の各種対物レンズを準備し交換しなければならない。さらに、位相物体を観察するためには位相板と絞り板の形状および大きさを共役にする必要があるが、従来の位相差顕微鏡装置では絞り板がコンデンサーに取り付けられているため、位相差用のコンデンサーを準備し交換しなければならない。そのうえ、対物レンズの倍率を変えるたびにそれに合わせてコンデンサーの絞り板も変えなければならない、等の不便さがあった。
【００１０】
一方、収束光位相差顕微鏡装置では、照明光の収束点を含み照明光の光軸に直交する面には、照明光による被検査物のフーリエ変換像すなわち回折像が形成される。この場合、対物レンズの前方にこの回折像を形成させることができるので、回折像そのものを観察したり、回折像に対して操作を加えて所望の処理を施したりすることができる。さらに、位相物体を観察するために、直接光もしくは回折像の中心付近以外に入射する回折光の位相を位相板により遅らせることができる。このとき、位相板が対物レンズの前方に配置されているため、従来の位相差顕微鏡装置に必ず必要な位相差用の対物レンズを準備し交換する必要がない。また、対物レンズの倍率を変えても位相板や位相板とともに用いる絞り板を変える必要がない。さらに、絞り板をコンデンサーに取り付けていないため、従来の位相差顕微鏡装置に必ず必要な位相差用のコンデンサーを準備し交換する必要もない。
【００１１】
回折像には被検査物の構造情報が集約されている。換言すると、被検査物の組織構造に応じた回折像が形成され、被検査物の組織構造が異なっていれば、その回折像も異なったものとなる。したがって、組織構造と回折像との関係が判っていれば、回折像から逆に被検査物の組織構造を知ることができる。本発明の収束光位相差顕微鏡装置は、微小なコントラストしか有さない位相物体の回折像と位相差像を同時に与えるものである。
【００１２】
対物レンズは、回折像面および被検査物のいずれにもに焦準（ピント）を合わせることができるようになっていることが望ましい。これにより、被検査物の光学像と回折像を共に観察することができ、被検査物の構造情報の取得を増やすことができる。
【００１３】
この収束光位相差顕微鏡装置は、回折像面の位置に配置され、照明光が被検査物において透過または反射した光の一部を選択的に遮蔽する空間絞りを備えることが望ましい。この場合、回折光の選択が自由にできるよう調整されているので、同一の被検査物について所望の回折光に応じた様々な位相差像を観察することができる。しかも位相板を備えているため、選択された回折光と直接光による被検査物の光学像の中の微小な屈折率分布を観察することができる。
【００１４】
この空間絞りは、所望の回折光を選択して対物レンズに入射させるためのものである。対物レンズの焦準を被検査物に合わせれば、選択された回折光と直接光の干渉によって形成された位相差像を観察することができる。しかも、回折光の選択が自由なので、同一の被検査物について所望の回折光に応じた様々な像を観察することができる。
【００１５】
この収束光位相差顕微鏡装置は、回折像面と被検査物との相対的な位置を任意に変更できる調整機構を備えている。通常は、収束する照明光の出射面となるコンデンサレンズの位置を変えることにより収束点の位置、すなわち回折像面の位置を変える。回折像面と被検査物との距離を変えると、回折像の大きさが変化する。距離を離すほど、回折像を大きくすることができ、回折像の微細なパターンを調べることができる。
【００１６】
この収束光位相差顕微鏡装置は、位相板が光の位相をπ／２すなわち波長にして四分の一波長遅らせるものであることが望ましい。光の位相を遅らせる量がπ／２でない場合に比べて屈折率分布によるコントラストを大きくすることができる。
【００１７】
さらにこの位相板が直接光の強度を減衰させる機能を合わせ有するとなお好ましい。直接光と回折光の干渉を調整して位相差像のコントラストをより高めることができるためである。
【００１８】
この収束光位相差顕微鏡装置は、単色化装置としてグリーンフィルターを用いると、本発明による位相差像を安価で簡便に得ることができる。
【００１９】
この収束光位相差顕微鏡装置は、空間絞りを通過した光の方向と対物レンズの光軸とを略一致させる調整機構を備えていることが望ましい。空間絞りにより光量が減少するが、この２つの光軸を略一致させることにより、ひずみの少ない明るい像を得ることができる。
【００２０】
このような収束光位相差顕微鏡装置を用いた本発明の顕微鏡観察方法の一つは、対物レンズの焦準を被検査物に合わせて被検査物を観察するものである。収束光を照明光として用いるので、極めてコントラストの高い、焦点深度の深い観察像を得ることができる。
【００２１】
本発明の他の収束光位相差顕微鏡観察方法は、収束点を含む対物レンズの光軸に直交する回折像面に対物レンズの焦準を合わせることにより、回折像面上に形成された被検査物の照射光による回折像を観察するものである。
【００２２】
被検査物について、回折像と組織構造との関係を予め取得しておけば、回折像を直接観察することにより、回折像のパターンの特徴から被検査物の組織構造を知ることができる。
【００２３】
本発明の他の収束光位相差顕微鏡観察方法は、最初に対物レンズの焦準を被検査物に合わせて被検査物を観察した後、対物レンズの焦準を回折像面上に形成された回折像に合わせて回折像を観察するか、もしくは、最初に対物レンズの焦準を回折面上に形成された回折像に合わせて回折像を観察した後、対物レンズの焦準を被検査物に合わせて被検査物を観察するものである。
【００２４】
光学像の観察だけでは分かりにくかった組織構造の全体的な特徴を把握したり、回折像の観察だけでは分かりにくかった回折像が由来する組織構造の詳細を知ることができる。
【００２５】
本発明の他の収束光位相差顕微鏡観察方法は、空間絞りを用いて回折像面上の所望の領域のみの光を通過させ、空間絞りを通過した光について対物レンズの焦準を被検査物に合わせて被検査物を観察するものである。
【００２６】
この結果、空間絞りで選択された回折光と直接光の干渉による被検査物の位相差像を観察することができる。回折光の選択が自由なので、同一の被検査物について回折光に応じた様々な像を観察することができる。これにより被検査物の組織構造をさらに詳しく知ることができる。
【００２７】
本発明の他の収束光位相差顕微鏡観察方法は、回折像面に対物レンズの焦準を合わせることにより、回折像面上に形成された被検査物の照射光による回折像を観察し、回折像の所望の領域と直接光の両者を通過させるように空間絞りを調整した後、対物レンズの焦準を被検査物に合わせることにより、空間絞りを通過した光によって被検査物を観察するものである。
【００２８】
回折像に基づいて回折光を選択してから像観察を行うので、いかなる回折光に基づく光学像かを知ることができる。これにより被検査物の組織構造をさらに詳しく知ることができる。
【００２９】
本発明の収束光位相差顕微鏡観察方法では、対物レンズが回折像面の近傍に位置したときに被検査物に焦準が合うように回折像面の位置を調整して被検査物を観察することが望ましい。回折像面は照射光が収束する位置なので、そこに対物レンズが置かれたときに回折光をロスすることなく最も像が明るくなるためである。
【００３０】
空間絞りの形状または回折像面上での位置を変えることにより、もしくは、対物レンズの光軸に対する照明光の光軸の角度を変えることにより、対物レンズによる被検査物の光学像形成にあずかる回折光を選択して被検査物を観察することができる。
【００３１】
本発明の顕微鏡観察方法では、空間絞りを通過した光の方向と対物レンズの光軸とを略一致させて被検査物を観察することが望ましい。空間絞りにより光量が減少するが、この２つの光軸を略一致させることにより、ひずみの少ない明るい像を得ることができる。
【００３２】
本発明の顕微鏡観察方法では、照明光の収束点の位置を対物レンズの光軸方向において変えることにより回折像の大きさを調整することができることが望ましい。距離を離すほど、回折像の大きさを大きくすることができ、回折像のより詳細な観察ができる。
【００３３】
本発明の顕微鏡観察方法は、高分子材料に対して好適に用いることができる。高分子材料の重要な組織構造について、従来の顕微鏡観察方法では得られなかった詳細な知見が得られる。
【００３４】
【発明の実施の形態】
図１は本発明の一実施形態である収束光位相差顕微鏡装置の基本構成を示す図である。光源１と単色化装置１７とコンデンサレンズ２によって、空間の一点４に収束する収束光を照明光として照射する照明手段３が構成されている。光源１から出力される光は、単色化装置１７により単色光となるようにする。
【００３５】
照明手段３の上方には、被検査物（標本）６を載置する被検査物載置台（ステージ）５が配置されている。ステージ５は、その中央に照明手段３からの照明光を透過する開口を有し、照明光はこの開口を通ってさらに上方の収束点４で収束する。これにより、収束点４を通り照明光の光軸７に垂直な平面８には、被検査物６の照明光によるフーリエ変換像すなわち回折像が形成される。ここでは、この平面８を回折像面と呼ぶことにする。
【００３６】
収束点４付近には位相板１５が設けられている。図２は位相板１５の平面図であり、回折光を透過する石英板等の中央にたとえば直径４５〜５０μｍの円板状の位相を変化させる材料が取り付けられている。この位相板１５は観察中でも容易に脱着可能となっている。
【００３７】
なお、位相板１５上の位相を変化させる材料の形状は必ずしも円板状でなくともよい。光源１とコンデンサレンズ２の間にある光源像結像位置付近に絞り板１６を挿入することにより、絞り板１６の形状に応じて方形、半円形、扇形、リング状等を適宜選択することができる。
【００３８】
コンデンサレンズ２は、ステージ５に対して光軸７の方向に移動可能となっている。コンデンサレンズ２を光軸７の方向に移動させることにより、ステージ５に載置された被検査物６と収束点４との距離、すなわち、被検査物６と回折像面８との距離を変化させることができる。
【００３９】
回折像面８上またはその近傍の位置には、回折像面８と平行に空間絞り９が設けられている。図３は空間絞り９の平面図であり、遮光板の中央に、たとえば直径数百ミクロンの円形開口が形成されている。この空間絞り９は、光軸７に直交する方向に移動可能となっており、回折像面８上に形成される回折像の観察視野を選択できる。また、この空間絞り９は観察途中でも、容易に脱着可能となっている。
【００４０】
なお、空間絞り９に形成する開口形状すなわち観察視野は、必ずしも円形でなくてもよい。目的に応じて方形、半円形、扇形等を適宜選択することができる。
【００４１】
空間絞り９のさらに上方には、対物レンズ１０、結像レンズ１１および接眼レンズ１２を備えた鏡筒１３が配置されている。この鏡筒１３の内部構成自体は、従来からある一般的なものであり、光軸７の方向に移動させることにより、焦準合わせを行うことができるようなっている。
【００４２】
しかし、焦準合わせのための鏡筒移動可能範囲は、従来の一般的な顕微鏡装置に比べて十分に長いことが必要である。すなわち、少なくとも被検査物６と回折像面８の両方に焦準合わせができるようになっている。
【００４３】
対物レンズ１０は、被検査物６に焦準を合わせたときの位置が空間絞り９よりも後方（上方）になるような焦点距離を有している。したがって、焦準合わせ動作において、空間絞り９が邪魔になることがない。
【００４４】
なお、図４に示すように、対物レンズ１０が空間絞り９に最も近づいたときに被検査物６に焦準が合うように、回折像面８の位置を調整すると、最も明るい像を得ることができる。
【００４５】
対物レンズ１０が捕らえた像は、結像レンズ１１の後方にある中間像位置１４に形成され、接眼レンズ１２はこの像を観察できるように焦準が調整されている。
【００４６】
本発明による被検査物としては、高分子材料（たとえば、ポリエチレン、ポリプロピレン等のポリマーフィルム）、生体材料、セラミックス、金属他を挙げることができるが、組織構造を観察し得るという点で、ポリマーフィルムは最も典型的な対象材料である。
【００４７】
【発明の効果】
以上のように、本発明の収束光位相差顕微鏡装置およびこれを用いた収束光位相差顕微鏡観察方法によれば、対物レンズの手前で一点に収束する照明光を用い、かつ収束点付近に位相板を配置しているので、被検査物の位相差顕微鏡像およびその回折像を対物レンズを光軸方向に移動するだけで選択的に観察できる。また、この収束光位相差顕微鏡装置は、回折像面と被検査物との相対的な位置を任意に変更できる調整機構を備えているので、回折像面と被検査物との距離を離すほど、回折像の大きさを大きくすることができ、回折像の微細なパターンを調べることができる。さらに、空間絞りを適当に挿入または移動させることにより、所望の回折光による被検査物の位相差顕微鏡像と回折像とを得ることができる。したがって、従来の光学顕微鏡装置では得られなかった微小な屈折率差しか有さない被検査物の構造に関する詳細な知見を得ることができる。
【図面の簡単な説明】
【図１】本発明の一実施形態である収束光位相差顕微鏡装置の構成を示す図
【図２】位相板１５を示す平面図
【図３】空間絞り９を示す平面図
【図４】対物レンズ１０を空間絞り９に近接させた状態の収束光位相差顕微鏡装置を示す図
【符号の説明】
１…光源、２…コンデンサレンズ、３…照明手段、４…収束点、５…被検査物載置台（ステージ）、６…被検査物、７…光軸、８…回折像面、９…空間絞り、１０…対物レンズ、１１…結像レンズ（必ずしも必要ではない）、１２…接眼レンズ、１３…鏡筒、１４…中間像位置、１５…位相板、１６…絞り板、１７…単色化装置[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a convergent light phase-contrast microscope apparatus and a convergent-light phase contrast microscope observation method suitable for observing the structure of various materials. Examples of materials that can be observed by the convergent light phase contrast microscope apparatus and the convergent light phase contrast microscope observation method of the present invention include polymer materials such as polyethylene films and polypropylene films, biomaterials such as plants and pathological tissues, and coating liquids. And suspensions such as emulsions and semiconductor materials.
[0002]
[Prior art]
Since the structure of various materials is closely correlated with their physical properties, it is important to accurately evaluate and analyze the structure. For this purpose, many techniques have been developed and utilized, but among them, the optical microscope apparatus is most commonly used as a method for observing the structure of materials because of its ease of use and the diversity of information obtained. It is a method.
[0003]
In the conventional optical microscope apparatus, as an illumination method for the inspection object, a Koehler illumination method in which parallel light is incident is usually used in order to uniformly irradiate the inspection object and increase the resolution of the image. . In addition, a phase contrast microscope apparatus in which a phase plate (phase plate) is disposed on the back focal plane of the objective lens and the contrast of the incident light is changed by changing the phase of direct light or diffracted light to interfere with each other has been conventionally used. Often used. Hereinafter, these are collectively referred to as a conventional microscope apparatus.
[0004]
[Problems to be solved by the invention]
However, observation with a conventional optical microscope apparatus is simply performed by forming an enlarged optical image of an object to be inspected with an objective lens and further enlarging it with an eyepiece. In other words, since only an image caused by the intensity (amplitude) of light reflected or transmitted by the inspection object is observed, it cannot be applied to an inspection object (phase object) having a very small refractive index difference. The phase contrast microscope apparatus described above is used as a means for solving this problem. However, in the conventional phase contrast microscope apparatus, the contrast is not always sufficient depending on the sample. Of course, the conventional phase contrast microscope apparatus could not observe the diffraction image of the phase object.
[0005]
[Means for Solving the Problems]
The convergent light phase contrast microscope apparatus and the convergent light phase contrast microscope observation method of the present invention have been made in order to solve such problems.
[0006]
The convergent light phase contrast microscope apparatus of the present invention includes an illuminating unit that irradiates convergent light that converges to one point in space as illumination light, a monochromator that substantially monochromatically illuminates the illumination light in front of an object to be inspected, and the illumination An inspection object mounting table on which the inspection object is mounted before the convergence point of light, and the illumination light is incident on the convergence point after the light transmitted or reflected by the inspection object is once converged on the convergence point. And an objective lens disposed on the front side of the objective lens, disposed on a diffraction image plane including the convergence point and orthogonal to the optical axis of the illumination light, and diffracted or scattered by the inspection object. Phase that delays the phase of diffracted light or scattered light that is diffracted or scattered by the object to be inspected and is diffracted or scattered by the inspection object without being near the center of the diffracted image. Board Phase plates), characterized in that it comprises an adjustment mechanism capable of arbitrarily changing the relative position between the said diffractive image surface inspection object.
[0007]
As described above, when the monochromatic convergent light is used as the illumination light instead of the parallel light, a phase contrast microscopic image having a very high contrast and a deep focal depth can be obtained. In addition, since a phase plate that delays the phase of incident light other than near the center of the direct light or diffraction image is provided, a clear phase-contrast microscope image can be obtained even for an inspection object (phase object) with a low refractive index. Can do.
[0008]
Here, the phase object refers to an object that causes a phase difference (difference in light wave traveling stage) to light transmitted or reflected by each part of the object but does not cause an intensity difference. Since there is no difference in intensity, each part of the object cannot be observed as a difference in brightness as it is. In this case, use the phase plate to change the phase of the direct light with respect to the diffracted light and then interfere with the diffracted light, or change the phase of the diffracted light with respect to the direct light and then interfere with the direct light. As a result, a difference in intensity occurs in the light transmitted or reflected by each part of the object, and observation can be performed. This is the principle of the phase contrast microscope.
[0009]
In the conventional optical microscope apparatus, the diffraction image is formed on the rear focal plane of the objective lens, that is, inside the lens barrel. Therefore, it cannot be observed unless the eyepiece is removed, and it is impossible to add operations. is there. Further, in the conventional phase-contrast optical microscope apparatus, the phase plate is attached to the objective lens as the diffraction image is formed inside the lens barrel, and various objective lenses for phase difference must be prepared and replaced. . Furthermore, in order to observe a phase object, it is necessary to conjugate the shape and size of the phase plate and the diaphragm plate. However, in the conventional phase-contrast microscope apparatus, the diaphragm plate is attached to a condenser. The capacitor must be prepared and replaced. In addition, each time the magnification of the objective lens is changed, the condenser diaphragm plate must be changed accordingly.
[0010]
On the other hand, in the convergent light phase contrast microscope apparatus, a Fourier transform image, that is, a diffraction image of the object to be inspected by the illumination light is formed on a surface that includes the convergence point of the illumination light and is orthogonal to the optical axis of the illumination light. In this case, since this diffraction image can be formed in front of the objective lens, the diffraction image itself can be observed, or a desired process can be performed by applying an operation to the diffraction image. Furthermore, in order to observe the phase object, the phase of the diffracted light incident on the direct light or other than near the center of the diffraction image can be delayed by the phase plate. At this time, since the phase plate is arranged in front of the objective lens, it is not necessary to prepare and replace the objective lens for the phase difference that is necessary for the conventional phase contrast microscope apparatus. Further, even if the magnification of the objective lens is changed, there is no need to change the phase plate or the diaphragm plate used together with the phase plate. In addition, since the diaphragm plate is not attached to the condenser, it is not necessary to prepare and replace a condenser for phase difference that is necessary for a conventional phase contrast microscope apparatus.
[0011]
In the diffraction image, the structure information of the inspection object is collected. In other words, a diffraction image corresponding to the tissue structure of the inspection object is formed. If the tissue structure of the inspection object is different, the diffraction image is also different. Therefore, if the relationship between the tissue structure and the diffraction image is known, the tissue structure of the object to be inspected can be known from the diffraction image. The convergent light phase contrast microscope apparatus of the present invention simultaneously provides a diffraction image and a phase contrast image of a phase object having only a minute contrast.
[0012]
It is desirable that the objective lens can be focused on both the diffraction image plane and the inspection object. Thereby, both the optical image and the diffraction image of the inspection object can be observed, and the acquisition of the structure information of the inspection object can be increased.
[0013]
The convergent light phase-contrast microscope apparatus is preferably provided with a spatial aperture that is arranged at the position of the diffraction image plane and selectively shields part of the light transmitted or reflected by the illumination light on the inspection object. In this case, since the adjustment is made so that the diffracted light can be freely selected, various phase difference images corresponding to the desired diffracted light can be observed for the same inspection object. In addition, since the phase plate is provided, it is possible to observe a minute refractive index distribution in the optical image of the inspection object by the selected diffracted light and direct light.
[0014]
This spatial aperture is for selecting desired diffracted light and making it enter the objective lens. If the focusing of the objective lens is adjusted to the object to be inspected, a phase difference image formed by interference between the selected diffracted light and direct light can be observed. Moreover, since the diffracted light can be freely selected, various images corresponding to the desired diffracted light can be observed for the same inspection object.
[0015]
This convergent light phase contrast microscope apparatus includes an adjustment mechanism that can arbitrarily change the relative position of the diffraction image plane and the inspection object. Normally, the position of the converging point, that is, the position of the diffraction image plane is changed by changing the position of the condenser lens that becomes the exit surface of the convergent illumination light. When the distance between the diffraction image plane and the inspection object is changed, the size of the diffraction image changes. As the distance is increased, the diffraction image can be enlarged, and a fine pattern of the diffraction image can be examined.
[0016]
In this convergent light phase contrast microscope apparatus, it is desirable that the phase plate delays the wavelength by ¼ / 2, that is, a wavelength, by a quarter wavelength. Compared with the case where the amount of delaying the phase of light is not π / 2, the contrast by the refractive index distribution can be increased.
[0017]
It is further preferable that this phase plate has a function of directly attenuating the intensity of light. This is because the contrast of the phase difference image can be further increased by adjusting the interference between the direct light and the diffracted light.
[0018]
In this convergent light phase contrast microscope apparatus, when a green filter is used as a monochromator, a phase difference image according to the present invention can be obtained inexpensively and easily.
[0019]
The convergent light phase contrast microscope apparatus preferably includes an adjustment mechanism that substantially matches the direction of light that has passed through the spatial aperture and the optical axis of the objective lens. Although the amount of light is reduced by the spatial stop, a bright image with little distortion can be obtained by making these two optical axes substantially coincident with each other.
[0020]
One of the microscope observation methods of the present invention using such a convergent-light phase-contrast microscope apparatus is to observe the inspection object by aligning the focus of the objective lens with the inspection object. Since converged light is used as illumination light, an observation image with a very high contrast and a deep focal depth can be obtained.
[0021]
Another method for observing a convergent light phase-contrast microscope of the present invention is to inspect an object formed on a diffraction image plane by focusing the objective lens on a diffraction image plane orthogonal to the optical axis of the objective lens including the convergence point. A diffraction image of an object irradiated with light is observed.
[0022]
If the relationship between the diffraction image and the tissue structure is acquired in advance for the inspection object, the tissue structure of the inspection object can be known from the characteristics of the pattern of the diffraction image by directly observing the diffraction image.
[0023]
According to another convergent light phase contrast microscope observation method of the present invention, after first focusing the objective lens on the inspection object and observing the inspection object, the focusing of the objective lens is formed on the diffraction image plane. Observe the diffraction image according to the diffraction image, or first observe the diffraction image by aligning the focus of the objective lens with the diffraction image formed on the diffraction surface, and then adjust the focus of the objective lens to the object to be inspected. The object to be inspected is observed at the same time.
[0024]
It is possible to grasp the overall characteristics of the tissue structure that was difficult to understand only by observing the optical image, or to know the details of the tissue structure from which the diffraction image that was difficult to understand only by observing the diffraction image.
[0025]
According to another method for observing a convergent light phase-contrast microscope of the present invention, the light of only a desired region on the diffraction image plane is allowed to pass using a spatial stop, and the focusing of the objective lens is focused on the light that has passed through the spatial stop. The object to be inspected is observed at the same time.
[0026]
As a result, it is possible to observe the phase difference image of the inspection object due to the interference between the diffracted light selected by the spatial aperture and the direct light. Since the selection of the diffracted light is free, various images corresponding to the diffracted light can be observed for the same inspection object. Thereby, the tissue structure of the object to be inspected can be known in more detail.
[0027]
The other convergent light phase contrast microscope observation method of the present invention observes the diffraction image by the irradiation light of the object to be inspected formed on the diffraction image plane by aligning the focus of the objective lens on the diffraction image plane. After the spatial aperture is adjusted so that both the desired area of the image and direct light pass through, the object is observed by the light passing through the spatial aperture by adjusting the focus of the objective lens to the inspection object It is.
[0028]
Since image observation is performed after selecting diffracted light based on the diffracted image, it is possible to know what diffracted light is based on the optical image. Thereby, the tissue structure of the object to be inspected can be known in more detail.
[0029]
In the converging-light phase-contrast microscope observation method of the present invention, when the objective lens is positioned in the vicinity of the diffraction image plane, the position of the diffraction image plane is adjusted so that the inspection object is focused and the inspection object is observed. It is desirable. This is because the diffracted image plane is a position where the irradiation light converges, so that when the objective lens is placed there, the image becomes brightest without losing the diffracted light.
[0030]
Diffraction that contributes to the optical image formation of an object to be inspected by the objective lens by changing the shape of the spatial aperture or the position on the diffraction image plane, or by changing the angle of the optical axis of the illumination light with respect to the optical axis of the objective lens The object can be observed by selecting light.
[0031]
In the microscope observation method of the present invention, it is desirable to observe the object to be inspected with the direction of the light that has passed through the space diaphragm and the optical axis of the objective lens substantially matched. Although the amount of light is reduced by the spatial stop, a bright image with little distortion can be obtained by making these two optical axes substantially coincident with each other.
[0032]
In the microscope observation method of the present invention, it is desirable that the size of the diffraction image can be adjusted by changing the position of the convergence point of the illumination light in the optical axis direction of the objective lens. As the distance is increased, the size of the diffraction image can be increased, and the diffraction image can be observed in more detail.
[0033]
The microscope observation method of the present invention can be suitably used for a polymer material. With respect to the important structure of the polymer material, detailed knowledge that cannot be obtained by the conventional microscope observation method can be obtained.
[0034]
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 is a diagram showing a basic configuration of a convergent light phase contrast microscope apparatus according to an embodiment of the present invention. The light source 1, the monochromator 17, and the condenser lens 2 constitute an illuminating unit 3 that irradiates convergent light that converges on a single point 4 in space as illumination light. The light output from the light source 1 is changed to monochromatic light by the monochromator 17.
[0035]
An inspection object mounting table (stage) 5 on which an inspection object (specimen) 6 is mounted is disposed above the illumination means 3. The stage 5 has an opening that transmits the illumination light from the illumination unit 3 at the center thereof, and the illumination light converges at a convergence point 4 further upward through this opening. As a result, a Fourier transform image, that is, a diffraction image is formed on the plane 8 that passes through the convergence point 4 and is perpendicular to the optical axis 7 of the illumination light. Here, this plane 8 is called a diffraction image plane.
[0036]
A phase plate 15 is provided in the vicinity of the convergence point 4. FIG. 2 is a plan view of the phase plate 15, and a disk-shaped material having a diameter of 45 to 50 μm, for example, is attached to the center of a quartz plate or the like that transmits diffracted light. This phase plate 15 can be easily detached even during observation.
[0037]
Note that the shape of the material that changes the phase on the phase plate 15 is not necessarily disc-shaped. By inserting the diaphragm plate 16 in the vicinity of the light source image forming position between the light source 1 and the condenser lens 2, a square shape, a semicircular shape, a sector shape, a ring shape, or the like can be appropriately selected according to the shape of the diaphragm plate 16. it can.
[0038]
The condenser lens 2 is movable in the direction of the optical axis 7 with respect to the stage 5. By moving the condenser lens 2 in the direction of the optical axis 7, the distance between the inspection object 6 placed on the stage 5 and the convergence point 4, that is, the distance between the inspection object 6 and the diffraction image plane 8 is changed. Can be made.
[0039]
A spatial stop 9 is provided in parallel with the diffraction image surface 8 at a position on or near the diffraction image surface 8. FIG. 3 is a plan view of the space stop 9, and a circular opening having a diameter of several hundred microns, for example, is formed in the center of the light shielding plate. The spatial diaphragm 9 is movable in a direction perpendicular to the optical axis 7 and can select an observation field of the diffraction image formed on the diffraction image surface 8. Further, the space stop 9 can be easily detached even during observation.
[0040]
The opening shape formed in the space stop 9, that is, the observation field of view, is not necessarily circular. A square shape, a semi-circle shape, a sector shape, or the like can be appropriately selected according to the purpose.
[0041]
A lens barrel 13 including an objective lens 10, an imaging lens 11, and an eyepiece lens 12 is disposed further above the spatial aperture 9. The internal structure itself of the lens barrel 13 is a conventional one, and can be focused by moving in the direction of the optical axis 7.
[0042]
However, the movable range of the lens barrel for focusing needs to be sufficiently longer than that of a conventional general microscope apparatus. That is, focusing can be performed at least on both the inspection object 6 and the diffraction image plane 8.
[0043]
The objective lens 10 has a focal length such that the position when the object 6 is focused on is behind (upward) the space stop 9. Therefore, the space stop 9 does not get in the way in the focusing operation.
[0044]
As shown in FIG. 4, when the position of the diffraction image plane 8 is adjusted so that the object 6 is focused when the objective lens 10 is closest to the space stop 9, the brightest image is obtained. Can do.
[0045]
The image captured by the objective lens 10 is formed at an intermediate image position 14 behind the imaging lens 11, and the eyepiece 12 is adjusted in focus so that this image can be observed.
[0046]
Examples of the inspected object according to the present invention include polymer materials (for example, polymer films such as polyethylene and polypropylene), biomaterials, ceramics, metals, and the like. Is the most typical target material.
[0047]
【The invention's effect】
As described above, according to the convergent-light phase-contrast microscope apparatus of the present invention and the convergent-light phase-contrast microscope observation method using the same, the illumination light that converges to one point before the objective lens is used, and the phase near the convergent point is obtained. Since the plate is disposed, the phase-contrast microscope image and the diffraction image of the inspection object can be selectively observed by simply moving the objective lens in the optical axis direction. In addition, this convergent light phase contrast microscope apparatus includes an adjustment mechanism that can arbitrarily change the relative position between the diffraction image plane and the inspection object, so that the distance between the diffraction image plane and the inspection object increases. The size of the diffraction image can be increased, and the fine pattern of the diffraction image can be examined. Furthermore, by appropriately inserting or moving the space stop, a phase contrast microscopic image and a diffraction image of the object to be inspected by desired diffracted light can be obtained. Therefore, it is possible to obtain detailed knowledge about the structure of the object to be inspected that has only a minute refractive index difference that cannot be obtained with the conventional optical microscope apparatus.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a convergent light phase contrast microscope apparatus according to an embodiment of the present invention. FIG. 2 is a plan view showing a phase plate. FIG. 3 is a plan view showing a spatial diaphragm. The figure which shows the convergence light phase-contrast microscope apparatus of the state which made the lens 10 adjoin to the space diaphragm 9
DESCRIPTION OF SYMBOLS 1 ... Light source, 2 ... Condenser lens, 3 ... Illuminating means, 4 ... Convergence point, 5 ... Inspection object mounting stage (stage), 6 ... Inspection object, 7 ... Optical axis, 8 ... Diffraction image plane, 9 ... Space Aperture, 10 ... objective lens, 11 ... imaging lens (not necessarily required), 12 ... eyepiece, 13 ... lens barrel, 14 ... intermediate image position, 15 ... phase plate, 16 ... aperture plate, 17 ... monochromator

Claims

Illumination means for irradiating convergent light that converges to one point in space as illumination light, a monochromator that substantially monochromatically illuminates the illumination light before the object to be inspected, and the object to be inspected before the convergence point of the illumination light An object mounting table on which an object is mounted, an objective lens arranged so that the light transmitted through or reflected by the object to be inspected is once converged on the convergence point, and incident light; Diffraction formed on the diffractive image plane that is located on the diffractive image plane that is forward and includes the convergence point and is orthogonal to the optical axis of the illumination light, and is not diffracted or scattered by the inspection object A phase plate (phase plate) that delays the phase of diffracted light or scattered light that is diffracted or scattered by direct light incident on the vicinity of the center of the image or by the object to be inspected other than near the center of the diffracted image; Optical microscope and wherein the further comprising an adjusting mechanism capable of arbitrarily changing the relative position of the object to be inspected with.

The optical microscope apparatus according to claim 1, wherein the objective lens is adapted to be focused on both the diffraction image plane and the inspection object.

The space stop which is arrange | positioned in the position immediately after the said phase plate, and selectively shields a part of said diffracted light which permeate | transmitted or reflected in the said to-be-inspected object is provided. The optical microscope apparatus according to item.

The optical microscope apparatus according to claim 1, wherein the phase plate delays the phase of light by π / 2.

The optical microscope apparatus according to claim 4, wherein the phase plate also has a function of attenuating the intensity of the direct light.

The optical microscope apparatus according to claim 1, wherein the substantially monochromator is a green filter.

The optical microscope apparatus according to any one of claims 3 to 6, further comprising an adjustment mechanism that substantially matches a direction of light that has passed through the spatial diaphragm and an optical axis of the objective lens.

The microscope observation method using the optical microscope apparatus according to claim 1, wherein the inspection object is observed by adjusting a focus of the objective lens to the inspection object.

2. The microscope observation method using the optical microscope apparatus according to claim 1, wherein the object lens is focused on a diffraction image plane that includes the convergence point and is orthogonal to the optical axis of the objective lens. And observing a diffraction image of the illumination light.

In the microscope observation method using the optical microscope apparatus according to claim 2, first, after focusing the objective lens on the inspection object and observing the inspection object, focusing of the objective lens is performed on the object Observe the diffraction image according to the diffraction image formed on the diffraction image surface, or first observe the diffraction image by aligning the focus of the objective lens with the diffraction image formed on the diffraction surface. And then observing the inspection object by aligning the focusing of the objective lens with the inspection object.

In the microscope observation method using the optical microscope apparatus as described in any one of Claims 3-7, only the said direct light and desired diffracted light are allowed to pass through using the said spatial stop, The light which passed the said spatial stop A method of observing the inspection object, wherein the object lens is focused on the inspection object to observe the inspection object.

The microscope observation method using the optical microscope apparatus according to any one of claims 3 to 7, wherein the objective lens is focused on the diffraction image surface, thereby forming the diffraction image surface on the diffraction image surface. After observing a diffraction image of the object to be inspected by the irradiation light and adjusting the spatial aperture so that only light in a desired region of the diffraction image passes, the focus of the objective lens is adjusted to the object to be inspected. Thus, the microscope observation method is characterized in that the inspection object is observed with the light passing through the space diaphragm.

The microscope observation method using the optical microscope apparatus according to any one of claims 3 to 7, wherein the object is focused when the objective lens is positioned in the vicinity of the diffraction image plane. A microscope observation method, wherein the inspection object is observed by adjusting a position of the diffraction image plane.

By changing the shape of the spatial diaphragm or the position of the spatial diaphragm on the diffraction surface, or by changing the angle of the optical axis of the illumination light with respect to the optical axis of the objective lens, the object by the objective lens is changed. The microscope observation method according to any one of claims 11 to 13, wherein the inspection object is observed by selecting light participating in optical image formation of the inspection object.

The microscope observation method according to any one of claims 11 to 13, wherein the inspection object is observed with a direction of light passing through the spatial aperture substantially matched with an optical axis of the objective lens. .

The size of the diffraction image is adjusted by changing the position of the convergence point of the illumination light in the optical axis direction of the objective lens, according to any one of claims 8 to 12, 14, or 15. The microscope observation method described.

The microscope observation method according to claim 8, wherein the object to be inspected is a polymer material.