Introduction: The average human heart beats ~2.8 billion times over a 70 year lifetime. In the failing heart, alterations in the contraction-relaxation cycle of the sarcomere can be visualized by high-speed time-lapse phase contrast microscopy. Here, we describe the methodology used to quantify changes in sarcomere length over time.
Methods and Results: Half-sarcomere measurements from spontaneously contracting (SPOC) bundles of human cardiomyocytes imaged by high-speed time-lapse phase contrast (Leica SP5 Multiphoton) microscopy. We also used the second harmonic feature of this microscope to identify the myosin A bands that are known to not change length during SPOC. The position of each sarcomere were analysed by frame-by-frame (FF) and difference-from-first (DF) analyses. FF analysis detects low-intensity, high-frequency changes in pixel intensity that correlate with contraction-relaxation phases of sarcomere length changes. The opposite intensity-frequency was detected by DF analysis, and is associated with the SPOC wave. The averaged sarcomere position was fitted to a sawtooth model where contraction and relaxation parameters were extracted.
Conclusion: We have previously demonstrated that SPOC measurements can sense changes in failing cardiomyocytes that are closely related to the disease phenotype. SPOC measures length changes of ~0.1 µm with a temporal acquisition of ~15 ms/frame. To our knowledge, no commercial software can track changes with such accuracy. These methods can be applied to other oscillatory/cyclic movements need to be measured.
This work was supported by a Postgraduate Scholarship (PB 12S 6939) from the National Heart Foundation of Australia.