Fast measurement of sarcomere length and cell orientation in Langendorff-perfused hearts using remote focusing microscopy.

Abstract

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. 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. 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. 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.