JP2000056228A - System for detection by wavelengths to be used for laser scanning microscope, and image recording method - Google Patents
System for detection by wavelengths to be used for laser scanning microscope, and image recording methodInfo
- Publication number
- JP2000056228A JP2000056228A JP11217811A JP21781199A JP2000056228A JP 2000056228 A JP2000056228 A JP 2000056228A JP 11217811 A JP11217811 A JP 11217811A JP 21781199 A JP21781199 A JP 21781199A JP 2000056228 A JP2000056228 A JP 2000056228A
- Authority
- JP
- Japan
- Prior art keywords
- filter
- wavelength transmission
- transmission filter
- detection
- wavelength
- 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.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は蛍光顕微鏡、特にレ
ーザ走査顕微鏡に使用されるバンドパスフィルタの自由
なプログラミングを可能にするための、短波長透過フィ
ルタと長波長透過フィルタとの組み合わせにおける可変
式または非連続式の直列的配置に関するものである。こ
れらのフィルタは新たに市販される蛍光着色用色素にも
適合させることができ有利である。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a variable type of combination of a short wavelength transmission filter and a long wavelength transmission filter for enabling free programming of a bandpass filter used in a fluorescence microscope, particularly a laser scanning microscope. Or, it relates to a discontinuous serial arrangement. These filters are advantageously adaptable to newly marketed fluorescent coloring dyes.
【0002】[0002]
【発明の実施の形態】図1は顕微鏡ユニットMおよびそ
れに連結された走査ヘッドSを図式化して描いたもので
ある。顕微鏡には、従来通り光線分離器ST1を通して
試料テーブルT上の目的物を照射する、照射光学系付光
源LQ1が配備されている。転向式ミラーS3は光源L
Q2から集光器KOを通じてなされる透過光照射を切り
換えるのに用いられる。観察は接眼レンズOKから鏡胴
レンズTLおよびミラーS1を通して行う。走査光路は
このミラーまたは光線分離器S1を通過してさらに走査
レンズSLおよび走査装置SCに通じている。FIG. 1 schematically shows a microscope unit M and a scanning head S connected thereto. The microscope is provided with a light source LQ1 with an irradiation optical system that irradiates a target object on the sample table T through the beam separator ST1 as in the related art. The turning mirror S3 is a light source L
It is used to switch the transmitted light irradiation performed from the collector Q2 through the condenser KO. Observation is performed from the eyepiece OK through the lens barrel lens TL and the mirror S1. The scanning light path passes through this mirror or beam splitter S1 and further to the scanning lens SL and the scanning device SC.
【0003】レーザLの光はシャッター装置V、結合光
学系EO、ミラーS2および光線分離器ST2を通って
走査装置SCの方向に導かれる。目的物からの反射光お
よび/または蛍光は、ダイクロイック光線分離器ST2
を通って視準光学系KO、ピンホールPHへと進み検出
器に到るが、その前には拡大図示してある本発明に基づ
くフィルタユニットFEが配置されている。The light of the laser L passes through a shutter device V, a coupling optical system EO, a mirror S2, and a beam splitter ST2, and is guided toward the scanning device SC. The reflected light and / or fluorescent light from the target object is output to the dichroic beam splitter ST2.
Through the collimation optical system KO and the pinhole PH to the detector, before which is arranged a filter unit FE according to the invention, which is shown enlarged and shown.
【0004】フィルタユニットFEは取換可能な短波長
透過フィルタSPと長波長透過フィルタLPとから成る
のが好ましい。それにより、通過帯幅およびその重点域
の位置を望み通りに調整できるからである。さらにこれ
に加えて、光学密度の高い励起波長だけを差し止めるノ
ッチフィルタNFを配置することもできる。それによっ
て蛍光に対する検出感度がさらに高まる。The filter unit FE preferably comprises a replaceable short wavelength transmission filter SP and a long wavelength transmission filter LP. Thereby, the pass band width and the position of the important region can be adjusted as desired. In addition to this, a notch filter NF that suppresses only the excitation wavelength having a high optical density can be provided. Thereby, the detection sensitivity for fluorescence is further increased.
【0005】上記フィルタユニットは、勿論複数の検出
管路に使用することもできる。その場合スペクトル領域
の大まかな予備的選択には、公知の方法により、例えば
ドイツ特許1970275A1のようなダイクロイック
光線分離器やレボルバー切換式分離器による検出管路毎
の区分けが可能であるが、但し微調整は本発明に基づく
フィルタユニットを通じて行う。The above-mentioned filter unit can of course be used for a plurality of detection lines. In this case, a rough preliminary selection of the spectral range can be made in a known manner, for example by means of a dichroic beam separator or a revolver-switching separator as in DE 197 275 A1, for each detection line, but with a slight difference. The adjustment takes place through the filter unit according to the invention.
【0006】図2a〜2dは本発明に基づくフィルタシ
ステムの例である。図2aは相前後して配置されたスラ
イド式フィルタFS1、FS2であり、それぞれ円形フ
ィルタF1、F2を有している。当システムでは異なっ
た二つのフィルタが光学軸Aを共有して組み合わされて
いる。FIGS. 2a to 2d are examples of a filter system according to the invention. FIG. 2a shows sliding filters FS1, FS2 arranged one behind the other, each having a circular filter F1, F2. In this system, two different filters are combined sharing the optical axis A.
【0007】図2bではフィルタF1、F2がそれぞれ
回転式円板FR1,FR2上に配置されており、光学軸
Aを共有して組み合わされている。最も有利なものは、
連続可変式エッジフィルタVF1、VF2を少なくとも
一つずつ組み合わせたものである。図2cには円形回転
式、図2dにはスライド式のものを図示してある。この
種の可変式エッジパスフィルタは例えばCOHERENT社から
取り寄せることができる。図2dには個々のフィルタ領
域に対応する透過曲線の経過が描かれている。VF2は
短波長SP用の透過フィルタ、VF1は長波長LP用の
透過フィルタである。In FIG. 2b, filters F1 and F2 are arranged on rotary disks FR1 and FR2, respectively, and are combined so as to share an optical axis A. The most advantageous is
This is a combination of at least one of the continuously variable edge filters VF1 and VF2. FIG. 2c shows a circular rotary type and FIG. 2d shows a sliding type. Such a variable edge pass filter can be obtained, for example, from COHERENT. FIG. 2d shows the course of the transmission curve corresponding to the individual filter areas. VF2 is a transmission filter for short wavelength SP, and VF1 is a transmission filter for long wavelength LP.
【0008】図3には二つの調整例を示してある。それ
より明らかなように、短波長透過フィルタと長波長透過
フィルタとを組み合わせて、相互移動調整することによ
り任意の帯幅を持つ検出波長透過用バンドパスフィルタ
を構成することができる。これは小さな波長帯幅単位で
全域を連続的に走査する波長可変式モノクロメータとし
て、試料内の検査対象である外生または内生蛍光団につ
いて完全な蛍光スペクトルの記録を可能にし、極めて有
用である。FIG. 3 shows two adjustment examples. As is clear from this, a short-wavelength transmission filter and a long-wavelength transmission filter are combined to adjust the mutual movement, whereby a bandpass filter for transmitting a detection wavelength having an arbitrary bandwidth can be configured. This is a wavelength tunable monochromator that continuously scans the entire area in small wavelength bandwidth units, enabling the recording of a complete fluorescence spectrum of the exogenous or endogenous fluorophore to be inspected in the sample, which is extremely useful. is there.
【0009】このように走査装置、試料テーブルおよび
検出器を調整することによってX、YおよびZ方向での
波長別、時間別の検出が可能になる。それによって5次
元(X、Y、Z、T、λ)の試料像が形成されることに
なる。 即ち、各画素毎に、また走査サイクルの各時間
毎にスペクトルを記録することが可能になる。By adjusting the scanning device, the sample table, and the detector in this manner, it becomes possible to perform detection by wavelength and by time in the X, Y, and Z directions. Thereby, a five-dimensional (X, Y, Z, T, λ) sample image is formed. That is, a spectrum can be recorded for each pixel and each time of a scanning cycle.
【0010】そのほか、本発明に基づくフィルタユニッ
トは照射面のスペクトル選択にも効果的に使用できる。
例えばシャッター装置の代わりとして、広幅なレーザ光
源を狭める目的に、またはHBO、HALランプにおけ
る波長の選択に使用できる。[0010] In addition, the filter unit according to the present invention can be effectively used for spectrum selection of an irradiation surface.
For example, as a substitute for a shutter device, it can be used for narrowing a wide laser light source or for selecting a wavelength in an HBO or HAL lamp.
【図1】顕微鏡ユニットMおよびそれに連結された走査
ヘッドSの図式FIG. 1 is a schematic diagram of a microscope unit M and a scanning head S connected thereto.
【図2】(a)スライド式フィルタ、(b)回転式円板
フィルタ、(c)連続可変円形回転式エッジフィルタ,
(d)連続可変スライド式FIG. 2 (a) a slide type filter, (b) a rotary disk filter, (c) a continuously variable circular rotary edge filter,
(D) Continuously variable slide type
【図3】短波長透過フィルタと長波長透過フィルタとの
調整例FIG. 3 shows an example of adjusting a short wavelength transmission filter and a long wavelength transmission filter.
【符号の説明】 M 顕微鏡ユニット S 走査ヘッド ST 光線分離器 LQ 光源 S2 転向式ミラー SC 走査装置 V シャッター EO 結像光学系 KO 視準光学系 PH ピンホール FE フィルタユニット SP 短波長透過フィルタ LP 長波長透過フィルタ NF ノッチフィルタ FS フィルタ FR 回転式フィルタ VF 連続可変式フィルタ[Description of Signs] M Microscope unit S Scan head ST Beam separator LQ light source S2 Turning mirror SC Scanning device V Shutter EO Imaging optical system KO Collimating optical system PH Pinhole FE Filter unit SP Short wavelength transmission filter LP Long wavelength Transmission filter NF Notch filter FS filter FR Rotary filter VF Continuously variable filter
───────────────────────────────────────────────────── フロントページの続き (72)発明者 ミカエル ストック (原語表記)Mic hael Stock ドイツ国 D−99510 アポルダ エルフ ルテルストラッセ 32 (原語表記)Er furter Str.32,D−99510 Apolda,Germany (72)発明者 ウルリッヒ サイモン (原語表記)Ul rich Simon ドイツ国 D−07751 ローゼンスタイン ブルグストラッセ 35 (原語表記)B urgstr.35,D−07751 Roth enstein,Germany (72)発明者 トルステン アントラック (原語表記) Torsten Antrack ドイツ国 D−07745 イエナ アンナ・ シイームセン・ストラッセ 20 (原語表 記)Anna−Siemsen−Str. 20, D−07745 Jena,Germ any ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Michael Stock (Original notation) Michael Stock Germany D-99510 Apolda Elf Luterstrasse 32 (Original notation) Er after Str. 32, D-99510 Apolda, Germany (72) Inventor Ulrich Simon (Original notation) Ulrich Simon Germany D-07751 Rosenstein Burgstrasse 35 (Original notation) Burgstr. 35, D-07751 Rotenstein, Germany (72) Inventor Torsten Antrac (Original notation) Torsten Anttrack Germany D-07745 Jena Anna Siamsen-Strasse 20 (Original description) Anna-Siemsen-Str. 20, D- 07745 Jena, German any
Claims (8)
短波長透過フィルタと少なくとも一つの長波長透過フィ
ルタとの、少なくとも一つの組み合わせから成る調整可
能なバンドパスフィルタとして、蛍光顕微鏡、特にレー
ザ走査顕微鏡に使用される制御可能な波長別検出のため
のシステム1. A fluorescence microscope, in particular a laser scanning microscope, as an adjustable bandpass filter comprising at least one combination of at least one short wavelength transmission filter and at least one long wavelength transmission filter arranged in the detection light path. For controllable wavelength-specific detection used in optics
性を持つ別なフィルタに取換可能および/またはその波
長特性が調整可能である、特許請求項1に基づくシステ
ム2. The system according to claim 1, wherein at least one filter is replaceable with another filter having another wavelength characteristic and / or its wavelength characteristic is adjustable.
長透過フィルタ用のフィルタ交換手段が備えられてい
る、特許請求項1または2に基づくシステム3. The system according to claim 1, further comprising filter changing means for a short wavelength transmission filter and / or a long wavelength transmission filter.
たは回転式円板装置である、特許請求項3に基づくシス
テム4. The system according to claim 3, wherein the filter changing means is a sliding device or a rotating disk device.
スライド式の連続移動可能な短波長透過フィルタおよび
/または長波長透過フィルタを有する、特許請求項1〜
4の一つに基づくシステム5. The method according to claim 1, comprising at least one rotatable and / or sliding continuously movable short wavelength transmission filter and / or long wavelength transmission filter.
System based on one of the four
段が取換可能である、特許請求項1〜5の一つに基づく
システム6. The system according to claim 1, wherein the filter and / or the filter changing means are replaceable.
段がモータ駆動式である、特許請求項1〜6の一つに基
づくシステム7. The system according to claim 1, wherein the filter and / or the filter changing means are motor-driven.
ムを、特にレーザ走査顕微鏡に使用して、検出ユニット
で検査結果を時間別、波長別に記録し、貯蔵ユニットに
記憶させる画像記録のための方法8. A system according to claim 1, wherein the inspection results are recorded by time and by wavelength in a detection unit and are stored in a storage unit by using a laser scanning microscope. Way for
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE1998135070 DE19835070B4 (en) | 1998-08-04 | 1998-08-04 | Arrangement for adjustable wavelength-dependent detection in a fluorescence microscope |
DE19835070.8 | 1998-08-04 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2000056228A true JP2000056228A (en) | 2000-02-25 |
JP4608688B2 JP4608688B2 (en) | 2011-01-12 |
Family
ID=7876336
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP21781199A Expired - Fee Related JP4608688B2 (en) | 1998-08-04 | 1999-07-30 | Laser scanning fluorescence microscope and image recording method |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP4608688B2 (en) |
DE (1) | DE19835070B4 (en) |
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