CN106338423A - 组织学染色的空间复用 - Google Patents

组织学染色的空间复用 Download PDF

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CN106338423A
CN106338423A CN201610534830.1A CN201610534830A CN106338423A CN 106338423 A CN106338423 A CN 106338423A CN 201610534830 A CN201610534830 A CN 201610534830A CN 106338423 A CN106338423 A CN 106338423A
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马修·古德曼
托德·霍夫曼
科迪·丹尼尔
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3SCAN Inc
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Abstract

本发明涉及用于通过来自一个或多个成像操作的虚拟图像的汇编来共定位显微术或组织学染色剂的方法。具体地，该方法减少了获得生物样品的多个标记的抗原或蛋白组织学图像所需的时间。该方法包括在由切片机对组织进行切片时，用刀口扫描显微镜对该组织进行成像，并通过所生成的图像使样品空间对准。通过该方法实现的样品空间对准允许在不同的样品切片间对比不同的抗原或蛋白二次或功能性染色剂组，从而允许对组织和细胞的同时二次染色。

Description

组织学染色的空间复用
交叉引用
本申请要求提交于2015年7月10日的美国临时申请号62/190,931的权益，该申请通过引用并入本文。
发明背景
本公开内容总体上涉及为了诊断或研究目的而对组织进行切片、成像和染色的方法。具体地，本公开内容涉及连续切片显微术，即，使用切片机对生物组织和其他材料样品进行的剖切，并且更具体地涉及对用免疫组织化学抗原染色的组织样品进行成像的方法。
免疫组织化学是将一组抗原施加于生物组织切段的过程。免疫组织化学染色常用于通过采用抗体测试组织样品中的某些抗原来鉴别异常细胞。抗体通常连接到使组织中的抗原变得在显微镜下可见的放射性物质或染料，并且该过程一般采用一组或一系列不同染色剂来进行，以检测各种癌细胞株。
本公开内容通过使用借助KESM技术而成为可能的新方法和过程，汇集和优化了传统上在免疫组织化学中使用的若干种不同的工作流程。通过介绍代表该领域当前技术水平的传统工作流程的以下三项研究，可以更好地理解本文所述的新颖而有用的方法。
发明内容
本公开内容总体上涉及用于使用切片机对物体进行成像并施加免疫组织化学染色剂的系统和方法，以便检测用于医学诊断或研究的某些生物标志物。具体地，本公开内容涉及通过使用刀口扫描显微镜(KnifeEdge Scanning Microscope)来剖切生物组织和其他材料样品并向单个切段施加染色剂来针对这些诊断使用连续切片显微术，以及使用通过刀口扫描技术实现的空间复用方法来对比整个样品上的各种染色剂和反应。
下文涉及用于使用多种造影剂来进行组织样品的快速病理学和/或组织学检查的技术。通过在组织操纵之前进行通过刀口扫描显微镜而成为可能的附加成像步骤，可以在造成物理或化学变形之前捕捉所成像物体的基本形状。由此，可以将变形的切片配准(register)回样品的原始坐标系中。这还可以提供在单个样品的许多连续切段间更好地共定位生物标志物的独特能力，并且可以在化学或机械处理之后通过使用中间成像步骤将单个切段彼此重新映射来产生作为整体的组织的更准确表示。这可以通过允许以较小的畸变在整个样品上比传统工作流程更快地对比多种造影剂而提供独特的优势。
结合以下描述和附图考虑，将会进一步领会和理解本公开内容的其他目标和优点。虽然以下描述可能包含描述本公开内容的特定实施方式的具体细节，但其不应被解释为对本发明范围的限制，而应当视为对优选实施方式的例示。对于本公开内容的每一方面，有可能存在如本文所提出的、本领域普通技术人员所知晓的众多变体。在不偏离本发明精神的情况下可在本公开内容范围内作出各种改变和修改。
援引并入
本说明书中提及的所有出版物、专利和专利申请均通过引用并入本文，程度如同具体地和个别地指出要通过引用而并入每一单个出版物、专利或专利申请。
附图说明
本发明的新颖特征在随附权利要求中具体阐述。通过参考阐述了利用本发明原理的说明性实施方式的以下详细描述以及附图，将会对本发明的特征和优点获得更好的理解，在附图中：
图1A为图示使用本领域现有技术的、典型的解剖学家工作流程的示意图。
图1B为图示使用本领域现有技术的、典型的组织学家或病理学家工作流程的示意图。
图1C为图示使用本领域现有技术的、典型的生物学家工作流程的示意图。
图1D1为图示在使用本领域现有技术的传统工作流程中如何会发生图像畸变的示意图。
图1D2为图示使用本文所述技术的空间复用如何通过与无畸变图像对准来改善对准图像堆栈的过程的示意图。
图2A为图示用于使用本文所述的空间复用方法对样品进行成像的步骤的示意图。
图2B为通过并排展示而图示主像(Primary image)与副像(Secondary image)的共配准的示意图。
图2C为图示通过数学组合而施加具有不同染色剂的若干个Z图像的共配准从而产生重合染色反应的合成图像的示意图。
具体实施方式
本文公开了与上述对组织样品进行切片、染色和成像的三种传统方法相比改善的系统和方法。本公开内容包括通过在剖切时或剖切之前引入借助KESM技术实现的一个或多个附加成像步骤，对前述三种工作流程的改善。
定义
“免疫组织化学”可以指通过使用标记抗体作为引起抗原-抗体相互作用的特异性试剂，将抗原或蛋白施加于组织切段之中，其中所述抗原-抗体相互作用可以通过诸如荧光染料或染色剂等标志物而可视化。
“连续切片显微术”可以指用切片机取得连续切段并且传统上通过将切片安装到玻璃上并染色来对其进行成像的做法。
“刀口扫描显微镜”或“KESM”可以指以自动化方式进行连续切片显微术的显微镜。参见美国专利号6,744,572。
“切段”或“切片”可以指借助于样品与刀之间的相对运动而从块面移取的单条连续材料。
“切片机”可以指对材料块进行精确切割以便从该块的表面移取或剖切非常薄的一层材料的装置。类似地，术语“切片术”可适用于切片机的操作。
“成像术”可以包括设计用于测量“图像”(即，光学或电子响应的空间图)的任何技术。这可以包括光学显微或电子显微技术。
“成像”一般可以指为了生成给定区域的可视化而进行的数据收集。
“配准”或“共配准”可以指从中对图像进行对准、拉伸和变形以使其相互匹配的计算步骤。参考连续切片显微术而言，该步骤可以校正来自切片、安装和化学处理的组织变形。
“复用”可以指通过在矩阵的两侧具有两个选择性寻址系统而在该矩阵内选择一个位置，从而仅需2N个选择器来寻址NA2个位置的方法。
“染色”可以指这样一种化学处理：其旨在通过包括但不限于贴附色素、遗传表达的荧光团或设计用于修饰待成像的目标结构的化学品等方法，来改变介质的全部或部分的光子响应。其可以包括但不限于传统的光学显微术染色剂、在免疫组织化学(IHC)和原位杂交(ISH)标记技术中使用的造影剂。
“分子诊断”可以指这样一种形式的化学测试或测定：其取得组织样品并鉴别生物标志物以作出诊断。
“变换”可以指将图像中的单个点从未染色图像到另一染色图像的重新映射。
“内插法(Interpolation)”可以包含用于对来源于另一数字域的数值进行选择性空间采样的方法。常用的方法可以包括“最近邻”内插法、线性、多项式或基于B样条(b-spline)的技术。这些方法一般用于计算经变换图像的离散“内插”值，或者用于对函数在给定空间坐标处的值取近似。
畸变
从切片机捕捉切段并将其安装到载玻片的过程可能使组织物理畸变。该畸变可能是薄而纤弱组织的翘曲、切段的折叠或者甚至撕裂。翘曲可能妨碍连续切段的简单对准，这是因为微观特征可能在切段间未恰当排列。通常，必须使用手动编辑与软件的组合来校正畸变，其中在相邻的图像上选择配准标记，并且软件对所述配准标记加以对准。该过程通常缓慢而困难，并且可能妨碍连续切片的更广泛采用。
染色和切片厚度
由于以下几个原因，控制切片厚度可能是重要的：
1.保持切段的结构/架构完整性以供后续操纵、染色、安装和处理。
2.确保切段对于所分辨的结构具有合适的厚度。切片应当足够厚以使得独特的特征包含在该切片内。在细胞表征中，切片通常为允许在平面内具有1-2层细胞的厚度。如果其过厚，则许多层的细胞可能掩盖其掩盖细胞水平细节的能力。类似地，如果切片过薄，则单细胞层内的相关特征可能被截断。
3.确保切片足够薄以使染色的化学动力学充分地标记相关联的特征。许多造影剂采用大分子，该大分子可能在其所能穿过以扩散并相应地标记的组织量方面受到限制。
类似地，可能依赖于基体材料的化学动力学的造影剂对于厚的或薄的切段可能有不同表现。
在组织学或病理学实验室中常见的做法是针对不同的染色/造影技术来切割不同厚度的切片。当需要快速结果(薄切片通常可以更快地染色)时，或者在诸如IHC或ISH法等染色中采用复杂的化学时，情况尤为如此。
解剖学家工作流程
存在许多原因令生物学家可能想要测量在三维空间体积上的组织性质。要使用本领域现有技术来做到这一点，切割出若干个切片、对其进行染色并将其彼此共配准。图1A为图示采用本领域现有技术的、典型的解剖学家工作流程100A的示图。在步骤111中，可将样品101切成一个或多个厚切段，诸如第一厚切段121A、第二厚切段121B和第三厚切段121C。可以在步骤131中对厚切段121A、121B、121C进行染色，并且继而可以在步骤141中对经染色的厚切段121A、121B、121C进行成像并将其彼此共配准。
组织学家/病理学家工作流程
类似地，当组织学家或病理学家尝试作出病变组织的诊断时，可以切割出若干个厚切段121A、121B、121C。继而，在步骤151中对其中一个切段进行染色、成像和检查。基于来自医疗专业人员的成像术输入，可以在最终作出步骤161中的诊断之前将该过程重复若干次，并且可以将所述厚切段中之一的诊断用于告知其他厚切段的染色、成像和检查。经染色的厚切段的诊断可以指示出步骤171中对患者的适当处理。图1B为图示采用本领域现有技术的、典型的组织学家或病理学家工作流程100B的示图。
生物学家工作流程
生物学研究人员通常采用免疫组织化学染色技术，在该技术中对一片特定组织进行染色、成像，继而从组织切段脱除染色剂。可以重复染色和脱除过程以便对组织样品片上的全组染色剂的免疫或抗原反应进行成像。图1C为图示采用本领域现有技术的、典型的生物学家工作流程100C的示图。可以在步骤181中对样品101进行切片并将其放置在载玻片中。在步骤191中，可以对切片/载玻片进行染色。在步骤1011中，可以对经染色的载玻片/切片进行成像。在步骤1031中，可以储存图像。在步骤1021中，可以去除染色剂，并且可以在步骤191中再次对载玻片/切片进行染色。
施加染色剂、成像、脱除染色剂、再染色、对该染色剂再成像、继而再一次再成像的这一过程被称为连续复用。由生物学家在免疫组织化学中常用的该方法是耗时的，完成全组的抗原染色通常耗时一周或更长时间。
另一方法是基于来自病理学家的检查，对下一切段进行鉴别染色。这可能导致相当长的周转时间，并且需要与医师增加互动。
所有这些方法均具有本发明的范围寻求解决的根本缺点：
·载玻片安装、染色和成像分别在切片中引入了独特的变形、畸变和伪影(artifact)，它们可能使得最终共配准过程变得困难、容易出错，并且耗时。
·往往存在大量的连续步骤，其中每个步骤均可能涉及大量的人工劳动和对细节的关注才能产生高质量的可靠结果。
·考虑到样品的3D性质，特征即使在相邻切片之间也可能变化，并且自动化特征提取和翘曲校正在较大的样品中是困难的。
本公开内容描述了用于通过引入由本文应用的KESM技术所实现的附加成像步骤来减少处理样品上的多种染色剂所需的人力并提高重构产物的准确性的系统和方法。
后处理畸变
在典型的连续切片显微术中，使用人工标注或算法来将切片对准，其中在重构之前校正安装过程中的翘曲。该过程通常涉及配准标记的选择，所述配准标记为相邻切段中出现的点。可以使用配准点来计算变换函数，该函数被应用于全部两个图像以使所述图像中的像素对准。由于堆叠中的每个图像与下一图像不同，因此在一系列图像中选择配准标记可能是困难的。特征也很少与切割平面完全垂直，因此如果针对配准标记选择了相同的特征，则该特征可能在切段之间空间漂移，并且因此例如位于斜面上的物体可能被误认为是垂直的。
如本文所公开，由于可以对完全相同的切段的两个图像(一个没有畸变，而一个或多个没有畸变)进行畸变后处理，因此可以改善图像的后处理。由于图像是相同切片的图像，因此相同的精确配准标记将会出现在所有的图像上，从而使得该选择过程更容易且更加一致。同样的现象还可以减少对于在相对于切割平面的斜面上延伸的圆柱状特征原本将会发生的空间漂移。图1D1和图1D2图示了采用本领域现有技术如何出现畸变(图1D1)，对比于如本文所公开的如何可以关于这些畸变来改善图像后处理(图1D2)。
如图1D1中所示，物体103可以存在于样品101中，该样品101可以切成两个切片105A和105B。对于每个切片105A、105B可以分别生成图像107A、107B，示出处于不同深度的物体103。
如图1D2中所示，物体103可以存在于样品101中，该样品101可以切成两个切片105A和105B。对于切片105A、105B中的每一个，可以生成主像和副像(诸如主切片105A的主像109A1和副像109A2，以及副切片105B的主像109B1和副像109B2)。
主成像
如本文所述，该方法中的第一步骤包括主成像(PI)步骤，该步骤可以在处理或后处理畸变之前捕捉切段的视图。这可以通过在进行切片之前对样品进行成像或通过在进行切片期间成像来实现。该主成像可以利用KESM来进行，KESM可以在切片操作期间在块面上捕捉未翘曲的图像。这些图像可以称为X_N。通过并入该PI步骤，副成像(SI)步骤可以具有对切片的实际原始形状和位置的直接锚定，这可以支持对样品染色反应的更好的配准和分析。图2A为图示空间复用方法200的成像步骤的示图。可以在从样品101切出薄切段221A之前，在主成像步骤211A中对样品101进行成像。可以在将样品进一步切成进一步的薄切段221B至221N之前进行进一步的主成像步骤211B至211N。可以收集主像并将其彼此配准以生成虚拟厚切片223。
如本文所述，在PI步骤(221A、221B、…221N)之后，可以将一个或多个切片安装在载波片上，并且继而可以在步骤231中将抗原或蛋白染色剂施加于样品切片以供进一步分析。
副成像
本公开内容的另一方面包括一个或多个副成像(SI)步骤241A、241B、…241N。在SI步骤中，可以在已经施加染色剂或全组染色剂231之后，使用KESM来对样品101的经染色切片进行成像。这些(一个或多个)步骤可以根据需要而重复多次，每当将新染色剂施加于样品时，对切片进行再成像。这些图像将被称为Y_n。
重构
本公开内容的另一方面描述了重构步骤。在该重构步骤中，可以采用各种计算来产生跨越不同的染色图像的重构图像。如图2A中所示，可以收集副像并将其彼此配准以生成共配准的染色剂243的堆叠。可以在数据采集或诊断步骤251中分析主虚拟厚切片223和染色切片242的共配准堆叠。
如本文所述，可以进行Y_n图像的计算变换以重构各个样品切片并将其彼此空间对准。可以将经染色Y_n图像的组映射回由X_n图像通过采用计算变换而建立的生物标志物坐标。这些重新映射的图像将被称为Z_n。这些计算变换可以包括但不限于：
a)“强求一致的(Procrustean)”变换，其可以包括拉伸、剪切、平移、旋转以及更一般的仿射变换，以在图像间映射可见变量或生物标志物。
b)采用基样条(basis spline)的“弹性片”型变换，或者其他非线性内插方案，以通过匹配物体的曲率来映射生物标志物，从而合并仿射变换和非仿射变换。
c)基于光学的校正，以将主成像与副成像的光学器件之间的差异纳入考虑。这可以包括透视校正、桶形校正、枕形校正和色度校正。
共配准
本公开内容的另一方面描述了共配准步骤，其中基于在重构步骤中确立的映射生物标志物，将通过KESM捕捉的来自PI步骤和SI步骤的图像彼此对比和对准。
如本文所述，可以将Z_n图像的堆叠叠加起来，以便共配准不同的染色剂。可以重复该步骤以完成全组的染色剂，并基于共配准的图像来对比结果。用于完成共配准步骤的方法可以包括：
a)Z_n图像的并排呈现、渲染或图像叠加。图2B图示了如何使用并排呈现方法将主像261和副像262共配准，并且可以将主像261和副像262示出为合成图像263。
b)若干个Z图像的数学组合，以产生能够示出不同的染色剂在何处存在和/或重合的合成图像或虚拟“切片”。图2C图示了如何可以通过数学组合来结合若干个图像271、272、273以产生合成图像274。
进一步实施方式
工作流程的附加实施方式可以与上述工作流程200相似，并在安装步骤与染色步骤之间具有附加的成像步骤。
以上的一个或多个描述描述了图2A中单个切片的处理。通过使用主成像信息，可以将多个切片跨Z平面与样品内的上方和下方的切片对准。通过使用这样的锚定，可以按以下方式替换上述的工作流程100A、100B、100C：
·解剖学家工作流程100A：“染色组”中的所有染色剂可以是相同的。可以将每个染色切片链接回其主像，并且可以将主像彼此链接起来。这既可以改善重构的质量，又可以使人为参与的部分最小化。
·组织学家/病理学家工作流程100B：可以在组内独立地运行大量的所要求的染色。可以将这些染色图像中的每一个再次链接回主像，并且可以将许多层/造影剂链接回总体样品。这可以使医疗专业人员将会参与的重复的次数最小化，并导致更快的诊断以及实验室分析的更快周转。
·生物学家工作流程100C：可以在交错的层上重复若干个不同的染色。以这种方式，可以在样品体积内复用染色剂，并且包括标记为a、b、c的3种染色剂的示例可能是这样：
a|b|c|a|b|c|a|b|c|a|b|c|a|b|c|a|b|c
这样的复用方法给出了标签在整个体积内的更大的3D分布，同时将每个切片栓系回总体块。另外，可以在相邻的部分之间内插染色信息。
这些实施方式还可以至少通过以下方式中的任一种来扩充：
1.可以将若干个通道图像组合成一个图像，从而产生虚拟的多通道组图像。
2.可以通过在主像中观察到的特征来确定最佳剖切厚度和染色剂数目，即，一些特征可能较大，厚切段可能是合适的，而其他特征较小并且可能需要薄切段。
3.不必在一次确定整个组。第一轮成像的输出可以用于确定第二轮。
4.可以关于染色剂化学来调整切段厚度，即，对于针对每种特定造影剂性质优化的复用染色剂，可以切割出较薄或较厚的切段。
5.基于主成像，可以对每个第n切片进行区别处理。这可能包括：
6.以针对所考虑的染色剂优化的厚度进行切片。
7.转向分子诊断或其他化学测定。
8.基于成像或切片缺陷或者切割片中样品的缺失而丢弃。
9.存档以符合监管规定。
10.如上所述，但替代地基于主成像步骤而作出切片处理决定。
虽然本文已经示出和描述了本公开内容的优选实施方式，但对于本领域技术人员将会显而易见的是，这样的实施方式只是以示例的方式提供的。本领域技术人员现将在不偏离本公开内容的范围的情况下想到许多更改、改变和替代。应当理解，在对本公开内容的发明的实践中可以采用针对本文所述的本公开内容实施方式的各种替代方案。以下权利要求旨在限定本发明的范围，并且因此涵盖这些权利要求范围内的方法和结构及其等效项。

Claims (14)

1.一种分析样品的方法，该方法包括：
生成所述样品的多个主像；
将所述样品切成多个切段；
对所述多个切段进行染色；
通过对所述多个染色切段进行成像而生成所述样品的多个副像；以及
将所述样品的所述多个副像彼此共配准。
2.如权利要求1所述的方法，其中样品包括组织样品。
3.如权利要求1所述的方法，其中在切出所述样品的单个切段之前生成所述样品的单个主像，所述样品的所述单个切段关联于所述单个主像。
4.如权利要求1所述的方法，还包括基于所述多个主像而生成所述样品的虚拟模型。
5.如权利要求4所述的方法，还包括将所述样品的共配准的多个副像与所述虚拟模型共配准。
6.如权利要求5所述的方法，还包括响应于所述样品的所述虚拟模型与所述共配准的多个副像的共配准而生成诊断。
7.如权利要求1所述的方法，还包括将单个主像与对应的单个副像共配准。
8.如权利要求7所述的方法，还包括基于所述单个主像来扭曲所述对应的单个副像。
9.如权利要求1所述的方法，用刀口扫描显微镜(KESM)对所述样品进行切片。
10.如权利要求1所述的方法，其中将所述样品切成多个切段包括对所述样品的块面进行切片。
11.如权利要求1所述的方法，还包括将所述多个切段安装到多个载玻片中。
12.如权利要求1所述的方法，其中用抗原或蛋白染色剂对所述多个切段进行染色。
13.如权利要求1所述的方法，还包括使用拉伸、剪切、平移、旋转、样条、非线性内插或光学校正变换中的一种或多种来扭曲所述副像。
14.如权利要求1所述的方法，还包括生成单个主像与单个副像的合成图像。
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