JPS62192632A - Microscope photometer - Google Patents
Microscope photometerInfo
- Publication number
- JPS62192632A JPS62192632A JP62031660A JP3166087A JPS62192632A JP S62192632 A JPS62192632 A JP S62192632A JP 62031660 A JP62031660 A JP 62031660A JP 3166087 A JP3166087 A JP 3166087A JP S62192632 A JPS62192632 A JP S62192632A
- Authority
- JP
- Japan
- Prior art keywords
- photometer
- microscope
- chopper wheel
- filters
- modulator
- 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.)
- Pending
Links
- 238000005286 illumination Methods 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 12
- 230000003287 optical effect Effects 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 230000004907 flux Effects 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims 1
- 230000005284 excitation Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 101001080808 Homo sapiens PH and SEC7 domain-containing protein 2 Proteins 0.000 description 4
- 102100027455 PH and SEC7 domain-containing protein 2 Human genes 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003157 biological pigment Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 101001080825 Homo sapiens PH and SEC7 domain-containing protein 1 Proteins 0.000 description 1
- 102100027472 PH and SEC7 domain-containing protein 1 Human genes 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0096—Microscopes with photometer devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J1/00—Photometry, e.g. photographic exposure meter
- G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
- G01J2001/4242—Modulated light, e.g. for synchronizing source and detector circuit
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/1738—Optionally different kinds of measurements; Method being valid for different kinds of measurement
- G01N2021/174—Optionally different kinds of measurements; Method being valid for different kinds of measurement either absorption-reflection or emission-fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6421—Measuring at two or more wavelengths
Abstract
(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、２つの異なる波長で試料の螢光または吸収を
測定するための顕微鏡測光器に関する。DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a microscopic photometer for measuring the fluorescence or absorption of a sample at two different wavelengths.
従来の技術
細胞生物学的研究に関しては、試料を標識するために、
極めて特殊な細胞特性もしくは試料特性の測定を許す生
物色素物質が次第に多く使用されている。標識の作用方
式は、化学的／生物学的反応の作用下で生物色素物質の
スペクトル挙動に変化が起ることを基礎としている。こ
れは第４図で略示しである。Conventional techniques for labeling samples for cell biological research include:
Biological pigments are increasingly being used that allow the measurement of very specific cellular or sample properties. The mode of action of the label is based on the fact that a change occurs in the spectral behavior of the biological pigment substance under the action of a chemical/biological reaction. This is shown schematically in FIG.
例えば標識物質ＦＵＲＡ−２は、被検細胞試料中の遊離
Ｃａイオンの濃度の変化に暴露さねている場合には、第
３図で略示した形で同物質の螢光挙動を変化させる。従
って細胞中の遊離カルシウムイオンの濃度を、顕微鏡測
光器で２つの励起波長を用いて螢光測光による測定によ
って特定することができる。For example, when the labeling substance FURA-2 is exposed to changes in the concentration of free Ca ions in a sample of cells to be tested, the fluorescent behavior of the substance changes in the manner shown schematically in FIG. The concentration of free calcium ions in cells can therefore be determined by fluorophotometric measurements using two excitation wavelengths in a microphotometer.
絶対強度のみ変化するが、スペクトル挙動は変らない従
来使用された標識に対して、最近の標識は相対測定の利
点を有する。従って例えば照明の装置特性および安定性
が省略される（係６（１９８５）、１４５〜１５７頁に
は、ライエン（Ｔｓｉｅｎ）等によって、２つの異なる
波長で試料を励起するために２個の光源および光源の後
に接続された２個の可変モノクロメータ−を有する顕微
鏡測光器が記載されている。両モノクロ）−９−＋７）
光はチヨツＡ −（Ｚｅｒｈａｃｋｅｒ ）を介して顕
微鏡の照明光路に順次入力される。In contrast to traditionally used labels, where only the absolute intensity changes, but the spectral behavior remains unchanged, modern labels have the advantage of relative measurements. Thus, for example, the device characteristics and stability of the illumination are omitted (Tsien et al. 6 (1985), pp. 145-157). A microscope photometer is described with two variable monochromators connected after the light source.
The light is sequentially input into the illumination optical path of the microscope via the Zerhacker.
この公知の装置は浪費的で高価であり、す↑に存在する
顕微鏡もしくは顕微鏡測光器にそのままあとから取付け
ることができない。This known device is tedious and expensive and cannot be directly retrofitted to existing microscopes or microphotometers.
また、モノクロメータ−およびチョツパーホイー／ｌ／
（Ｚｅｒｈａｃｋｅｒｒａｄ） ｏ代りに１対の回転
干渉フイルタ−を使用することも提案された。このよう
な照明装置は例えば米国特許第３４９７６９０号１他の
目的に関して記載されている。Also, monochromator and chopper wheel/l/
(Zerhackerrad) It has also been proposed to use a pair of rotating interference filters instead. Such a lighting device is described, for example, in US Pat. No. 3,497,690 1 for other purposes.
しかし前記両手段は根本的に、２つの異なる波長に従う
信号が測光器の検出器によって順次（二検出されるとい
う欠点を有する。この検出方式は、信号形が十分に保存
されていなければならず、従って広い増幅帯域幅の要求
される純粋な切換方法である。しかし広い増幅帯域幅に
よって測定系が環境光の障害および検出器ならびに増幅
器の雑音に対して敏感になる。従ってこのような方法の
検出範囲は限定されている。However, both said means fundamentally have the disadvantage that signals following two different wavelengths are detected sequentially by the detector of the photometer. This detection method requires that the signal shape be well preserved. , thus requiring a wide amplification bandwidth, is a pure switching method.However, a wide amplification bandwidth makes the measurement system sensitive to ambient light disturbances and detector and amplifier noise. Detection range is limited.
本発明の課題は、２つの異なる波長で極めて鋭敏な測定
を許し、この際このよう、な測定に必要な照明装置を大
きな費用なしにかつ容易に、すｆに存在する顕微鏡（＝
あとから適合させることのできる顕微鏡測光器を提供す
ること〕ある。The object of the invention is to allow extremely sensitive measurements at two different wavelengths, and to easily and without great expense provide the illumination equipment necessary for such measurements, which are present in the microscope (=
It is an object of the present invention to provide a microscope photometer that can be adapted later.
問題点を解決するための手段
前記課題は本発明により、ランプ（ランジノ１ウジング
）の代りに顕微鏡に取付は可能の照明ブロックが設けら
れていて、同照明ブロックが少なくとも２個のフイルタ
−およびこれらの２個のフィルタ′−を透過する光を異
なる周波数フ同時に変調する装置を包含しかつ試料から
放射する光の強度測定のために使用される検出器（光電
子増倍管）の後の検出チャンネルに、変調−装置の周波
数に同調される２個の狭帯域増幅器が並列接続で配置さ
れていることによって解決される。Means for Solving the Problems According to the present invention, an illumination block that can be attached to a microscope is provided instead of a lamp (a one-piece housing), and the illumination block is connected to at least two filters and the like. a detection channel after the detector (photomultiplier tube) which contains a device for simultaneously modulating the light transmitted through two filters to different frequencies and is used for measuring the intensity of the light emitted by the sample; This is achieved by arranging two narrowband amplifiers in parallel, which are tuned to the frequency of the modulator.
前記手段によって、異なる波長での励起（二対応する信
号が測光器の検出装置によって同時に検出され、並行的
に評価される。検出エレクトロニクスの２個の並行分岐
における狭帯域の信号処理（二より高い信号／雑音比が
得られ、その結果検出感度が著しく改善される。By said means, excitation at different wavelengths (two corresponding signals are detected simultaneously by the detection device of the photometer and evaluated in parallel; narrowband signal processing in two parallel branches of the detection electronics (two higher A signal/noise ratio is obtained, resulting in a significantly improved detection sensitivity.
さらに照明装置は変調可能な構成群として従来常用のラ
ンプハウジングの代りに顕微鏡測光器に容易に取付ける
ことができる。分割の異なる数列の孔路（Ｓｐｕｒ）を
有するチョツ・ぐ−ホイールまたは２つの異なる周波数
を有する両波長の光を同時に変調するための別個に制御
可能の元シャッターを設けるのが有利である。Furthermore, the illumination device can be easily mounted as a modulator on the microscope photometer instead of the conventional lamp housing. It is advantageous to provide a separately controllable source shutter for simultaneously modulating the light of both wavelengths with two different frequencies or a double wheel with a number of rows of holes of different divisions.
照明ブロックには、異なる波長の光を分離するために使
用されるフイルタ−、例えば干渉フイルターが有利には
交換可能に配置されており、これによって励起波長を種
々の標識物質のスペクトル挙動に適合させることができ
る。Filters, for example interference filters, which are used to separate light of different wavelengths are advantageously arranged interchangeably in the illumination block, thereby adapting the excitation wavelength to the spectral behavior of the various labeling substances. be able to.
本発明の他の利点は、次の実施例の説明から明らか（＝
なる。Other advantages of the invention are apparent from the following description of the embodiments (=
Become.
実施例
第６図で全構造を略示した測光器は、試料３から放射す
る、例えば螢光光束の検出のための検出器として光電子
増倍管２を有する従来の型の顕微鏡１から成る。試料３
を２つの異なる波長を有する光を励起するためには、変
調装置５を包含する照明ブロック生が用いられる。照明
ブロック生は第１・〜３図につい工さらに詳細に記載す
る。Embodiment The photometer, the complete structure of which is shown schematically in FIG. 6, consists of a microscope 1 of conventional type with a photomultiplier tube 2 as a detector for the detection of e.g. a fluorescent light flux emitted by a sample 3. Sample 3
In order to excite light with two different wavelengths, an illumination block generator containing a modulator 5 is used. The construction of the lighting block is described in more detail in Figures 1 to 3.
変調装置５は制御装置６によって制御され、２つの異な
る周波数ｆ１およびｆ２を有する照明光の２つの部分光
束の強度を変調する。２つの周波数はまたｒｅｆｌ’お
よびｒｅｆ ２を付した導線を介して参照周波数として
２個の並列狭帯域増幅器ＰＳＤｌおよびＰＳＤ２に伝送
されるが、これら増幅器のインプットは光電子増倍管２
の後に接続された前増幅器７のアウトプットに結合され
ている。A modulation device 5 is controlled by a control device 6 and modulates the intensity of two partial beams of illumination light with two different frequencies f1 and f2. The two frequencies are also transmitted as reference frequencies via conductors labeled refl' and ref 2 to two parallel narrowband amplifiers PSDl and PSD2, whose inputs are connected to photomultiplier tube 2.
is coupled to the output of a preamplifier 7 connected after the preamplifier 7.
狭帯域増幅器ＰＳＤＩおよびＰＳＤ２は、その都度の参
照周波数ｒｅｆ ｌおよびｒｅｆ ２で動作する（ロッ
クイン法＝ １ｏｃｋ −ｉｎ −Ｔｅｃｈｎｉｋ）位
相選択整流器を包含する。この場合には、光電子増倍管
２から送出された信号Ｅ（その時間的経過を第７図で示
す）から、２つの異なる励起波長１の試料３の螢光光束
の強度に相応する２つの別々の信号Ａ１およびＡ２が、
狭帯域増幅器ＰＳＤ１およびＰＳＤ２のアウトプットに
発生する。信号Ａ１およびＡ２は多重装量（Ｍｕｌｔｉ
ｐｌｅｘｅｒ ） ８ ニよツーｉｃ受信され、ｌｆ−
ログ−デジタル変換器９での変換後に、例えば試料３中
に含有されたＣａ イオンの濃度を引算するためにコ
ンピュータ１ｏに伝送される。The narrowband amplifiers PSDI and PSD2 include phase-selective rectifiers operating at the respective reference frequencies ref l and ref 2 (lock-in method = 1ock-in-Technik). In this case, from the signal E sent out from the photomultiplier tube 2 (its time course is shown in FIG. 7), two signals corresponding to the intensities of the fluorescent light beams of the sample 3 at two different excitation wavelengths 1 are obtained. Separate signals A1 and A2 are
occurs at the outputs of narrowband amplifiers PSD1 and PSD2. Signals A1 and A2 are multiplexed (Multi
plexer) 8 Niyo two IC received, lf-
After the conversion in the log-to-digital converter 9, it is transmitted to the computer 1o, for example in order to subtract the concentration of Ca 2 contained in the sample 3.
変調装置５の制御装置６と多重装置８およびコンピュー
ター１０の動作周波数との間の同期化は本発明（−よる
この装置の場合には必要ない。Synchronization between the control device 6 of the modulator 5 and the operating frequencies of the multiplexer 8 and the computer 10 is not necessary in the case of this device according to the invention.
第１ａ図には、使用される照明ブロック手が詳細に図示
しである。同ブロックは密閉ハウジング２６から成る。FIG. 1a shows a detailed illustration of the lighting blocks used. The block consists of a closed housing 26.
このハウジングは従来そこに取付けられた顕微鏡ランプ
の代りに、慣用の支持部材（Ａｕｆｎａｍｅｓｃｈｗａ
Ｉｂｅ ）１１によって顕微鏡測光器のスタンド２５
に取付けられている。ハウジング２６の内部には、高出
力光源１２、例えば相互に正反対側で対向している２個
のランプコレクター１３および１４を有する水銀高圧ラ
ンプが存在している。ランプ１２から放射する２つの光
束は、変向鏡１５および１６によって異なるスにクトル
透過率を有する各１個の励起フイルタ−１７および１８
に送られる。フイルタ−１７および１８は例えば、第３
図でＡ１およびＡ２で示しである２つの波長の近傍、例
えば３４０ ｎｍおよび３８０ ｎｍにその透過領域を
有する干渉フイルタ−ｆある。This housing is equipped with a conventional supporting member (Aufnameschwa) instead of the microscope lamp previously mounted thereon.
Microscope photometer stand 25 by Ibe) 11
installed on. Inside the housing 26 there is a high-power light source 12, for example a mercury high-pressure lamp with two lamp collectors 13 and 14 diametrically opposed to each other. The two beams emanating from the lamp 12 are passed through deflection mirrors 15 and 16 into excitation filters 17 and 18 each having a different transmittance.
sent to. Filters 17 and 18 are, for example, third
There is an interference filter f having its transmission region in the vicinity of two wavelengths, for example 340 nm and 380 nm, denoted A1 and A2 in the figure.
フイルタ−１７および１８を透過する、波長の異なる部
分光は、モーター２０によって駆動されるチョッパ−ホ
イール１９によって、周期的にではあるが異なる波長を
もって変調される。The partial lights of different wavelengths passing through the filters 17 and 18 are periodically modulated with different wavelengths by a chopper wheel 19 driven by a motor 20.
第１ｂ図は該チョッパ−ホイールの拡大部分を示す。同
図から分割の異なる２列の孔路２３および２４が認めら
れる。２本の部分光はチョッパ−ホイール１９による変
調後にビームスプリッタ−２１を介して顕微鏡測光器の
照明光路に一緒に入力される。 城励起光
の方形変調の場合には、狭帯増幅器ＰＳＤＩおよびＰＳ
Ｄ２による基本周波数ｆ１およびｆ２に基く信号の検出
を妨害する可能性のある調波信号が発生する。このよう
な妨害現象は、チョッ・ぐ−ホイールの両孔跡（２３お
よび２４）の分割比、ひいては周波数ｆｌ／ｆ２の比を
奇数的に選択することによって減少させることができる
。この手段によって、例えば周波数ｆ１の調波が周波数
ｆ２に対する検出に影響を与えることが妨止される。ま
た、励起光の強度を少なくともほぼ正弦波状に変調する
操作Ｃ二液えると、調波は初めから閉止される。正弦波
変調は、チョッパーホイールの平面における照明光路の
光束断面をチョツ・ξ−ホイールの孔の寸法に適当（二
適合させることによって得られる。Figure 1b shows an enlarged section of the chopper wheel. From the figure, two rows of holes 23 and 24 with different divisions can be seen. After modulation by the chopper wheel 19, the two partial beams are input together via a beam splitter 21 into the illumination beam path of the microscope photometer. In the case of square modulation of the pump light, narrowband amplifiers PSDI and PS
Harmonic signals are generated that can interfere with D2's detection of signals based on fundamental frequencies f1 and f2. Such disturbance phenomena can be reduced by an odd selection of the division ratio of the two hole traces (23 and 24) of the chopping wheel and thus of the frequency fl/f2. By this measure, for example, harmonics of frequency f1 are prevented from influencing the detection for frequency f2. Moreover, when the intensity of the excitation light is modulated at least approximately sinusoidally in the operation C, the harmonics are closed from the beginning. The sinusoidal modulation is obtained by suitably adapting the beam cross-section of the illumination beam path in the plane of the chopper wheel to the size of the hole in the chopper wheel.
また第２図のによる実施例の照明ブロックは、焦点距離
の異なる２個のコレクター（３３および３４）を有して
おり、これらのコレクターは、チョッパーホイールの２
種類の孔路上の孔の大きさに応じて異なる直径を有する
２つの円形領域が形成されるように、ランプ３２の光を
チョッパーホイール２９の平面（＝収束させる。Furthermore, the illumination block of the embodiment shown in FIG. 2 has two collectors (33 and 34) with different focal lengths, and these collectors
The light of the lamp 32 is focused on the plane of the chopper wheel 29 (=convergent) so that two circular areas with different diameters are formed on the different hole tops.
チョッノξ−ホイール２９の後の元の方向における２個
のレン！：３５および３６は、２つの異なる部分光路を
照明ブロック内で再び相互に調整するのに役立つ。残り
の構成部材は第１図による実施例の同部材と同様なので
ここで記載するのを省く。Chono ξ - two lenses in the original direction after the wheel 29! :35 and 36 serve to mutually align the two different partial beam paths again within the illumination block. The remaining components are similar to those of the embodiment according to FIG. 1 and will not be described here.
第３図には、第１図および第２図の照明ブロックとは別
の実施例を図示しである。第３図の照明ブロック１０４
は、そのハウジング１２４内：ニモーターによって駆動
されるチョッパ−ホイールの代りに、２個の別々に制御
可能の電気光学的光シヤツター１１９および１２０から
形成された変調装置１０５を包含する。該元シャッター
はフイルタ−１１７および１１８を透過する２つの部分
光を相互に独立的に変調する。FIG. 3 shows a different embodiment from the illumination block shown in FIGS. 1 and 2. Lighting block 104 in FIG.
contains in its housing 124 a modulation device 105 formed from two separately controllable electro-optic light shutters 119 and 120 instead of a chopper wheel driven by a motor. The original shutter modulates the two partial lights transmitted through filters 117 and 118 independently of each other.
でき、るだけ高い変調周波数を得ることができるために
、光シヤツターとして例えばカールセルが使用されてい
る。In order to obtain a modulation frequency as high as possible, a Karl cell, for example, is used as an optical shutter.
２個の元シャッターを有する前記手段は、シャッター開
放時間もしくは変調周波数が自由（二選択可能であって
、２本の照明光路に関して相互に独立的に調整可能であ
るという利点を提供する。この場合には変調周波数は検
出チャンネルで信号／雑音比に関して最適化されうる。Said means with two original shutters offers the advantage that the shutter opening time or the modulation frequency is freely selectable and can be adjusted independently of each other with respect to the two illumination beam paths. The modulation frequency can be optimized with respect to signal/noise ratio in the detection channel.
第１ａ図は、本発明による測光器の照明ブロックの第一
実施例の略示断面図、第１ｂ図は第１ａ図におけるチョ
ッパ−ホイール１９の拡大部分上面図、第２図は該照明
ブロックの第二実施例の略示断面図、第３図は該照明ブ
ロックの第三実施例の略示断面、第奎図は化学的−生物
学的反応の作用下での標識物質の吸光度のスペクトル変
化を示すグラフ、第３図は２種類のＣａ２＋濃度に対す
る生物色素標識物質ＦＵＲＡ−２の励起スペクトルを示
すグラフ、第６図は検出チャンネルを含む全測光器の構
成系統図、第７図は検出器２のアウトプットにおける信
号Ｅの時間的経過を示すグラフｔある：
１・・・顕微鏡、２・・・検出器、３・・・試料、牛、
１０４・・・照明ブロック、５，１０５・・・変調装置
、１７．１８ ； １１７．１１８・・・フイルタ−、
１９・・・チョッノξ−ホイール、２３．２４・・・孔
路、１１９．１２０・・・光シヤツター、ＰＳＤＩ。
ＰＳＤ２・・・狭帯域増幅器
４゛′明７’ ｕ ′’／
Ｆｉｇ、２
Ｆｉｇ、７FIG. 1a is a schematic cross-sectional view of a first embodiment of the illumination block of a photometer according to the invention, FIG. 1b is an enlarged top view of a chopper wheel 19 in FIG. 1a, and FIG. FIG. 3 is a schematic cross-sectional view of the third embodiment of the illumination block; FIG. Figure 3 is a graph showing the excitation spectrum of the biological dye labeling substance FURA-2 for two types of Ca2+ concentrations, Figure 6 is a diagram of the entire photometer including the detection channel, and Figure 7 is the detector. There is a graph t showing the time course of the signal E at the output of 2: 1...microscope, 2...detector, 3...sample, cow,
104... Lighting block, 5, 105... Modulator, 17.18; 117.118... Filter,
19... Chonno ξ-wheel, 23.24... Hole path, 119.120... Optical shutter, PSDI. PSD2...Narrowband amplifier 4''u''/ Fig, 2 Fig, 7
Claims (1)
るための顕微鏡測光器において、 −ランプ（ランプハウジング）の代りに顕微鏡に取付け
可能の照明ブロック（４、１０４）が設けられており、 −照明ブロックが少なくとも２個のフイルタ−（１７、
１８；１１７、１１８）および２個のフイルターを透過
し、同時に異なる周波数を有する光を変調する装置（５
、１０５）を包含し、 −かつ試料から放射する光の強度測定のために使用され
る検出器（光電子増倍管２）の後の検出チャンネルに、
変調装置（５、１０ ５）の周波数に同調された２個の狭帯域増幅器（ＰＳＤ
１、ＰＳＤ２）が並列接続で配置されていることを特徴
とする前記顕微鏡測光器。 ２、フイルター（１７、１８；１１７、１１８）が交換
可能である特許請求の範囲第１項記載の測光器。 ３、変調装置（５）が分割の異なる数列の孔跡（２３、
２４）を有するチョッパーホイール（１９）である特許
請求の範囲第１項記載の測光器。 ４、照明ブロックが、光をチョッパーホイールの平面に
集束する２本の結像光路を有する特許請求の範囲第３項
記載の測光器。 ５、チョッパーホイールの平面における２本の光路の光
束直径が異つている特許請求の範囲第４項記載の測光器
。 ６、変調装置（１０５）が別個に制御可能の２個の光シ
ャッター（１１９、１２０）から成る特許請求の範囲第
１項記載の測光器。[Claims] 1. In a microscope photometer for measuring the fluorescence or absorption of a sample at two different wavelengths: - an illumination block (4, 104) attachable to the microscope in place of the lamp (lamp housing); - the lighting block comprises at least two filters (17,
18; 117, 118) and a device (5) that transmits through two filters and simultaneously modulates light having different frequencies.
, 105) - and in the detection channel after the detector (photomultiplier tube 2) used for measuring the intensity of the light emitted by the sample,
Two narrowband amplifiers (PSD) tuned to the frequency of the modulator (5, 10 5)
1. The microscope photometer characterized in that PSDs (2) are arranged in parallel connection. 2. The photometer according to claim 1, wherein the filters (17, 18; 117, 118) are replaceable. 3. The modulator (5) creates hole traces (23,
24) A photometer according to claim 1, wherein the photometer is a chopper wheel (19) having a chopper wheel (19). 4. A photometer according to claim 3, wherein the illumination block has two imaging optical paths that focus the light onto the plane of the chopper wheel. 5. The photometer according to claim 4, wherein the two optical paths in the plane of the chopper wheel have different luminous flux diameters. 6. Photometer according to claim 1, wherein the modulator (105) comprises two separately controllable optical shutters (119, 120).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3604815.1 | 1986-02-15 | ||
| DE19863604815 DE3604815A1 (en) | 1986-02-15 | 1986-02-15 | Microscope photometer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62192632A true JPS62192632A (en) | 1987-08-24 |
Family
ID=6294162
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62031660A Pending JPS62192632A (en) | 1986-02-15 | 1987-02-16 | Microscope photometer |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS62192632A (en) |
| DE (1) | DE3604815A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02120056U (en) * | 1989-03-15 | 1990-09-27 | ||
| JPH0372238A (en) * | 1989-08-12 | 1991-03-27 | Res Dev Corp Of Japan | Highly sensitive microscopic multi-wavelength spectroscope |
| JP2003501615A (en) * | 1998-11-04 | 2003-01-14 | ザ リサーチ ファウンデーション オブ ステイト ユニヴァーシティ オブ ニューヨーク | Sample analysis with continuous quantum timing code |
| JP2004132760A (en) * | 2002-10-09 | 2004-04-30 | Shimadzu Corp | Method and device for analyzing sample containing a plurality of fluorescent materials |
| JP2011107160A (en) * | 2007-06-21 | 2011-06-02 | Gen-Probe Inc | Multichannel optical measuring apparatus |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3915421C2 (en) * | 1989-05-11 | 1995-03-02 | Bayer Ag | Device for measuring the fluorescence excitation of biological cells at two different wavelengths |
| DE4115401C2 (en) * | 1991-05-10 | 1994-04-14 | Rainer Dr Uhl | Fluorescence measuring device for determining the ion concentration of an examination object, which is colored with a fluorescent dye, the excitation maximum of which changes as a function of the ion concentration to be determined |
| DE4228366C2 (en) * | 1992-08-26 | 1995-05-24 | Rainer Dr Uhl | Fluorescence measuring device |
| DE4338531A1 (en) * | 1993-11-11 | 1995-05-18 | Leica Lasertechnik | Device for multi-color illumination of preparations |
| US5672515A (en) * | 1995-09-12 | 1997-09-30 | Optical Sensors Incorporated | Simultaneous dual excitation/single emission fluorescent sensing method for PH and pCO2 |
| DE10231667A1 (en) | 2002-07-12 | 2004-01-22 | Olympus Biosystems Gmbh | Lighting device and optical object inspection device |
| JP2004163413A (en) | 2002-08-28 | 2004-06-10 | Carl-Zeiss-Stiftung Trading As Carl Zeiss | Microscope system and microscope inspection method |
| GB0525072D0 (en) | 2005-12-09 | 2006-01-18 | Enigma Diagnostics Ltd | Fluorescence-based detection methods and apparatus |
| US20150049328A1 (en) * | 2012-06-11 | 2015-02-19 | Protectlife International Biomedical Inc. | Biochemical analyzing system and light module thereof |
| CN111398230A (en) * | 2019-03-27 | 2020-07-10 | 上海交通大学 | Time-gated fluorescence imaging system |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS49106382A (en) * | 1973-02-09 | 1974-10-08 | ||
| JPS5340541A (en) * | 1976-09-25 | 1978-04-13 | Olympus Optical Co Ltd | Quantitative detecting microscope |
| JPS53149088A (en) * | 1977-06-01 | 1978-12-26 | Hitachi Ltd | Optical absorption measuring system |
| JPS56501174A (en) * | 1979-08-23 | 1981-08-20 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5419263B2 (en) * | 1973-10-17 | 1979-07-13 | ||
| DE2614181C3 (en) * | 1976-04-02 | 1981-08-20 | Leybold-Heraeus Gmbh, 5000 Koeln | Method for measuring the optical absorbance of samples and device for carrying out the method |
| US4100416A (en) * | 1977-03-02 | 1978-07-11 | Block Engineering, Inc. | Serum fluorescence suppression |
-
1986
- 1986-02-15 DE DE19863604815 patent/DE3604815A1/en active Granted
-
1987
- 1987-02-16 JP JP62031660A patent/JPS62192632A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS49106382A (en) * | 1973-02-09 | 1974-10-08 | ||
| JPS5340541A (en) * | 1976-09-25 | 1978-04-13 | Olympus Optical Co Ltd | Quantitative detecting microscope |
| JPS53149088A (en) * | 1977-06-01 | 1978-12-26 | Hitachi Ltd | Optical absorption measuring system |
| JPS56501174A (en) * | 1979-08-23 | 1981-08-20 |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02120056U (en) * | 1989-03-15 | 1990-09-27 | ||
| JPH0372238A (en) * | 1989-08-12 | 1991-03-27 | Res Dev Corp Of Japan | Highly sensitive microscopic multi-wavelength spectroscope |
| JP2003501615A (en) * | 1998-11-04 | 2003-01-14 | ザ リサーチ ファウンデーション オブ ステイト ユニヴァーシティ オブ ニューヨーク | Sample analysis with continuous quantum timing code |
| JP2004132760A (en) * | 2002-10-09 | 2004-04-30 | Shimadzu Corp | Method and device for analyzing sample containing a plurality of fluorescent materials |
| JP2011107160A (en) * | 2007-06-21 | 2011-06-02 | Gen-Probe Inc | Multichannel optical measuring apparatus |
| US9458451B2 (en) | 2007-06-21 | 2016-10-04 | Gen-Probe Incorporated | Multi-channel optical measurement instrument |
| US10086342B2 (en) | 2007-06-21 | 2018-10-02 | Gen-Probe Incorporated | Multi-channel optical measurement instrument |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3604815C2 (en) | 1988-12-08 |
| DE3604815A1 (en) | 1987-08-20 |
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