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.

译文

理由:鞘管长度(SL)是心脏机械功能的关键指标,但是当前的成像技术在完整的活组织中无法在细胞水平上明确测量和表征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和区域细胞取向的方法,该方法是一种新兴的成像方式,可以捕获样品中任意倾斜平面的光。
方法和结果:我们提出了一种协议,该协议通过对2个与细胞共享主轴线的倾斜平面进行成像,从视野中的用户选择区域中明确,快速地确定细胞方向。我们展示了该技术在用膜染料di-4-ANEPPS加载的Langendorff灌注心脏中建立单细胞SL的有效性。
结论:远程聚焦显微镜可以测量复杂的2光子数据集中的细胞方向,而无需捕获完整的z堆栈。该技术可以快速评估健康和患病心脏实验制剂中的SL。

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