JP6822765B2 - 分子イメージングおよび関連する方法 - Google Patents
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- JP6822765B2 JP6822765B2 JP2015561338A JP2015561338A JP6822765B2 JP 6822765 B2 JP6822765 B2 JP 6822765B2 JP 2015561338 A JP2015561338 A JP 2015561338A JP 2015561338 A JP2015561338 A JP 2015561338A JP 6822765 B2 JP6822765 B2 JP 6822765B2
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Description
本出願は、参照により本明細書に組み込まれる、2013年3月6日出願の米国特許仮出願第61/851,276号の利益を主張する。
本発明は、一般に、単一分子、または単一分子の1つ以上のコレクションのイメージングと、イメージングに関連する方法とに関する。
解像度(r)=λ/(2NA) (1)
解像度(r)=0.61λ/NA (2)
解像度(r)=1.22λ/(NA(obj)+NA(cond)) (3)、
ここで「r」は、解像度(2つの目標物間の最小解像可能距離)であり、「NA」は、顕微鏡の開口数に関する一般的な用語であり、「λ」は、イメージング波長であり、「NA(obj)」が対物レンズの開口数に等しく、「NA(cond)」は、集光レンズの開口数である。
以下の材料、機器および一般的な方法は、本発明の方法の諸態様を示すものである。これらは、開示される発明(単数または複数)を決して限定するものではない。
オリゴマー・プローブは、典型的に、然るべきソフトウェア・パッケージ、例えば、www.singlemoleculefish.comにおいてバイオサーチテクノロジーズ(Biosearch Technologies)を通じて利用可能なProbe Designerを用いて設計される。プローブは、自動化されたDNA/RNAシンセサイザー、例えば、Biosearch8700上を含む、任意の適切な方法で合成されてもよい。
細胞試料を調製するための方法の非限定の例は、次の通りである。www.singerlab.org/protocolsにオンラインで公開されたSinger Lab Protocol(シンガーラボ・プロトコル)を参照。
0.5%ゼラチン中のカバーガラス:カバーガラスを0.1N HCl中で20分間煮沸することにより一箱分のカバーガラスを滅菌する。2回蒸留した水(「DDW:doubly distilled water」中でカバーガラスを数回リンスおよび洗浄する。ゼラチン(1.0g)を秤量して200mlのDDWに加える。結果として生じた混合物を攪拌し、温めて溶解を完了する。滅菌したカバーガラスをゼラチン溶液に移して、20分間オートクレーブする。10×PBSストック:500mlの10×PBSに250μLのDEPCを加える。混合物を溶解させるために攪拌し、次にオートクレーブする。1M MgCl2ストック:MgCl2(20.3g)を秤量して、DDWに加える。洗浄溶液(PBSM):100mlの10×PBSストックに5mlの1M MgCl2ストックを加える。結果として生じた混合物をDDWで1Lに希釈する。抽出溶媒(PBST):100mlの10×PBSストックに5mlのTriton X−100を加える。結果として生じた混合物をDDWで1Lに希釈し、静かに攪拌して溶解を完了する。固定液(4% PFA):10mlバイアルの20%パラホルムアルデヒド・ストックに5mlの10×PBSストックを加える。結果として生じた混合物をDDWで50mlに希釈する。
細胞を標準的な条件下で成長させて、ペトリ皿におけるゼラチン化したカバーガラス上に播種する。任意の処理ステップ、例えば、飢餓および刺激を行う。細胞を氷冷PBSMで手短かに洗浄する。細胞を室温においてPBST中で60秒間抽出する。細胞を氷冷PBSMで2回手短かに洗浄する。細胞を室温においてPFA固定液で20分間固定する。細胞を氷冷PBSMで2回手短かに洗浄する。固定済カバーガラスをPBSM中に4℃で使用まで貯蔵する。
洗浄溶液(PBSM):100mlの10×PBSストックに5mlの1M MgCl2ストックを加える。結果として生じた混合物をDDWで1Lに希釈する。プレ/ポストハイブリダイゼーション洗浄液(50%ホルムアミド/2×SSC):250mlホルムアミドに50mlの20×SSCストックを加える。結果として生じた混合物をDDWで500mLに希釈する。プローブ競合溶液(ssDNA/tRNA):50μlの10mg/ml断片化サケ精子DNAに50μlの10mg/ml大腸菌tRNAを加える。ハイブリダイゼーション用バッファー:60μlのDDWに20μlのBSAおよび20μlの20×SSCストックを加える。低塩濃度洗浄溶液(2×SSC):50mlの20×SSCストックに450mlのDDWを加える。核染色溶液(DAPI):100mlの10×PBSストックに50μlの10mg/ml DAPIストック(10mgを1.0mlのDDWに加えることにより固体から調製)を加える。結果として生じた混合物をDDWで1Lに希釈し、振盪してDAPIを溶解させる。封入剤:適切なキットの要素、例えば、Prolongキット(Molecular Probes)を調製するか、または等価な方法を用いる。
カラーコーディングおよび複数の転写物の検出に先立って、ハイブリダイゼーションの検査を行う。転写部位を示すために2つの高輝度色素を用いる。次に、色素の組み合わせを用いて各遺伝子に任意のカラーコードを割り当て、1つずつ検査を行う。固定したカバーガラスを、ピンセットを用いてコプリン・ジャー中に垂直に配置する。固定した細胞を再水和し、室温においてPBSM中で10分間洗浄する。細胞をプレハイブリダイゼーション溶液中で10分間平衡化する。アッセイされることになる標的の異なる組み合わせごとに、一定分量のオリゴヌクレオチド・プローブ混合物をチューブに加える。競合溶液を100倍超でプローブ混合物(単数または複数)に加える。混合物を真空乾燥する。乾燥したペレットを10μlホルムアミドに再懸濁して、チューブを加熱ブロック上に85℃で5〜10分間置き、その後即座に氷上に置く。10μlのハイブリダイゼーション用バッファーを各チューブに加えて、20μlの反応体積を確保する。ガラス・プレートをパラフィルムで包み、反応のための作用空間に充てる。カバーガラスを重ねることなく各体積を覆って配置できるように、各20μlの反応体積を十分遠く離してプレート上に点在させる。カバーガラスをプレハイブリダイゼーション溶液から取り出して、過剰な液体を拭い取る。各カバーガラスを、細胞側を下にしてプレート上に点在するハイブリダイゼーション・ミックス上に置く。反応を封じ込めるために、プレートおよびカバーガラスをパラフィルムの別の層で包む。ハイブリダイゼーション後にカバーガラスを2回洗浄するのに十分な量のプレハイブリダイゼーション溶液とともにプレートを37℃で3時間インキュベートする。カバーガラスの除去を可能にするためにパラフィルムの上部層を除去して下部層を外す。カバーガラスを予熱した洗浄液とともにコプリン・ジャー中に戻して37℃で20分間インキュベートする。洗浄液を変えて、20分間繰り返す。溶液を2×SSCに変えて、室温で10分間インキュベートする。溶液をPBSMに変えて、室温で10分間インキュベートする。溶液を調製したDAPIに変えて、室温で1分間インキュベートし、次にPBSMで洗浄することにより核を対比染色する。PBSMを変えて、取り付けまで室温で保持する。新たに調製した退色防止封入剤を用い、各カバーガラスを細胞側を下にしてスライドガラス上に取り付ける。過剰な液体を拭い取り、スライドを−20℃で保存する。
TOP1(トポイソメラーゼ(DNA)1)の発現をA549細胞におけるFISHによって分析し、SAOシステム(20×)を用いて画像化/定量化した。SAOイメージング条件は、次の通りである、すなわち、500mW主出力(532nm);500ms露出/フレーム。SAO画像の一部を図7に示す。画像は、およそ100個の細胞を含み、mRNAは、画像中に高輝度/白色/緑色ドットとして現れる。画像内の20個の細胞のサンプリングは、次のmRAカウントを示した、すなわち、56;59;58;54;69;60;63;54;74;65;95;52;60;85;66;67;46;36;65;53。図8は、TOP1 mRNAのSAO画像の関心領域の選択を示し、スポット強度および品質に基づく選択処理グラフを含む。
HER2の発現をMCF7細胞(ヒト乳腺癌細胞株)におけるFISHによって分析し、SAOシステム(20×)を用いて画像化/定量化した。SAOイメージング条件は、次の通りである、すなわち、500mW主出力(532nm);500ms露光/フレーム。結果を図9に示す。右上の画像は、100個超の細胞を含み、mRNAは、画像中に高輝度/白色ドットとして現れる。他の画像は、およそ20個の細胞を示す選択部である。20個の細胞のサンプリングは、次のカウントを示した、すなわち、62、61、71、97、74、66、69、48、58、87、37、92、103、80、90、21、37、109、57、122(平均72)。
FKBP5の発現をA549細胞(ヒト肺腺癌細胞株)におけるFISHによって分析した。図10は、標準的な蛍光顕微鏡(60×/1.41 NA0.1油浸)からの2つの画像を示す。「Minus Dex」とラベルされた画像は、24nMデキサメタゾンの添加によるアップレギュレーションより前の細胞(およそ13個の細胞)を示す;画像「Plus Dex」は、8時間にわたる24nMデキサメタゾンの添加後の細胞(およそ14個の細胞)を示す。大きい方の、ほぼ卵形の構造は、細胞核であり、個別の検出分子が核中および周りの高輝度/白色ドットとして示される。図11は、SAOイメージング・デバイス(20×)を備えるシステムを用いた2つの画像(得られた全画像の1/10)を示す。「Minus Dex」とラベルした画像は、24nMデキサメタゾンの添加によるアップレギュレーションより前の細胞(およそ50個の細胞)を示し、画像「Plus Dex」は、8時間にわたる24nMデキサメタゾンの添加後の細胞(およそ50個の細胞)を示す。大きい方の、ほぼ卵形の構造は、細胞核であり、個別の検出分子が核中および周りの高輝度/白色ドットとして示される。
Claims (4)
- 細胞の複製DNA鎖へのBrdUの取り込みを用いた細胞増殖のイメージングの方法であって、前記方法は、
a)複数の生細胞を含む試料調製物を得るステップと、
b)ある量のBrdUを前記試料調製物に供給して、実質的な量の前記BrdUが、増殖している細胞中に取り込まれることを可能にする期間にわたって、前記供給されたBrdUを前記試料調製物とともにインキュベートするステップと、
c)1つ以上の蛍光基を備えるある量の抗BrdU抗体を前記試料調製物に供給して、実質的な量の前記抗体の、前記複製DNA中に取り込まれた前記BrdUへの結合を可能にする期間にわたって、前記供給された抗体を前記試料調製物とともにインキュベートするステップと、
d)合成開口光学系を実現するデバイスを備えるシステムを用いて前記BrdUに結合された抗体を画像化することによって、前記BrdUに結合された抗体を検出するステップと、
を備え、
前記デバイスを備える前記システムは、少なくとも1×105μm2の画像化面積にわたって450nmより良好な解像度を提供する
方法。 - 前記画像化面積は、少なくとも1×106μm2である、請求項1に記載の方法。
- 前記画像化面積内のフルオロフォアの密度は、μm当たり1,000分子未満である、請求項1に記載の方法。
- 前記BrdUに結合された抗体を検出するステップは、単一の検出ステップにおいて生じ、データは、検出設定を何も変更しないで収集される、請求項1に記載の方法。
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