Miller Christine E, Thompson Robert P, Bigelow Michael R, Gittinger George, Trusk Thomas C, Sedmera David
Department of Mechanical Engineering, Bucknell University, Lewisburg, PA 17837, USA.
Microsc Microanal. 2005 Jun;11(3):216-23. doi: 10.1017/S1431927605050464.
Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 microm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 microm was achieved with a 10x dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5x objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.
共聚焦显微镜可对组织进行光学切片，从而无需进行物理切片及后续配准即可获得组织结构的三维图像。然而，诸如组织不透明度、光穿透性和探测器灵敏度等实际因素通常将可用成像深度限制在200微米。随着更新、更强大系统的出现，我们试图将这些限制扩展到由物镜工作距离所决定的范围。我们采用全组织免疫组化染色，随后用苯甲醇 - 苯甲酸苄酯（BABB）进行透明处理，以可视化三维心肌结构。使用10倍干物镜实现了对整个鸡胚心脏直至1.5毫米深度的共聚焦成像，体素尺寸为3微米。为了筛查先天性心脏缺陷，我们用荧光标记的聚-L-赖氨酸对心内膜进行染色，并使用5倍物镜对经BABB透明处理的心脏进行成像，深度可达2毫米。用Hoechst核染料染色的全组织标本的双光子成像在整个29期心脏中都产生了清晰的图像，且没有明显的信号衰减。因此，目前可用的系统能够对固定样本进行共聚焦成像，达到以前无法达到的深度，当前的限制因素是物镜工作距离、抗体穿透性、样本自发荧光和不完全透明处理。
