Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Abstract

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.