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

Circ Res. 2013 Sep 13;113(7):863-70. doi: 10.1161/CIRCRESAHA.113.301704. Epub 2013 Jul 30.


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.

Keywords: instrumentation; multiphoton fluorescence microscopy; optical imaging; sarcomeres.

MeSH terms

  • Animals
  • Female
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Myocardial Reperfusion / methods*
  • Rats
  • Rats, Sprague-Dawley
  • Rats, Wistar
  • Sarcomeres / ultrastructure*