Abstract
Recent advances in intravital microscopy have provided insight into dynamic biological events at the cellular level in both healthy and pathological tissue. However, real-time in vivo cellular imaging of the beating heart has not been fully established, mainly due to the difficulty of obtaining clear images through cycles of cardiac and respiratory motion. Here we report the successful recording of clear in vivo moving images of the beating rat heart by two-photon microscopy facilitated by cardiothoracic surgery and a novel cardiac stabiliser. Subcellular dynamics of the major cardiac components including the myocardium and its subcellular structures (i.e., nuclei and myofibrils) and mitochondrial distribution in cardiac myocytes were visualised for 4–5 h in green fluorescent protein-expressing transgenic Lewis rats at 15 frames/s. We also observed ischaemia/reperfusion (I/R) injury-induced suppression of the contraction/relaxation cycle and the consequent increase in cell permeability and leukocyte accumulation in cardiac tissue. I/R injury was induced in other transgenic mouse lines to further clarify the biological events in cardiac tissue. This imaging system can serve as an alternative modality for real time monitoring in animal models and cardiological drug screening, and can contribute to the development of more effective treatments for cardiac diseases.
Highlights
Intravital confocal and two-photon microscopy have been used in combination with fluorescence molecular imaging probes in cancer research, immunology, and neuroscience to investigate biological processes at the cellular level in living organisms[1,2,3,4,5]
We developed a two-photon microscopy system equipped with a custom-built cardiac tissue stabiliser (Fig. 1a) facilitated by cardiothoracic surgery for imaging a beating heart in vivo and successfully recorded clear, real-time, hours-long videos of beating rat hearts with and without regional cardiac I/R
The coverglass attached to a custom-made suction device was placed over the anterior left ventricle (LV) wall of rats in a dark box with the intravital microscope system, which was composed of a two-photon upright microscope equipped with a 25× water-dipping objective lens (Fig. 1b)
Summary
Intravital confocal and two-photon microscopy have been used in combination with fluorescence molecular imaging probes in cancer research, immunology, and neuroscience to investigate biological processes at the cellular level in living organisms[1,2,3,4,5]. There have been no reports of real-time in vivo imaging of cardiac tissue dynamics under ischaemia/reperfusion (I/R) conditions by intravital microscopy at subcellular resolution[20,21,22,23,24]. We developed a two-photon microscopy system equipped with a custom-built cardiac tissue stabiliser (Fig. 1a) facilitated by cardiothoracic surgery for imaging a beating heart in vivo and successfully recorded clear, real-time, hours-long videos of beating rat hearts with and without regional cardiac I/R.
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