Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy
Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy
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DOI：
10.1161/circresaha.113.301704
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发表时间：
2013-09-13
影响因子：
20.1
通讯作者：
Bub, Gil
中科院分区：
文献类型：
--
作者：
Botcherby, Edward J.;Corbett, Alex;Bub, Gil
理论基础：肌节长度(SL)是心脏机械功能的一个关键指标，但目前的成像技术在细胞水平上明确测量和表征完整的活组织的SL的能力有限。目的：我们开发了一种使用远程聚焦显微镜测量SL和区域细胞方向的方法，这是一种新兴的成像方式，可以从样本中的任意倾斜平面捕获光。方法和结果：我们提出了一种协议，通过成像与细胞共享共同长轴的两个倾斜平面，明确并快速地确定视野中用户选择的区域的细胞方向。我们证明了该技术在负载膜染料di-4-ANEPPS的朗宁多夫灌流心脏中建立单细胞SL的有效性。结论：远程聚焦显微镜可以在复杂的双光子数据集中测量细胞方向，而不需要捕获完整的z堆栈。这项技术可以快速评估健康和患病心脏实验制剂中的SL。
Rationale: Sarcomere length (SL) is a key indicator of cardiac mechanical function, but current imaging technologies are limited in their ability to unambiguously measure and characterize SL at the cell level in intact, living tissue.Objective: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample.Methods and Results: We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS.Conclusions: Remote focusing microscopy can measure cell orientation in complex 2-photon data sets without capturing full z stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.