Related Experiment Video For calcium signaling
Updated: Mar 29, 2026
Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta
Published on: June 10, 2015
Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes
Samir Awasthi1, Leighton T Izu1, Ziliang Mao1
1From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
Rationale:
Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes.
Objective:
To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.
Methods And Results:
Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy.
Conclusions:
Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.
