CN101304682B - 用于提供一个或多个生物结构的多模态显微成像的装置和方法 - Google Patents

用于提供一个或多个生物结构的多模态显微成像的装置和方法 Download PDF

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CN101304682B
CN101304682B CN2006800405482A CN200680040548A CN101304682B CN 101304682 B CN101304682 B CN 101304682B CN 2006800405482 A CN2006800405482 A CN 2006800405482A CN 200680040548 A CN200680040548 A CN 200680040548A CN 101304682 B CN101304682 B CN 101304682B
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吉列尔莫·J·蒂尔尼
德维尔·叶林
本杰明·J·瓦科奇
吴汪烈
布雷特·尤金·鲍马
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Abstract

可以提供根据本发明的示范性实施例的方法和设备。例如，可以基于第一模态提供与从至少一个样本的至少一个区域接收的第一信号相关联的第一数据，并且可以基于不同于所述第一模态的第二模态提供与从所述至少一个样本接收的第二信号相关联的第二数据。可以接收与基准相关联的第三数据。基于所述第一、第二和第三数据可以生成进一步的数据。另外，可以获得与从所述至少一个样本接收的第二信号相关联的第三数据。所述第三数据中的每一个可以基于不同于所述第一模态和所述第二模态的进一步的模态，并且可以基于所述第三数据来进一步确定所述进一步的数据。进一步，所述第一模态可以是谱编码模态，并且所述第二模态可以是非谱编码模态。

Description

用于提供一个或多个生物结构的多模态显微成像的装置和方法
相关申请的交叉引用
本申请基于并要求2005年9月29日提交的序列号为60/721,802的美国专利申请的权益，其整体内容通过引用结合于此。
技术领域
本发明一般涉及用于提供一个或多个生物结构的多模态显微成像的装置和方法，并且具体地涉及例如使用谱编码共焦显微(“SECM”)、荧光SECM、光学相干断层扫描(“OCT”)、谱域(“SD”)-OCT、光频域干涉测量(“OFDI”)和光相干显微(“OCM”)过程来实施生物样品的反射和/或荧光显微。
背景技术
基因变化的分子基础和表型之间的关系的确定一般利用了生物样品的微观结构的准确的二维和三维表征。然而，运动和小的尺度使得许多活体生物样品更加难以评估。
光学技术提供了以高分辨率对生物样品进行成像的潜力。对于某些应用，基于内源性对照的光学成像相对于需要外源性试剂的技术是有利的，因为这样的有利过程可以允许在少量准备的情况下，以样品的天然状态并且在多个时间点对样品进行分析。作为例子，在此描述了用于使胚胎心脏微观结构可视化的几种内源性对照成像模态：如D.huang等人的“Opticalcoherence tomography”，Science 254，pp.1178-1181(1991)中描述的光学相干断层扫描(“OCT”)的两种示范性形式、如S.A.Boppart等人的“Investigation of developing embryonic morphology using opticalcoherence tomography”，Dev Biol 177，pp.54-63(1996)中描述的时域光学相干断层扫描(“TD-OCT”)以及如M.A.Choma等人的“Sensitivityadvantage of swept source and Fourier domain optical coherencetomography”，Optics Express 11，pp.2183-2189(2003)和S.H.Yun等人的“High-speed optical frequency-domain imaging”，Optics Express 11，pp.2953-2963(2003)中描述的光频域成像(“OFDI”)。
可以提供并使用另外的例子，包括两种反射显微技术，例如在E.Beaurepaire等人的“Full-field optical coherence microscopy”，OpticsLetters 23，pp.244-246(1998)、A.Dubois等人的“Ultrahigh-resolutionfull-field optical coherence tomography”，Appl Opt 43，pp.2874-2883(2004)和G.Moneron等人的“Stroboscopic ultrahigh-resolution full-fieldoptical coherence tomography”，Opt Lett 30，pp.1351-1353(2005)中描述的全场光学相干显微(“FFOCM”)，以及如G.J.Tearney等人的“Spectrally encoded confocal microscopy”，Optics Letters 23，pp.1152-1154(1998)和C.Boudoux等人的“Rapid wavelength-swept spectrallyencoded confocal microscopy”，Optics Express 13，pp.8214-8221(2005)中描述的谱编码共焦显微(“SECM”)。
例如，TDOCT技术可以使用低相干干涉测量来获得具有大约10μm的分辨率和高达2mm的深度处的横截面图像。参见：S.A.Boppart等人的“Noninvasive assessment of the developing Xenopus cardiovascularsystem using optical coherence tomography”，Proc Natl Acad Sci USA 94，pp.4256-4261(1997)；S.Yazdanfar等人的“High resolution imaging of invivo cardiac dynamics using color Doppler optical tomography”，OpticsExpress 1，pp.424-431(1997)；T.M.Yelbuz等人的“Optical coherencetomography：a new high-resolution imaging technology to study cardiacdevelopment in chick embryos”，Circulation 106，pp.2771-2774(2002)；V.X.D.Yang等人的“High speed，wide velocity dynamic range Doppleroptical coherence tomography(Part II)：Imaging in vivo cardiac dynamicsof Xenopus laevis”，Optics Express 11，pp.1650-1658(2003)；以及W.Luo等人的“Three-dimensional optical coherence tomography of theembryonic murine cardiovascular system”，Journal of biomedical optics11，021014(2006)。
示范性OFDI技术可以被认为是TDOCT技术的衍生，其能够以显著更高的帧率获取图像，如R.Huber等人的“Three-dimensional andC-mode OCT imaging with a compact，frequency swept laser source at1300nm”，Optics Express 13，pp.10523-10538(2005)中描述的那样。OFDI技术中的高速度能够实现真实四维(4D)显微(例如作为时间函数的三维显微)。全场光学相干显微(“FFOCM”)技术能够利用低相干干涉测量和较高的数值孔径物镜来获得全三维的亚细胞水平的分辨率。这样的FFOCM技术很可能显著慢于OFDI技术。示范性SECM技术可以具有反射共焦显微的形式，使用该技术可以以比使用FFOCM技术可能获得的速度显著更高的速度获得具有微米等级分辨率的二维图像。
虽然这些自然对照过程中的每一个都可以单独地用于对胚胎心脏的微观结构进行成像，但是当组合时，这些过程可以提供一组强有力的工具，用于早期心肌形态和动态二维、三维和四维表征。将这些不同的模态组合成一个单个显微镜装置可以具有另外的优点，比如(a)比较不同格式、不同分辨率和视场的图像、(b)同时获取结构信息和功能信息和/或(c)可以使用一个仪器而不需要移动或改变样品来完成这些任务。
发明内容
本发明的目的之一是要克服现有技术系统的某些缺陷和缺点(包括上面在此描述的那些)，并且提供示范性实施例，该示范性实施例提供了一个或多个生物结构的多模态显微成像。这样的示范性实施例可以使用谱编码共焦显微(“SECM”)、荧光SECM、光学相干断层扫描(“OCT”)、谱域(“SD”)-OCT、光频域干涉测量(“OFDI”)和光相干显微(“OCM”)过程来实施生物样品的反射和/或荧光显微。
例如，生物样品的分析一般使用其微观结构和功能的可视化，优选地具有对样品的微小改变。根据本发明的一个示范性实施例，可以在单个显微镜装置中提供多个不同成像模态的组合。根据本发明的某些示范性实施例的每种示范性技术可以提供不同且互补的成像能力，包括高速(例如每秒1000帧)和高轴向分辨率(4-16μm)的体内横截面成像、体内真实四维成像、体外具有各向同性细胞分辨率(例如1-2μm)的三维显微以及体内二维亚细胞成像。当组合时，这些示范性成像模态可以实现生物样品的形态和动态的更加完整的图像。
因此，本发明的示范性实施例包括用于获取多模态微观数据的装置和方法。例如，根据一个示范性实施例，可以使用独特宽带宽或快速波长扫描源与插在扫描机构和成像透镜之间的光学器件的组合。可以例如在不移动样品的情况下，同时和/或连续地获取数据。例如，可以共同配准从不同模态获得的数据，以便能够并排和/或在彼此顶部重叠地显示。可以用互补的方式从所有的数据集中获得定量信息。
因此，可以提供根据本发明的示范性实施例的方法和设备。例如，可以基于第一模态提供与从至少一个样本的至少一个区域接收的第一信号相关联的第一数据，并且可以基于不同于所述第一模态的第二模态提供与从所述至少一个样本接收的第二信号相关联的第二数据。可以接收与基准相关联的第三数据。基于所述第一、第二和第三数据可以生成进一步的数据。另外，可以获得与从所述至少一个样本接收的第二信号相关联的第三数据。所述第三数据中的每一个可以基于不同于所述第一模态和所述第二模态的进一步的模态，并且可以基于所述第三数据来进一步确定所述进一步的数据。进一步，所述第一模态可以是谱编码模态，并且所述第二模态可以是非谱编码模态。
在本发明的另一个示范性实施例中，所述第一模态可以是荧光成像。可以提供显微镜装置和/或射束扫描装置。射束扫描装置可以配置成将电磁辐射转送到所述至少一个区域。进一步，可以作为所述进一步的数据的函数产生二维图像和/或三维图像。可以基本上同时获得所述第一和第二数据。另外，所述第一和第二数据可以与所述样本上的近似相同位置相关联，和/或可以通过使用所述第一和第二数据中的另一个来获得。
根据本发明的进一步的示范性实施例，可以在探针和/或单个罩中提供设备。还可以使用这样的示范性设备和方法获得谱编码显微信息以及明视场、暗视场、相位反差、极化、上反射(epireflectance)和/或反射显微信息。可以进一步使用这样的示范性设备和方法从所述第一模态改变到所述第二模态。可以获得与具有多个波长的源装置提供的信号相关联的光学相干断层扫描信息。可以提供多个检测器以检测作为波长函数的第二和第三信号之间的谱干涉。
可以获得与波长随时间变化的源装置提供的信号相关联的光学相干断层扫描信息。基于所述第一和第二数据可以生成至少一个图像。另外，可以基于所述第一数据生成第一图像，并且可以基于所述第二数据生成第二图像。所述第一和第二图像可以作为所述第一和第二数据的函数而彼此相关联。可以获得光学相干断层扫描信息和/或光频域干涉测量信息。
结合所附的权利要求书，当阅读以下对本发明实施例的详细描述时，本发明的其它特征和优点将会变得明显。
附图说明
结合示出了本发明的示意性实施例的附图，从以下详细描述中，本发明的进一步的目的、特征和优点将会变得明显，其中：
图1是使用宽带宽源的示范性SECM系统的示意图；
图2是示范性SD-OCT系统的示意图；
图3是使用宽带宽源的示范性OCM系统的示意图；
图4是使用宽带宽源的示范性FFOCM系统的示意图；
图5是使用宽带宽源的示范性荧光SECM系统的示意图；
图6是使用波长调谐源的示范性SECM系统的示意图；
图7是使用波长调谐/调制源的示范性OFDI系统的示意图；
图8是使用波长调制/调谐源的示范性OCM系统的示意图；
图9是使用波长调制/调谐源的示范性FFOCM系统的示意图；
图10是根据本发明的第一示范性实施例的使用宽带宽源的示范性组合SECM/SD-OCT/OCM系统的示意图；
图11是根据本发明的第二示范性实施例的使用宽带宽源的示范性组合SECM/SD-OCT/FFOCM系统的示意图；
图12是根据本发明的特殊示范性实施例的示范性多模态显微镜滑动器的示意图；
图13是根据本发明的第三示范性实施例的使用波长调谐源的示范性组合SECM/OFDI/OCM系统的示意图；
图14是根据本发明的第三示范性实施例的使用波长调谐源的示范性组合SECM/OFDI/FFOCM系统的示意图；
图15a-15m是使用TDOCT和OFDI过程的示范性实施例在体内获得的光滑爪蟾(Xenopus laevis)心脏(阶段49)的各种示范性图像；
图16a-16m是使用FFOCM过程的示范性实施例在体外获得的爪蟾心脏的各种示范性三维图像；
图17a-17h是使用SECM过程的示范性实施例在体内获得的示范性高分辨率共焦图像；
图18a-18e是使用根据本发明的方法和装置的示范性实施例获得的爪蟾心脏中的动脉瘤扩张的示范性图像；以及
图19a-19x是使用根据本发明的方法和装置的示范性实施例获得的由乙醇暴露引起的异常心脏形成的示范性图像。
贯穿附图，除非另外声明，否则相同的标号和字符用于指示图示实施例的相同特征、元件、部件或部分。此外，虽然现在将参考附图详细地描述本发明，但是这将结合示意性实施例进行。能够对描述的实施例进行改变和修改，而不认为脱离了如所附权利要求所限定的本发明的真实范围和精神。
具体实施方式
示范性SECM技术能够提供组织或生物样品的亚细胞水平分辨率的图像。SECM图像可替选地表示来自样本的荧光或来自样本的反射。图1描绘了使用宽带源的示范性SECM装置的示意图。在这个示范性配置中，准单色或宽带光100照射可替选地为分束器的环行器110。在一个实施例中，这个环行器或分束器是光纤耦合的。光纤的核心可以充当用于共焦显微系统的针孔。光纤可替选地具有多重包覆，其传输光，使得例如激发样本的光为单模而收集的光则为多模。来自这个元件的光可以入射在扫描机构115上，该扫描机构115扫描射束的角度，以便在样本上产生一个或多个横向扫描。扫描机构可替选地为共振扫描仪、检流计扫描仪、多边形扫描镜、声光扫描仪等等中的一种。望远镜设备可以用于将扫描轴成像到物镜130的后焦平面。来自扫描机构的光然后可以被引向波长分散元件120如传输衍射光栅、棱镜、光栅棱镜、双棱镜光栅棱镜(DP-GRISM)等等。这个示范性元件可以分散宽带宽源中的不同波长，以便以取决于波长的变化的角度在物镜130上入射。
在一个示范性实施例中，透镜可以具有产生小焦斑的数值孔径，或者可替选地透镜具有大于0.2的高NA。物镜130将每个波长区域聚焦到样本上，其中样本160上的每个波长区域可以定位在不同的空间位置。对于衍射光栅和物镜，这些示范性元件可以在样本上形成波长编码线140，其中线上的每个位置由不同的波长区域编码。来自样本160的光可以被反射返回通过图1的示范性系统。散焦光可以被光纤的包覆滤去，而聚焦(例如共焦)光则被传输返回通过环行器/分束器110到达分光计，该分光计测量返回的光145的谱含量。通过测量在图像上形成一条线的这个谱，解码作为空间位置函数的共焦传送。针对扫描机构115的每个角位置形成连续的线，从而形成谱编码共焦显微图像。
图2描绘了示范性谱域OCT系统的示意图。与示范性SECM系统相反，示范性SD-OCT可以通过使用傅立叶域中的相干光栅来提供生物样品的横截面图像。SD-OCT图像典型地可以具有较低的分辨率(大约3-10μm)，并且可以具有较大的视场(几个毫米)。在这个示范性SD-OCT系统中，宽带宽或准单色源200可以被输入到干涉仪中，该干涉仪可以是基于光纤的。光纤耦合的光可以被传输到环行器210和分束器220。当耦合到环行器210中时，光可以优选地被分束器220随后分离，以便光的一部分可以被传输到基准臂225，一部分被传输到样本臂235。来自基准臂225的光可以从镜230(例如基准)反射到分束器220或者可替选地被传输返回到分束器220。在一个示范性实施例中，分束器220可以配置成将大多数的光传输到样本臂235。来自样本臂光纤的光然后可以被引向透镜和扫描机构240。扫描机构可以以任意的一维或二维模式扫描样本臂235的光。可以将光从扫描机构传输到透镜250，该透镜250在一个示范性实施例中可以具有如此的NA，以至于共焦参数足够大，以允许在生物样品或样本260中进行横截面成像。
从样本传送的光可以被传输返回通过设备到达环行器/分束器210，并且被引向分光计280。例如通过在中央处理单元或计算机290中对谱干涉信号进行背景扣除、将λ空间重新映射到k空间以及逆傅立叶变换，可以重构组织之内的作为深度的函数的反射(A线)。针对每个扫描机构位置获得连续的A线，从而重构样本的横截面图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(“STFT”)、多普勒敏感SD-OCT和极化敏感SD-OCT从样本中获得谱信息的能力在内，也可以用于从生物样品中提取额外信息，诸如吸收、流动和双折射。
图3描绘了示范性光学相干显微(“OCM”)系统的示意图。示范性OCM系统可以使用共焦显微和OCT技术的组合，这是有利的，因为两种这样的示范性技术的轴点扩散函数可以相乘，以便提供更大程度的光学分割。在OCM系统的一个示范性实施例中，来自宽带宽源的光可以被输入到调制元件310中，以便调制频率逼近干涉仪中的谱干涉的频率。这种示范性调制元件可以是Michelson干涉仪、脉冲成形设备、谱过滤器等等中的一种。该调制也可以随时将谱相位转移某个量，以便连续的谱可以被减去以仅提取谱干涉项。在调制元件之后，光可以被传输到环行器/分束器320，然后如果使用的是环行器的话则到达分束器330。光可以再次被传输到基准臂335和样本臂345。来自基准臂335的光被镜340反射。来自样本臂345的光可以被传输到x-y扫描仪350，该x-y扫描仪350可替选地为共振扫描仪、检流计扫描仪、多边形扫描镜、声光扫描仪等等中的一种或其组合。
来自扫描仪350的光可以被引向物镜355，以便可以在样本之内扫描紧密焦斑。可替选地以任意的三维扫描物镜或样本360，以便于从样本之内的不同部分进行数据收集。光被从样本360传输返回到环行器/分束器320并随后到达检测设备。在一个实施例中，检测器为分光计，并且通过以与示范性SD-OCT所进行的类似的方式从样本中获得A线来获得OCM数据。在谱调制实施例中，检测器可替选地为同步于源调制元件310的光电二极管或其它单个检测器。示范性锁定或扣除技术可以用于提取OCM信号。
全场光学相干显微术典型地为自由空间干涉测量技术，其使用宽带宽源来获得生物样品的横向高分辨率光学片段。图4A描绘了示范性FFOCM系统的示意图，其中宽带宽光400被传输到分束器410。光被分离到样本臂423和基准臂422中。根据一个示范性实施例，基准臂422中的光可以被引向415基准物镜420并到达能够轴向移动的镜425。样本臂423中的光可以被引向样本物镜430并到达样本440。在一个示范性实施例中，基准和样本物镜420、430具有类似特性。
在另一个示范性实施例中，可以用浸液对物镜420、430进行优化，该浸液具有类似于样本的折射率。样本可以耦合到提供任意三维移动的台443。使用透镜445可以将来自基准臂422和样本臂423的光成像到CCD摄影机450上。CCD摄影机445检测由样本臂422和基准臂423的干涉产生的条纹。针对基准臂镜425的不同位置可以典型地检测多个图像。可以算术组合示范性图像，以便从样本之内的光学分段中提取信息。
在如图4B所示的FFOCM系统的另一个示范性实施例中，宽带宽光源451可以耦合到调制元件如Michelson干涉仪或其它干涉仪(例如Mach-Zehnder、Sagnac)或谱改变单元中。对于Michelson干涉仪的情况，来自源451的光可以被传输到分束器452。光然后可以被分离到用于示范性臂A 453和臂B 455的两个臂中。来自臂A 453的光被传输到镜并被反射返回到分束器。来自臂B 455的光同样地被传输到镜456，并被反射返回到分束器452。臂A中的路径长度La和臂B中的路径长度Lb之间的差|La-Lb|可以被设置为基本上等于第二干涉仪中的基准和样本臂之间的路径长度差。臂A或B中的至少一个可以配置成改变路径长度以在其中的光中产生相移。在一个示范性实施例中，通过移动镜中的一个或快速扫描光延迟线可以改变路径长度。通过压电换能器、检流计、线性马达等等可以驱使移动。可替选地，通过声光调制器或电光调制器中的一种可以生成路径长度变化。
基准和样本臂光两者可以在分束器处组合，并被传输到具有分束器459的另一个静态干涉仪，该分束器459将光分别分开到基准臂458和样本臂457中。来自臂457、458两者的光可以分别照射基本上类似的物镜460、470。在基准臂458中，基准物镜460可以被带到聚焦在反射器465上，而在样本臂中，样本物镜470则将样本臂光聚焦在样本480上或之内。样本480或样本物镜470可以安装到台481，所述台481能够在手动控制或计算机控制之下任意三维地移动样本480。
基准臂458和样本臂457的路径长度之间的路径长度差可以基本上等于第一干涉仪的|La-Lb|。分别来自基准和样本臂458、457的光可以在分束器459处组合，并且经由透镜485成像到CCD阵列490或检测器的阵列上。通过由CCD 490在镜456移动的同时或者在镜456的不同位置处获取的图像的线性组合可以生成FFOCM图像或数据。通过CPU 495提供FFOCM图像的处理、显示和存储。使用累积或平均来增加信噪比。
图5描绘了配置成荧光检测并且使用宽带宽源的SECM系统的示范性实施例。例如，来自源500的光可以被传输到分束器510，该分束器510将光分离到两个路径515和520中。两个臂/路径都终止在镜420和525上，其中至少一个臂具有随时改变的路径长度或相位。从两个臂530返回的光可以耦合到分束器510，并且被引向535包含光栅或色散元件540和物镜550的SECM探针。如在此讨论的那样，光栅和物镜550的布置将谱编码线560聚焦在可以安装到三维台的样品562上或之内。样本之内的荧光可以被照射的光激发，传输返回通过物镜550，被另一个透镜565成像到检测器570上。检测到的光可以由处理装置(例如CPU)580数字化并转换成图像中的线。可以在射束扫描机构537的不同位置处生成图像中的另外的线。通过具有照射相同移动镜520的窄带宽源的示范性干涉仪521，可以校正移动镜中的非线性。
图6描绘了使用波长调谐源600的SECM系统的示范性实施例的示意图。例如，源600可以耦合到环行器/分束器610中。根据一个示范性实施例，来自分束器610的光经由光纤被传输到扫描仪，该扫描仪可替选地还包含将扫描轴投影到物镜625的后焦平面的望远镜透镜成像系统。来自扫描机构的光被传输到色散元件620(诸如衍射光栅、棱镜、GRISM或DP-GRISM等等)。来自620的光被传输到可以将射束聚焦在样本635之内的优选地具有高NA的物镜625。在任何时间点，来自波长扫描源600的一个波长可以照射样本的不同部分。随着扫描源600的波长随着时间变化，可以沿着样本635之内的线630扫描射束。从样本635传送的光可以被传输返回分别通过元件625、620和615，由光纤或针孔进行空间滤波，并被传输返回到环行器/分束器610。来自分束器610的光可以被引向检测器640，并且由处理装置(例如CPU)650进行数字化、显示和数字存储。在波长调谐源的一次全扫描之后获得图像中的单根线。可以在扫描机构的不同位置处获取线以形成图像。从样本传送的由波长调谐源600激发的荧光可替选地可以由检测器660检测以形成荧光图像。
图7描绘了示范性OFDI系统的示意图。在这个示范性OFDI系统的一个示范性实施例中，波长调谐源可以耦合到基于光纤的环行器705和分束器705。来自环行器705的光可以被传输到分束器705，该分束器705在优选实施例中配置成将大多数的光发送到样本臂725。向基准臂715转送的这种分离的光可以由反射器720终止，并被发送返回到分束器710和环行器705。样本臂725中的光被传输到扫描机构730和成像透镜735，该成像透镜735具有足够低的NA以允许生物样品740的横截面成像。光从基准镜720和样本740反射，在环行器705处重组，并且由光纤750引向检测器设备755，该检测器设备755在示范性实施例中可以包含双平衡检测器。
光由检测器设备755数字化，并且数字信号被传输到CPU 760。以与使用示范性SD-OCT系统/过程的处理类似的方式处理谱干涉，例如扣除背景，将λ空间转换到k空间，以及进行逆傅立叶变换以产生A线。可以作为扫描机构位置的函数获取A线，从而产生横截面OFDI图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(“STFT”)、复谱域处理、多普勒敏感OFDI和极化敏感OFDI从样本中获得谱信息的能力在内，也可以用于从生物样品中提取额外信息，诸如吸收、流动和双折射。
图8描绘了使用波长调谐/调制源的OCM系统的示范性实施例的示意图。例如，波长调制装置805可以在源上产生谱图案例如谱的正弦调制，其可以随时变化以对应于样本和基准臂之间的干涉产生的谱干涉调制。来自源800和/或调制装置805的光可以耦合到光纤环行器/分束器810中，并随后被传输到优选地将大多数的光引向样本臂830的分束器815。
基准臂820中的光被引向基准反射器825或传输元件。样本臂830中的光可以被传输到x-y扫描仪，该x-y扫描仪可以包括检流计、共振扫描仪、多边形扫描仪、声光扫描仪、电光扫描仪等等中的一种或多种。来自扫描仪的光优选地被传输到望远镜837和优选地具有高NA的物镜840。物镜840将光聚焦在优选地附着到三维台847的样本845之内。光从样本返回通过元件840、837和835，并且优选地耦合返回到样本臂831中的光纤或针孔的核心中以滤去散焦光。光被引向环行器810并被传输到检测器855，被数字化并被传输到CPU 860。
在一个示范性实施例中，通过以与使用示范性OFDI系统和过程进行的方式类似的方式从样本中获得A线可以获得OCM数据。例如，在示范性谱调制系统和过程中，检测器可以同步于源调制元件805。在这种情况下可以使用锁定或扣除技术来提取OCM信号。通过针对x-y扫描机构835的每个位置获取数据可以生成示范性图像。从样本传送的荧光可以进一步借助于分色镜或滤光器853和第二检测器865来检测。
图9描绘了使用波长调谐/调制源900的FFOCM系统的示范性实施例。光源可以在其带宽之上被调谐，或者可替选地被调制以包含与干涉仪的谱干涉调制提供的频率基本上类似的谱调制频率。来自源900的光可以耦合到分束器905，并且分别被引向由各自物镜920、930终止的样本臂910和基准臂915。基准臂物镜920将基准臂光聚焦到反射器上，光随后返回到分束器905。样本臂光由930聚焦到样品935上或之内。从样本传送的光在905处与基准臂光组合，并且由透镜940成像到CCD阵列950上。图像可以针对波长扫描源的每个波长或源的不同调制模式而被获得，并且由CPU 960进行算术组合以重构示范性FFOCM光学片段。
根据本发明的一个示范性实施例，上述示范性系统及其可替选的示范性实施例可以组合以形成多模态成像系统。通过以下可以提供系统和/或装置的这种示范性组合：生产分开的系统，并且配置它们的光学器件，以便它们可以从生物样品的相同部分中获得图像。可以在这样的组合模态系统中提供不同的波长、扫描和检测机构。可替选地，可以使用它们共享以提供更加有效、成本效率的设备的许多共同部件来实施不同的装置。
图10描绘了根据本发明的示范性实施例的多模态系统的示意图，其使用宽带宽源1000和分光计1080来提供同时且共同配准的SD-OCT、OCM、SECM和荧光SECM数据和/或图像。例如，来自宽带宽源1000的光可以可替选地耦合到谱调制单元1005中。来自谱调制单元1005的光耦合到环行器1010和分束器1015中。如果使用了环行器，则来自环行器1010的光被传输到优选地将大多数的光引向样本的分束器1015。基准臂1020中的光被传输到基准反射器1025，该基准反射器1025可以移动或改变1020的路径长度，和/或可以是不可移动的。如果允许基准臂移动，则使用现有技术中已知的处理，使用示范性SD-OCT装置和/或过程，可以实施传统时域OCT(例如TD-OCT)装置和/或过程，或者可以获得复谱域。
样本臂1030中的光被传输到滤光/分色/WDM设备1035，该设备在从分束器到样本的方向上传输样本臂光。来自1035的光被引向能够以高速或慢速在两个方向上扫描射束的射束扫描机构1040。射束扫描机构1040还可以包含望远镜，用于将扫描仪成像到透镜1055的后焦平面上。来自扫描机构1040的光可以被传输到包含多个光学元件的滑动器1045。例如，当滑动器1045位于不同位置时，可以实施SD-OCT、OCM、SECM和/或荧光OCM装置/过程中的一个或多个或其组合。来自滑动器1045的光可以被传输到物镜1055，该物镜1055在一个实施例中安装到能够改变物镜的透镜旋转台。滑动器1045和/或旋转台1050可以处在计算机控制之下，用于成像模态的自动选择。光由物镜1055聚焦到样本1060上或之内，所述样本1060可以安装到计算机控制的三维平移台1065。反射的光被传输返回通过设备到达1010，其然后将光重新引向分光计。使用在此描述的装置和/或过程处理检测到的反射光以形成示范性SD-OCT、OCM、SECM图像。
如图10所示，荧光可以经由滤光/分色镜/WDM设备1035被重新引向第二检测器到达第二检测器1075。来自1075的荧光用于重构生物样本1060的荧光共焦图像。在使用不可见近红外光的情况下，可见瞄准射束可以耦合到示范性系统中，与近红外光共同入射，以允许成像位置的可视化。可替选地或者另外，借助于显微镜上的可替选的成像端口，可以提供研究中的样品的白光图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(STFT)、多普勒敏感SD-OCT和极化敏感SD-OCT从样本中获得谱信息的能力在内，也可以用于从生物样品中获得额外信息，诸如吸收、流动和双折射。
在图11中描绘了配置成从其它三个模态中以不同的波长根据本发明提供SD-OCT、OCM、SECM和FFOCM图像和数据的可替选的示范性多模态实施例。在这个示范性实施例中，宽带宽源1100可替选地耦合到谱调制单元1105。来自谱调制单元1105的光耦合到环行器1110和分束器1115中。如果使用了环行器1110，则来自环行器1110的光可以被传输到优选地将大多数的光引向样本的分束器1115。基准臂1120中的光被传输到基准反射器1125，该基准反射器1125可以是固定的，和/或可以改变基准臂1120的路径长度。在允许基准臂1120移动的情况下，通过现有技术中已知的方法，对于SD-OCT可以使用示范性传统时域OCT(TD-OCT)过程或复谱域。样本臂1130中的光被传输到能够以高速或慢速在两个方向上扫描射束的射束扫描机构1135。射束扫描机构1135还可以包括望远镜，用于将扫描仪成像到透镜1160的后焦平面上。来自扫描机构1135的光被传输到二色分离器/WDM 1140，其传输用于SD-OCT、OCM和SECM模态的激发光并且可以反射FFOCM光。
例如，类似于如图3所示的示范性FFOCM系统可以经由1140耦合到射束路径中。来自1140的光被引向包含多个光学元件的滑动器1150；当滑动器可以位于不同位置时，提供SD-OCT、OCM、SECM或FFOCM中的一种或其组合。来自滑动器1150的光被传输到物镜1160，该物镜1160在一个实施例中安装到能够改变物镜的透镜旋转台1155。滑动器1150和/或旋转台1155可以处在计算机控制之下，用于成像模态的自动选择。光由物镜1160聚焦到样本1165上或之内，所述样本1165可以安装到计算机控制的三维平移台1170。反射的光被传输返回通过设备到达环行器1110，其然后将光重新引向分光计。通过在此描述的方法可以处理检测到的反射光以形成示范性SD-OCT、OCM、SECM图像。可以经由滤光/分色镜/WDM设备1140将FFOCM光重新引向FFOCM系统1175。
在使用不可见近红外光的情况下，可见瞄准射束可以耦合到图11中示出的示范性系统中，与近红外光共同入射，以允许成像位置的可视化。可替选地或者另外，借助于显微镜上的可替选的成像端口，可以提供研究中的样品的白光图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(STFT)、多普勒敏感SD-OCT和极化敏感SD-OCT从样本中获得谱信息的能力在内，也可以用于从生物样品中提取额外信息，诸如吸收、流动和双折射。
图12描绘了根据本发明的可以用于多模态成像的滑动器的布置的示范性实施例。例如，光学元件可以包含在壳体1200中，该壳体1200可以手动平移，或者可以在计算机或自动控制之下平移。每个滑动器位置可以终止于提供一个或多个成像模态的不同滑动器位置1205、1210、1230、1260。滑动器位置1205、1210、1230、1260可以耦合到物镜旋转台。在一个示范性实施例中，滑动器位置1205不包含光学元件(空气)或光学元件窗口。在这个示范性配置中，显微镜被配置成执行FFOCM。对于滑动器位置1210，透镜设备1212和1213可以配置成扩展射束并照射包含包围透射光栅1220的两个棱镜1215和1225的DP-GRISM。这个示范性配置提供了进行SECM成像的能力。使用扫描跨越样本的谱编码线的扫描机构也可以在这个位置实施示范性OCM过程。对于滑动器位置1230，透镜设备1240、1250可以配置成使用或不使用射束放大来对射束角度进行成像。这个滑动器位置1230可以使用示范性SDOCT过程提供成像。对于滑动器位置1260，透镜设备1270、1280被配置成扩展扫描射束以允许使用示范性OCM过程进行成像。
虽然多模态成像系统的某些实施例已使用了宽带宽源，但是组合系统的示范性实施例还可以包括波长调谐/调制源和单个或多个检测器配置，并且这样的示范性实施例在图13中示出。例如，在图13中，波长调谐/调制源1300耦合到环行器1305和分束器1310中。如果使用了环行器，则来自环行器1305的光被传输到优选地将大多数的光引向样本的分束器1310。基准臂1315中的光被传输到基准反射器1320，该基准反射器1320可以是固定的，或相反可以改变1315的路径长度。在允许基准臂移动的情况下，通过现有技术中已知的方法，传统示范性时域OCT(TD-OCT)过程可以被提供或者复谱域可以被使用用于实现OFDI模态。样本臂1325中的光被传输到滤光/分色/WDM设备1330，该设备在从分束器到样本的方向上传输样本臂光。来自1330的光被引向能够以高速或慢速在两个方向上扫描射束的x-y射束扫描机构1335。
射束扫描机构1335还可以包括望远镜，用于将扫描仪成像到透镜1353的后焦平面上。来自扫描机构1335的光被传输到包含多个光学元件的滑动器1340；当滑动器位于不同位置时，可以提供OFDI、OCM、SECM或荧光OCM模态中的一种或其组合。来自滑动器1340的光被传输到物镜1353，该物镜1353在一个实施例中安装到能够改变物镜的透镜旋转台1350。滑动器1340和/或旋转台1350可以是手动的，处在计算机控制之下，用于成像模态的自动选择。光由物镜1353聚焦到样本1355上或之内，所述样本1355可以安装到计算机控制的三维平移台1360。反射的光被传输返回通过设备到达1305，其将光重新引向检测器设备1380，该检测器设备1380适合于检测OFDI、波长调谐OCM或SECM信号、图像和/或数据。通过上述方法由CPU 1385处理检测到的反射光以形成示范性OFDI、OCM、SECM图像。
荧光可以经由滤光/分色镜/WDM设备1330被重新引向第二检测器到达第二检测器1370。来自1370的荧光用于重构生物样本1355的荧光共焦图像。在使用不可见近红外光的情况下，可见瞄准射束可以耦合到系统中，与近红外光共同入射，以允许成像位置的可视化。可替选地或者另外，借助于显微镜上的可替选的成像端口，可以提供研究中的样品的白光图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(STFT)、多普勒敏感SD-OCT和极化敏感SD-OCT从样本中获得谱信息的能力在内，也可以用于从生物样品中提取额外信息，诸如吸收、流动和双折射。
在图14中描绘了根据本发明的系统的另一个示范性多模态实施例，该系统配置成提供OFDI、OCM、SECM和FFOCM图像、数据和其它信息，其中从其它三种模态中以不同波长提供FFOCM信号。在这个示范性实施例中，波长调谐源1400可替选地耦合到谱调制单元1405。来自谱调制单元1405的光耦合到环行器1410和分束器1415中。如果使用了环行器1410，则来自环行器1410的光被传输到优选地将大多数的光引向样本的分束器1415。基准臂1420中的光被传输到基准反射器1425，该基准反射器1425可以是固定的，或者可以相反地改变1420的路径长度。在允许基准臂1420移动的情况下，通过现有技术中已知的方法，对于OFDI数据可以提供传统时域OCT(TD-OCT)过程和模态或者可以获得复谱域。样本臂1430中的光被传输到能够以高速或慢速在两个方向上扫描射束的射束扫描机构1435。射束扫描机构1435还可以包含望远镜，用于将扫描仪成像到透镜1465的后焦平面上。来自扫描机构1435的光被传输到二色分离器/WDM 1445，其传输用于OFDI、OCM和SECM的激发光但是反射FFOCM光。
类似于图3和/或图4的(一个或多个)系统的示范性FFOCM系统可以经由二色分离器/WDM 1445耦合到射束路径中。来自二色分离器/WDM 1445的光被引向包含多个光学元件的滑动器1455；当滑动器1455位于不同位置时，提供OFDI、OCM、SECM或FFOCM数据和/或图像中的一种或其组合。来自滑动器1455的光被传输到物镜1465，该物镜1465在一个示范性实施例中安装到能够改变物镜的透镜旋转台1460。滑动器1455和/或旋转台1460可以处在计算机控制之下，用于成像模态的自动选择。光可以由物镜1465聚焦到样本1470上或之内，所述样本1470可以安装到计算机控制的三维平移台1475。
反射的光被传输返回通过设备到1410，其将光重新引向分光计。通过在此描述的方法处理检测到的反射光以形成OFDI、OCM、SECM图像。可以经由滤光/分色镜/WDM设备1445将FFOCM光重新引向FFOCM系统1450。在使用不可见近红外光的情况下，可见瞄准射束可以耦合到示范性系统中，与近红外光共同入射，以允许成像位置的可视化。可替选地或者另外，借助于显微镜上的可替选的成像端口，可以提供研究中的样品的白光图像。现有技术中已知的可替选的示范性实施例，包括通过谱干涉的短时傅立叶变换(STFT)、多普勒敏感OFDI和极化敏感OFDI从样本中获得谱信息的能力在内，也可以用于从生物样品中提取额外信息，诸如吸收、流动和双折射。
在本发明的另一个示范性实施例中，显微镜可以配置成允许从样本的两侧进行成像。例如，可以从样本之上执行SDOCT、SECM和OCM过程，而可以用从下面照射样本的成像透镜执行FFOCM过程。在这样的示范性配置中，样本可以安装在显微镜载玻片和薄盖玻片之间，以允许从两侧进行成像。
在此描述的示范性系统可以用各种不同的格式、速度、分辨率、视场和对比机制来提供生物样品的多模态成像。每个图像数据集可以是二维或三维的，并且可以共同配准到其它各个成像模态的数据集。现有技术中已知的计算机处理方法可以用于以各种不同的成像格式显示不同的数据集，所述成像格式包括三维体积可视化、四维表示以及处理的二维、三维和四维数据集，其中处理设备被配置成使所关心的重要区域突出。任何一个或多个数据集可以相对于其它数据集而被显示，并且可以从个别数据集的组合中导出全面的全部包括的数据集。可以在数据集的二维、三维和四维环境中从数据集导出定量信息。图像数据也可以与生物样品的传统荧光或明视场图像组合。
例子
以下提供的是实施以研究使用根据本发明的示范性多个成像模态来对发育中的光滑爪蟾心脏进行成像的例子。
示范性方法
Bench-Top示范性OCT和OFDI系统
在示范性TDOCT配置中，借助于低相干反射测量进行轴向测距，其中在时间上连续地探测各个深度点。使用了以1.3μm为中心的宽带宽(50nm)源，提供组织中大约10μm的轴向分辨率(n＝1.4)。帧率为每秒20(2kHz A线速率、100×500像素)。
示范性OFDI过程和系统可以使用其中所有深度点被同时获取的频域测量。这种技术提供了信噪比(SNR)方面的数百倍改进，如M.A.Choma等人的“Sensitivity advantage of swept source and Fourier domainoptical coherence tomography”，Optics Express 11，pp.2183-2189(2003)以及S.H.Yun等人的“High-speed optical frequency-domain imaging”，Optics Express 11，pp.2953-2963(2003)中描述的那样。示范性OFDI系统和过程可以使用快速扫描的波长可调谐的激光器作为光源。扩展空腔半导体激光器使用腔内谱过滤器，如M.A.Choma等人的“Sensitivityadvantage of swept source and Fourier domain optical coherencetomography”，Optics Express 11，pp.2183-2189(2003)以及C.Boudoux等人的“Rapid wavelength-swept spectrally encoded confocalmicroscopy”，Optics Express 13，pp.8214-8221(2005)中描述的那样。
激光器特征为高达64kHz的扫描重复率、以1320nm为中心的111nm的宽调谐范围和30mW的高平均输出功率(组织上为7mW)。轴向分辨率为组织中10μm。系统进一步包括声光移频器(25MHz)，用于去除频域反射测量中固有的深度简并，如S.H.Yun等人的“Removing thedepth-degeneracy in optical frequency domain imaging with frequencyshifting”，Optics Express 12，pp.4822-4828(2004)中描述的那样。实施极化分集检测以消除基于光纤的OFDI系统中的极化伪像。双平衡光接收器用于通过减少激光强度噪声来改善成像灵敏度。用2通道模拟到数字转换器以14位的分辨率以100MHz的采样率对光接收器输出进行数字化。
示范性TDOCT和高速OFDI配置被结合到解剖光显微镜中。扫描系统包括准直透镜(5mm射束直径)、用于横向扫描的两个同步检流计扫描仪、聚焦透镜(50mm焦距)以及朝着样本向下偏转射束的小镜。对于示范性TDOCT和OFDI配置，横向分辨率为16μm，其中共焦参数为330μm。
通过在逐帧的基础上从收缩末期的心脏表面位置减去舒张末期的心脏表面位置，直接从体积数据中确定与局部心脏活动相关联的位移。使用颜色检查表来显示位移。通过使用OsiriX软件来完成体积再现和三维可视化。
使用具有200nm的调谐范围、以1250nm为中心的激光源来执行高分辨率OFDI过程，其中两个半导体光学放大器用作增益媒介，如W.Y.Oh等人的“Wide tuning range wavelength-swept laser with twosemiconductor optical amplifiers”，IEEE Photonics Technology Letters17，pp.678-680(2005)中描述的那样。实现了组织中4μm的轴向分辨率。使用NA＝0.2的物镜，横向分辨率为2μm。使用20kHz的A线速率(每帧500A线)，成像速率为每秒40帧。执行极化分集和双平衡检测，并且用2通道模拟到数字转换器以12位的分辨率以10MHz的采样率对光接收器输出进行数字化。
示范性FFOCM系统
例如，FFOCM是一种使用二维并行检测来提供生物样品之内的反射光的亚细胞分辨率图像的干涉测量技术，如A.Dubois等人的“Ultrahigh-resolution full-field optical coherence tomography”，ApplOpt 43，pp.2874-2883(2004)和A.Dubois等人的“Three-dimensionalcellular-level imaging using full-field optical coherence tomography”，Phys Med Biol 49，pp.1227-1234(2004)中描述的那样。示范性FFOCM系统使用来自氙弧灯的空间非相干宽带光来照射样本和使用两个等同的NA＝0.3的水浸显微镜物镜的Linnik干涉显微镜的基准镜。用具有以650nm为中心的谱响应的CMOS区域扫描摄影机拍摄干涉图像。横向分辨率为2μm，并且轴向分辨率为1.1μm。对于近似700μm×700μm的横向视场，获取时间为每帧2秒。通过以1μm增量将样本移动通过焦点来获得三维数据。通过使用OsiriX软件完成体积再现和可视化。
示范性SECM系统
例如，SECM是一种反射共焦显微技术，其使用了近红外光，与使用可见光的共焦显微镜相比，允许更深地穿透到组织中，如R.R.Anderson等人的“The optics of human skin”，J Invest Dermatol 77，pp.13-19(1981)中描述的那样。示范性SECM技术与传统激光扫描共焦显微的不同之处在于它将不同的波长投影到样本上的不同位置上，如G.J.Tearney等人的“Spectrally encoded confocal microscopy”，Optics Letters23，pp.1152-1154(1998)中描述的那样。从样本返回的谱的快速获取使得能够高速重构图像。在SECM系统中，如C.Boudoux等人的“Rapidwavelength-swept spectrally encoded confocal microscopy”，OpticsExpress 13，pp.8214-8221(2005)中描述的那样，近红外中的来自快速波长调谐源的光(中心波长＝1.32μm，瞬时线宽＝0.1nm，总带宽＝70nm，重复率高达15.7kHz)，被校准到衍射光栅上(每毫米1100线)，并且使用1.2NA的60x的物镜(Olympus UPlanApo/IR 60X/1.20W)来聚焦。多模光纤用于信号收集，导致0.9μm的横向分辨率和2.5μm的轴向分辨率。以每秒10帧获取包括500×500像素的图像。最大成像深度被限制到物镜的280μm工作距离。
样品制备、乙醇处理和组织结构
从Nasco(Fort Atkinson，Wisconsin)购买光滑爪蟾蛙。根据Massachusetts General Hospital Subcommittee对Research Animal Care的批准协议执行动物手续。胚胎是通过体外受精获得的，在0.1x的Marc’smodified Ringer’s(MMR)培养基中孵化(如J.Newport等人的“A majordevelopment transition in early Xenopus embryos：Characterization andtiming of cellular changes at the midblastula stage”，Cell 30，pp.675-686，1982中描述的那样)，并且根据Nieuwkoop and Faber表划分阶段。(参见P.D.Nieuwkoop和J.Faber的Normal table of Xenopus laevis，Daudin，North-Holland Publishing Company，Amsterdam，1967)。
在0.1X MMR(体积/体积)中进行乙醇处理，不久之后是Mid BlastulaTransition(中囊胚过渡)(如R.Yelin等人的“Ethanol exposure affectsgene expression in the embryonic organizer and reduces retinoic acidlevels”，Dev Biol 279，pp.193-204(2005)中描述的那样)，直到成像为止。在体内成像之前，使用0.02％的3氧基苯甲酸乙酯(A-5040，Sigma)使胚胎麻醉。对于TDOCT和OFDI成像技术和系统，将胚胎安置在1.5％的琼脂糖凝胶板上，其中它们的腹侧面朝上，用麻醉工作液覆盖。对于用示范性SECM系统进行成像，将胚胎放置在盖玻片上，以它们的腹侧躺在麻醉缓冲器中，并且从下面进行成像。在MEMFA(0.1M MOPS[pH7.4]，2mM EGTA，1mM MgSO4和3.7％的甲醛)中定影大于一个小时之后，通过示范性FFOCM过程和/或系统进行的体外成像开始。在成像之前，将被定影的胚胎转移到具有1x PBS(8gr NaCl，0.2gr KCl，1.44grNa2HPO4，0.24gr KH2PO4)的培养皿中，其中它的腹侧面朝上，由粘土支撑。
在Karnovsky’s Fixative(KII)中另外定影和在tEpon-812(Tousimis)中嵌入之后，获得Plastic Histology(塑料组织结构)片段。1μm厚的片段在Reichert Ultracut Microtome上切割，并且用硼酸盐缓冲器(Tousimis)中的亚甲基蓝/甲苯胺蓝进行染色。石蜡片段(5μm厚)用Hematoxylin & Eosin(苏木精和伊红)染色。
示范性结果
在体内用OFDI技术对胚胎心脏进行四维成像
对跳动心脏的快速体积成像使得能够评估心跳周期期间的三维形态和功能。与提供体内横截面成像(如图15a和15b所示)的TDOCT相比，示范性OFDI系统和过程能够以高得多的帧率进行成像，使得可以对跳动心脏进行四维成像而没有心脏门控。以每秒20个三维数据集的速率获取爪蟾心脏(阶段49)的体积OFDI图像(如图15c-15g所示)。在收缩末期，OFDI过程的使用表明心室处在其最小体积；心房和动脉干(TA)的体积相反地处在它们的最大值(如图15c和15d所示)。在舒张末期，心室膨胀到其最大体积，而心房和TA的体积则处在它们的最小值(如图15e和15f所示)。取自四维数据集的心脏的三维再现(如图15g所示)对应于在其解剖之后的相同心脏的明视场照片(如图15h所示)。
对体内胚胎心脏的高分辨率OFDI过程
虽然示范性OFDI系统能够进行四维成像，但是存在需要较高分辨率来识别细微形态和功能异常的情况。为了增加分辨率，使用宽带(例如200nm)波长扫描源在体内获得阶段49的爪蟾心脏的OFDI横截面(如图15i-15m所示)，如W.Y.Oh等人的“Wide tuning rangewavelength-swept laser with two semiconductor optical amplifiers”，IEEEPhotonics Technology Letters 17，pp.678-680(2005)中描述的那样。与前面描述的TDOCT和OFDI过程和系统的16μm的横向分辨率和10μm的轴向分辨率相比，高分辨率OFDI结果的横向和轴向分辨率分别为2μm和4μm。用高分辨率OFDI过程和系统可以清楚地分辨三室爪蟾心脏之内的细节，包括房室瓣动态(如图15i-15k所示)、心室收缩和小梁动态(图15m)。还可以看到单独的血细胞，从心房通过房室瓣流向心室(如图15k所示)。
在体外使用FFOCM过程对胚胎心脏进行高分辨率三维成像
示范性FFOCM过程和系统提供了用几乎各向同性的细胞水平的分辨率对胚胎心脏的微观结构进行成像的能力。体积FFOCM图像横跨700×700×1000μm(轴向)的视场。横向和轴向分辨率分别为2μm和1.1μm。获取时间对于单个面片段为2秒，对于整个体积为33分钟。与使用示范性TDOCT或OFDI过程或系统生成的相比，爪蟾心脏(阶段49)的示范性FFOCM片段允许更加详细地可视化心室小梁(如图16a和16c所示)、螺旋瓣(如图16b和16d所示，参见箭头)以及部分心房间隔(如图16d所示，参见箭头)。心脏的部分透明的体积再现(如图16e-16h所示)，揭示了成角的TA(如图16e所示)、主动脉弓(如图16f和16g所示)以及心房的薄壁(如图16g和16h所示)在它们的三维环境中的循环压缩结构。剖视图(如图16e所示)示出了精细的三维内部结构，包括小梁(如图16i和16j所示)和房室瓣(如图16k所示)。紧接于相同胚胎的相应组织结构片段(如图16m所示)示出的房室瓣的放大图(如图16l所示)展示了它的双尖形态。
在体内用SECM过程对胚胎心脏进行高速成像
示范性SECM过程和系统提供了可与跟FFOCM相关联的那些相比较的横向分辨率，但是是以较高的帧率，使得能够在体内对心脏进行显微。以10/s的帧率、220×220μm的视场以及分别为1.2和6μm的横向和轴向分辨率，使用示范性SECM过程和系统在体内对爪蟾心肌(阶段49)进行成像。最大穿透深度为280μm。通过TD-OCT(如图15a和15b所示)和FFOCM(如图16a-16m所示)过程和系统可视化的示范性的相同蝌蚪(阶段49)，示出了在腹部表面之下近似280μm的房室瓣的薄尖(如图17a所示)以及包含小梁内空间之内的单独血细胞的心室和TA的部分(如图17c所示)。SECM图像与相应的组织结构片段很好地相关(如图17b和17d所示)。来自不同蝌蚪(阶段47)的一系列帧展示了从TA到主动脉分叉的以单细胞水平观看的调节血流的闭合时(如图17e所示)和开启时(如图17f和17g所示)的螺旋瓣。血细胞在小梁之内也是明显的(如图17h所示)。能够观察到可以表示核和细胞器的单独肌细胞之内的细胞内特征。
爪蟾胚胎中的动脉瘤扩张
在胚胎(阶段47)中的一个中，已识别了从TA壁发出的突起。以两个不同的深度在体内获得的SECM片段(如图18a和18b所示)揭示了它的囊形形状，它相对于螺旋瓣的位置，以及单独的血细胞通过该缺陷的流动。在体内使用示范性TDOCT过程和系统也观察到了这种异常性(如图18a所示，参见插图)。胚胎然后被定影并用示范性FFOCM过程和系统进行成像。FFOCM片段(如图18c所示)和FFOCM体积数据集的三维再现(如图18d所示)示出了整个心脏的环境方面的扩张。在传统的明视场显微之下难以看到(如图18e所示)、但是使用示范性TDOCT、FFOCM和SECM过程和系统清楚地可视的这个突起可以表示心脏中的TA的囊形动脉瘤扩张，其否则显现为具有正常的表型。
由乙醇暴露引起的心脏异常性
心血管畸形可以由遗传(如K.L.Clark等人的“Transcription factorsand congenital heart defects”，Annu Rev Physiol 68，pp.97-121(2006)中描述的那样)和产生畸形的因素(如S.M.Mone等人的“Effects ofenvironmental exposures on the cardiovascular system：prenatal periodthrough adolescence”，Pediatrics 113，pp.1058-1069(2004)中描述的那样)造成。乙醇是众所周知的致畸物；怀孕期间的人类胚胎暴露于酒精(乙醇)与胎儿酒精综合症(FAS)相关联。(参见K.L.Jones等人的“Recognitionof the fetal alcohol syndrome in early infancy”，Lancet 2，pp.999-1001(1973)和J.D.Chaudhuri的“Alcohol and the developing fetus—a review”，Med Sci Monit 6，pp.1031-1041(2000))。一个估计表明具有FAS的小孩中的54％具有心脏缺陷。(参见E.L.Abel的Fetal Alcohol Syndrome，Medical Economics Books，Oradell，NJ，1990)。
为了研究乙醇对爪蟾心脏发育的致畸效应，将胚胎从中囊胚过渡(阶段8.5)暴露于不同浓度的乙醇(0.5％-2.5％)。(参见R.Yelin等人的“Ethanol exposure affects gene expression in the embryonic organizerand reduces retinoic acid levels”，Dev Biol 279，pp.193-204(2005))。在相同的条件下发育但不暴露于乙醇的同属用作对照。在发育过程期间，我们使用示范性TDOCT过程和系统拍摄胚胎的心脏区域以识别和定量评估致畸效应的程度。我们没有观察到0.5％的乙醇处理组(n＝16)和对照组(n＝42)之间的形态差异。被定义为与对照相比在形态方面具有显著变化的完全成熟的中等致畸效应，在暴露于1％乙醇的少数(25％)胚胎中(n＝28)和暴露于1.5％乙醇的大多数(74％)胚胎中(n＝27)被发现。被定义为心管和/或不完全成熟的严重异常循环的严重效应，在2.0％和2.5％组中的所有胚胎中(分别为n＝17、n＝7)都被发现。心脏活动在所有的胚胎中都是明显的，即使在具有最严重畸形的胚胎中也是如此。
使用示范性TDOCT过程和系统，已从对照、0.5％、1.5％、和2.0％的乙醇处理组的每一个中选择了蝌蚪(阶段48)以展示典型的表型(如图19a-19d所示)。通过识别部分心房间隔(如图19a-19d所示，参见右边图像，用箭头标记隔膜)和房室瓣的存在，可以确定四个蝌蚪的心脏处在晚期发育阶段。TDOCT图像提供了1.5％和2.0％组中被破坏的循环的第一个迹象。进一步观察到的是来自1.5％和2.0％组中心室之内的较低TDOCT信号，这可归因于这些胚胎中减少的血流。在图19e-19h中示出了在体内取自腹面的蝌蚪照片。
在体外用示范性FFOCM系统和过程获取的数据的三维再现允许以高分辨率评估心肌结构，揭示对照和0.5％的蝌蚪之间的相似性，并且清楚地示出来自1.5％和2.0％组的蝌蚪中的有缺陷心管循环(如图19i-19l所示)。通过FFOCM体积数据集的片段展示了与对照(如图19m所示)和0.5％的(如图19n所示)胚胎相比较的1.5％的(如图19o所示)和2.0％的(如图19p所示)胚胎中的较小的畸变的TA和螺旋瓣(用箭头标记)。与对照和0.5％的组相比较，心包浮肿在1.5％和2.0％的组中出现(如图19o、19p、19s和19t所示)。乙醇还影响了心室；对照的(如图19q所示)和0.5％的(如图19r所示)心脏中发育的小梁与以下形成反差：1.5％的组中欠发育的小梁(如图19s所示)以及暴露于2.0％的乙醇的胚胎中具有稀疏矮小小梁的大心室空腔(如图19t所示)。相应的组织结构片段证实了我们的一些发现，包括较大乙醇暴露的胚胎中欠发育的小梁(如图19u-19x所示)。
示范性结果的讨论
发育生物研究中的共同范例是操纵遗传型和监视表型。形态是表型的重要方面。在心脏中，即使轻微的形态和动态异常性也可能对于适当的心肌功能是决定性的。识别二维和三维中的细微形态和动态变化的能力可以显著地改善这个范例的灵敏度。
在爪蟾蝌蚪中，心脏结构如心肌壁、隔膜和瓣可能仅有几个细胞那么厚。评估形态表型不仅需要分辨这样的精细结构，而且还需要使跳动心脏之内的微观特征可视化的能力，其中典型的位移速度为1mm/s的数量级。如果成像速度足够高，则可以在心跳周期之内的不同时刻获得胚胎心脏的三维图像。这种示范性四维成像允许动态生理参数的可靠测量，诸如心搏量和喷血分数以及瓣对向、刚度和模块性，这在人类病理生理学方面具有密切模拟。高分辨率和高速度不是对心脏有效成像的仅有要求。在爪蟾胚胎中，心脏从腹部表面之下在200μm和800μm之间延伸。有效的成像方法因此也应当能够在这些深度进行成像而没有信号和分辨率的显著损失。
使用组织结构片段的三维再现已在体外研究并详细描述了发育光滑爪蟾心脏的形态。(参见T.J.Mohun等人的“The morphology of heartdevelopment in Xenopus laevis”，Dev Biol 218，74-88(2000))。然而对于组织的研究，样本制备和切片使得难以保存结构真实。结果，对完整心脏在它们的天然环境中进行成像是优选的。使用多种非侵入成像模态如微MRI(参见D.L.Kraitchman等人的“In vivo magnetic resonance imagingof mesenchymal stem cells in myocardial infarction”，Circulation 107，pp.2290-2293(2003)和F.Wiesmann等人的“Developmental changes ofcardiac function and mass assessed with MRI in neonatal，juvenile，andadult mice”，Am J Physiol Heart Circ Physiol 278，pp.H652-657(2000))、微CT(参见M.Malyar等人的“Relationship between arterial diameterand perfused tissue volume in myocardial microcirculation：a micro-CT-based analysis”，Am J Physiol Circ Physiol 286，pp.H2386-2392(2004)和C.T.Badea等人的“4-D micro-CT of the mouse heart”，Mol Imaging 4，pp.110-116(2005))、超声(参见S.Srinivasan等人的“Noninvasive，in uteroimaging of mouse embryonic heart development with 40-MHzechocardiography”，Circulation 98，pp.912-918(1998))和PET(参见L.W.Dobrucki等人的“Molecular cardiovascular imaging”，Curr CardiolRep 7，pp.130-135(2005)和L.Stegger等人的“Monitoring left ventriculardilation in mice with PET”，J Nucl Med 46，pp.1516-1521(2005))，已展示了对体内心脏的结构成像。
光学技术使得能够以较高分辨率对胚胎心脏进行成像。共焦显微已用于在体外对早期爪蟾心脏发育进行成像(如S.J.Kolker等人的“Confocalimaging of early heart development in Xenopus laevis”，Dev Biol 218，pp.64-73(2000)中描述的那样)，并且用于在体内研究心内流体力在斑马鱼胚胎心脏发生中的作用(如J.R.Hove等人的“Intracardiac fluid forces arean essential epigenetic factor for embryonic cardiogenesis”，Nature 421，pp.172-177(2003)中描述的那样)。多普勒TDOCT过程和系统用于研究爪蟾蝌蚪中的血流，允许组织表面之下的定量速度测量。(参见J.R.Hove等人的“Intracardiac fluid forces are an essential epigenetic factor forembryonic cardiogenesis”，Nature 421，pp.172-177(2003)和V.X.D.Yang、M.L.Gordon、E.Seng-Yue等人的“High speed，wide velocitydynamic range Doppler optical coherence tomography(Part II)：Imagingin vivo cardiac dynamics of Xenopus laevis”，Optics Express 11，pp.1650-1658(2003))。由于其有限的成像速度，使用TDOCT的三维心脏成像以前主要仅在体外展示。(参见S.A.Boppart等人的“Noninvasiveassessment of the developing Xenopus cardiovascular system using opticalcoherence tomography”，Proc Natl Acad Sci USA 94，pp.4256-4261(1997)、T.M.Yelbuz等人的“Optical coherence tomography：a newhigh-resolution imaging technology to study cardiac development in chickembryos”，Circulation 106，pp.2771-2774(2002)和W.Luo等人的“Three-dimensional optical coherence tomography of the embryonicmurine cardiovascular system”，Journal of biomedical optics 11，021014(2006))。
门控或后获取同步技术已用于避开传统成像方法的有限速度，使得能够重构心跳周期中的不同阶段的胚胎心脏的三维图像。(参见M.W.Jenkins等人的“4D embryonic cardiography using gated optical coherencetomography”，Optics Express 14，pp.736-748(2006)和M.Liebling等人的“Four-dimensional cardiac imaging in living embryos viapostacquisition synchronization of nongated slice sequences”.J BiomedOpt 10，054001(2005))。对于实验中的一些，我们使用了TDOCT，因为它在我们的实验室中更容易得到，然而示范性OFDI过程和系统能够以高得多的速度提供示范性TDOCT过程和系统的全部的功能性。示范性OFDI过程和系统提供了对跳动心脏的实时的真实四维成像而不需要心脏门控，并且被发现可用于评估心跳周期期间的心肌壁位移(如图15c-15f所示)。
通过修改OFDI光源，我们还能够用更高的轴向分辨率(4μm)实施实时横截面成像，使得能够使瓣动态(如图15i-15k所示)和单细胞血流可视化。对于胚胎心脏的亚细胞水平分辨率成像，我们调查研究了示范性FFOCM和SECM过程和系统的使用。发现FFOCM模态能够用各向同性的细胞分辨率(1-2μm)提供高质量三维成像。SECM模态展示了可与FFOCM模态相比较的分辨率，但是能够以更高的速度进行成像，使得能够以亚细胞水平使体内的肌细胞、血液和瓣活动可视化。表1概括了每个过程的不同能力，突出了它们的互补性质。
表1用于胚胎心脏的光学成像的内生对照模态的比较。灰色阴影的单元指示具有最好横向分辨率、轴向分辨率和帧率特性的成像技术。
Figure S2006800405482D00261
*包括TDOCT和OFDI模态。
示范性TDOCT和FFOCM过程和系统的大穿透深度允许通过作为乙醇致畸表型的部分而发育的心包浮肿对心脏进行成像。我们的初步结果表明乙醇干扰了心脏循环的过程(图19i-l)，与对鹌鹑的研究一致。(参见W.O.Twal等人的“Retinoic acid reverses ethanol-inducedcardiovascular abnormalities in quail embryos”，Alcohol Clin Exp Res 21，pp.1137-1143(1997))。这项工作中报告的TA尺寸的减少由Cavierrs和Smith预测(参见M.F.Cavieres等人的“Genetic and developmentalmodulation of cardiac deficits in prenatal alcohol exposure”，Alcohol ClinExp Res 24，pp.102-109(2000))，但未被观察到。可以认为这里描述的欠发育的心室小梁(如图19q-t所示)以前尚未发育。由于在爪蟾和斑马鱼(Danio rerio)中，心室小梁充当His-Purkinje系统的功能等价物(参见D.Sedmera等人的“Functional and morphological evidence for a ventricularconduction system in zebrafish and Xenopus hearts”，Am J Physiol HeartCirc Physiol 284，pp.H1152-1160(2003))，所以欠发育小梁的确定与已在乙醇处理的鹌鹑(参见W.O.Twal等人的“Retinoic acid reversesethanol-induced cardiovascular abnormalities in quail embryos”，AlcoholClin Exp Res 21，pp.1137-1143(1997))和斑马鱼胚胎(参见J.Bilotta等人的“Ethanol exposure alters zebrafish development：a novel model offetal alcohol syndrome”，Neurotoxicol Teratol 26，pp.737-743(2004))中报告的较慢心率相关联。由乙醇处理引起的主动血液循环的中断(W.O.Twal等人的“Retinoic acid reverses ethanol-induced cardiovascularabnormalities in quail embryos”，Alcohol Clin Exp Res 21，pp.1137-1143(1997)和X.Wang等人的“Japanese medaka(Oryzias latipes)：developmental model for the study of alcohol teratology”，Birth DefectsRes B Dev Reprod Toxicol 77，pp.29-39(2006))可以解释信号从心脏空腔之内的丢失，这也与确定一致。
尽管它们有相对高的穿透深度，由于在这些较早阶段的高度散射，传统光学成像过程中没有一种能够在心脏器官的初起(心管形成，阶段39)时对心脏进行成像。然而，随着胚胎变得光学透明，初始的心脏活动(阶段35)被观察到，并且获得了室形成(大约阶段40)的初起时的详细结构图像。尤其是对于FFOCM和SECM模态，难以将组织结构与微观数据集相匹配。胚胎在被处理和嵌入时十分脆弱，使得形态的保存成为挑战。进而，图像应当用10μm数量级的精度与组织结构配准，这用传统切片技术难以实现。
对于根据本发明的示范性实施例的成像过程，通过内生散射生成对照。进而，分子成像可能对于将基因和蛋白质表达与表型相关是重要的。这样一来，在此描述的示范性系统和方法就可以用于对荧光标签和分子种类进行成像。已描述了通过修改源和检测电子器件经由谱编码可以实施荧光成像。(参见J.T.Motz等人的“Spectral-and frequency-encodedfluorescence imaging”，Opt Lett 30，pp.2760-2762(2005))。荧光SECM过程和系统中使用的相同原理可以同样地用于内窥镜的双光子和第二谐波成像。使用示范性TDOCT、OFDI和FFOCM过程和系统中使用的相干检测，可能难以直接检测荧光。然而，对于OCT模态已经描述了几种分子对照方法。(参见C.Yang的“Molecular contrast optical coherencetomography：a review”，Photochem Photobiol 81，pp.215-237(2005)和S.A.Boppart等人的“Optical probes and techniques for molecular contrastenhancement in coherence imaging”，J Biomed Opt 10，41208(2005))。
这项工作中提出的天然对照光学成像模态允许从不同的有利位置对胚胎心脏进行评估。组合OFDI、SECM和FFOCM模态可以发挥它们的力量(参见表1)并提供获得更加全面的形态和功能的心肌表型的能力。同样可以将这种多模态范例扩展到其它系统和动物模型。由于这些非侵入成像技术并不改变样品，所以它们可以顺序地或并行地使用。进而，虽然我们在这项工作中使用了分开的成像系统，但是没有根本性的降碍阻止将它们组合成使用单个波长扫描源的一个成像系统。(参见：S.H.Yun等人的“High-speed optical frequency-domain imaging”，Optics Express 11，pp.2953-2963(2003)；C.Boudoux等人的“Rapid wavelength-sweptspectrally encoded confocal microscopy”’Optics Express 13，pp.8214-8221(2005)；以及W.Y.Oh等人的“Wide tuning range wavelength-sweptlaser with two semiconductor optical amplifiers”，IEEE PhotonicsTechnology Letters 17，pp.678-680(2005))。
前述仅仅表明了本发明的原理。考虑到在此的教导，对所描述的实施例的各种修改和变更对本领域技术人员而言将会是明显的。事实上，根据本发明的示范性实施例的装置、系统和方法，可以与任何OCT系统、OFDI系统、SD-OCT系统或其它成像系统一起使用，并且例如与2004年9月8日提交的国际专利申请PCT/US2004/029148、2005年11月2日提交的美国专利申请No.11/266,779和2004年7月9日提交的美国专利申请No.10/501,276中描述的那些一起使用，这些专利申请的整体内容通过引用结合于此。这样一来，将会意识到的是，本领域技术人员将会设计众多的系统、装置和方法，它们尽管没有在此明确地示出或描述，但却体现了本发明的原理，并从而处在本发明的精神和范围之内。另外，就上面尚未明确地将现有技术知识通过引用结合于此而言，明确地以其整体结合于此。上面引用在此的所有公布都以其整体通过引用结合于此。

Claims (32)

1.一种用于成像至少一个样本的设备，包括：
至少一个第一装置，其配置成提供基于第一模态的与从所述至少一个样本的至少一个区域接收的第一信号相关联的第一数据，和基于不同于所述第一模态的第二模态的与从所述至少一个样本接收的第二信号相关联的第二数据，其中所述至少一个第一装置进一步配置成接收与基准相关联的第三数据，其中所述第一装置基本上同时提供所述第一和第二数据；以及
至少一个第二装置，其配置成基于所述第一、第二和第三数据生成进一步的数据。
2.根据权利要求1所述的设备，其中，所述第一模态为谱编码共焦显微术。
3.根据权利要求1所述的设备，其中，所述第二模态为荧光成像。
4.根据权利要求1所述的设备，进一步包括显微镜装置，其与所述第一和第二装置相关联。
5.根据权利要求1所述的设备，进一步包括射束扫描装置，其配置成将电磁辐射转送到所述至少一个区域。
6.根据权利要求1所述的设备，其中，所述至少一个第二装置生成作为所述进一步的数据的函数的(i)二维图像或(ii)三维图像中的至少一种。
7.根据权利要求1所述的设备，其中，所述第一和第二数据与所述至少一个样本上的近似相同位置相关联。
8.根据权利要求1所述的设备，其中，所述第一数据或所述第二数据中的至少一个是通过使用所述第一和第二数据中的另一个来获得的。
9.根据权利要求1所述的设备，其中，在探针或单个罩中的至少一个中提供所述第一和第二装置。
10.根据权利要求1所述的设备，其中，所述第一和第二装置包括公共部件。
11.根据权利要求10所述的设备，其中，在波长扫描源装置中提供所述公共部件。
12.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得谱编码显微信息。
13.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得明视场、暗视场、相位反差、极化、上反射或反射显微信息中的至少一种。
14.根据权利要求1所述的设备，进一步包括配置成从所述第一模态改变到所述第二模态的进一步的装置。
15.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得与具有多个波长的源装置提供的信号相关联的光学相干断层扫描信息，并且进一步包括多个检测器，其配置成检测作为波长函数的所述第二和第三信号之间的谱干涉。
16.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得与波长随时间变化的源装置提供的信号相关联的光学相干断层扫描信息。
17.根据权利要求1所述的设备，其中，所述至少一个第一装置进一步配置成接收与基准相关联的第三数据，并且所述至少一个第二装置配置成生成作为所述第三数据的函数的所述进一步的数据。
18.根据权利要求1所述的设备，进一步包括：
至少一个第三装置，其配置成基于以前获得的所述第一数据或所述第二数据中的至少一个来控制所述至少一个第一装置或所述至少一个第二装置中的至少一个。
19.根据权利要求1所述的设备，进一步包括：
至少一个第四装置，其配置成基于所述第一和第二数据生成图像。
20.根据权利要求1所述的设备，进一步包括：
至少一个第五装置，其配置成生成基于所述第一数据的至少一个第一图像和基于所述第二数据的至少一个第二图像，其中所述第一和第二图像作为所述第一和第二数据的函数而彼此相关联。
21.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得光学相干断层扫描信息。
22.根据权利要求1所述的设备，其中，所述至少一个第一装置配置成获得光频域干涉测量信息。
23.根据权利要求1所述的设备，其中，代替所述第三数据与所述基准相关联，所述第三数据与从所述至少一个样本接收的所述第二信号相关联，并且其中所述第三数据中的每一个基于与所述第一模态和所述第二模态不同的进一步的模态。
24.根据权利要求23所述的设备，其中，所述至少一个第一装置配置成获得光学相干断层扫描信息。
25.根据权利要求23所述的设备，其中，所述至少一个第一装置配置成获得光学相干显微信息。
26.根据权利要求23所述的设备，其中，所述至少一个第一装置配置成获得全场光学相干显微信息。
27.根据权利要求1所述的设备，其中所述第一模态是谱编码模态，并且其中所述第二模态是非谱编码模态。
28.根据权利要求1所述的设备，其中，所述第一模态包括与所述至少一个样本之内的第一平面相关联的信息，并且所述第二模态包括与所述至少一个样本之内的不同于所述第一平面的第二平面相关联的信息。
29.一种用于成像至少一个样本的方法，包括：
提供基于第一模态的与从所述至少一个样本的至少一个区域接收的第一信号相关联的第一数据，和基于不同于所述第一模态的第二模态的与从所述至少一个样本接收的第二信号相关联的第二数据，其中基本上同时提供所述第一和第二数据；
接收与基准相关联的第三数据；以及
基于所述第一、第二和第三数据生成进一步的数据。
30.根据权利要求29所述的方法，其中，代替所述第三数据与所述基准相关联，所述第三数据与从所述至少一个样本接收的所述第二信号相关联，其中所述第三数据中的每一个基于与所述第一模态和所述第二模态不同的进一步的模态。
31.根据权利要求29所述的方法，其中所述第一模态是谱编码模态，并且其中所述第二模态是非谱编码模态。
32.根据权利要求29所述的方法，其中，所述第一模态包括与所述至少一个样本之内的第一平面相关联的信息，并且所述第二模态包括与所述至少一个样本之内的不同于所述第一平面的第二平面相关联的信息。
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