JP7254643B2 - 補正係数を用いた顕微鏡画像化法 - Google Patents
補正係数を用いた顕微鏡画像化法 Download PDFInfo
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- 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
- G02B21/008—Details of detection or image processing, including general computer control
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- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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Description
light efficient optically sectioning microscope; Journal of Microscopy 189: 114-117)及びWilson他による刊行物(Wilson, T. et al. (1996), Confocal
microscopy by aperture correlation; OPTICS
LETTERS 21: 1879-1881)は、それを用いて複合画像及び広視野画像から共焦点信号を抽出できる可能性を提案している。
共焦点信号=複合信号-n*広視野信号
結果として、実際には存在しない試料の凹部又は凸部が計算されることになる。
共焦点信号=複合信号-n*広視野信号
に従って算出された。試料1の表面Aoの位置は、矢印及び点線によって示される。さらに、x軸にわたる強度の最大値が示されている。x軸は、z軸に直交し、表面Aoに略平行に延在する。
本発明による方法を使用した場合、金属塗料(Metalliclack)を有する試料1の表面Aoは、ゼロ位置の周りに約1.5から2μmの変動幅のトポグラフィで認識され表示される(図7)。対照的に、従来技術による方法を使用して作成される試料1のトポグラフィは、約5~10μmを超える変動幅を有していた(図8)。
2 凹部
Ao (試料1の)表面
Cl 複合信号
Imax 強度最大、強度ピーク
WF 広視野信号
xCl (複合信号Clの)振幅値
xWF (広視野の信号WFの)振幅値
zn (補正面の)位置
Claims (5)
- 顕微鏡画像化法であって、
試料を照明放射で照射する照射ステップ及び前記照明放射によって生じた検出放射を、第1時点では広視野信号として、第2時点では複合信号として、検出軸に沿って捕捉するステップであって、前記複合信号は共焦点信号及び広視野信号の重ね合せによって形成されている、ステップと、
前記広視野信号を前記複合信号から減算することによって前記共焦点信号を抽出するステップであって、その際、補正係数を用いる、抽出ステップと、
実行される画像化ごとに及び/又は画像化される試料ごとに現在の補正係数を求めるステップと、
それぞれの前記現在の補正係数を用いて、前記共焦点信号を抽出するステップと、
を含み、
前記検出軸に沿って前記試料の表面からある距離において選択される補正面において、前記現在の補正係数を求めるために、それぞれ少なくとも1つの補正広視野画像及び補正複合画像を捕捉し、それらの画像データを求め、求められた前記画像データに基づいて現在の補正係数を求め、
前記補正面の前記距離は、前記試料の表面が前記補正面を通過しないほどの大きさに選択されている、
顕微鏡画像化方法。 - 前記補正平面の前記距離は、前記検出放射を捕捉するために用いられる対物レンズの点分布関数の半値幅の4~6倍の範囲から選択される、
請求項1記載の顕微鏡画像化方法。 - それぞれ少なくとも1つの補正広視野画像及び補正複合画像は、補正面内において現在の焦点面の前及び後で捕捉される、
請求項1又は2記載の顕微鏡画像化方法。 - 前記現在の補正係数は、前記補正広視野画像及び前記補正複合画像の平均輝度から求められる、
請求項1乃至3いずれか1項記載の顕微鏡画像化方法。 - 前記の補正画像の各画素対に対して現在の補正係数が求められ、前記各画素対は、前記補正広視野画像の特定の画素と、前記補正複合画像の対応する画素とによって形成される、
請求項1乃至4いずれか1項記載の顕微鏡画像化方法。
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DE102018210606.1A DE102018210606A1 (de) | 2018-06-28 | 2018-06-28 | Mikroskopisches Abbildungsverfahren unter Verwendung eines Korrekturfaktors |
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JP2000275540A (ja) | 1999-03-24 | 2000-10-06 | Olympus Optical Co Ltd | 共焦点顕微鏡 |
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GB9603788D0 (en) * | 1996-02-22 | 1996-04-24 | Isis Innovation | Confocal microscope |
CN100356163C (zh) * | 1996-08-16 | 2007-12-19 | Ge保健尼亚加拉公司 | 用于分析井板、凝胶和斑点的数字成像系统 |
DE19824460A1 (de) * | 1998-05-30 | 1999-12-02 | Zeiss Carl Jena Gmbh | Anordnung und Verfahren zur mikroskopischen Erzeugung von Objektbildern |
GB9901365D0 (en) * | 1999-01-22 | 1999-03-10 | Isis Innovations Ltd | Confocal microscopy apparatus and method |
US6426835B1 (en) * | 1999-03-23 | 2002-07-30 | Olympus Optical Co., Ltd. | Confocal microscope |
US6341035B1 (en) * | 1999-07-09 | 2002-01-22 | Olympus Optical Co., Ltd. | Confocal microscope |
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US10154785B2 (en) * | 2012-01-18 | 2018-12-18 | University Of Utah Research Foundation | Devices and systems for fluorescence imaging of tissue |
US9449377B2 (en) * | 2012-10-09 | 2016-09-20 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Imaging methods and computer-readable media |
DE102013218795A1 (de) * | 2013-09-19 | 2015-03-19 | Carl Zeiss Microscopy Gmbh | Laserscanningmikroskop und Verfahren zur Korrektur von Abbildungsfehlern insbesondere in der hochauflösenden Scanning-Mikroskopie |
DE102014004249A1 (de) | 2014-03-24 | 2015-09-24 | Carl Zeiss Microscopy Gmbh | Konfokales Mikroskop mit Aperturkorrelation |
CN103926228B (zh) * | 2014-04-28 | 2016-03-02 | 江苏天宁光子科技有限公司 | 一种激光扫描共焦荧光显微内窥成像系统 |
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CN107228846B (zh) * | 2017-05-10 | 2019-09-17 | 暨南大学 | 基于离轴光束焦面共轭的荧光成像光切片方法和装置 |
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JP2000275540A (ja) | 1999-03-24 | 2000-10-06 | Olympus Optical Co Ltd | 共焦点顕微鏡 |
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CN110658617A (zh) | 2020-01-07 |
DE102018210606A1 (de) | 2020-01-02 |
JP2020003793A (ja) | 2020-01-09 |
US11238575B2 (en) | 2022-02-01 |
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