JP2007193213A - Total reflection fluorescence microscope - Google Patents

Total reflection fluorescence microscope Download PDF

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JP2007193213A
JP2007193213A JP2006012727A JP2006012727A JP2007193213A JP 2007193213 A JP2007193213 A JP 2007193213A JP 2006012727 A JP2006012727 A JP 2006012727A JP 2006012727 A JP2006012727 A JP 2006012727A JP 2007193213 A JP2007193213 A JP 2007193213A
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total reflection
objective lens
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Kenichi Kusaka
健一 日下
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Olympus Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a total reflection fluorescence microscope that can efficiently acquire a fluorescent light by eliminating loss of the fluorescent light excited by total reflection illumination to the utmost. <P>SOLUTION: The microscope includes a light source 11, a condenser lens 12 for condensing light from the light source 11, an objective lens 13, a barrier filter 14 for extracting the fluorescent light from the light out of a sample 16, and an imaging device 18. The microscope further includes: a relay lens 17 wherein a position P1' which relays a backside focal position of the objective lens 13, and which is conjugate with the backside focal position P1 of the objective lens 13 is arranged so as to match a condensing point from the light source 11 by the condenser lens 12; a mirror 20 which is arranged at the position conjugate with a pupil position of the objective lens 13 through the relay lens 17, and which is arranged at the condensing point for reflecting only the light condensed at the condensing point; and an opening member 21 for passing the light from the sample 16 passing through the relay lens 17 toward the imaging device 18 in the vicinity of an arrangement position of the mirror 20. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、全反射蛍光顕微鏡に関する。   The present invention relates to a total reflection fluorescent microscope.

近年、生体細胞の機能解析等において、特に、細胞膜の機能を観察するための顕微鏡として、細胞膜およびその近傍からの全反射蛍光画像を取得する全反射蛍光顕微鏡（ＴＩＲＦＭ：Ｔｏｔａｌ Ｉｎｔｅｒｎａｌ Ｒｅｆｌｅｃｔｉｏｎ Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｍｉｃｒｏｓｃｏｐｙ）が注目されている。   In recent years, as a microscope for observing the function of a cell membrane in a function analysis of a living cell, a total reflection fluorescence microscope (TIRFM: Total Internal Fluorescence Microscope) that acquires a total reflection fluorescence image from the cell membrane and its vicinity has been used. Attention has been paid.

全反射蛍光顕微鏡では、レーザ光を、集光レンズを介して対物レンズ内の瞳（後側焦点位置の領域）における極く一部の所定領域に集光させ、対物レンズを介してガラス表面近傍の試料のみを対象に局所的に斜めに全反射照明させる。そして、このときにガラスと試料の境界面で試料側に数１００ｎｍ以下の僅かな範囲にしみ出すエバネッセント光を用いて試料からの蛍光を励起する。全反射蛍光顕微鏡によれば、バックグラウンド・ノイズ（散乱光など）が極めて低いため、微弱な蛍光での観察を行うことができる。
従来、この種の全反射蛍光顕微鏡としては、例えば、次の特許文献１に記載のように、ダイクロイックミラーを用いた全反射蛍光顕微鏡がある。 In a total reflection fluorescent microscope, laser light is condensed on a very small part of the pupil (region at the rear focal position) in the objective lens through a condenser lens, and near the glass surface through the objective lens. Only the sample of the sample is locally totally reflected and illuminated obliquely. At this time, the fluorescence from the sample is excited using evanescent light that oozes out to a slight range of several hundred nm or less on the sample side at the interface between the glass and the sample. According to the total reflection fluorescence microscope, since background noise (scattered light or the like) is extremely low, observation with weak fluorescence can be performed.
Conventionally, as this type of total reflection fluorescent microscope, there is a total reflection fluorescent microscope using a dichroic mirror, for example, as described in Patent Document 1 below.

図７は従来の特許文献１に記載されているタイプの全反射蛍光顕微鏡の要部概略構成図である。
図７に示した全反射蛍光顕微鏡は、光源５１と、集光レンズ５２と、対物レンズ５３と、バリアフィルタ５４を有している。
集光レンズ５２は、光源５１から出射されたレーザ光を対物レンズ５３内の瞳（後側焦点位置の領域）における光軸からシフトした極く一部のスポット状の所定領域Ｐ１に集光させるように設けられている。対物レンズ５３は、集光レンズ５２を介して集光されたレーザ光をガラス５５表面近傍の試料（標本）５６のみを対象に局所的に斜めに全反射照明するように構成されている。 FIG. 7 is a schematic configuration diagram of the main part of a total reflection fluorescent microscope of the type described in Patent Document 1.
The total reflection fluorescent microscope shown in FIG. 7 includes a light source 51, a condenser lens 52, an objective lens 53, and a barrier filter 54.
The condensing lens 52 condenses the laser light emitted from the light source 51 onto a very small spot-shaped predetermined region P1 shifted from the optical axis in the pupil (region at the rear focal position) in the objective lens 53. It is provided as follows. The objective lens 53 is configured to locally and totally totally illuminate the laser beam collected through the condenser lens 52 only on the sample (specimen) 56 near the surface of the glass 55.

また、集光レンズ５２と対物レンズ５３との間には、集光レンズ５２を経たレーザ光を対物レンズ５３に導くとともに、全反射照明により励起され、対物レンズ５３を経た蛍光の有効光束領域をカバーする大きさの入射面を有し、標本５６からの入射光のうち、標本５６で全反射され、対物レンズ５３を経た発散途中の戻り光をカットし、所望の蛍光を透過するダイクロイックミラー５７が設けられている。
バリアフィルタ５４は、ダイクロイックミラー５７を透過した光のうち、僅かに残る蛍光以外の光を反射し、蛍光のみを撮像素子等の蛍光検出手段５８側に透過するように構成されている。 In addition, between the condensing lens 52 and the objective lens 53, the laser light that has passed through the condensing lens 52 is guided to the objective lens 53, and an effective luminous flux region of fluorescence that is excited by total reflection illumination and passes through the objective lens 53 A dichroic mirror 57 which has an incident surface of a size to cover, cuts the return light in the middle of divergence, which is totally reflected by the sample 56 from the sample 56, passes through the objective lens 53, and transmits desired fluorescence. Is provided.
The barrier filter 54 is configured to reflect a small amount of light other than fluorescence remaining among the light transmitted through the dichroic mirror 57 and transmit only the fluorescence to the fluorescence detection means 58 side such as an image sensor.

このように構成された全反射蛍光顕微鏡では、光源５１から出射したレーザ光は、集光レンズ５２を介して収束光となり、途中、ダイクロイックミラー５７で反射され、対物レンズ５３内の瞳におけるスポット状の所定領域Ｐ１に集光し、対物レンズ５３を介して標本５６を全反射照明する。すると、全反射照明する際にしみ出したエバネッセント光により標本５６から蛍光が励起される。励起された蛍光は、対物レンズ５３を経てダイクロイックミラー５７を透過し、バリアフィルタ５４を介して僅かに残る所望の蛍光波長以外の光が除去され、結像レンズ６２、蛍光検出手段５８を介して取得される。また、標本５６で全反射された戻り光は、対物レンズ５３内の瞳におけるスポット状の所定領域Ｐ１とは異なる所定領域Ｐ２に集光し、対物レンズ５３を経てダイクロイックミラー５７で反射されて、蛍光検出手段５８への入射が阻止される。
これにより、全反射照明によって励起された所望の蛍光のみが取得される。 In the total reflection fluorescent microscope configured as described above, the laser light emitted from the light source 51 becomes convergent light via the condenser lens 52, and is reflected by the dichroic mirror 57 on the way, and is spot-like at the pupil in the objective lens 53. The sample 56 is condensed on the predetermined region P1 and the sample 56 is totally reflected through the objective lens 53. Then, the fluorescence is excited from the specimen 56 by the evanescent light that oozes out during the total reflection illumination. Excited fluorescence passes through the dichroic mirror 57 through the objective lens 53, and light other than the desired fluorescence wavelength remaining through the barrier filter 54 is removed, and through the imaging lens 62 and the fluorescence detection means 58. To be acquired. The return light totally reflected by the specimen 56 is condensed on a predetermined area P2 different from the spot-shaped predetermined area P1 in the pupil in the objective lens 53, reflected by the dichroic mirror 57 through the objective lens 53, Incident to the fluorescence detection means 58 is blocked.
Thereby, only the desired fluorescence excited by the total reflection illumination is acquired.

しかし、ダイクロイックミラーは、分光特性の立ち上がりの波長幅がある程度広くなる。
このため、図７に示したタイプの全反射蛍光顕微鏡の場合、所望波長領域以外の光の透過を完全に阻止するような透過率特性を有するダイクロイックミラーを用いると、全反射された戻り光とともに観察に必要な蛍光も相当量カットされてしまう。これでは、得られる微弱な蛍光の光量がより少なくなり、観察画像が暗くなって観察し難くなる。
一方、所望波長領域の光を極力透過させるような透過率特性を有するダイクロイックミラーを用いると、所望波長領域以外の不要な光も相当量透過させてしまう。これでは、微弱な蛍光に対して他の強い光がノイズとなって観察画像が白っぽくなり観察し難くなる。
このように、ダイクロイックミラーを用いた、特許文献１に記載されているようなタイプの全反射蛍光顕微鏡には、得られる微弱な蛍光の光量のロスが大きく、或いは、不要な光が混入する度合いが高く観察に難くなるという問題があった。 However, in the dichroic mirror, the wavelength width at which the spectral characteristics rise is widened to some extent.
For this reason, in the case of the total reflection fluorescent microscope of the type shown in FIG. 7, when a dichroic mirror having a transmittance characteristic that completely blocks transmission of light outside the desired wavelength region is used, together with the totally reflected return light, A considerable amount of fluorescence necessary for observation is also cut off. This reduces the amount of weak fluorescent light obtained and makes the observation image dark and difficult to observe.
On the other hand, when a dichroic mirror having a transmittance characteristic that transmits light in a desired wavelength region as much as possible is used, a considerable amount of unnecessary light outside the desired wavelength region is transmitted. In this case, other strong light with respect to weak fluorescence becomes noise, and the observation image becomes whitish and difficult to observe.
As described above, the total reflection fluorescent microscope of the type described in Patent Document 1 using the dichroic mirror has a large loss of the amount of weak fluorescent light obtained or a degree in which unnecessary light is mixed. There was a problem that it was difficult to observe.

しかるに、ダイクロイックミラーを用いない全反射蛍光顕微鏡が、例えば、次の特許文献２に提案されている。
図８は従来の特許文献２に記載されているタイプの全反射蛍光顕微鏡の要部概略構成図である。
図８に示した全反射蛍光顕微鏡では、集光レンズ５２と対物レンズ５３との間に、集光レンズ５２を経た収束途中のレーザ光を対物レンズ５３の瞳（後側焦点位置の領域）における光軸からシフトした極く一部のスポット状の所定領域Ｐ１に導くミラー５９と、標本５６からの入射光のうち、標本５６で全反射され、対物レンズ５３を経た発散途中の戻り光をカットするためのミラー６０とが、全反射照明により励起され対物レンズ５３を経た蛍光の有効光束領域内の一部の所定領域Ｐ２に、それぞれ別個に配置して構成されている。 However, a total reflection fluorescence microscope that does not use a dichroic mirror is proposed in, for example, the following Patent Document 2.
FIG. 8 is a schematic configuration diagram of a main part of a total reflection fluorescent microscope of the type described in Patent Document 2.
In the total reflection fluorescence microscope shown in FIG. 8, the laser beam that is in the process of convergence after passing through the condenser lens 52 is passed between the condenser lens 52 and the objective lens 53 in the pupil (region of the rear focal position) of the objective lens 53. Of the incident light from the sample 56 and the mirror 59 guided to a very small part of the spot-like region P1 shifted from the optical axis, the reflected light that is totally reflected by the sample 56 and passes through the objective lens 53 is cut off. The mirror 60 is configured to be separately arranged in a predetermined area P2 in the effective luminous flux area of the fluorescence excited by the total reflection illumination and passed through the objective lens 53.

このように構成された全反射蛍光顕微鏡では、光源５１から出射したレーザ光は、集光レンズ５２を介して収束光となり、途中、ミラー５９で反射され、対物レンズ５３内の瞳におけるスポット状の所定領域Ｐ１に集光し、対物レンズ５３を介して標本５６を全反射照明する。すると、全反射照明する際にしみ出したエバネッセント光により標本５６から蛍光が励起される。励起された蛍光は、対物レンズ５３を経て、ミラー５９，６０で遮断される以外、バリアフィルタ５４に入射し、バリアフィルア５４を介して僅かに残る所望の蛍光波長以外の光が除去され、結像レンズ６２、蛍光検出手段５８を介して取得される。また、標本５６で全反射された戻り光は、対物レンズ５３内の瞳におけるスポット状の所定領域Ｐ１とは異なる所定領域Ｐ２に集光し、対物レンズ５３を経てミラー６０で反射されることによって蛍光検出手段５８とは異なる方向に偏向されて、蛍光検出手段５８への入射が阻止される。
従って、図８に示した特許文献２に記載されたようなタイプの全反射蛍光顕微鏡によれば、全反射照明により励起された蛍光の光束有効領域全体に図７に示したようなダイクロイックミラーを配置しないで済むので、その分、観察に必要な蛍光がカットされずに済み、微弱な蛍光の観察に有利となる。
特開２００１−２７２６０６号公報 特開２００３−１４００５２号公報 In the total reflection fluorescent microscope configured as described above, the laser light emitted from the light source 51 becomes convergent light via the condenser lens 52, and is reflected by the mirror 59 on the way, and is spot-shaped at the pupil in the objective lens 53. The light is condensed on the predetermined area P 1 and the sample 56 is totally reflected and illuminated through the objective lens 53. Then, the fluorescence is excited from the specimen 56 by the evanescent light that oozes out during the total reflection illumination. Excited fluorescence passes through the objective lens 53 and is blocked by the mirrors 59 and 60, and enters the barrier filter 54. Light other than the desired fluorescence wavelength remaining through the barrier filter 54 is removed to form an image. Obtained through the lens 62 and the fluorescence detection means 58. The return light totally reflected by the specimen 56 is condensed on a predetermined region P2 different from the spot-shaped predetermined region P1 in the pupil in the objective lens 53, and reflected by the mirror 60 through the objective lens 53. The light is deflected in a direction different from that of the fluorescence detection means 58, and the incidence to the fluorescence detection means 58 is blocked.
Therefore, according to the total reflection fluorescent microscope of the type described in Patent Document 2 shown in FIG. 8, the dichroic mirror as shown in FIG. 7 is provided on the entire effective luminous flux region excited by total reflection illumination. Since it is not necessary to arrange them, the fluorescence necessary for observation is not cut accordingly, which is advantageous for observation of weak fluorescence.
JP 2001-272606 A Japanese Patent Laid-Open No. 2003-140052

しかし、図８に示したような特許文献２に記載されたタイプの全反射顕微鏡では、集光レンズ５２を介して対物レンズ５３の瞳におけるスポット状の所定領域Ｐ１に結像させるまでの途中の収束光路と、全反射照明により励起され対物レンズ５３を経た蛍光の有効光束領域内とが重なる所定領域にミラー５９，６０が配置されている。そして、ミラー５９は、集光レンズ５２を経た収束途中のレーザ光束を、対物レンズ５３の瞳におけるスポット状の所定領域Ｐ１に導くために相当量の面積を必要とする。また、ミラー６０は、標本５６で全反射され対物レンズ５３内の瞳の所定領域Ｐ２で集光され、対物レンズ５３を経た発散途中の戻り光をカットするために相当量の面積を必要とする。このため、全反射照明により励起された蛍光の有効光束領域において相当面積の光束領域が、ミラー５９やミラー６０によって遮られてしまうことになる。このため、特許文献２に記載された全反射顕微鏡を用いても、観察に必要な蛍光が相当量カットされ、光量のロスが多く出てしまい、微弱な蛍光を効率良く取得することが出来なかった。   However, in the total reflection microscope of the type described in Patent Document 2 as shown in FIG. 8, it is in the middle until an image is formed on the spot-shaped predetermined region P <b> 1 in the pupil of the objective lens 53 via the condenser lens 52. Mirrors 59 and 60 are arranged in a predetermined area where the convergent light path and the fluorescent effective light beam area excited by total reflection illumination and passed through the objective lens 53 overlap. The mirror 59 requires a considerable amount of area in order to guide the converging laser light beam that has passed through the condenser lens 52 to the spot-like predetermined region P1 in the pupil of the objective lens 53. Further, the mirror 60 is totally reflected by the sample 56 and collected in a predetermined region P2 of the pupil in the objective lens 53, and requires a considerable amount of area in order to cut the return light in the middle of the divergence passing through the objective lens 53. . For this reason, in the effective luminous flux region of the fluorescence excited by the total reflection illumination, the luminous flux region having a considerable area is blocked by the mirror 59 and the mirror 60. For this reason, even if the total reflection microscope described in Patent Document 2 is used, a considerable amount of fluorescence necessary for observation is cut, a large amount of light loss occurs, and weak fluorescence cannot be efficiently acquired. It was.

本発明は、上記課題を鑑みてなされたものであり、全反射照明により励起された蛍光のロスを極力無くして、蛍光を効率よく取得可能な全反射蛍光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 the above problems, and an object of the present invention is to provide a total reflection fluorescence microscope capable of efficiently acquiring fluorescence while minimizing loss of fluorescence excited by total reflection illumination.

上記目的を達成するため、本発明による全反射蛍光顕微鏡は、光源と、前記光源からの光を集光させる集光レンズと、対物レンズと、標本からの光から蛍光を抽出するバリアフィルタと、前記バリアフィルタを透過した光の像を撮像する撮像素子を有する全反射蛍光顕微鏡において、前記対物レンズの後側焦点位置をリレーし、該対物レンズの後側焦点位置と共役な位置が前記集光レンズによる光源からの集光点に一致するように配置されたリレーレンズと、前記集光点に配置され、該集光点に集光された光線を反射する反射部材と、前記反射部材の配置位置近傍に、前記リレーレンズを経た前記標本からの光を前記撮像素子に向けて通過させる開口部材を有することを特徴としている。   In order to achieve the above object, a total reflection fluorescence microscope according to the present invention includes a light source, a condensing lens for condensing light from the light source, an objective lens, and a barrier filter for extracting fluorescence from light from a specimen, In a total reflection fluorescent microscope having an image sensor that captures an image of light that has passed through the barrier filter, a rear focal position of the objective lens is relayed, and a position conjugate with the rear focal position of the objective lens is the condensing light. A relay lens arranged so as to coincide with a condensing point from a light source by the lens, a reflecting member arranged at the condensing point and reflecting a light beam condensed at the condensing point, and an arrangement of the reflecting member In the vicinity of the position, there is provided an opening member that allows light from the sample that has passed through the relay lens to pass toward the imaging element.

また、本発明の全反射蛍光顕微鏡においては、前記開口部材が、透過率９０％以上のガラスで構成され、前記反射部材が、前記開口部材のガラス面上に設けられているのが好ましい。   Moreover, in the total reflection fluorescence microscope of this invention, it is preferable that the said opening member is comprised with the glass of 90% or more of transmittance | permeability, and the said reflection member is provided on the glass surface of the said opening member.

また、本発明の全反射蛍光顕微鏡においては、標本で全反射された光の前記撮像素子への到達を阻止する阻止手段を、該標本で全反射された光が集光する位置又は該集光する位置と共役な位置に備えるのが好ましい。   In the total reflection fluorescent microscope of the present invention, the blocking means for blocking the light totally reflected by the specimen from reaching the image sensor is a position where the light totally reflected by the specimen is collected or the light collection. It is preferable to provide at a position conjugate with the position to be performed.

また、本発明の全反射蛍光顕微鏡においては、前記反射部材が、前記集光点に対応した大きさの径を持つ点状の反射面を有しているのが好ましい。   In the total reflection fluorescent microscope of the present invention, it is preferable that the reflecting member has a point-like reflecting surface having a diameter corresponding to the condensing point.

また、本発明の全反射蛍光顕微鏡においては、前記反射部材が、前記集光点に対応した大きさの径を持ち、かつ、前記集光点の位置から通常の落射照明をする場合における光軸にかけて延びたライン状の反射面を有しているのが好ましい。   In the total reflection fluorescent microscope of the present invention, the reflecting member has a diameter corresponding to the condensing point, and an optical axis in the case of normal epi-illumination from the position of the condensing point. It is preferable to have a line-like reflecting surface extending over the distance.

また、本発明の全反射蛍光顕微鏡においては、前記反射部材が、ダイクロイックミラーであるのが好ましい。   In the total reflection fluorescent microscope of the present invention, it is preferable that the reflecting member is a dichroic mirror.

また、本発明の全反射蛍光顕微鏡においては、前記阻止手段が、遮光部材であるのが好ましい。   In the total reflection fluorescent microscope of the present invention, it is preferable that the blocking means is a light shielding member.

また、本発明の全反射蛍光顕微鏡においては、前記阻止手段が、ダイクロイックミラーであるのが好ましい。   In the total reflection fluorescent microscope of the present invention, it is preferable that the blocking means is a dichroic mirror.

本発明の全反射蛍光顕微鏡によれば、全反射照明により励起された蛍光のロスを極力無くして、蛍光を効率よく取得可能な全反射蛍光顕微鏡が得られる。   According to the total reflection fluorescence microscope of the present invention, it is possible to obtain a total reflection fluorescence microscope capable of efficiently acquiring fluorescence while minimizing the loss of fluorescence excited by total reflection illumination.

図１は本発明の一実施形態にかかる全反射蛍光顕微鏡の概略構成図、図２は図１の全反射蛍光顕微鏡に用いられる開口部材と開口部材近傍に設けられる反射部材とを示す説明図である。   FIG. 1 is a schematic configuration diagram of a total reflection fluorescence microscope according to an embodiment of the present invention, and FIG. 2 is an explanatory view showing an opening member used in the total reflection fluorescence microscope of FIG. 1 and a reflection member provided in the vicinity of the opening member. is there.

本実施形態の全反射蛍光顕微鏡は、光源１１と、集光レンズ１２と、対物レンズ１３と、バリアフィルタ１４と、撮像素子を備えた撮像手段１８と、リレーレンズ１７と、ミラー１９と、反射部材２０と、開口部材２１と、阻止手段２２を有している。なお、図中、１５はカバーガラス、１６は標本、６１はイマージョンオイル、２３は標本１６で励起された蛍光を撮像素子上に結像するための撮像レンズである。
光源１１は、レーザ装置で構成されている。なお、レーザ装置は、所定波長を出力するように構成されたもの、所望の波長のレーザを適宜選択して出力されるように構成されたもののいずれも適用できる。
集光レンズ１２は、光源１１からのレーザ光を対物レンズ１３内の瞳（後側焦点位置の領域）における光軸からシフトした極く一部のスポット状の所定領域Ｐ１と共役な所定位置Ｐ１’に集光するように配置されている。
対物レンズ１３は、瞳面におけるスポット状の所定領域Ｐ１に集光された光源１１からの入射光をカバーガラス１５表面近傍の標本１６のみを対象に局所的に斜めに全反射照射するとともに、標本１６で全反射された戻り光を瞳面における所定のスポット状の所定領域Ｐ１とは異なる所定領域Ｐ２に集光する。また、リレーレンズ１７、撮像レンズ２３と相俟って、標本１６で励起された蛍光を撮像素子１８の撮像面上で結像するように配置されている。
バリアフィルタ１４は、標本１６からの光のうち所望の蛍光波長のみを透過し、それ以外の波長の光を除去するように構成されている。 The total reflection fluorescent microscope of the present embodiment includes a light source 11, a condenser lens 12, an objective lens 13, a barrier filter 14, an image pickup means 18 provided with an image pickup device, a relay lens 17, a mirror 19, and a reflection. The member 20, the opening member 21, and the blocking means 22 are included. In the figure, 15 is a cover glass, 16 is a specimen, 61 is immersion oil, and 23 is an imaging lens for imaging the fluorescence excited by the specimen 16 on the imaging element.
The light source 11 is composed of a laser device. As the laser device, either a laser device configured to output a predetermined wavelength or a laser device configured to select and output a laser having a desired wavelength can be applied.
The condensing lens 12 has a predetermined position P1 conjugate with a very small part of the spot-shaped predetermined area P1 obtained by shifting the laser light from the light source 11 from the optical axis in the pupil (rear focal position area) in the objective lens 13. It is arranged to condense.
The objective lens 13 irradiates the incident light from the light source 11 focused on the spot-shaped predetermined region P1 on the pupil plane locally and totally totally with respect to only the sample 16 near the surface of the cover glass 15, and the sample. The return light totally reflected at 16 is condensed on a predetermined area P2 different from the predetermined spot-shaped predetermined area P1 on the pupil plane. Further, in combination with the relay lens 17 and the imaging lens 23, the fluorescence excited by the specimen 16 is arranged on the imaging surface of the imaging element 18.
The barrier filter 14 is configured to transmit only a desired fluorescence wavelength in the light from the specimen 16 and remove light of other wavelengths.

リレーレンズ１７は、対物レンズ１３内の瞳（後側焦点位置の領域）を、集光レンズ２３による光源１１からの集光点にリレーし、対物レンズ１３内の瞳におけるスポット状の所定領域Ｐ１と共役な位置Ｐ１’が、集光レンズ１２により集光される、光源１１からの集光点に一致するように配置されている。
反射部材２０は、リレーレンズ１７を介した対物レンズ１３内の瞳におけるスポット状の所定領域Ｐ１と共役な位置Ｐ１’(即ち、上記光源１１からの集光点に一致した位置)に配置され、集光レンズ１２により集光される、光源１１からの集光点に集光された光線を反射するように構成されている。そして、反射部材２０は、図２に示すように、前記集光点に対応した大きさの径を持ち、且つ、集光点の位置から光軸の中心にかけて延びたライン状に形成されたミラー面を有している。
開口部材２１は、透過率９０％以上のガラスで構成されており、リレーレンズ１７を経た標本１６からの光を撮像素子１８に向けて透過させるようになっている。また、反射部材２０は、開口部材２１のガラス面に配置されている。
阻止部材２２は、遮光部材で構成されており、標本１６で全反射された戻り光が集光される対物レンズ１３の瞳における所定領域Ｐ２に配置されている。なお、遮光部材２２の配置は、対物レンズ内に限定されるものではなく、対物レンズ１３内の瞳における所定領域Ｐ２と共役な位置であれば、どこに配置してもよい。例えば、開口部材２１のガラス面上又はその近傍に、対物レンズ１３内の瞳における所定領域Ｐ２と共役な位置が位置する場合には、遮光部材２２を開口部材２１のガラス面上又はその近傍に配置してもよい。 The relay lens 17 relays the pupil (region of the rear focal position) in the objective lens 13 to a condensing point from the light source 11 by the condensing lens 23, and a spot-like predetermined region P 1 in the pupil in the objective lens 13. And a position P1 ′ conjugate with the condensing lens 12 are arranged so as to coincide with a condensing point from the light source 11 that is condensed by the condensing lens 12.
The reflecting member 20 is disposed at a position P1 ′ conjugate with the spot-like predetermined region P1 in the pupil in the objective lens 13 via the relay lens 17 (that is, a position that coincides with the condensing point from the light source 11). It is configured to reflect the light beam collected by the condensing lens 12 and condensed at the condensing point from the light source 11. As shown in FIG. 2, the reflecting member 20 has a diameter corresponding to the condensing point, and is a mirror formed in a line shape extending from the condensing point position to the center of the optical axis. Has a surface.
The opening member 21 is made of glass having a transmittance of 90% or more, and transmits light from the specimen 16 that has passed through the relay lens 17 toward the image sensor 18. The reflecting member 20 is disposed on the glass surface of the opening member 21.
The blocking member 22 is composed of a light blocking member, and is disposed in a predetermined region P2 in the pupil of the objective lens 13 where the return light totally reflected by the sample 16 is collected. The arrangement of the light shielding member 22 is not limited to the objective lens, and may be arranged anywhere as long as the position is conjugate with the predetermined region P2 in the pupil in the objective lens 13. For example, when a position conjugate with the predetermined region P2 in the pupil in the objective lens 13 is positioned on or near the glass surface of the aperture member 21, the light shielding member 22 is positioned on or near the glass surface of the aperture member 21. You may arrange.

このように構成された本実施形態の全反射蛍光顕微鏡によれば、光源１１から出射したレーザ光は、集光レンズ１２を介して、反射部材２０上の所定スポットＰ２に集光し、反射部材２０で反射される。反射部材２０で反射された光は、リレーレンズ１７を介して収束光となり、ミラー１９で反射された後、対物レンズ１３の瞳におけるスポット状の所定領域Ｐ１に集光する。集光した光は、対物レンズ１３を介して、カバーガラス１５の表面近傍の標本１６を局所的に斜めに全反射照明する。標本１６に照射された光のほとんどは、標本１６で全反射される。また、照射された光のうち、僅かな光が、カバーガラス１５と標本１６の境界面で標本１６側に数１００ｎｍ以下の僅かな範囲にエバネッセント光としてしみ出して標本１６を励起する。励起された標本１６からは蛍光が発する。標本１６で全反射された戻り光は、対物レンズ１３の瞳における所定領域Ｐ２に集光され、所定領域Ｐ２に設けられた遮光部材２２で遮光される。標本１６から発した蛍光は、対物レンズ１３、ミラー１９、リレーレンズ１７を経て、開口部材２０に到達する。開口部材２０に到達した光は、ミラー１９が設けられたライン状の領域を除いて開口部材２０を透過し、バリアフィルタ１４を介して所望の蛍光波長以外の光が除去されるとともに、所望の蛍光のみが抽出され、撮像レンズ２３を経て、撮像素子１８の撮像面上に結像される。撮像素子１８は結像された蛍光像を撮像する。撮像した蛍光画像は、全反射顕微鏡において一般的に用いられている画像処理手段、画像表示装置等（図示省略）を介して観察される。   According to the total reflection fluorescence microscope of the present embodiment configured as described above, the laser light emitted from the light source 11 is condensed on the predetermined spot P2 on the reflection member 20 via the condenser lens 12, and is reflected on the reflection member. 20 is reflected. The light reflected by the reflecting member 20 becomes convergent light via the relay lens 17, is reflected by the mirror 19, and then converges on a spot-like predetermined region P 1 in the pupil of the objective lens 13. The condensed light illuminates the sample 16 near the surface of the cover glass 15 locally and totally totally through the objective lens 13. Most of the light applied to the specimen 16 is totally reflected by the specimen 16. Further, of the irradiated light, a small amount of light oozes out as evanescent light to a slight range of several hundred nm or less on the sample 16 side at the boundary surface between the cover glass 15 and the sample 16 to excite the sample 16. Fluorescence is emitted from the excited specimen 16. The return light totally reflected by the specimen 16 is condensed on a predetermined area P2 in the pupil of the objective lens 13, and is blocked by the light shielding member 22 provided in the predetermined area P2. The fluorescence emitted from the specimen 16 reaches the aperture member 20 through the objective lens 13, the mirror 19, and the relay lens 17. The light that has reached the aperture member 20 is transmitted through the aperture member 20 except for the line-shaped region where the mirror 19 is provided, and light other than the desired fluorescence wavelength is removed through the barrier filter 14 and the desired light is removed. Only fluorescence is extracted and imaged on the imaging surface of the image sensor 18 through the imaging lens 23. The image sensor 18 captures the formed fluorescent image. The captured fluorescent image is observed through image processing means, an image display device, etc. (not shown) generally used in a total reflection microscope.

このとき、本実施形態の全反射蛍光顕微鏡によれば、対物レンズ１３の瞳位置と共役な位置が、集光レンズ１２の集光点と一致するようにリレーレンズ１７を設け、且つ、その集光点に集光された光線を反射するように反射部材２０を配置したので、反射部材２０の径を集光レンズ１２の回折限界程度にまで最小限に小さくすることができる。そして、反射部材２０の配置位置近傍に開口部材２０を設けてリレーレンズ１７を経た試料１６からの光を通過させるようにしたので、試料からの光のうちミラー１９で遮られる光量のロスを最小にして、ほぼ最大光量の蛍光を取得することができる。   At this time, according to the total reflection fluorescent microscope of the present embodiment, the relay lens 17 is provided so that the position conjugate with the pupil position of the objective lens 13 coincides with the condensing point of the condensing lens 12, and Since the reflecting member 20 is disposed so as to reflect the light beam collected at the light spot, the diameter of the reflecting member 20 can be reduced to the minimum as much as the diffraction limit of the condensing lens 12. Since the opening member 20 is provided in the vicinity of the arrangement position of the reflecting member 20 so as to allow the light from the sample 16 that has passed through the relay lens 17 to pass therethrough, the loss of the amount of light blocked by the mirror 19 among the light from the sample is minimized. In this way, it is possible to obtain the fluorescence with the maximum amount of light.

この点に関し、従来の特許文献２に記載のようなタイプの全反射蛍光顕微鏡における全反射蛍光の有効光束径に対するミラーで遮光される面積の割合をシミュレートして、本発明と比較説明する。
ここでは、図８に示した全反射蛍光顕微鏡の構成において、対物レンズ５３、ミラー５９，６０、集光レンズ５２は、それぞれ次のような諸元値とした。これらの値は、この種の全反射蛍光顕微鏡において一般的に用いられる値である。
(1)対物レンズ５３の焦点距離：３ｍｍ（無限遠対物レンズとし、焦点距離が１８０ｍｍの結像レンズ（ここでは、集光レンズ５２）と組み合わせたときに倍率が６０倍となる対物レンズ）、
(2)対物レンズ５３の瞳位置からミラー５９，６０までの距離：６０ｍｍ、
(3)対物レンズ５３の開口数ＮＡ：１．４５、
(4)対物レンズ５３の視野数：Φ１１ｍｍ（半径で５．５ｍｍ）、
(5)集光レンズ５２の焦点距離：１８０ｍｍ In this regard, the ratio of the area shielded by the mirror with respect to the effective luminous flux diameter of the total reflection fluorescence in the type of total reflection fluorescence microscope of the type described in Patent Document 2 will be simulated and compared with the present invention.
Here, in the configuration of the total reflection fluorescent microscope shown in FIG. 8, the objective lens 53, the mirrors 59 and 60, and the condenser lens 52 have the following specification values, respectively. These values are values generally used in this type of total reflection fluorescent microscope.
(1) Focal length of the objective lens 53: 3 mm (an objective lens having an infinite objective and a magnification of 60 times when combined with an imaging lens having a focal length of 180 mm (here, the condenser lens 52)),
(2) Distance from the pupil position of the objective lens 53 to the mirrors 59 and 60: 60 mm,
(3) Numerical aperture NA of the objective lens 53: 1.45,
(4) Number of fields of the objective lens 53: Φ11 mm (radius is 5.5 mm),
(5) Focal length of condenser lens 52: 180 mm

対物レンズ５３の瞳径は、上記(1)と(3)より、
３ｍｍ（対物レンズの焦点距離）×１．４５（ＮＡ）×２＝Φ８．７ｍｍ
である。
従って、ミラー５９，６０の位置での対物レンズ５３の有効光束径は、上記(2)、(4)、(5)より、
Φ８．７ｍｍ（対物レンズ５３の瞳径）＋２×５．５ｍｍ（最大像高）／１８０ｍｍ（集光レンズ５２の焦点距離）×６０ｍｍ（ミラー５９，６０までの距離）
＝Φ１２．３７ｍｍ
となる。
これを図４に概念的に示す。
なお、ここでのミラー５９，６０の位置での対物レンズ５３の有効光束径は、便宜上、光軸に対し垂直な面でのものとして算出してある。実際には、ミラー５９，６０は、夫々光軸に対し４５の角度で傾斜して設けられている。 From the above (1) and (3), the pupil diameter of the objective lens 53 is
3 mm (focal length of objective lens) × 1.45 (NA) × 2 = Φ8.7 mm
It is.
Therefore, the effective luminous flux diameter of the objective lens 53 at the positions of the mirrors 59 and 60 is as follows from the above (2), (4) and (5).
Φ8.7 mm (pupil diameter of the objective lens 53) + 2 × 5.5 mm (maximum image height) / 180 mm (focal length of the condenser lens 52) × 60 mm (distance to the mirrors 59 and 60)
= Φ12.37mm
It becomes.
This is conceptually illustrated in FIG.
Note that the effective luminous flux diameter of the objective lens 53 at the positions of the mirrors 59 and 60 here is calculated on the plane perpendicular to the optical axis for convenience. Actually, the mirrors 59 and 60 are provided at an angle of 45 with respect to the optical axis.

次に、ミラー５９，６０の位置での全反射照明光の光線径及び全反射照明された戻り光の光線径は、それぞれ、
２×５．５ｍｍ（最大像高）／１８０ｍｍ（集光レンズ５２の焦点距離）×６０ｍｍ（ミラー５９，６０までの距離）＝Φ３．６７ｍｍ
となる。
これを図５に概念的に示す。 Next, the light beam diameter of the total reflection illumination light at the positions of the mirrors 59 and 60 and the light beam diameter of the return light subjected to the total reflection illumination are respectively
2 × 5.5 mm (maximum image height) / 180 mm (focal length of condenser lens 52) × 60 mm (distance to mirrors 59 and 60) = Φ3.67 mm
It becomes.
This is conceptually illustrated in FIG.

これらのことから、ミラー５９，６０の位置での全反射照明光線径及び全反射照明された戻り光の光線径の対物レンズ５３の有効光束径に対する比率は、
２×（３．６７／１２．３７）2＝０．１７６
となる。
ここで、ミラー５９，６０は全反射照明されて励起された蛍光のみを観察し、それ以外の光を除去するためにミラー面に入射した光を透過させることはない。
このため、図８の全反射蛍光顕微鏡を基にシミュレートした図４，図５の構成では、ミラー５９，６０により約１８％もの光量ロスが生じることになる。
また、対物レンズ５３の瞳位置とは異なる位置であって、対物レンズ５３の有効光束径内にミラー５９，６０を配置するため、ミラー５９，６０により視野の欠け、解像力の劣化も生じる。 From these facts, the ratio of the total reflection illumination light beam diameter at the positions of the mirrors 59 and 60 and the light beam diameter of the return light subjected to the total reflection illumination to the effective light beam diameter of the objective lens 53 is:
2 × (3.67 / 12.37) 2 = 0.176
It becomes.
Here, the mirrors 59 and 60 observe only the fluorescent light that is excited by being totally reflected and does not transmit the light incident on the mirror surface in order to remove other light.
For this reason, in the configuration of FIGS. 4 and 5 simulated based on the total reflection fluorescence microscope of FIG.
In addition, since the mirrors 59 and 60 are disposed at positions different from the pupil position of the objective lens 53 and within the effective light beam diameter of the objective lens 53, the mirrors 59 and 60 cause the field of view to be lost and the resolution to deteriorate.

また、実際には、全反射照明観察を行う顕微鏡においては、照明光の標本面への入射角度を変えることにより、全反射照明と通常の落射照明とに切り替えることができるようにすることが望まれる。通常の落射照明を行うためには、照明光を対物レンズに向けて反射する反射部材を対物レンズの光軸上に配置されていることが必要となる。このため、図６(a)に示す２つのミラー５９，６０に変えて、図６(b)に示すように、全反射照明光線径ａ及び全反射照明された戻り光の光線径ｂに加えて、対物レンズの有効光束径の中心領域ｃをもカバー可能な細長形状のミラー６１を備える必要がある。
このようにすると、ミラーによる光量の損失の割合が更に大きくなり、視野の欠けもより一層大きくなってしまう。 In fact, in a microscope that performs total reflection illumination observation, it is desirable to be able to switch between total reflection illumination and normal epi-illumination by changing the angle of incidence of illumination light on the specimen surface. It is. In order to perform normal epi-illumination, it is necessary that a reflecting member that reflects the illumination light toward the objective lens is disposed on the optical axis of the objective lens. Therefore, instead of the two mirrors 59 and 60 shown in FIG. 6A, as shown in FIG. 6B, in addition to the total reflection illumination beam diameter a and the total reflection illumination beam diameter b. Thus, it is necessary to provide an elongated mirror 61 that can also cover the central region c of the effective light beam diameter of the objective lens.
If it does in this way, the ratio of the loss of the light quantity by a mirror will become still larger, and the loss of visual field will become still larger.

これに対し、本実施形態の全反射蛍光顕微鏡では、対物レンズ１３の瞳位置と共役な位置に前記集光点に集光された光線を反射するように反射部材２０を配置したので、図３(a)に示すように、反射部材２０の径を集光レンズ１２の回折限界程度（例えば、Φ２０μｍ程度）まで最小限に小さくすることができる。その場合、通常の落射照明を考慮せずに全反射照明のみを考慮すれば、反射部材２０はほぼ点状の大きさで足り、反射部材２０の位置での全反射照明光線径の対物レンズ１３の有効光束径に対する比率を、２％程度に抑えることができる。
しかも、本実施形態の全反射蛍光顕微鏡によれば、標本１６で全反射された光の撮像素子への到達を阻止する阻止手段としての遮光部材２２を、標本１６で全反射された光が集光する位置（所定領域Ｐ２）に備えたので、開口部材２１のガラス面上には反射部材２０以外に設けずに済ますことができ、より一層光量のロスを抑えることができ、かつ、視野かけのない観察像を得ることができる。
また、全反射照明と通常の落射照明とに切り替えることができるように顕微鏡を構成したとしても、反射部材２０は、集光点に対応した大きさの径を持つライン形状に形成すれば足りるので、図４〜図６、図８を用いて説明した特許文献２に記載されたタイプの全反射蛍光顕微鏡と比較して、光量のロスを格段と抑えることができ、かつ、視野かけのない観察像を得ることができる（図３(b)）。
なお、本実施形態の全反射蛍光顕微鏡において、遮光部材２２を、開口部材２１近傍における、標本１６で全反射された光が集光する位置（所定領域Ｐ２）と共役な位置に設けた場合も、遮光部材２２の径を、反射部材２０と同様、対物レンズ１３の回折限界程度（例えば、Φ２０μｍ程度）まで最小限に小さくすることができる。 On the other hand, in the total reflection fluorescent microscope of the present embodiment, the reflecting member 20 is arranged so as to reflect the light beam collected at the condensing point at a position conjugate with the pupil position of the objective lens 13, so that FIG. As shown in (a), the diameter of the reflecting member 20 can be minimized to the diffraction limit of the condenser lens 12 (for example, about Φ20 μm). In that case, if only the total reflection illumination is considered without considering the normal epi-illumination, the reflecting member 20 needs only to have a dot-like size, and the objective lens 13 having the total reflection illumination beam diameter at the position of the reflection member 20 is sufficient. Can be suppressed to about 2%.
Moreover, according to the total reflection fluorescence microscope of the present embodiment, the light totally reflected by the specimen 16 is collected by the light blocking member 22 as a blocking means for preventing the light totally reflected by the specimen 16 from reaching the imaging device. Since it is provided at the light-emitting position (predetermined region P2), it is not necessary to provide other than the reflection member 20 on the glass surface of the opening member 21, and the loss of light quantity can be further suppressed, and the field of view is reduced An observation image with no image can be obtained.
Further, even if the microscope is configured so that it can be switched between total reflection illumination and normal epi-illumination, the reflection member 20 need only be formed in a line shape having a diameter corresponding to the focal point. Compared with the total reflection fluorescent microscope of the type described in Patent Document 2 described with reference to FIGS. 4 to 6 and FIG. 8, the loss of light amount can be remarkably suppressed, and observation with no field of view is possible. An image can be obtained (FIG. 3 (b)).
In the total reflection fluorescent microscope of the present embodiment, the light shielding member 22 may be provided at a position conjugate with the position (predetermined region P2) where the light totally reflected by the sample 16 is collected in the vicinity of the opening member 21. The diameter of the light shielding member 22 can be reduced to the minimum to the diffraction limit of the objective lens 13 (for example, about Φ20 μm), similarly to the reflection member 20.

さらに、本実施形態の全反射蛍光顕微鏡において、反射部材２０を、ダイクロイックミラーで構成するとさらに好ましい。例えば、ダイクロイックミラーを、全反射照明により励起された蛍光波長のみを透過し、それ以外の波長の透過を阻止するような透過率特性を有するように構成する。そのようにすれば、全反射された戻り光とともに観察に必要な蛍光もダイクロイックミラーで相当量カットされたとしても、ダイクロイックミラーを透過した蛍光の光量分が、反射部材２０を通常のミラーで構成した場合に比べてより多く撮像素子１８で得られる。このため、蛍光光量のロスをより一層少なくすることができる。   Furthermore, in the total reflection fluorescent microscope of the present embodiment, it is more preferable that the reflection member 20 is formed of a dichroic mirror. For example, the dichroic mirror is configured to have a transmittance characteristic that transmits only the fluorescence wavelength excited by the total reflection illumination and blocks the transmission of other wavelengths. By doing so, even if a considerable amount of fluorescence necessary for observation along with the totally reflected return light is cut by the dichroic mirror, the amount of fluorescent light transmitted through the dichroic mirror constitutes the reflecting member 20 by a normal mirror. Compared to the case, the image sensor 18 can obtain more. For this reason, the loss of the amount of fluorescence can be further reduced.

本発明の全反射蛍光顕微鏡は、全反射照明による生体細胞からの非常に微弱な蛍光を極力明るく観察することが求められる生物学、医学、薬学の分野において有用である。   The total reflection fluorescence microscope of the present invention is useful in the fields of biology, medicine, and pharmacology where it is required to observe extremely weak fluorescence from living cells by total reflection illumination as much as possible.

本発明の一実施形態にかかる全反射蛍光顕微鏡の概略構成図である。It is a schematic block diagram of the total reflection fluorescence microscope concerning one Embodiment of this invention. 図１の全反射蛍光顕微鏡に用いられる開口部材と開口部材近傍に設けられる反射部材とを示す説明図である。It is explanatory drawing which shows the aperture member used for the total reflection fluorescence microscope of FIG. 1, and the reflective member provided in the aperture member vicinity. 図１の全反射蛍光顕微鏡における開口部材及び反射部材の位置での対物レンズの有効光束領域と、全反射照明光の光線領域及び全反射照明された戻り光の光線領域との関係を示す説明図であり、(a)は全反射蛍光観察のみを行う顕微鏡として構成した場合、(b)は全反射と落射蛍光観察の両方を兼用可能な顕微鏡として構成した場合、(c)は(b)の顕微鏡において、遮光部材を、開口部材近傍における、所定領域Ｐ２と共役な位置に設けた場合を示している。Explanatory drawing which shows the relationship between the effective light beam area | region of the objective lens in the position of an aperture member and a reflection member in the total reflection fluorescence microscope of FIG. When (a) is configured as a microscope that only performs total reflection fluorescence observation, (b) is configured as a microscope that can be used for both total reflection and epifluorescence observation, (c) is (b) In the microscope, the light shielding member is provided at a position conjugate with the predetermined region P2 in the vicinity of the opening member. 本発明の比較例にかかる、図８に示した全反射蛍光顕微鏡における対物レンズの瞳位置での瞳径と、反射部材（ミラー５９，６０）の位置での対物レンズの有効光束径の関係を示す説明図である。The relationship between the pupil diameter at the pupil position of the objective lens in the total reflection fluorescence microscope shown in FIG. 8 and the effective luminous flux diameter of the objective lens at the position of the reflecting members (mirrors 59, 60) according to the comparative example of the present invention is shown. It is explanatory drawing shown. 本発明の比較例にかかる、図８に示した全反射蛍光顕微鏡における対物レンズの瞳位置での瞳径と、反射部材（ミラー５９，６０）の位置での全反射照明光の光線径及び全反射照明された戻り光の光線径との関係を示す説明図である。The pupil diameter at the pupil position of the objective lens in the total reflection fluorescent microscope shown in FIG. 8 according to the comparative example of the present invention, the beam diameter of the total reflection illumination light at the position of the reflecting member (mirrors 59, 60), and the total It is explanatory drawing which shows the relationship with the beam diameter of the return light by which reflection illumination was carried out. 本発明の比較例にかかる、図８に示した全反射蛍光顕微鏡における反射部材（ミラー５９，６０）の位置での対物レンズの有効光束領域と、反射部材（ミラー５９，６０）の位置での全反射照明光の光線領域及び全反射照明された戻り光の光線領域との関係を示す説明図であり、(a)は全反射蛍光観察のみを行う顕微鏡として構成する場合、(b)は全反射と落射蛍光観察の両方を兼用可能な顕微鏡として構成する場合を示している。The effective luminous flux area of the objective lens at the position of the reflecting member (mirrors 59, 60) in the total reflection fluorescent microscope shown in FIG. 8 and the position of the reflecting member (mirrors 59, 60) according to the comparative example of the present invention. It is an explanatory diagram showing the relationship between the light ray region of the total reflection illumination light and the light ray region of the return light that has been totally reflected illumination, (a) is configured as a microscope that only performs total reflection fluorescence observation, (b) is the total This shows a case where the microscope is configured to be used for both reflection and epifluorescence observation. 従来の特許文献１に記載されているタイプの全反射蛍光顕微鏡の要部概略構成図である。It is a principal part schematic block diagram of the type of total reflection fluorescence microscope of the type currently described in the patent document 1. FIG. 従来の特許文献２に記載されているタイプの全反射蛍光顕微鏡の要部概略構成図である。It is a principal part schematic block diagram of the type of total reflection fluorescence microscope of the type currently described in the patent document 2. FIG.

符号の説明Explanation of symbols

１１ 光源
１２ 集光レンズ
１３ 対物レンズ
１４ バリアフィルタ
１５ ガラス
１６ 標本
１７ リレーレンズ
１８ 蛍光検出手段
１９ ミラー
２０ 反射部材
２１ 開口部材（ガラス板）
２２ 遮光部材
２３ 撮像レンズ
５１ 光源
５２ 集光レンズ
５３ 対物レンズ
５４ バリアフィルタ
５５ ガラス
５６ 標本
５７ ダイクロイックミラー
５８ 蛍光検出手段
５９，６０ ミラー
６１ イマージョンオイル
６２ 結像レンズ DESCRIPTION OF SYMBOLS 11 Light source 12 Condensing lens 13 Objective lens 14 Barrier filter 15 Glass 16 Sample 17 Relay lens 18 Fluorescence detection means 19 Mirror 20 Reflective member 21 Opening member (glass plate)
22 light shielding member 23 imaging lens 51 light source 52 condensing lens 53 objective lens 54 barrier filter 55 glass 56 specimen 57 dichroic mirror 58 fluorescence detection means 59, 60 mirror 61 immersion oil 62 imaging lens

Claims (8)

光源と、前記光源からの光を集光させる集光レンズと、対物レンズと、標本からの光から蛍光を抽出するバリアフィルタと、前記バリアフィルタを透過した光の像を撮像する撮像素子を有する全反射蛍光顕微鏡において、
前記対物レンズの後側焦点位置をリレーし、該対物レンズの後側焦点位置と共役な位置が前記集光レンズによる光源からの集光点に一致するように配置されたリレーレンズと、
前記集光点に配置され、該集光点に集光された光線を反射する反射部材と、前記反射部材の配置位置近傍に、前記リレーレンズを経た前記標本からの光を前記撮像素子に向けて通過させる開口部材を有することを特徴とする全反射蛍光顕微鏡。 A light source, a condensing lens for condensing light from the light source, an objective lens, a barrier filter for extracting fluorescence from the light from the sample, and an image sensor for capturing an image of the light transmitted through the barrier filter In a total reflection fluorescence microscope,
Relay the rear focal position of the objective lens, and a relay lens arranged such that a position conjugate with the rear focal position of the objective lens coincides with a condensing point from a light source by the condenser lens;
A reflecting member that is disposed at the condensing point and reflects a light beam condensed at the condensing point, and light from the sample that has passed through the relay lens is directed toward the imaging element in the vicinity of the arrangement position of the reflecting member. And a total reflection fluorescent microscope characterized by having an opening member that allows the light to pass through. 前記開口部材が、透過率９０％以上のガラスで構成され、
前記反射部材が、前記開口部材のガラス面上に設けられていることを特徴とする請求項１に記載の全反射蛍光顕微鏡。 The opening member is made of glass having a transmittance of 90% or more,
The total reflection fluorescence microscope according to claim 1, wherein the reflection member is provided on a glass surface of the opening member. 標本で全反射された光の前記撮像素子への到達を阻止する阻止手段を、該標本で全反射された光が集光する位置又は該集光する位置と共役な位置に備えたこと特徴とする請求項１又は２に記載の全反射蛍光顕微鏡。   A blocking means for blocking the light totally reflected by the sample from reaching the image sensor at a position where the light totally reflected by the sample is collected or a position conjugate with the position where the light is collected; The total reflection fluorescence microscope according to claim 1 or 2. 前記反射部材が、前記集光点に対応した大きさの径を持つ点状の反射面を有していることを特徴とする請求項１〜３のいずれかに記載の全反射蛍光顕微鏡。   The total reflection fluorescent microscope according to claim 1, wherein the reflection member has a point-like reflection surface having a diameter corresponding to the condensing point. 前記反射部材が、前記集光点に対応した大きさの径を持ち、かつ、前記集光点の位置から通常の落射照明をする場合における光軸にかけて延びたライン状の反射面を有していることを特徴とする請求項１〜３のいずれかに記載の全反射蛍光顕微鏡。   The reflective member has a diameter corresponding to the condensing point, and has a linear reflecting surface extending from the position of the condensing point to the optical axis when performing normal epi-illumination. The total reflection fluorescent microscope according to claim 1, wherein the total reflection fluorescence microscope is provided. 前記反射部材が、ダイクロイックミラーであることを特徴とする請求項１〜５のいずれかに記載の全反射蛍光顕微鏡。   The total reflection fluorescent microscope according to claim 1, wherein the reflection member is a dichroic mirror. 前記阻止手段が、遮光部材であることを特徴とする請求項１〜６のいずれかに記載の全反射蛍光顕微鏡。   The total reflection fluorescent microscope according to claim 1, wherein the blocking means is a light shielding member. 前記阻止手段が、ダイクロイックミラーであることを特徴とする請求項１〜６のいずれかに記載の全反射蛍光顕微鏡。   The total reflection fluorescence microscope according to claim 1, wherein the blocking means is a dichroic mirror.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017522603A (en) * 2014-07-22 2017-08-10 ライカ マイクロシステムズ シーエムエス ゲゼルシャフト ミット ベシュレンクテル ハフツングＬｅｉｃａ Ｍｉｃｒｏｓｙｓｔｅｍｓ ＣＭＳ ＧｍｂＨ Method and apparatus for microscopic examination of a sample
CN107783267A (en) * 2016-08-30 2018-03-09 北京大学 Micro- amplification system

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017522603A (en) * 2014-07-22 2017-08-10 ライカ マイクロシステムズ シーエムエス ゲゼルシャフト ミット ベシュレンクテル ハフツングＬｅｉｃａ Ｍｉｃｒｏｓｙｓｔｅｍｓ ＣＭＳ ＧｍｂＨ Method and apparatus for microscopic examination of a sample
CN107783267A (en) * 2016-08-30 2018-03-09 北京大学 Micro- amplification system
CN107783267B (en) * 2016-08-30 2024-04-26 北京大学 Microscopic amplifying system

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