JPH07199074A - Confocal microscope - Google Patents
Confocal microscopeInfo
- Publication number
- JPH07199074A JPH07199074A JP33466293A JP33466293A JPH07199074A JP H07199074 A JPH07199074 A JP H07199074A JP 33466293 A JP33466293 A JP 33466293A JP 33466293 A JP33466293 A JP 33466293A JP H07199074 A JPH07199074 A JP H07199074A
- Authority
- JP
- Japan
- Prior art keywords
- sample
- light
- illumination
- scanning
- epi
- 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
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
- G02B21/0044—Scanning details, e.g. scanning stages moving apertures, e.g. Nipkow disks, rotating lens arrays
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- 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)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は走査型共焦点顕微鏡に関
し、特に落射照明時に対物レンズを交換することなく容
易に観察位置を特定できる共焦点顕微鏡に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning confocal microscope, and more particularly to a confocal microscope which can easily identify an observation position without changing the objective lens during epi-illumination.
【０００２】[0002]
【従来の技術】従来の走査型共焦点顕微鏡（以下、単に
共焦点顕微鏡と呼ぶ。）は複数ピンホールを有する円板
を回転させ、この円板にレーザの平行光を入射して前記
各ピンホールを通過した光が試料上を走査するようにし
たものである。また、前記ピンホールと同一パターンの
マイクロレンズを有する円板を前記ピンホールを有する
円板と同期させて回転させることにより光利用効率を改
善することができる。2. Description of the Related Art A conventional scanning confocal microscope (hereinafter, simply referred to as a confocal microscope) rotates a disc having a plurality of pinholes, and collimates a laser beam to the disc to make each pin. The light passing through the hole scans the sample. Further, the light utilization efficiency can be improved by rotating the disk having the microlenses having the same pattern as the pinhole in synchronization with the disk having the pinhole.
【０００３】図２はこのような従来のマイクロレンズを
付加した共焦点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はレーザ、２は落射照明用光源、３
はミラー、４は複数のマイクロレンズを有する円板（以
下、円板４と呼ぶ。）、５はカメラ、６はレンズ、７は
バリアフィルタ、８はダイクロイックミラー、９は前記
マイクロレンズと同一パターンのピンホールを有する円
板（以下、円板９と呼ぶ。）、１０は対物レンズ、１１
は試料である。FIG. 2 is a block diagram showing an example of a confocal microscope to which such a conventional microlens is added. In FIG. 2, 1 is a laser, 2 is a light source for epi-illumination, and 3 is
Is a mirror, 4 is a disc having a plurality of microlenses (hereinafter referred to as disc 4), 5 is a camera, 6 is a lens, 7 is a barrier filter, 8 is a dichroic mirror, and 9 is the same pattern as the microlens. A disc having pinholes (hereinafter referred to as disc 9), 10 is an objective lens, 11
Is a sample.
【０００４】落射照明時に、落射照明用光源２の出力光
はミラー３、ダイクロイックミラー８及び対物レンズ１
０を介して試料１１に照射される。During epi-illumination, the output light of the epi-illumination light source 2 is a mirror 3, a dichroic mirror 8 and an objective lens 1.
The sample 11 is irradiated via 0.
【０００５】試料１１からの反射光は対物レンズ１０を
介してダイクロイックミラー８に入射される。ダイクロ
イックミラー８においてこの入射光は反射されバリアフ
ィルタ７及びレンズ６を介してカメラ５に入射される。The reflected light from the sample 11 is incident on the dichroic mirror 8 via the objective lens 10. This incident light is reflected by the dichroic mirror 8 and enters the camera 5 via the barrier filter 7 and the lens 6.
【０００６】共主点観察時にはミラー３は取り除かれ、
円板４及び９が挿入される。レーザ１の出力光は円板４
に入射され、入射光の内マイクロレンズを透過した光は
ダイクロイックミラー８を介して円板９に入射される。When observing the principal points, the mirror 3 is removed,
The discs 4 and 9 are inserted. Output light of laser 1 is disk 4
The light that has been incident on and has passed through the microlens of the incident light is incident on the disc 9 via the dichroic mirror 8.
【０００７】また、円板９に入射された光の内ピンホー
ルを通過した光は対物レンズ１０を介して試料１１に照
射される。The light that has passed through the inner pinhole of the light incident on the disk 9 is applied to the sample 11 via the objective lens 10.
【０００８】試料１１からの反射光は対物レンズ１０を
介して円板９に入射され、前記ピンホールと同一のピン
ホールを通過してダイクロイックミラー８に入射され
る。ダイクロイックミラー８においてこの入射光は反射
されバリアフィルタ７及びレンズ６を介してカメラ５に
入射される。The reflected light from the sample 11 is incident on the disk 9 through the objective lens 10, passes through the same pinhole as the pinhole, and is incident on the dichroic mirror 8. This incident light is reflected by the dichroic mirror 8 and enters the camera 5 via the barrier filter 7 and the lens 6.
【０００９】ここで、図２に示す従来例の動作を説明す
る。先ず、試料１１上の観察位置を探す時には、円板４
及び９を取り除き、ミラー３を挿入して落射照明用光源
２の出力光を試料１１に照射する。この時対物レンズ１
０を低倍率にして大きな視野で観察位置を探し、大まか
の見当を付けた後、対物レンズ１０を高倍率にして観察
位置を特定する。The operation of the conventional example shown in FIG. 2 will be described. First, when searching for the observation position on the sample 11, the disk 4
And 9 are removed, the mirror 3 is inserted, and the output light of the epi-illumination light source 2 is applied to the sample 11. At this time, the objective lens 1
The observation position is searched for in a large field of view with 0 as the low magnification, and after roughly estimating, the objective lens 10 is set to the high magnification to specify the observation position.
【００１０】次に、ミラー３を取り除き円板４及び９を
挿入し、共焦点系に切り換えて試料１１上の前記観察位
置での観察を行う。Next, the mirror 3 is removed, the disks 4 and 9 are inserted, and the confocal system is switched to perform observation at the observation position on the sample 11.
【００１１】[0011]
【発明が解決しようとする課題】しかし、図２に示す従
来例において、共焦点系では分解能は向上するがスライ
ス画像となるため全体像が分かりにくいため、一旦共焦
点系に切り換えて後に観察位置を見失った場合には、再
びミラー３を挿入し円板４及び９を取り除き落射照明に
し、さらに対物レンズ１０を低倍率に切り換えて観察位
置を特定し直さなければならない場合が多い。However, in the conventional example shown in FIG. 2, the resolution is improved in the confocal system, but the whole image is difficult to understand because it becomes a slice image. When the user loses sight, it is often necessary to insert the mirror 3 again, remove the disks 4 and 9 to make epi-illumination, and switch the objective lens 10 to a low magnification to respecify the observation position.
【００１２】また、共焦点系ではスライスによって光量
が低下するためレーザ１には強い励起光のものを用いて
おり、この結果、試料１１の蛍光退色が生じ易くなるた
め共焦点系と落射照明を何回も切り換えるのは好ましく
ない。Further, in the confocal system, since the light quantity is reduced by slicing, strong excitation light is used for the laser 1. As a result, fluorescence fading of the sample 11 is likely to occur, so that the confocal system and the epi-illumination are used. It is not preferable to switch it many times.
【００１３】さらに、対物レンズ１０は水浸若しくは油
浸の物が多いため、倍率切り換えの際に大きく位置がず
れたり、交換に時間がかかり面倒な作業であると言った
問題点がある。従って本発明の目的は、同一の対物レン
ズを用いて観察位置の特定が容易な共焦点顕微鏡を実現
することにある。Further, since the objective lens 10 is often soaked with water or oil, there is a problem in that the position is greatly displaced when switching magnifications, and replacement is time-consuming and troublesome. Therefore, an object of the present invention is to realize a confocal microscope in which the observation position can be easily specified by using the same objective lens.
【００１４】[0014]
【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、走査型共焦点顕微鏡において、
複数の開孔を有する円板を用いて照明光を走査させる走
査手段と、試料の観察位置を特定するための落射照明手
段と、前記走査手段と前記落射照明手段との出力光を切
り換え、この切り換えられた出力光を前記試料に照射
し、前記試料からの反射光若しくは蛍光を前記走査手段
を介させると共に観察倍率を高倍率にして出力若しくは
直接出力する切り換え手段と、この切り換え手段の出力
光を観察する観察手段とを備えたことを特徴とするもの
である。In order to achieve such an object, in the present invention, in a scanning confocal microscope,
Scanning means for scanning the illumination light using a disc having a plurality of apertures, epi-illumination means for specifying the observation position of the sample, and switching the output light of the scanning means and the epi-illumination means, Switching means for irradiating the sample with switched output light, outputting reflected light or fluorescence from the sample through the scanning means, and outputting or directly outputting a high magnification of observation magnification, and output light of this switching means And observing means for observing.
【００１５】[0015]
【作用】共焦点系である走査手段と試料の観察位置を特
定する落射照明手段とを設け、切り換え手段を用いて走
査手段と落射照明手段とを切り換えると共に共焦点系の
時にはリレーレンズを透過させることにより、同一の対
物レンズを用いて観察位置の特定ができる。The scanning means, which is a confocal system, and the epi-illumination means for specifying the observation position of the sample are provided, and the switching means is used to switch between the scanning means and the epi-illumination means, and the relay lens is transmitted when the confocal system is used. As a result, the observation position can be specified using the same objective lens.
【００１６】[0016]
【実施例】以下本発明を図面を用いて詳細に説明する。
図１は本発明に係る共焦点顕微鏡の一実施例を示す構成
ブロック図である。ここで、１，２，４〜７及び９〜１
１は図２と同一符号を付してある。The present invention will be described in detail below with reference to the drawings.
FIG. 1 is a configuration block diagram showing an embodiment of a confocal microscope according to the present invention. Where 1, 2, 4-7 and 9-1
1 is given the same reference numeral as in FIG.
【００１７】図１において１２は励起フィルタ、１３及
び１６はダイクロイックミラー、１４及び１７はミラ
ー、１５はリレーレンズである。In FIG. 1, 12 is an excitation filter, 13 and 16 are dichroic mirrors, 14 and 17 are mirrors, and 15 is a relay lens.
【００１８】また、１，４，９及び１６は走査手段５０
を、２及び１２は落射照明手段５１を、１０，１３〜１
５及び１７は切り換え手段５２を、５〜７は観察手段５
３をそれぞれ構成している。Further, reference numerals 1, 4, 9 and 16 are scanning means 50.
2 and 12 are epi-illumination means 51, 10, 13-1
5 and 17 are switching means 52, and 5 to 7 are observing means 5.
3 respectively.
【００１９】レーザ１の出力光は円板４に入射され、入
射光の内一のマイクロレンズを透過した光はダイクロイ
ックミラー１６を介して円板９に入射される。The output light of the laser 1 is incident on the disc 4, and the light that has passed through one of the microlenses of the incident light is incident on the disc 9 via the dichroic mirror 16.
【００２０】また、円板９に入射された光の内一のピン
ホールを通過した光はミラー１７、ミラー１４及び対物
レンズ１０を介して試料１１に照射される。The light that has passed through one of the pinholes of the light incident on the disk 9 is applied to the sample 11 via the mirror 17, the mirror 14 and the objective lens 10.
【００２１】試料１１からの反射光は対物レンズ１０、
ミラー１４及びミラー１７を介して円板９に入射され、
前記ピンホールと同一のピンホールを通過してダイクロ
イックミラー１６に入射される。ダイクロイックミラー
１６においてこの入射光は反射されリレーレンズ１５、
バリアフィルタ７及びレンズ６を介してカメラ５に入射
される。The reflected light from the sample 11 is the objective lens 10,
It is incident on the disk 9 through the mirror 14 and the mirror 17,
The light enters the dichroic mirror 16 through the same pinhole as the pinhole. This incident light is reflected by the dichroic mirror 16, and the relay lens 15,
The light enters the camera 5 via the barrier filter 7 and the lens 6.
【００２２】一方、落射照明用光源２の出力光は励起フ
ィルタ１２、ダイクロイックミラー１３及び対物レンズ
１０を介して試料１１に照射される。On the other hand, the output light of the epi-illumination light source 2 is applied to the sample 11 via the excitation filter 12, the dichroic mirror 13 and the objective lens 10.
【００２３】試料１１からの反射光は対物レンズ１０を
介してダイクロイックミラー１３に入射される。ダイク
ロイックミラー１３においてこの入射光は反射されバリ
アフィルタ７及びレンズ６を介してカメラ５に入射され
る。The reflected light from the sample 11 enters the dichroic mirror 13 via the objective lens 10. The incident light is reflected by the dichroic mirror 13 and enters the camera 5 via the barrier filter 7 and the lens 6.
【００２４】ここで、図１に示す実施例の動作を説明す
る。切り換え手段５２はダイクロイックミラー１３及び
ミラー１４を図面垂直方向に移動させることにより、光
路へのダイクロイックミラー１３及びミラー１４の挿入
及び取り除きを行う。The operation of the embodiment shown in FIG. 1 will be described. The switching means 52 moves the dichroic mirror 13 and the mirror 14 in the vertical direction in the drawing to insert and remove the dichroic mirror 13 and the mirror 14 into the optical path.
【００２５】先ず、試料１１上の観察位置を探す時に
は、切り換え手段５２によりダイクロイックミラー１３
を光路に挿入し、ミラー１４を光路から取り除く。即
ち、落射照明用光源２の出力光は励起フィルタにより波
長が選択され、円板４及び９を介さずに試料１１に照射
され、試料１１からの蛍光も円板４及び９を介さずにカ
メラ５に戻って来るため対物レンズ１０により観察位置
を特定することができる。First, when the observation position on the sample 11 is searched, the dichroic mirror 13 is switched by the switching means 52.
Is inserted into the optical path and the mirror 14 is removed from the optical path. That is, the wavelength of the output light of the epi-illumination light source 2 is selected by the excitation filter, and the sample 11 is irradiated without passing through the discs 4 and 9, and the fluorescence from the sample 11 is also passed through the camera without passing through the discs 4 and 9. Since it returns to 5, the observation position can be specified by the objective lens 10.
【００２６】次に、切り換え手段５２によりダイクロイ
ックミラー１３を光路から取り除き、ミラー１４を光路
に挿入することにより共焦点系に切り換える。この際、
共焦点系の光路には落射照明時よるも高倍率の観察倍率
になるリレーレンズ１５が付加されている。Next, the dichroic mirror 13 is removed from the optical path by the switching means 52 and the mirror 14 is inserted into the optical path to switch to the confocal system. On this occasion,
A relay lens 15 is added to the optical path of the confocal system so that the observation magnification is high even when the epi-illumination is used.
【００２７】この結果、共焦点系である走査手段５０と
試料の観察位置を特定する落射照明手段５１とを設け、
切り換え手段５２を用いて走査手段５０と落射照明手段
５１とを切り換えると共に共焦点系の時にはリレーレン
ズ１５を透過させることにより、同一の対物レンズ１０
を用いて観察位置の特定が容易になる。As a result, the scanning means 50 which is a confocal system and the epi-illumination means 51 for specifying the observation position of the sample are provided,
The switching means 52 is used to switch between the scanning means 50 and the epi-illumination means 51, and when the confocal system is used, the relay lens 15 is allowed to pass therethrough, whereby the same objective lens 10 is provided.
It becomes easy to specify the observation position by using.
【００２８】即ち、落射照明の観察倍率を共焦点系の観
察倍率よりも下げることにより、分解能は低いが焦点深
度が深い落射照明と分解能は高いが焦点深度が浅い共焦
点との特徴を生かすことができ、対物レンズの交換によ
る位置ずれがなく、短時間で観察位置の特定が可能な
る。That is, by making the observation magnification of the epi-illumination lower than that of the confocal system, the characteristics of the epi-illumination having a low resolution but a deep depth of focus and the confocal having a high resolution but a shallow depth of focus can be utilized. Therefore, the observation position can be specified in a short time without any displacement due to replacement of the objective lens.
【００２９】なお、図１に示す実施例ではカメラ５を用
いているが、レンズ６を接眼レンズとして直接観察して
も良い。また、図１に示す実施例ではマイクロレンズを
有する円板４を用いているが、円板４は省略しても良
い。また、図１に示す実施例では蛍光により観察位置を
特定しているが、反射照明や透過照明でも良い。また、
図１に示す実施例ではピンホールを用いているが、ピン
ホールのみならず入射光が通過する開孔であれば形状等
に係わりなく用いることが可能である。Although the camera 5 is used in the embodiment shown in FIG. 1, the lens 6 may be directly observed as an eyepiece. Further, although the disc 4 having the microlenses is used in the embodiment shown in FIG. 1, the disc 4 may be omitted. Further, in the embodiment shown in FIG. 1, the observation position is specified by fluorescence, but reflected illumination or transmitted illumination may be used. Also,
Although the pinhole is used in the embodiment shown in FIG. 1, not only the pinhole but also an opening through which incident light passes can be used regardless of the shape or the like.
【００３０】[0030]
【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。共焦点系である
走査手段と試料の観察位置を特定する落射照明手段とを
設け、切り換え手段を用いて走査手段と落射照明手段と
を切り換えると共に共焦点系の時には高倍率のリレーレ
ンズを透過させることにより、同一の対物レンズを用い
て観察位置の特定が容易な共焦点顕微鏡が実現できる。As is apparent from the above description,
The present invention has the following effects. A confocal system scanning means and epi-illumination means for specifying the observation position of the sample are provided, and the switching means is used to switch between the scanning means and epi-illumination means, and a confocal system allows a high-magnification relay lens to pass through. As a result, it is possible to realize a confocal microscope in which the observation position can be easily specified using the same objective lens.
【図１】本発明に係る共焦点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 an embodiment of a confocal microscope according to the present invention.
【図２】従来の共焦点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 showing an example of a conventional confocal microscope.
１ レーザ ２ 落射照明用光源 ３，１４，１７ ミラー ４ マイクロレンズを有する円板 ５ カメラ ６ レンズ ７ バリアフィルタ ８，１３，１６ ダイクロイックミラー ９ ピンホールを有する円板 １０ 対物レンズ １１ 試料 １２ 励起フィルタ １５ リレーレンズ ５０ 走査手段 ５１ 落射照明手段 ５２ 切り換え手段 ５３ 観察手段 1 Laser 2 Light Source for Epi-illumination 3, 14, 17 Mirror 4 Disc with Micro Lens 5 Camera 6 Lens 7 Barrier Filter 8, 13, 16 Dichroic Mirror 9 Disc with Pinhole 10 Objective Lens 11 Sample 12 Excitation Filter 15 Relay lens 50 scanning means 51 epi-illumination means 52 switching means 53 observing means
Claims (1)
査手段と、 試料の観察位置を特定するための落射照明手段と、 前記走査手段と前記落射照明手段との出力光を切り換
え、この切り換えられた出力光を前記試料に照射し、前
記試料からの反射光若しくは蛍光を前記走査手段を介さ
せると共に観察倍率を高倍率にして出力若しくは直接出
力する切り換え手段と、 この切り換え手段の出力光を観察する観察手段とを備え
たことを特徴とする共焦点顕微鏡。1. In a scanning confocal microscope, a scanning means for scanning illumination light using a disc having a plurality of apertures, an epi-illumination means for specifying an observation position of a sample, and the scanning means. The output light from the epi-illumination means is switched, the switched output light is applied to the sample, the reflected light or fluorescence from the sample is passed through the scanning means, and the observation magnification is increased to a high output or directly. A confocal microscope comprising switching means for outputting and observing means for observing the output light of the switching means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33466293A JP3262147B2 (en) | 1993-12-28 | 1993-12-28 | Confocal microscope |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33466293A JP3262147B2 (en) | 1993-12-28 | 1993-12-28 | Confocal microscope |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07199074A true JPH07199074A (en) | 1995-08-04 |
| JP3262147B2 JP3262147B2 (en) | 2002-03-04 |
Family
ID=18279865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33466293A Expired - Lifetime JP3262147B2 (en) | 1993-12-28 | 1993-12-28 | Confocal microscope |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3262147B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5969846A (en) * | 1996-11-28 | 1999-10-19 | Olympus Optical Co., Ltd. | Confocal microscope |
| JP2008064671A (en) * | 2006-09-08 | 2008-03-21 | Yokogawa Electric Corp | Drug discovery screening system |
| JP2008185432A (en) * | 2007-01-30 | 2008-08-14 | Yokogawa Electric Corp | Drug discovery screening apparatus |
| US8275226B2 (en) | 2008-12-09 | 2012-09-25 | Spectral Applied Research Ltd. | Multi-mode fiber optically coupling a radiation source module to a multi-focal confocal microscope |
| US8670178B2 (en) | 2009-12-08 | 2014-03-11 | Spectral Applied Research Inc. | Imaging distal end of multimode fiber |
-
1993
- 1993-12-28 JP JP33466293A patent/JP3262147B2/en not_active Expired - Lifetime
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5969846A (en) * | 1996-11-28 | 1999-10-19 | Olympus Optical Co., Ltd. | Confocal microscope |
| JP2008064671A (en) * | 2006-09-08 | 2008-03-21 | Yokogawa Electric Corp | Drug discovery screening system |
| JP2008185432A (en) * | 2007-01-30 | 2008-08-14 | Yokogawa Electric Corp | Drug discovery screening apparatus |
| US8275226B2 (en) | 2008-12-09 | 2012-09-25 | Spectral Applied Research Ltd. | Multi-mode fiber optically coupling a radiation source module to a multi-focal confocal microscope |
| US9134519B2 (en) | 2008-12-09 | 2015-09-15 | Spectral Applied Reseach Inc. | Multi-mode fiber optically coupling a radiation source module to a multi-focal confocal microscope |
| US8670178B2 (en) | 2009-12-08 | 2014-03-11 | Spectral Applied Research Inc. | Imaging distal end of multimode fiber |
| US8922887B2 (en) | 2009-12-08 | 2014-12-30 | Spectral Applied Research Inc. | Imaging distal end of multimode fiber |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3262147B2 (en) | 2002-03-04 |
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