JP3244100B2 - Two-photon excitation microscope - Google Patents
Two-photon excitation microscopeInfo
- Publication number
- JP3244100B2 JP3244100B2 JP19309394A JP19309394A JP3244100B2 JP 3244100 B2 JP3244100 B2 JP 3244100B2 JP 19309394 A JP19309394 A JP 19309394A JP 19309394 A JP19309394 A JP 19309394A JP 3244100 B2 JP3244100 B2 JP 3244100B2
- Authority
- JP
- Japan
- Prior art keywords
- sample
- photon excitation
- objective lens
- light
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、２光子励起顕微鏡に関
し、特に光の利用効率が良く、高速走査が可能な２光子
励起顕微鏡に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a two-photon excitation microscope and, more particularly, to a two-photon excitation microscope which can efficiently use light and can perform high-speed scanning.
【０００２】[0002]
【従来の技術】２光子励起法とは「レーザ顕微鏡研究会
第１２回講演会論文集」２８頁〜３５頁の「２光子励起
蛍光顕微鏡法による細胞内カルシウム濃度の３次元計
測」に記載されているように、試料に対して単光子励起
の励起光の２倍の波長を持つ光子を同時に２光子照射す
ることにより、前記試料から蛍光分子を励起する方法で
ある。2. Description of the Related Art The two-photon excitation method is described in "Two-Dimensional Measurement of Intracellular Calcium Concentration by Two-Photon Excitation Fluorescence Microscopy" on pages 28-35 in "Transactions of the Laser Microscopic Society 12th Lecture Meeting". As described above, this method is to excite fluorescent molecules from the sample by simultaneously irradiating the sample with two photons having a wavelength twice as long as the excitation light of the single-photon excitation.
【０００３】このような２光子励起は単位体積当たり大
量の光子を必要とするため対物レンズの焦点位置でしか
生じない。言い換えれば、共焦点ピンホールを用いるこ
となく共焦点顕微鏡と同様に試料の断面積を得ることが
できる。[0003] Such two-photon excitation requires a large amount of photons per unit volume and occurs only at the focal position of the objective lens. In other words, the cross-sectional area of the sample can be obtained similarly to a confocal microscope without using a confocal pinhole.
【０００４】図２はこのような従来の２光子励起顕微鏡
の一例を示す構成ブロック図である。図２において１は
パルスレーザ等の２光子励起が可能なレーザ、２はダイ
クロイックミラー、３及び４は走査ミラー、５は対物レ
ンズ、６は試料、７は光電子増倍管等の光検出器であ
る。FIG. 2 is a configuration block diagram showing an example of such a conventional two-photon excitation microscope. In FIG. 2, 1 is a laser capable of two-photon excitation such as a pulse laser, 2 is a dichroic mirror, 3 and 4 are scanning mirrors, 5 is an objective lens, 6 is a sample, and 7 is a photodetector such as a photomultiplier tube. is there.
【０００５】レーザ１の出力光はダイクロイックミラー
２に入射され、ダイクロイックミラー２からの反射光は
走査ミラー３、走査ミラー４及び対物レンズ５を介して
試料６に入射される。[0005] The output light of the laser 1 is incident on a dichroic mirror 2, and the reflected light from the dichroic mirror 2 is incident on a sample 6 via a scanning mirror 3, a scanning mirror 4 and an objective lens 5.
【０００６】試料６で生じた蛍光は対物レンズ５、走査
ミラー４及び走査ミラー３を介してダイクロイックミラ
ー２に入射され、試料６からの蛍光はダイクロイックミ
ラー２を透過して光検出器７に入射される。The fluorescence generated from the sample 6 is incident on the dichroic mirror 2 via the objective lens 5, the scanning mirror 4, and the scanning mirror 3, and the fluorescence from the sample 6 is transmitted through the dichroic mirror 2 and is incident on the photodetector 7. Is done.
【０００７】ここで、図２に示す従来例の動作を説明す
る。走査ミラー３及び４を機械的に動かすことにより、
レーザ１の出力光は試料６上を走査する。この時、前述
の２光子励起により試料６では蛍光が生じる。Here, the operation of the conventional example shown in FIG. 2 will be described. By mechanically moving the scanning mirrors 3 and 4,
The output light of the laser 1 scans on the sample 6. At this time, fluorescence is generated in the sample 6 by the two-photon excitation described above.
【０００８】試料６で生じた蛍光は対物レンズ５、走査
ミラー４及び走査ミラー３を介してダイクロイックミラ
ー２に入射されるが、蛍光の波長はレーザ１の出力光の
波長と異なるためダイクロイックミラー２を透過して光
検出器７で検出される。また、前述のように２光子励起
は対物レンズの焦点位置でしか生じないため、ピンホー
ルを用いることなく共焦点効果を得ることができる。The fluorescence generated by the sample 6 is incident on the dichroic mirror 2 via the objective lens 5, the scanning mirror 4 and the scanning mirror 3, but the wavelength of the fluorescence is different from the wavelength of the output light of the laser 1, so that the dichroic mirror 2 And is detected by the photodetector 7. Further, as described above, since the two-photon excitation occurs only at the focal position of the objective lens, the confocal effect can be obtained without using a pinhole.
【０００９】[0009]
【発明が解決しようとする課題】しかし、図２に示す従
来例では走査方法として走査ミラー３及び４を用いてい
るため高速走査は困難である。また、構造上、試料６上
を１点の走査光によって走査するため試料６から戻って
くる蛍光の光強度が弱いと言った問題点もある。従って
本発明の目的は、高速走査が可能で、光の利用効率の良
い２光子励起顕微鏡を実現することにある。However, in the conventional example shown in FIG. 2, high-speed scanning is difficult because the scanning mirrors 3 and 4 are used as a scanning method. In addition, there is also a problem in that the light intensity of the fluorescence returning from the sample 6 is weak because the scanning is performed on the sample 6 with one point of scanning light. Therefore, an object of the present invention is to realize a two-photon excitation microscope capable of high-speed scanning and having high light use efficiency.
【００１０】[0010]
【課題を解決するための手段】このような目的を達成す
るために、本発明では、試料に対して単光子励起の場合
の励起光の２倍の波長を持つ光子を同時に２光子照射し
て前記試料からの蛍光を観測する２光子励起顕微鏡にお
いて、２光子励起が可能なレーザと、このレーザの出力
光が入射され、複数のマイクロレンズを有し回転するこ
とにより前記試料を走査する回転板と、前記マイクロレ
ンズの焦点に中間像位置を一致させ前記出力光を前記試
料に集光させる対物レンズと、前記複数のマイクロレン
ズからの出力光を透過若しくは反射させ、前記試料の前
記対物レンズの焦点位置で生じる蛍光を分離するダイク
ロイックミラーと、このダイクロイックミラーで分離さ
れた前記蛍光が入射される撮像装置若しくは接眼装置と
を備えたことを特徴とするものである。In order to achieve the above object, according to the present invention, a sample is irradiated with two photons simultaneously having two times the wavelength of the excitation light in the case of single-photon excitation. In a two-photon excitation microscope for observing fluorescence from the sample, a laser capable of two-photon excitation and an output light of the laser are incident and have a plurality of microlenses .
A rotating plate that scans the sample, an objective lens that matches an intermediate image position to the focal point of the microlens, and condenses the output light on the sample, and transmits or reflects output light from the plurality of microlenses. A dichroic mirror that separates fluorescence generated at the focal position of the objective lens of the sample, and an imaging device or eyepiece device into which the fluorescence separated by the dichroic mirror is incident. is there.
【００１１】[0011]
【作用】複数のマイクロレンズを設けた回転板を回転さ
せて試料上を走査することにより、高速走査が可能にな
り、光の利用効率も良くなる。By rotating a rotating plate provided with a plurality of microlenses to scan on a sample, high-speed scanning becomes possible and light use efficiency is improved.
【００１２】[0012]
【実施例】以下本発明を図面を用いて詳細に説明する。
図１は本発明に係る２光子励起顕微鏡の一実施例を示す
構成ブロック図である。図１において１ａは２光子励起
が可能なレーザ、２ａはダイクロイックミラー、５ａは
対物レンズ、６ａは試料、８は複数のマイクロレンズを
有する回転板、９はレンズ、１０はＣＣＤカメラ等の撮
像装置である。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings.
FIG. 1 is a configuration block diagram showing one embodiment of a two-photon excitation microscope according to the present invention. In FIG. 1, 1a is a laser capable of two-photon excitation, 2a is a dichroic mirror, 5a is an objective lens, 6a is a sample, 8 is a rotating plate having a plurality of microlenses, 9 is a lens, and 10 is an imaging device such as a CCD camera. It is.
【００１３】回転板８に設けられているマイクロレンズ
は、例えば、本願出願人の出願に係る「特願平３−２８
６１１２号（特開平５−１２７０９０号公報）」に記載
したように配列されているものである。The microlenses provided on the rotating plate 8 are described, for example, in Japanese Patent Application No. Hei.
No. 6112 (JP-A-5-127090) .
【００１４】レーザ１ａの出力光は回転板８に入射さ
れ、回転板８上に設けられたマイクロレンズ（以下、単
にマイクロレンズと呼ぶ。）を透過した光はダイクロイ
ックミラー２ａ及び対物レンズ５ａを介して試料６ａに
入射される。The output light of the laser 1a is incident on the rotating plate 8, and the light transmitted through a micro lens (hereinafter simply referred to as a micro lens) provided on the rotating plate 8 passes through the dichroic mirror 2a and the objective lens 5a. Incident on the sample 6a.
【００１５】試料６ａで生じた蛍光は対物レンズ５ａを
介してダイクロイックミラー２ａに入射され、ダイクロ
イックミラー２ａで反射された試料６ａからの蛍光はレ
ンズ９を介して撮像装置１０に入射される。The fluorescent light generated by the sample 6a is incident on the dichroic mirror 2a via the objective lens 5a, and the fluorescent light from the sample 6a reflected by the dichroic mirror 2a is incident on the imaging device 10 via the lens 9.
【００１６】ここで、図１に示す実施例の動作を説明す
る。また、前記マイクロレンズは回転板８上に複数個設
けられているが、説明を簡単にするため図１中”イ”に
示すマイクロレンズについてのみ説明する。Here, the operation of the embodiment shown in FIG. 1 will be described. Although a plurality of the micro lenses are provided on the rotating plate 8, only the micro lens indicated by "A" in FIG. 1 will be described for the sake of simplicity.
【００１７】図１中”イ”に示すマイクロレンズは対物
レンズ５ａの中間像位置である図１中”ロ”の点にレー
ザ１ａの出力光を集光させるような位置関係で設けられ
ている。即ち、図１中”ロ”の点にマイクロレンズの焦
点を一致させるようにマイクロレンズを設ける。The microlens indicated by "A" in FIG. 1 is provided in such a positional relationship as to converge the output light of the laser 1a at the point "B" in FIG. 1, which is the intermediate image position of the objective lens 5a. . That is, a microlens is provided so that the focal point of the microlens coincides with the point "b" in FIG.
【００１８】また、対物レンズ５ａの他方の焦点は試料
６ａ上にあり、回転板８を回転させることにより試料６
ａ上を高速で走査することができる。The other focal point of the objective lens 5a is located on the sample 6a.
a can be scanned at high speed.
【００１９】前述のように２光子励起は対物レンズ５ａ
の焦点位置でのみ生じ、発生した蛍光はダイクロイック
ミラー２ａにおいて分離され撮像装置１０に入射されて
観測される。As described above, the two-photon excitation is performed by the objective lens 5a.
Is generated only at the focal position, and the generated fluorescence is separated by the dichroic mirror 2a and is incident on the imaging device 10 and observed.
【００２０】この結果、マイクロレンズを設けた回転板
８を回転させて試料６ａ上を走査することにより、走査
ミラー３及び４で走査する場合と比較して高速走査をす
ることが可能となる。As a result, by rotating the rotating plate 8 provided with the microlenses and scanning the sample 6a, high-speed scanning can be performed as compared with the case where scanning is performed by the scanning mirrors 3 and 4.
【００２１】また、回転板８には複数のマイクロレンズ
が設けられており、それぞれのマイクロレンズにより走
査された蛍光が同時に撮像装置１０に入射されることに
なるので、試料６ａから戻ってくる蛍光の光強度が従来
例と比較して強くなり光の利用効率が向上する。The rotating plate 8 is provided with a plurality of microlenses, and the fluorescent light scanned by each of the microlenses is simultaneously incident on the image pickup device 10, so that the fluorescent light returning from the sample 6a. The light intensity is higher than in the conventional example, and the light use efficiency is improved.
【００２２】なお、光の利用効率が向上することによ
り、撮像装置１０の代わりに接眼装置を用いて直接肉眼
で観測することも可能である。By improving the light use efficiency, it is possible to directly observe the image with the naked eye using an eyepiece instead of the image pickup apparatus 10.
【００２３】[0023]
【発明の効果】以上説明したことから明らかなように、
本発明によれば次のような効果がある。複数のマイクロ
レンズを設けた回転板を回転させて試料上を走査するこ
とにより、高速走査が可能で、光の利用効率の良い２光
子励起顕微鏡を実現することができる。As is apparent from the above description,
According to the present invention, the following effects can be obtained. By rotating a rotating plate provided with a plurality of microlenses and scanning the sample, high-speed scanning is possible, and a two-photon excitation microscope with high light use efficiency can be realized.
【図１】本発明に係る２光子励起顕微鏡の一実施例を示
す構成ブロック図である。FIG. 1 is a configuration block diagram showing one embodiment of a two-photon excitation microscope according to the present invention.
【図２】従来の２光子励起顕微鏡の一例を示す構成ブロ
ック図である。FIG. 2 is a configuration block diagram illustrating an example of a conventional two-photon excitation microscope.
１，１ａ レーザ ２，２ａ ダイクロイックミラー ３，４ 走査ミラー ５，５ａ 対物レンズ ６，６ａ 試料 ７ 光検出器 ８ 回転板 ９ レンズ １０ 撮像装置 Reference Signs List 1, 1a Laser 2, 2a Dichroic mirror 3, 4 Scanning mirror 5, 5a Objective lens 6, 6a Sample 7 Photodetector 8 Rotating plate 9 Lens 10 Imaging device
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 21/62 - 21/74 G02B 21/00 ＪＩＣＳＴファイル（ＪＯＩＳ)──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. 7 , DB name) G01N 21/62-21/74 G02B 21/00 JICST file (JOIS)
Claims (1)
２倍の波長を持つ光子を同時に２光子照射して前記試料
からの蛍光を観測する２光子励起顕微鏡において、 ２光子励起が可能なレーザと、 このレーザの出力光が入射され、複数のマイクロレンズ
を有し回転することにより前記試料を走査する回転板
と、 前記マイクロレンズの焦点に中間像位置を一致させ前記
出力光を前記試料に集光させる対物レンズと、 前記複数のマイクロレンズからの出力光を透過若しくは
反射させ、前記試料の前記対物レンズの焦点位置で生じ
る蛍光を分離するダイクロイックミラーと、 このダイクロイックミラーで分離された前記蛍光が入射
される撮像装置若しくは接眼装置とを備えたことを特徴
とする２光子励起顕微鏡。1. A two-photon excitation microscope for simultaneously irradiating a sample with two photons having a wavelength twice as long as the excitation light in the case of single-photon excitation and observing fluorescence from the sample. A possible laser, a rotating plate that receives the output light of the laser, scans the sample by rotating with a plurality of microlenses, and matches the intermediate image position to the focal point of the microlens, and outputs the output light. An objective lens that focuses on the sample, a dichroic mirror that transmits or reflects output light from the plurality of microlenses, and separates fluorescence generated at a focal position of the objective lens of the sample, A two-photon excitation microscope, comprising: an imaging device or an eyepiece device to which the fluorescence is incident.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19309394A JP3244100B2 (en) | 1994-08-17 | 1994-08-17 | Two-photon excitation microscope |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19309394A JP3244100B2 (en) | 1994-08-17 | 1994-08-17 | Two-photon excitation microscope |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0854340A JPH0854340A (en) | 1996-02-27 |
| JP3244100B2 true JP3244100B2 (en) | 2002-01-07 |
Family
ID=16302120
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP19309394A Expired - Lifetime JP3244100B2 (en) | 1994-08-17 | 1994-08-17 | Two-photon excitation microscope |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3244100B2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6025917A (en) * | 1996-09-24 | 2000-02-15 | Laboratory Of Molecular Biophotonics | Polarization characteristic measuring method and apparatus |
| JP2001506015A (en) * | 1997-12-22 | 2001-05-08 | マックス―プランク―ゲゼルシャフト・ツール・フェルデルング・デア・ヴィッセンシャフテン・エー・ファウ | Scanning microscope that optically excites samples at multiple sample locations simultaneously |
| DE19801139B4 (en) * | 1998-01-14 | 2016-05-12 | Till Photonics Gmbh | Point Scanning Luminescence Microscope |
| JP3099063B2 (en) * | 1998-12-28 | 2000-10-16 | 大阪大学長 | Multiphoton microscope |
| JP2001272343A (en) * | 2000-03-23 | 2001-10-05 | Olympus Optical Co Ltd | Double resonance absorption microscope |
| CN101365937B (en) * | 2005-10-11 | 2015-01-14 | Bt成像股份有限公司 | Method and system for inspecting indirect bandgap semiconductor structure |
| CN103558193B (en) * | 2013-10-24 | 2015-09-09 | 深圳先进技术研究院 | A kind of Two Photon Fluorescence |
-
1994
- 1994-08-17 JP JP19309394A patent/JP3244100B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| 田名綱 健雄 外，マイクロレンズを付加したピンホール走査型高速共焦点スキャナ，レーザ顕微鏡研究会，日本，１９９４年 １月１３日，Ｐａｇｅ６０−６７ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0854340A (en) | 1996-02-27 |
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