JP2007192819A - レアな事象の検知における強調された染料比率弁別のための新規な免疫的細胞の着色方法 - Google Patents
レアな事象の検知における強調された染料比率弁別のための新規な免疫的細胞の着色方法 Download PDFInfo
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Abstract
【解決手段】細胞を含むサンプルを準備する方法であって、細胞を含む第1の材料をスライドの上に配置し、第1のタイプの抗体を追加し、第2のタイプの抗体を追加し、第1のマーカーを、第1のタイプの抗体及び第2のタイプの抗体の1つと関連付け、第2のマーカーを、第1のタイプの抗体及び第2のタイプの抗体の1つと対応付けるステップを含み、準備されたサンプルが真のポジティブ画像事象から殆どの偽ポジティブを弁別し除去するためのフィルタリングを可能とするために十分なスペクトラム解像度を有する信号を放射するように構成され、更に、興味の対象である細胞であるかを決定するための、より高い解像度の検知方法によって確認される方法。
【選択図】図1
Description
12 サンプル
14 生物学的滲み
16 スライド
18 ステージ
20 線形的に移動可能なトラック
22 モータ
26 線形移動
28 矢印
30 光パイプ
32 第1の端部
34 第2の端部
36 矩形の入力開口(aperture)
38 出力開口
40 走査放射(光)源
42 レーザ
44 励起光(放射ビーム)
46 ガルバノメータ(galvanometer)
48 矢印
50 光学的合焦レンズ
52 矢印
54 サンプル12の軌跡
56 電気的制御ユニット
58 信号検知器
60 第1のレンズ
62 ブロッキング・フィルタ
64 励起光
66 光検知器アレンジメント
68 ディスプレイ
70 プライマリの抗体(1stAB)
72 細胞ターゲット
74 セカンダリの抗体
76 フルオロフォア
78 フルオロフォア
80 電気的制御ユニット
82 EX_555
84 EM_488
86 EM_555
90 フルオロフォア(Alexa 488)
92 プライマリの抗体
94 関心の対象の細胞
96 セカンダリ抗体結合されたフルオロフォア(secondary antibody conjugated fluorphore)(Alexa 555)
98 光検知器
100 非結合された(non-conjugated)プライマリの抗体
102 関心の対象の細胞
104 第2の抗体
106 酵素わさびペルオキシダーゼ(horse radish peroxidase:HRP)
110 マーカー
112 過酸化水素H2O2
114 ティラミド混合物(tyramide mixture)
114’ 短命の自由基(short-lived free-radical)のティラマイド・フォーム
116 他の染料(例えば、Alexa 488)
120 第1のタイプ(プライマリ)の抗体
122 関心の対象の細胞
124 単一のフルオロフォア染料
126 2番目(2nd)のタイプの(セカンダリの)抗体
150 放射カーブ
Claims (4)
- 興味の対象である潜在的な細胞(potential cells of interest)を含む画像事象(imaging events)を検知することが意図される、レーザ・ベースの走査システムで使用するために、興味の対象である潜在的な細胞を含むサンプルを準備する方法であって、
前記興味の対象である潜在的な細胞を含む第1の材料を、スライドの上に配置し、
前記興味の対象である潜在的な細胞をターゲット化する(target)ように構成された、少なくとも第1のタイプの抗体を追加し、
前記第1のタイプの抗体をターゲット化するように構成された第2のタイプの抗体を追加し、
少なくとも第1のマーカー又はタグを、前記第1のタイプの抗体及び前記第2のタイプの抗体の少なくとも1つと関連付け(associating)、そして、
第2のマーカー又はタグを、前記第1のタイプの抗体及び前記第2のタイプの抗体の少なくとも1つと対応付ける、
ステップを含み、
前記レーザ・ベースの走査システムによって動作されたときに、前記準備されたサンプルが、真のポジティブ画像事象から、殆どの偽ポジティブを弁別し、除去するためのフィルタリングを可能とするために十分なスペクトラム解像度を有する信号を放射するように構成され、フィルタリングされた画像化事象が、次に、それらが真に、興味の対象である細胞であるかを決定するための、より高い解像度の検知方法によって確認される(confirmed)方法。 - 前記第1のマーカー又はタグの量、及び前記第2のマーカーの量が、等しくない、請求項1に記載の方法。
- 前記第1のマーカー又はタグが、前記第1のタイプの抗体と関連付けられ、前記第2のタイプのマーカー又はタグが、前記第2のタイプの抗体と関連付けられている、請求項1に記載の方法。
- 興味の対象である潜在的な細胞を含むサンプルを走査するための、レーザ走査システムであって、
スライドであって、当該スライド上にサンプルが配置されるスライドをサポートするイメージャ・ステージ、
励起光波長において励起光を放射する光源、及び、放射された蛍光を、前記サンプルに衝突するコリメートされた励起光に変換する光学系であって、前記励起光と前記サンプルの間の相互作用が、当該励起光の波長とは異なった波長においてルミネセンス光を生成するものであり、
前記ルミネセンス光の少なくとも一部を収集するようにされた入力開口、及び、
前記入力開口と光学的に連絡し、前記入力開口によって収集された前記ルミネセンス光を放射する出力開口、
を備え、
獲得された画像化事象に対して、偽のポジティブと真のポジティブの間を弁別するために十分なスペクトル的な解像度を有する信号を放射するために前記サンプルが準備され、当該真のポジティブが、興味の対象である細胞の検知を確認するものである、
レーザ走査システム。
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US11/333,731 US7842465B2 (en) | 2006-01-17 | 2006-01-17 | Immunocytostaining methods for enhanced dye ratio discrimination in rare event detection |
US11/333,731 | 2006-01-17 |
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JP2007192819A true JP2007192819A (ja) | 2007-08-02 |
JP5512916B2 JP5512916B2 (ja) | 2014-06-04 |
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EP (1) | EP1808689B1 (ja) |
JP (1) | JP5512916B2 (ja) |
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---|---|---|---|---|
US8921102B2 (en) | 2005-07-29 | 2014-12-30 | Gpb Scientific, Llc | Devices and methods for enrichment and alteration of circulating tumor cells and other particles |
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