TW201709150A - 組織學染色之空間多工 - Google Patents
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Abstract
以下係關於藉由組裝來自一或多個成像操作之虛擬影像使顯微術或組織學染色共定位之方法。具體而言,該方法減少獲得生物樣品之經多重標記之抗原或蛋白質之組織學影像所需之時間。該方法包括使組織當藉由薄片切片機將其切片時利用刀口掃描顯微鏡成像並藉由所生成之該等影像使該等樣品空間對準。該方法所實現之樣品之空間對準容許跨越不同樣品切片比較一組不同抗原或蛋白質次級或功能性染色,藉此允許組織及細胞之同時次級染色。
Description
本申請案主張於2015年7月10日提出申請之美國臨時申請案第62/190,931號的權利,該申請案以引用方式併入本文中。
本揭示內容概言之係關於用於診斷或研究目的之對組織進行切片、成像及染色之方法。具體而言,本揭示內容係關於連續切片顯微術,即,使用薄片切片機對生物組織及其他材料樣品進行切片,且更特定而言係關於使經免疫組織化學抗原染色之組織樣品成像之方法。
免疫組織化學係其中將一組抗原施加至生物組織之切片之方法。免疫組織化學染色通常用來鑑別異常細胞,其採用抗體來測試組織之樣品中之某些抗原。抗體通常連接至放射性物質或染料,此使得組織中之抗原在顯微鏡下變得可見,且此方法通常以一組或一系列不同染色來實施以檢測各種癌細胞株。
本揭示內容藉由使用利用KESM技術使之成為可能之新穎方法及製程彙集並最佳化免疫組織化學中傳統上使用之若干不同工作流程。藉由呈現代表當前現有技術之傳統工作流程之以下三項研究,可更好的理解本文所述之新穎及可用方法。
本揭示內容概言之係關於利用薄片切片機及應用免疫組織化學染色使目標物成像之系統及方法,以檢測某些生物標記物用於醫療診
斷或研究。具體而言,本揭示內容係關於使用連續切片顯微術用於該等診斷,其係藉由以下實施:使用刀口掃描顯微鏡對生物組織及其他材料樣品進行切片並施加染色至個別切片,並使用由刀口掃描技術所實現之空間多工方法以跨越樣品比較各種染色及反應。
下文涉及使用多種對比劑對組織樣品進行快速病理學及/或組織學檢查之技術。藉由在可由刀口掃描顯微鏡進行組織處置之前實施額外成像步驟,可在物理或化學變形發生之前捕獲所成像物體之基本形狀。由此,可將變形切片配準回至樣品之初始坐標系中。此亦可呈現使生物標記物跨越單一樣品之多個連續切片更好的共定位之獨特能力,且在化學或機械處理之後,可藉由使用中間成像步驟以使個別切片彼此重新映射來作為整體產生組織之更精確表示。此可藉由允許較傳統工作流程更快速地跨越樣品快速比較多種對比劑、同時具有較少失真而提供獨特優點。
當結合以下說明及隨附圖式進行考量時,將進一步瞭解及理解本揭示內容之其他目的及優點。雖然以下說明可含有闡述本揭示內容之具體實施例之特定細節,但不應將其解釋為對本揭示內容範圍之限制而是解釋為較佳實施例之例示。對於本揭示內容之每一態樣,熟習此項技術者所已知之本文所建議之許多變化形式皆係可能的。可在不背離本發明精神下在本揭示內容之範圍內作出各種各樣之改變及修改。
本說明書中所提及之所有公開案、專利及專利申請案均以引用方式併入本文中,其併入程度如同明確地及個別地指示將每一個別公開案、專利或專利申請案以引用方式併入。
100A‧‧‧典型解剖學家之工作流程/解剖學家工作流程/工作流程
100B‧‧‧典型組織學家或病理學家之工作流程/組織學家或病理學家工作流程/工作流程
100C‧‧‧典型生物學家之工作流程/生物學家工作流程/工作流程
101‧‧‧樣品
103‧‧‧目標物
105A‧‧‧切片/初級切片
105B‧‧‧切片/次級切片
107A‧‧‧影像
107B‧‧‧影像
109A1‧‧‧初級影像
109A2‧‧‧次級影像
109B1‧‧‧初級影像
109B2‧‧‧次級影像
141‧‧‧步驟
151‧‧‧步驟
161‧‧‧步驟
171‧‧‧步驟
181‧‧‧步驟
191‧‧‧步驟
211A‧‧‧初級成像步驟
211B‧‧‧初級成像步驟
211N‧‧‧初級成像步驟
221A至221N‧‧‧薄切片/PI步驟
223‧‧‧初級虛擬厚切片/虛擬厚切片
231‧‧‧步驟/染色
231A至231N‧‧‧染色1至N
241A‧‧‧次級成像(SI)步驟
241B‧‧‧次級成像(SI)步驟
241N‧‧‧次級成像(SI)步驟
243‧‧‧經共配準之染色
251‧‧‧數據收集或診斷步驟
261‧‧‧初級影像
262‧‧‧次級影像
263‧‧‧複合影像
271‧‧‧影像
272‧‧‧影像
273‧‧‧影像
274‧‧‧複合影像
1011‧‧‧步驟
1021‧‧‧步驟
1031‧‧‧步驟
本發明之新穎特徵特別闡釋於隨附申請專利範圍中。參閱闡釋
其中利用本發明原理之說明性實施例的下文詳細說明及附圖將會更好地瞭解本發明之特徵及優點,在附圖中:圖1A係圖解說明使用當前現有技術之典型解剖學家之工作流程之示意圖。
圖1B係圖解說明使用當前現有技術之典型組織學家或病理學家之工作流程圖之示意圖。
圖1C係圖解說明使用當前現有技術之典型生物學家之工作流程圖之示意圖。
圖1D1係圖解說明使用當前現有技術在傳統工作流程中如何發生影像失真之示意圖。
圖1D2係藉由使未失真之影像對準圖解說明使用本文所述技術之空間多工如何改良使影像堆疊對準之製程之示意圖。
圖2A係圖解說明使用本文所述之空間多工方法使樣品成像之步驟之示意圖。
圖2B係圖解說明藉由並排呈現使初級及次級影像共配準之示意圖。
圖2C係圖解說明使具有藉由數學組合所施加之不同染色之若干Z影像共配準以產生重合染色反應之複合影像之示意圖。
本文藉由比較如上文所述對組織樣品進行切片、染色及成像之三種傳統方法來揭示經改良之系統及方法。本揭示內容包括藉由在切片時或在此之前引入KESM技術所實現之一或多個額外成像步驟對上述三種工作流程之改良。
「免疫組織化學」可係指抗原或蛋白質在組織切片中之應用,其藉由使用經標記抗體作為引起抗原-抗體相互作用之特異性試劑來
實施,該相互作用可藉由標記物(例如螢光染料或染色劑)可視化。
「連續切片顯微術」可係指用薄片切片機獲得連續切片並以傳統方式藉由將切片安裝至載片並染色來使其成像之實踐。
「刀口掃描顯微鏡」或「KESM」可係指以自動化方式實施連續切片顯微術之顯微鏡。參見美國專利第6,744,572號。
「切片(section或slice)」可係指藉助樣品與刀之間之相對運動自塊面(block face)移除之連續材料之單一條帶。
「薄片切片機」可係指其中將材料塊精確地切割以使得自塊之表面移除或切下極薄材料層之裝置。類似地,術語「顯微切片法」可應用於薄片切片機之操作。
「成像術」可包括經設計以量測「影像」(光學或電子反應之空間映射)之任何技術。此可包括光學或電子顯微術技術。
「成像」通常可係指數據收集,以生成既定區域之可視化。
「配準」或「共配準」可係指其中使影像對準、拉伸及變形以彼此匹配之計算步驟。關於連續切片顯微術,此步驟可校正切片、安裝及化學處理所致之組織變形。
「多工(multiplex或multiplexing)」可係指藉由在矩陣之兩側具有兩個選擇性定址系統(由此僅需要2N個選擇器來定址NA2個位置)以在矩陣內選擇一個位置之方法。
「染色」可係指藉由包括(但不限於)附接顏料、基因表現之螢光團或經設計以改質欲成像之靶結構之化學品之方法的化學處理,其旨在改變全部或一部分介質之光子反應。此可包括(但不限於)傳統光學顯微鏡染色、免疫組織化學(IHC)及原位雜交(ISH)標記技術中所用之對比劑。
「分子診斷」可係指獲得組織之樣品並鑑別生物標記物以做出診斷之化學測試或分析之形式。
「轉換」可係指將影像中之單點自未染色影像重新映射至另一經染色影像。
「內插法」可涵蓋源於另一數域之數值的選擇性空間取樣之方法。常用方法可包括「最近鄰」內插法、線性、多項式或基於b-樣條曲線之技術。該等通常用於計算經轉換影像之離散「內插」值,或估算在既定空間坐標處之函數值。
自薄片切片機獲取切片並將其安裝至載玻片之製程可物理上失真組織。失真可係薄及脆弱組織之翹曲、切片之摺疊或甚至撕裂。翹曲可妨礙連續切片之簡單對準,此乃因微觀特徵不能自切片至切片適當地排列。通常,必須使用手動編輯與軟體之組合對失真進行校正,其中在毗鄰影像上選擇配準標記且軟體使配準標記對準。此過程通常緩慢且艱難,且可阻礙連續切片之更廣泛採用。
出於以下若干原因,控制切片厚度可甚為重要:
1.維持切片之結構/架構完整性用於後續處置、染色、安裝及處理。
2.確保切片具有對於所解析之結構適當之厚度。切片應足夠厚以使得獨特特徵位於切片以內。在細胞特性分析中,切片之厚度通常容許1-2層細胞於平面中。若其太厚,則許多層細胞會掩蓋細胞層面的細節。類似地,若切片太薄,則會截斷單一細胞層內之相關特徵。
3.確保切片對於染色之化學動力學而言足夠薄以充分標記相關特徵。許多對比劑採用大分子,此可限制其能夠穿過組織擴散並相應地標記之該組織的量。
可依賴於基礎材料之化學動力學之類似對比劑對於厚或薄切片
可表現不同。
在組織學或病理學實驗室中切割不同厚度之切片用於不同染色/顯影技術係常見實踐。此在需要快速結果(通常薄切片可更快染色)或在如IHC或ISH方法中之染色中採用複雜化學物質時尤其如此。
存在許多原因使得生物學家希望跨越三維空間之三維體積來量測組織性質。為使用當前現有技術來實施,將切割若干切片,將其染色並彼此共配準。圖1A係圖解說明採用當前現有技術之典型解剖學家之工作流程100A之圖解。在步驟111中,可將樣品101切片為一或多個厚切片,例如第一厚切片121A、第二厚切片121B及第三厚切片121C。厚切片121A、121B、121C可在步驟131中染色且經染色之厚切片121A、121B、121C可然後在步驟141中成像並彼此共配準。
類似地,當組織學家或病理學家試圖對病變組織做出診斷時,可切割若干個厚切片121A、121B、121C。然後,在步驟151中將一者進行染色、成像並檢查。基於來自醫療技術人員之影像輸入,此過程在步驟161中做出最終診斷之前可重複若干次,且對厚切片之一者之診斷可用以告知對其他厚切片進行染色、成像及檢查。對經染色厚切片之診斷可在步驟171中指示患者之適當治療。圖1B係圖解說明採用當前現有技術之典型組織學家或病理學家之工作流程100B之圖解。
生物學研究人員通常採用免疫組織化學染色技術,其中將組織之特定片進行染色、成像,然後將染色自組織之切片完全剝除。染色及剝除製程可重複進行以使在組織之樣品片上之整組染色之免疫或抗原反應成像。圖1C係圖解說明採用當前現有技術之典型生物學家之工作流程100C之圖解。樣品101可在步驟181中經切片並置於載玻片
中。在步驟191中,可對切片及/或載玻片進行染色。在步驟1011中,可使經染色之載玻片及/或切片成像。在步驟1031中,可儲存影像。在步驟1021中,染色可去除且載玻片/切片可在步驟191中再次染色。
此進行染色、成像、剝除染色、再染色、使染色再成像、然後再一次再成像之過程稱為連續多工。生物學家在免疫組織化學中常用之此方法耗費時間,通常需要一周或更久來完成整組之抗原染色。
另一方法係基於病理學家之檢查對下一切片之鑑別染色。此可導致相當長之周轉時間以及需要增加醫師之互動。
所有該等方法具有本揭示內容之範圍尋求解決之以下根本缺點:
˙載玻片安裝、染色及成像各自在切片中引入獨特變形、失真及假影,此可造成共配準之最終製程難以進行,易於產生誤差且費時。
˙通常存在大量連續步驟,若欲產生高品質之可靠結果,則每一步驟可涉及顯著人類勞動及對細節之關注。
˙鑒於樣品之3D性質,特徵可跨越甚至毗鄰切片而改變,且在較大樣品中難以自動化特徵提取及翹曲校正。
本揭示內容闡述藉由引入由如本文中所應用之KESM技術所實現之額外成像步驟來減少處理樣品上之多重染色所需要之人力並增加所重構產物之精確度之系統及方法。
在典型連續切片顯微術中,使用人類注釋或演算法使切片對準,其中在重構之前校正安裝期間之翹曲。此製程通常涉及配準標記之選擇,其係跨越毗鄰切片存在之點。配準點可用來計算轉換函數,其應用於兩個影像以使影像中之像素對準。跨越一系列影像選擇配準標記可甚為困難,此係由於堆疊中之影像之每一者彼此不同。特徵亦很少恰好交叉垂直於切割平面,故若選擇相同特徵用於配準標記,特
徵可跨越切片空間上漂移且因此(例如)在斜坡處之目標物可誤解為係垂直的。
如本文所揭示,影像之後處理可由於失真之後處理可在完全相同切片之兩個影像上實施而改良,一者無失真,則一或多者均無失真。由於影像係同一切片之影像,故完全相同之配準標記將存在於所有影像上,此使得選擇製程更容易且更一致。同樣現象亦可減小圓柱形特徵原本將發生之相對於切割平面在傾斜處行進之空間漂移。圖1D1及圖1D2圖解說明採用當前現有技術如何發生失真(圖1D1)及與此相比影像後處理可如何關於本文所揭示之該等失真進行改良(圖1D2)。
如圖1D1中所示,目標物103可存在於樣品101中,該樣品可切成兩個切片105A及105B。可生成分別針對每一切片105A、105B之影像107A、107B,其顯示不同深度處之目標物103。
如圖1D2中所示,目標物103可存在於樣品101中,該樣品可切成兩個切片105A及105B。可生成切片105A、105B之每一者之初級及次級影像(例如初級切片105A之初級影像109A1及次級影像109A2,及於次級切片105B之初級影像109B1及次級影像109B2)。
如本文所述,此方法中之第一步驟包含初級成像(PI)步驟,其可在處理或後處理失真之前捕獲切片之視圖。此可藉由在切片之前使樣品成像或藉由在切片期間成像來完成。初級成像可利用KESM來實施,其可在切片操作期間在塊面上捕獲未翹曲之影像。該等影像可稱為X_N。藉由併入此PI步驟,次級成像(SI)步驟可直接錨定切片之實際初始形狀及位置,此可使得能夠更好的配準及分析樣品之染色反應。圖2A係圖解說明空間多工方法200之成像步驟之圖解。樣品101可在初級成像步驟211A中成像,然後自樣品101切薄切片221A。在將
樣品進一步切片為進一步薄切片221B至221N之前可實施其他初級成像步驟211B至211N。可收集初級影像並使其彼此配準以生成虛擬厚切片223。
如本文所述,在PI步驟(221A、221B、...221N)之後,可在步驟231中將一或多個切片置於載玻片上,接著將抗原或蛋白質染色施加至樣品切片(以產生染色231A、231B、...231N)用於進一步分析。
本揭示內容之另一態樣包括次級成像(SI)步驟241A、241B、...241N。在SI步驟中,在施加一次染色或整組染色231之後可使用KESM來使樣品101之經染色切片成像。該等步驟可根據視需要重複多次,每次使切片再成像,將新的染色施加至樣品。該等影像將稱為Y_n。
本揭示內容之另一態樣闡述重構步驟。在重構步驟中,可採用各種計算來產生跨越不同染色影像之重構影像。如圖2A中所示,可收集次級影像並使其彼此配準以生成經共配準染色243之堆疊。初級虛擬厚切片223及經染色切片243之共配準堆疊可在數據收集或診斷步驟251中進行分析。
如本文所述,可實施Y_n影像之計算轉換以重構各個樣品切片並使其彼此空間對準。該組經染色Y_n影像可藉由採用計算轉換映射返回至藉由X_n影像所確立之生物標記物坐標。該等經重新映射之影像將稱為Z_n。該等計算轉換可包括(但不限於):
a.「普羅克拉斯提斯(Procrustean)」轉換,其可包括拉伸、剪切、平移、旋轉及更通常仿射轉換以跨越影像映射可見變量或生物標記物。
b.「彈性薄片」型轉換,其採用基礎樣條曲線或其他非線性
內插方案以藉由匹配目標物之曲率、併入仿射及非仿射轉換二者來映射生物標記物。
c.基於光學之校正以慮及初級與次級成像之光學間之差異。此可包括透視、桶形、枕形及色彩校正。
本揭示內容之另一態樣闡述共配準步驟,其中比較來自PI及SI步驟由KESM所捕獲之影像並基於在重構步驟中確立之經映射生物標記物使其彼此對準。
如本文所述,可使Z_n影像之堆疊重疊以使不同染色共配準。可重複此步驟以完成整組染色並基於共配準影像比較結果。用於完成共配準步驟之方法可包括:
a.Z_n影像之並排呈現、算繪或影像疊置。圖2B圖解說明初級影像261及次級影像262如何使用並排呈現方法共配準,且可顯示為複合影像263。
b.若干個Z影像之數學組合以產生能夠顯示不同染色存在及/或重合之位置的複合影像或虛擬「切片」。圖2C圖解說明若干個影像271、272、273可如何藉由數學組合進行組合以產生複合影像274。
工作流程之額外實施例可類似於上文之工作流程200,其在安裝與染色步驟之間具有額外成像步驟。
以上說明闡述圖2A中之單一切片之處理。使用初級成像資訊,多個切片可跨越Z平面與樣品內之上及之下之切片對準。使用此錨定,上文所述之工作流程100A、100B、100C可以以下方式替代:
˙解剖學家工作流程100A:「染色組」中之所有染色可相同。每一經染色切片可鏈接回至其初級成像且初級影像彼此鏈接。此可改良重構之品質以及最小化人類參與之分數。
˙組織學家/病理學家工作流程100B:大量所請求染色可在組內獨立地運行。該等經染之影像之每一者可再次鏈接回至初級成像,且許多層/對比劑可鏈接回至總體樣品。此可最小化醫療專業人員將參與其中之迭代次數,並導致更快診斷及實驗室分析之更快周轉。
˙生物學家工作流程100C:若干不同染色可在交插層上重複進行。以此方式,可在樣品體積上使染色多工,且包括標記為a、b、c之3種染色之實例看起來可如下:a|b|c|a|b|c|a|b|c|a|b|c|a|b|c|a|b|c
此多工方法給出標記跨越體積之較大3D分佈,同時將每一切片系鏈回至整體塊。此外,染色資訊可跨越毗鄰切片進行內插。
該等實施例亦可以至少以下方式之任一者擴充:
1.可將若干通道影像合併為一者,此產生虛擬多通道面板影像。
2.最佳切片厚度及染色之數量可藉由在初級成像中所觀測之特徵來確定,即一些特徵可較大,且厚切片可係適宜的,而其他特徵較小且可需要薄切片。
3.不需要立刻測定定整個組。可使用第一輪成像之輸出來確定第二輪。
4.切片厚度可關於染色化學進行調整,即可切割較薄或較厚切片用於針對每一具體對比劑之特徵最佳化之多工染色。
5.基於初級成像,可對每第n個切片進行不同處理。此可包括:
6.以對於所討論之染色最佳之厚度切片。
7.轉至分子診斷或另一化學分析。
8.基於成像或切片缺陷或切片中缺少樣品而丟棄。
9.為遵守法規而歸檔。
10.如上述,但基於初級成像步驟做出切片處理決定。
儘管本文中已顯示並闡述本揭示內容之較佳實施例,但熟習此項技術者將顯而易見此等實施例僅係以實例方式提供。熟習此項技術者在不背離本揭示內容之範圍下現將想出許多變化、改變及替換。應理解,對本文所述之本揭示內容之實施例的多種替代可在實踐本揭示內容之發明中採用。以下申請專利範圍意欲界定本發明之範圍並藉此覆蓋該等申請專利範圍及其等效內容範圍內之方法及結構。
101‧‧‧樣品
211A‧‧‧初級成像步驟
211B‧‧‧初級成像步驟
211N‧‧‧初級成像步驟
221A至221N‧‧‧薄切片/PI步驟
223‧‧‧初級虛擬厚切片/虛擬厚切片
231‧‧‧步驟/染色
231A至231N‧‧‧染色1至N
241A‧‧‧次級成像(SI)步驟
241B‧‧‧次級成像(SI)步驟
241N‧‧‧次級成像(SI)步驟
243‧‧‧經共配準之染色
251‧‧‧數據收集或診斷步驟
Claims (14)
- 一種分析樣品之方法,該方法包含:生成該樣品之複數個初級影像;將該樣品切成複數個切片;使該複數個切片染色;藉由使該複數個經染色切片成像生成該樣品之複數個次級影像;及使該樣品之該複數個次級影像彼此共配準。
- 如請求項1之方法,其中樣品包含組織樣品。
- 如請求項1之方法,其中該樣品之個別初級影像係在對該樣品之個別切片進行切片之前生成,該樣品之該個別切片與該個別初級影像相關。
- 如請求項1之方法,其進一步包含基於該複數個初級影像生成該樣品之虛擬模型。
- 如請求項4之方法,其進一步包含使該樣品之該經共配準之複數個次級影像共配準至該虛擬模型。
- 如請求項5之方法,其進一步包含因應該樣品之該虛擬模型與該經共配準之複數個次級影像之共配準生成診斷。
- 如請求項1之方法,其進一步包含使個別初級影像與相應個別次級影像共配準。
- 如請求項7之方法,其進一步包含基於該個別初級影像使該相應個別次級影像失真。
- 如請求項1之方法,該樣品係利用刀口掃描顯微鏡(KESM)進行切片。
- 如請求項1之方法,其中將該樣品切成複數個切片包含對該樣品 之塊面進行切片。
- 如請求項1之方法,其進一步包含將該複數個切片安裝於複數個載玻片中。
- 如請求項1之方法,其中該複數個切片利用抗原或蛋白質染色進行染色。
- 如請求項1之方法,其進一步包含使用拉伸、剪切、平移、旋轉、樣條曲線、非線性內插或光學校正轉換中之一或多者來使該等次級影像失真。
- 如請求項1之方法,其進一步包含生成個別初級影像與個別次級影像之複合影像。
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KR20170007181A (ko) | 2017-01-18 |
JP6841609B2 (ja) | 2021-03-10 |
JP2017021028A (ja) | 2017-01-26 |
US20170011511A1 (en) | 2017-01-12 |
US10839509B2 (en) | 2020-11-17 |
US20210073980A1 (en) | 2021-03-11 |
EP3115766A1 (en) | 2017-01-11 |
CN106338423A (zh) | 2017-01-18 |
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