JP2010139675A - Fluorescence microscope - Google Patents

Fluorescence microscope Download PDF

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JP2010139675A
JP2010139675A JP2008315265A JP2008315265A JP2010139675A JP 2010139675 A JP2010139675 A JP 2010139675A JP 2008315265 A JP2008315265 A JP 2008315265A JP 2008315265 A JP2008315265 A JP 2008315265A JP 2010139675 A JP2010139675 A JP 2010139675A
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fluorescence
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JP5190886B2 (en
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Koichi Kajiyama
康一 梶山
Michinobu Mizumura
通伸 水村
Kazue Hashimoto
和重 橋本
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V Technology Co Ltd
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<P>PROBLEM TO BE SOLVED: To simplify structure and to reduce an influence of surface reflection of fluorescence and exciting light generated near the surface of an observation object to facilitate the observation of a deep part of the observation object. <P>SOLUTION: This fluorescence microscope irradiating a fluorescent material 8 inside the observation object 3 with laser beams L1 to excite the fluorescent material 8 and observing fluorescence L2 generated from the fluorescent material 8, is provided with: an excitation illuminating device 1 generating the laser beams L1 to irradiate the observation object 3; and an observation device 2 observing fluorescence L2 radiated in a direction different from the optical axis direction of the excitation illuminating device 1 with fluorescence L2 generated inside the observation object 3 being excited by the laser beams L1. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

本発明は、観察物内部の蛍光物質に励起光を照射して励起させ、蛍光物質で発生する蛍光を観察する蛍光顕微鏡に関し、詳しくは、構造が簡単であると共に、観察物表面近傍で発生する蛍光や励起光の表面反射の影響を軽減して観察物の深部の観察を容易にしようとする蛍光顕微鏡に係るものである。   The present invention relates to a fluorescence microscope for observing fluorescence generated in a fluorescent material by irradiating the fluorescent material inside the observed material with excitation light, and more specifically, having a simple structure and occurring near the surface of the observed material. The present invention relates to a fluorescence microscope that reduces the influence of fluorescence and surface reflection of excitation light to facilitate observation of the deep part of an observation object.

従来の蛍光顕微鏡は、光源からの光を対物レンズを介して観察物上に導き、観察物内部の蛍光物質が励起されて発生する蛍光を上記対物レンズを介してＣＣＤカメラに導いて観察可能にし、ＣＣＤカメラでの測光及び撮像のタイミングに合わせて制御手段により光源の点灯及び消灯を制御するものとなっていた（例えば、特許文献１参照）。   A conventional fluorescence microscope guides light from a light source onto an observation object through an objective lens, and guides fluorescence generated by excitation of a fluorescent substance inside the observation object to the CCD camera through the objective lens so that observation is possible. The light source is turned on and off by the control means in accordance with the timing of photometry and imaging by the CCD camera (see, for example, Patent Document 1).

また、他の蛍光顕微鏡は、多光子励起走査型レーザ顕微鏡であり、パルスレーザ光を出射する光源装置と、光源装置から発せられたパルスレーザ光を標本に照射し、標本において発せられた蛍光を観察する観察装置本体と、標本における観察面の深さに応じて、標本に照射するパルスレーザ光の光量を調節する制御装置とを具備するものとなっている（例えば、特許文献２参照）。
特開２００５−１９５９４０号公報 特開２００７−２６３７３０号公報 Another fluorescence microscope is a multi-photon excitation scanning laser microscope, which irradiates a specimen with a light source device that emits pulsed laser light, and pulse laser light emitted from the light source device, and emits fluorescence emitted from the specimen. An observation apparatus main body to be observed and a control apparatus that adjusts the amount of pulsed laser light applied to the specimen in accordance with the depth of the observation surface of the specimen (see, for example, Patent Document 2).
JP 2005-195940 A JP 2007-263730 A

しかし、このような従来の蛍光顕微鏡において、上記特許文献１に記載の蛍光顕微鏡は、同一の対物レンズを介して光源からの光を観察物上に導くと共に、観察物内部で発生する蛍光をＣＣＤカメラに導くようにしているため、観察物表面近傍で発生する蛍光や光源光の観察物表面における反射の影響を排除することができなかった。したがって、これら観察物表面近傍で発生する蛍光や光源光の表面反射光がノイズとなってＳ／Ｎを劣化させ、観察対象物の深部で発生する微弱な蛍光を容易に検出することができない場合があった。   However, in such a conventional fluorescence microscope, the fluorescence microscope described in Patent Document 1 guides the light from the light source onto the observation object through the same objective lens, and converts the fluorescence generated inside the observation object to the CCD. Since the light is guided to the camera, it is impossible to eliminate the influence of the fluorescence generated near the surface of the observation object and the reflection of the light source light on the observation object surface. Therefore, when the fluorescent light generated near the surface of the observation object or the surface reflected light of the light source light becomes noise and deteriorates the S / N, it is not possible to easily detect the weak fluorescence generated in the deep part of the observation object. was there.

また、上記特許文献２に記載の多光子励起走査型レーザ顕微鏡は、焦点でのみ蛍光物質を励起することができるため観察物の深部の観察が可能であるが、装置の構造が複雑で大型化することから高価になること、顕微鏡の光軸調整に多大な時間を必要とすること等の問題がある。   In addition, the multiphoton excitation scanning laser microscope described in Patent Document 2 can excite a fluorescent material only at the focal point, so that it is possible to observe a deep portion of an observation object. However, the structure of the apparatus is complicated and the size is increased. Therefore, there are problems such as being expensive and requiring a lot of time to adjust the optical axis of the microscope.

そこで、本発明は、このような問題点に対処し、構造が簡単であると共に、観察物表面近傍で発生する蛍光や励起光の表面反射の影響を軽減して観察物の深部の観察を容易にしようとする蛍光顕微鏡を提供することを目的とする。   Therefore, the present invention addresses such problems, has a simple structure, and reduces the influence of surface reflection of fluorescence and excitation light generated near the surface of the object, making it easy to observe the deep part of the object. An object is to provide a fluorescent microscope to be manufactured.

上記目的を達成するために、本発明による蛍光顕微鏡は、観察物内部の蛍光物質に励起光を照射して励起させ、蛍光物質で発生する蛍光を観察する蛍光顕微鏡であって、前記励起光を発生して前記観察物に照射する励起照明装置と、前記励起光によって励起されて前記観察物内部で発生する蛍光にて、前記励起照明装置の光軸方向と異なる方向に放射された蛍光を観察する観察装置と、を備えたものである。   In order to achieve the above object, a fluorescence microscope according to the present invention is a fluorescence microscope that observes fluorescence generated in a fluorescent substance by irradiating the fluorescent substance in an observation object with excitation light and exciting the excitation substance. Observation of fluorescence emitted in a direction different from the optical axis direction of the excitation illumination device by the excitation illumination device generated and irradiated on the observation object and the fluorescence generated inside the observation object by being excited by the excitation light An observation device.

このような構成により、励起照明装置で励起光を発生して観察物に照射し、該励起光によって観察物内部の蛍光物質が励起されて発生する蛍光にて、励起照明装置の光軸方向と異なる方向に放射された蛍光を観察装置で観察する。   With such a configuration, the excitation illumination device generates excitation light and irradiates the observation object, and the fluorescence generated by the excitation light exciting the fluorescent substance inside the observation object Fluorescence emitted in different directions is observed with an observation device.

また、前記励起照明装置は、励起光をシート状に整形して前記観察物に照射するものである。これにより、励起照明装置で励起光をシート状に整形して観察物に照射する。   Further, the excitation illumination device shapes the excitation light into a sheet shape and irradiates the observation object. Thereby, the excitation light is shaped into a sheet shape by the excitation illumination device and irradiated to the observation object.

さらに、前記観察物は、前記励起照明装置及び観察装置に対して相対的に前記シート状の励起光の法線方向に移動可能にされたものである。これにより、観察物を励起照明装置及び観察装置に対して相対的にシート状の励起光の法線方向に移動する。   Further, the observation object is movable in the normal direction of the sheet-like excitation light relative to the excitation illumination device and the observation device. Accordingly, the observation object is moved in the normal direction of the sheet-like excitation light relative to the excitation illumination device and the observation device.

そして、前記シート状の励起光の法線方向に放射された蛍光を観察可能に前記観察装置を設けたものである。これにより、観察装置でシート状の励起光の法線方向に放射された蛍光を観察する。   And the said observation apparatus is provided so that the fluorescence radiated | emitted in the normal line direction of the said sheet-like excitation light can be observed. Thereby, the fluorescence emitted in the normal direction of the sheet-like excitation light is observed by the observation apparatus.

請求項１に係る発明によれば、観察物内部で発生する蛍光にて、励起照明装置の光軸方向と異なる方向に放射された蛍光を観察するようにしているので、観察物表面近傍で発生する蛍光と観察物深部に存在する観察対象の蛍光物質から発生する蛍光とを互いに異なる位置に観察することができる。したがって、従来技術における観察対象の蛍光物質から発生する蛍光が観察物表面近傍で発生する蛍光や励起光の表面反射光と重なって観察しがたいという問題を解消することができ、観察物の深部まで容易に観察することができる。また、観察物内部で発生する蛍光にて励起照明装置の光軸方向と異なる方向に放射された蛍光を観察するように観察装置を配置しただけであるので、構造が簡単であり、従来の励起照明装置及び観察装置を利用して構成することができる。したがって、装置を安価に製造することができる。   According to the first aspect of the present invention, since the fluorescence emitted inside the observation object is observed in the direction different from the optical axis direction of the excitation illumination device, it is generated near the surface of the observation object. The fluorescence generated from the fluorescent substance to be observed existing in the deep part of the observation object can be observed at different positions. Therefore, it is possible to solve the problem that the fluorescence generated from the fluorescent material to be observed in the prior art overlaps with the fluorescence generated in the vicinity of the surface of the observation object or the surface reflected light of the excitation light and is difficult to observe. Can be easily observed. In addition, since the observation device is simply arranged to observe the fluorescence emitted inside the observation object in the direction different from the optical axis direction of the excitation illumination device, the structure is simple and the conventional excitation is performed. An illumination device and an observation device can be used. Therefore, the device can be manufactured at low cost.

また、請求項２に係る発明によれば、シート状の励起光と同一平面内の蛍光物質の断層像を観察することができる。この場合、励起光を走査して二次元像を得る必要がないので、励起光の照射から断層像の表示までの時間を短縮することができる。   Moreover, according to the invention which concerns on Claim 2, the tomographic image of the fluorescent substance in the same plane as sheet-like excitation light can be observed. In this case, since it is not necessary to scan the excitation light to obtain a two-dimensional image, the time from irradiation of the excitation light to the display of the tomographic image can be shortened.

さらに、請求項３に係る発明によれば、シート状の励起光の法線に略直交する蛍光物質の複数の断層像を観察することができる。したがって、これら複数の断層像に基づいて蛍光物質の三次元像を観察することができる。   Furthermore, according to the invention which concerns on Claim 3, the some tomogram of the fluorescent substance substantially orthogonal to the normal line of sheet-like excitation light can be observed. Therefore, a three-dimensional image of the fluorescent material can be observed based on the plurality of tomographic images.

そして、請求項４に係る発明によれば、シート状の励起光の法線方向に放射された蛍光を観察するようにしているので、観察対象の蛍光物質から発生する蛍光と観察物表面近傍で発生する蛍光とを明確に分離して観察することができる。したがって、観察対象の蛍光物質から発生する蛍光をより容易に観察することができる。   According to the invention of claim 4, since the fluorescence emitted in the normal direction of the sheet-like excitation light is observed, the fluorescence generated from the fluorescent material to be observed and the vicinity of the surface of the observation object are observed. The generated fluorescence can be clearly separated and observed. Therefore, the fluorescence generated from the fluorescent substance to be observed can be observed more easily.

以下、本発明の実施形態を添付図面に基づいて詳細に説明する。図１は本発明による蛍光顕微鏡の実施形態の概略構成を示す正面図である。この蛍光顕微鏡は、観察物内部の蛍光物質に励起光を照射して励起させ、蛍光物質で発生する蛍光を観察するもので、励起照明装置１と、観察装置２とを備えて構成されている。   Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. FIG. 1 is a front view showing a schematic configuration of an embodiment of a fluorescence microscope according to the present invention. This fluorescence microscope is for irradiating excitation light to an inside of an observation object by exciting it and observing fluorescence generated in the fluorescent material, and is configured to include an excitation illumination device 1 and an observation device 2. .

上記励起照明装置１は、励起光を発生して観察物３に照射するものであり、レーザ光源４と、ビーム整形手段５とを備えている。ここで、レーザ光源４は、所定波長の励起光としてのレーザ光Ｌ１を放射するものである。また、ビーム整形手段５は、レーザ光源４から放射された断面円形状のレーザ光Ｌ１を厚みの薄いシート状のレーザ光Ｌ１に整形して射出するもので、凸状シリンドリカルレンズ６と凹状シリンドリカルレンズ７とを組み合わせて構成されている。   The excitation illumination device 1 generates excitation light and irradiates the observation object 3, and includes a laser light source 4 and beam shaping means 5. Here, the laser light source 4 emits laser light L1 as excitation light having a predetermined wavelength. The beam shaping means 5 shapes the laser beam L1 having a circular cross section emitted from the laser light source 4 into a thin sheet-like laser beam L1 and emits it. The convex cylindrical lens 6 and the concave cylindrical lens are emitted. 7 in combination.

観察位置において、上記励起照明装置１の光軸と光軸を略直交させて観察装置２が設けられている。この観察装置２は、レーザ光Ｌ１の照射によって観察物３内部で観察対象の蛍光物質８が励起されて発生するレーザ光Ｌ１の波長と異なる波長の蛍光Ｌ２にて、シート状のレーザ光Ｌ１の法線方向に放射される蛍光Ｌ２を観察するものであり、光軸上に観察物３側から対物レンズ９と結像レンズ１０とをこの順に備えて、励起照明装置１のシート状のレーザ光Ｌ１に照射された蛍光物質８の断層像を観察カメラ１１の二次元撮像面に結像するようになっている。また、観察カメラ１１の前面には、レーザ光Ｌ１の透過を遮断し、蛍光物質８で発生する蛍光Ｌ２を選択的に透過させるバンドパスフィルター１２が設けられている。なお、本実施形態においては、対物レンズ９は、その焦点Ｆが励起照明装置１のシート状のレーザ光Ｌ１と略同一平面内に位置するように設けられている。   At the observation position, the observation device 2 is provided with the optical axis of the excitation illumination device 1 and the optical axis substantially orthogonal to each other. The observation apparatus 2 is configured to emit the sheet-like laser light L1 with the fluorescence L2 having a wavelength different from the wavelength of the laser light L1 generated when the fluorescent substance 8 to be observed is excited inside the observation object 3 by irradiation with the laser light L1. This is for observing the fluorescence L2 emitted in the normal direction. The objective lens 9 and the imaging lens 10 are provided in this order from the observation object 3 side on the optical axis, and the sheet-like laser light of the excitation illumination device 1 is provided. A tomographic image of the fluorescent material 8 irradiated to L1 is formed on the two-dimensional imaging surface of the observation camera 11. In addition, a band-pass filter 12 that blocks the transmission of the laser light L1 and selectively transmits the fluorescence L2 generated by the fluorescent material 8 is provided on the front surface of the observation camera 11. In the present embodiment, the objective lens 9 is provided so that its focal point F is located in substantially the same plane as the sheet-like laser light L1 of the excitation illumination device 1.

次に、このように構成された蛍光顕微鏡の動作について説明する。
レーザ光源４から放射されたレーザ光Ｌ１は、図２（ａ）に示すように、正面視にて、凸状シリンドリカルレンズ６と凹状シリンドリカルレンズ７との組み合わせから成るビーム整形手段５の集光点に集光し、厚みが薄くされる。一方、同図（ｂ）に示すように、平面視においては、レーザ光源４から放射されたレーザ光Ｌ１は、ビーム整形手段５をそのまま通過する。したがって、レーザ光源４から放射された断面円形状のレーザ光Ｌ１は、ビーム整形手段５により厚みの薄いシート状に整形されることになる。 Next, the operation of the thus configured fluorescence microscope will be described.
As shown in FIG. 2A, the laser light L1 emitted from the laser light source 4 is a condensing point of the beam shaping means 5 composed of a combination of a convex cylindrical lens 6 and a concave cylindrical lens 7 in front view. The light is condensed and the thickness is reduced. On the other hand, as shown in FIG. 4B, the laser light L1 emitted from the laser light source 4 passes through the beam shaping means 5 as it is in a plan view. Therefore, the laser beam L1 having a circular cross section emitted from the laser light source 4 is shaped into a thin sheet by the beam shaping means 5.

シート状に整形されたレーザ光Ｌ１は、励起照明装置１を射出して観察物３に照射する。さらに、レーザ光Ｌ１は、観察物３内部に侵入し、シート状レーザ光Ｌ１と略同一平面内に存在する蛍光物質８を励起させて蛍光Ｌ２を発生させる。   The laser light L1 shaped into a sheet is emitted from the excitation illumination device 1 and irradiates the observation object 3. Further, the laser light L1 enters the observation object 3 and excites the fluorescent substance 8 existing in substantially the same plane as the sheet-like laser light L1 to generate fluorescence L2.

一方、蛍光物質８がシート状レーザ光Ｌ１に照射されて発生した蛍光Ｌ２は、シート状レーザ光Ｌ１の法線方向に設けられた観察装置２の対物レンズ９内に取り込まれる。このとき、対物レンズ９は、その焦点Ｆがシート状レーザ光Ｌ１と略同一平面内に位置するように設けられているので、対物レンズ９に取り込まれた蛍光Ｌ２は、平行光となって観察カメラ１１側に進行する。そして、結像レンズ１０により、観察カメラ１１の撮像面上に集光される。これにより、シート状レーザ光Ｌ１と略同一平面内の蛍光物質８の断層像が観察カメラ１１によって撮像されることになる。   On the other hand, the fluorescence L2 generated by irradiating the fluorescent material 8 to the sheet-like laser light L1 is taken into the objective lens 9 of the observation apparatus 2 provided in the normal direction of the sheet-like laser light L1. At this time, since the objective lens 9 is provided so that its focal point F is located in substantially the same plane as the sheet-like laser beam L1, the fluorescence L2 taken into the objective lens 9 is observed as parallel light. Proceed to the camera 11 side. Then, the light is condensed on the imaging surface of the observation camera 11 by the imaging lens 10. As a result, a tomographic image of the fluorescent material 8 in substantially the same plane as the sheet-like laser beam L1 is picked up by the observation camera 11.

この場合、図３に示すように、観察カメラ１１の視野Ａ内には、観察対象の蛍光物質８が発生する蛍光Ｌ２以外に、該蛍光物質８の側方にて観察物３の表面近傍に存在する蛍光性物質１３が発生する蛍光Ｌ２も同時に観察されることがある。しかしながら、観察対象の蛍光物質８から発生する蛍光Ｌ２も蛍光性物質１３から発生する蛍光Ｌ２も同一平面内の互いに異なる位置に観察されるため、観察対象の蛍光物質８から発生する蛍光Ｌ２が蛍光性物質１３から発生する蛍光Ｌ２の影響を受けて観察が困難となることはない。したがって、観察カメラ１１に接続された図示省略のモニター画面上で、観察対象の蛍光物質８に注目して観察することができ、観察物３の深部に存在する蛍光物質８の断層像を容易に観察することができる。   In this case, as shown in FIG. 3, in the field of view A of the observation camera 11, in addition to the fluorescence L <b> 2 generated by the fluorescent material 8 to be observed, near the surface of the observation object 3 on the side of the fluorescent material 8. The fluorescence L2 generated by the existing fluorescent substance 13 may be observed at the same time. However, since the fluorescence L2 generated from the fluorescent material 8 to be observed and the fluorescence L2 generated from the fluorescent material 13 are observed at different positions in the same plane, the fluorescence L2 generated from the fluorescent material 8 to be observed is fluorescent. Observation is not difficult under the influence of the fluorescence L2 generated from the luminescent substance 13. Therefore, on the monitor screen (not shown) connected to the observation camera 11, observation can be performed while paying attention to the fluorescent material 8 to be observed, and a tomographic image of the fluorescent material 8 existing deep in the observation object 3 can be easily obtained. Can be observed.

このように、本発明の蛍光顕微鏡によれば、従来、観察物３の表面から５０μｍ程度の深さまでしか観察できなかったものが、約４００μｍ以上の深部まで鮮明に観察することができる。   As described above, according to the fluorescence microscope of the present invention, what can conventionally be observed only from the surface of the observation object 3 to a depth of about 50 μm can be clearly observed to a depth of about 400 μm or more.

ここで、図１において矢印で示すように、観察物３を励起照明装置１及び観察装置２に対して相対的にシート状のレーザ光Ｌ１の法線方向、即ち観察装置２の光軸方向に移動させれば、観察装置２の光軸に略直交する蛍光物質８の複数の断層像を観察することができる。したがって、これら複数の断層像に基づいて蛍光物質８の三次元像を観察することが可能となる。   Here, as shown by an arrow in FIG. 1, the observation object 3 is positioned relative to the excitation illumination device 1 and the observation device 2 in the normal direction of the sheet-like laser light L 1, that is, in the optical axis direction of the observation device 2. If moved, a plurality of tomographic images of the fluorescent substance 8 substantially perpendicular to the optical axis of the observation device 2 can be observed. Therefore, a three-dimensional image of the fluorescent material 8 can be observed based on the plurality of tomographic images.

なお、上記実施形態においては、シート状のレーザ光Ｌ１を観察物３に照射する場合について説明したが、本発明はこれに限られず、レーザ光Ｌ１を観察物３内の蛍光物質８に集光させてもよい。このとき、レーザ光Ｌ１の集光点と観察装置２の対物レンズ９の焦点Ｆとが合致するように観察装置２を設けるとよい。この場合、観察物３を励起照明装置１及び観察装置２に対して相対的に直交三軸方向に移動させると、蛍光物質８の三次元像を観察することができる。   In the above-described embodiment, the case where the observation object 3 is irradiated with the sheet-like laser light L1 has been described. However, the present invention is not limited to this, and the laser light L1 is focused on the fluorescent material 8 in the observation object 3. You may let them. At this time, it is preferable to provide the observation device 2 so that the condensing point of the laser beam L1 and the focal point F of the objective lens 9 of the observation device 2 coincide. In this case, when the observation object 3 is moved in the orthogonal three-axis direction relative to the excitation illumination device 1 and the observation device 2, a three-dimensional image of the fluorescent substance 8 can be observed.

また、上記実施形態においては、観察装置２がシート状のレーザ光Ｌ１の法線方向に放射された蛍光Ｌ２を観察するように配置された場合について説明したが、本発明はこれに限られず、レーザ光Ｌ１によって励起されて観察物３内部で発生する蛍光Ｌ２にて、励起照明装置１の光軸方向と異なる方向に放射される蛍光Ｌ２が観察可能であれば如何なる位置に配置されてもよい。   Moreover, in the said embodiment, although the case where the observation apparatus 2 was arrange | positioned so that the fluorescence L2 radiated | emitted in the normal line direction of the sheet-like laser beam L1 was demonstrated, this invention is not limited to this, The fluorescence L2 that is excited by the laser light L1 and is generated inside the observation object 3 may be arranged at any position as long as the fluorescence L2 emitted in a direction different from the optical axis direction of the excitation illumination device 1 can be observed. .

そして、上記実施形態においては、レーザ光源４を使用する場合について説明したが、本発明はこれに限られず、ＬＥＤ等の他の光源を使用してもよい。   In the above embodiment, the case where the laser light source 4 is used has been described. However, the present invention is not limited to this, and other light sources such as LEDs may be used.

本発明による蛍光顕微鏡の実施形態の概略構成を示す正面図である。It is a front view which shows schematic structure of embodiment of the fluorescence microscope by this invention. 本発明による蛍光顕微鏡に使用されるビーム整形手段の作用を示す説明図であり、（ａ）は正面図、（ｂ）は平面図である。It is explanatory drawing which shows the effect | action of the beam shaping means used for the fluorescence microscope by this invention, (a) is a front view, (b) is a top view. 本発明による蛍光顕微鏡の観察装置で観察される蛍光物質及び蛍光性物質の断層像を示す説明図である。It is explanatory drawing which shows the tomographic image of the fluorescent substance observed with the observation apparatus of the fluorescence microscope by this invention, and a fluorescent substance.

符号の説明Explanation of symbols

１…励起照明装置
２…観察装置
３…観察物
５…ビーム整形手段
８…観察対象の蛍光物質
Ｌ１…レーザ光（励起光）
Ｌ２…蛍光 DESCRIPTION OF SYMBOLS 1 ... Excitation illumination apparatus 2 ... Observation apparatus 3 ... Observation object 5 ... Beam shaping means 8 ... Fluorescent substance of observation object L1 ... Laser beam (excitation light)
L2 ... Fluorescence

Claims (4)

観察物内部の蛍光物質に励起光を照射して励起させ、蛍光物質で発生する蛍光を観察する蛍光顕微鏡であって、
前記励起光を発生して前記観察物に照射する励起照明装置と、
前記励起光によって励起されて前記観察物内部で発生する蛍光にて、前記励起照明装置の光軸方向と異なる方向に放射された蛍光を観察する観察装置と、
を備えたことを特徴とする蛍光顕微鏡。 A fluorescence microscope for irradiating excitation light to the fluorescent substance inside the observation object and exciting it, and observing the fluorescence generated in the fluorescent substance,
An excitation illumination device that generates the excitation light and irradiates the observation object;
An observation device for observing fluorescence emitted in a direction different from the optical axis direction of the excitation illumination device with fluorescence generated inside the observation object by being excited by the excitation light;
A fluorescence microscope comprising: 前記励起照明装置は、励起光をシート状に整形して前記観察物に照射するものであることを特徴とする請求項１記載の蛍光顕微鏡。   The fluorescence microscope according to claim 1, wherein the excitation illumination device is configured to irradiate the observation object with excitation light shaped into a sheet shape. 前記観察物は、前記励起照明装置及び観察装置に対して相対的に前記シート状の励起光の法線方向に移動可能にされたことを特徴とする請求項２記載の蛍光顕微鏡。   The fluorescence microscope according to claim 2, wherein the observation object is movable in a normal direction of the sheet-like excitation light relative to the excitation illumination device and the observation device. 前記シート状の励起光の法線方向に放射された蛍光を観察可能に前記観察装置を設けたことを特徴とする請求項２又は３記載の蛍光顕微鏡。   The fluorescence microscope according to claim 2 or 3, wherein the observation device is provided so that fluorescence emitted in a normal direction of the sheet-like excitation light can be observed.
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