JP2021090439A - 三次元核酸含有マトリックスの立体撮像のための方法と装置 - Google Patents
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Abstract
Description
本願は2015年11月03日に出願された米国特許仮出願第62/250182号の優先権を主張し、全ての目的のために前記仮出願の全体をここに参照により本明細書に援用する。
政府の権利に関する説明
本発明の分野
実施例1
図2は本開示の自動配列決定−三次元撮像デバイスの動作関連構成要素の模式的な斜視図である。筐体要素を含むその他の構成要素が前記デバイスに付け加えられ得ることを理解すべきである。図2に示されるように、前記自動配列決定−三次元撮像デバイスは試料からの光を受け取るためのピンホールアレイとカメラ検出器を含むレーザー共焦点ヘッド格納容器を含む。かかるハードウェア配置によって三次元核酸含有マトリックスの立体撮像が可能になる。前記デバイスはレーザー共焦点ヘッド格納容器に向かう(to)反射ベースのオートフォーカス付きの光路をさらに含む。前記デバイスは、1種類以上の試薬を試料に供給するように機能する加圧フルイディクスブロック上の温度制御フルイディクスヘッドをさらに含む。作動可能に接続されたバルクフルイディクスバルブと試料から液体を除去するための吸引チューブとが配備される。温度制御及びXY移動を行うシステム付きの試料ステージが配備され、スライドグラス試料ホルダーと共に示されている。リニアエンコーダ付きのステージ移動用軌道は、作動可能なように前記ステージに接続されている。無酸素(アルゴン)環境のための格納容器ドアが、図示されていない格納容器の残りの部分と共に配備される。前記デバイスは防振基部の上に設置されている。
参照文献
Drmanac, R., Sparks, A.B., Callow, M.J., Halpern, A.L., Burns, N.L., Kermani, B.G., Carnevali, P., Nazarenko, I., Nilsen, G.B., Yeung, G., et al. (2010). Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 327, 78-81.
Islam, S., Kjallquist, U., Moliner, A., Zajac, P., Fan, J.B., Lonnerberg, P., and Linnarsson, S. (2011). Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21, 1160-1167.
Larsson, C., Grundberg, I., S[oe]derberg, O., and Nilsson, M. (2010). In situ detection and genotyping of individual mRNA molecules. Nature methods 7, 395-397.
Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A.K., Landegren, U., and Nilsson, M. (2004). In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nature methods 1, 227-232.
Shendure, J., Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon, J.P., Rosenbaum, A.M., Wang, M.D., Zhang, K., Mitra, R.D., and Church, G.M. (2005). Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728- 1732.
Claims (8)
- 三次元重合マトリックス内で複数の核酸を増幅してアンプリコンを作製すること、
前記アンプリコンを前記マトリックスに共有結合すること、
検出可能な標識で前記複数のアンプリコンが標識される光学的配列決定法を用いて、前記複数のアンプリコンを配列決定すること、及び
前記複数のアンプリコンを立体撮像して前記複数のアンプリコンの三次元撮像データを作成すること;ここで、光強度データは三次元立体画像に加工されること
を含む、三次元重合マトリックス内の複数の核酸を分析する方法。 - 前記複数の核酸が生体試料に含まれており、前記マトリックスを形成する材料が前記生体試料に導入される、請求項1に記載の方法。
- 前記複数の核酸が細胞に含まれており、前記マトリックスを形成する材料が前記細胞に導入される、請求項1に記載の方法。
- 前記複数の核酸が組織試料に含まれており、前記マトリックスを形成する材料が前記組織試料に導入される、請求項1に記載の方法。
- 前記細胞内の前記複数のアンプリコンの相対的位置を前記三次元撮像データによって特定する、請求項1に記載の方法。
- 蛍光in situ配列決定法を用いて前記複数のアンプリコンを配列決定する、請求項1に記載の方法。
- 前記複数の核酸が、3D構造化照明法、選択的平面照明顕微鏡法、光シート顕微鏡法、放出光処理法、ピンホール共焦点顕微鏡法を用いる立体撮像、開口相関共焦点顕微鏡法を用いる立体撮像、スライスからの立体復元を用いる立体撮像、デコンボリューション顕微鏡法を用いる立体撮像、収差補正多焦点顕微鏡法を用いる立体撮像、及びデジタルホログラフィック顕微鏡法を用いる立体撮像のうちの1つ以上を用いて立体撮像される、請求項1に記載の方法。
- 三次元核酸含有マトリックス用の試料ホルダーを含む、多軸ステージ又は位置決めシステム;
前記ステージに作動可能に接続されており、増幅及び配列決定のための熱サイクリングに有用な時間及び温度をプログラムすることができる、加熱又は冷却装置;
1種類以上の試薬を収容するための1つ以上のリザーバーと流体連結しており、プログラムされた量の液体試薬の前記試料ホルダーへの分注をプログラムすることができる、前記試料ホルダーに1種類以上の試薬を分注するために配置された流体ディスペンサー;
前記流体ディスペンサーに作動可能に接続されており、前記流体ディスペンサーを介して前記1つ以上のリザーバーから1種類以上の試薬を押し出す又は引き抜くポンプ;
1つ以上の光軸を含む光学アセンブリ;
受光できるように前記試料ホルダーに通じて配置されており、前記核酸試料の三次元立体画像へと加工される光強度シグナルを受光する、1つ以上の検出器;並びに
前記試料ホルダーへの試薬の導入、前記試料ホルダーの熱サイクリング、並びに画像の検出及び取得を、自動化し制御するための、ソフトウェアを備えた1つ以上のマイクロプロセッサ
を含む、自動配列決定及び立体撮像デバイス。
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US11542554B2 (en) | 2023-01-03 |
JP2019501635A (ja) | 2019-01-24 |
HK1252878A1 (zh) | 2019-06-06 |
US20190241950A1 (en) | 2019-08-08 |
WO2017079406A1 (en) | 2017-05-11 |
US10266888B2 (en) | 2019-04-23 |
EP3371329A4 (en) | 2019-06-19 |
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