Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

Abstract

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.

Dynamic Myofibrillar Remodeling in Live Cardiomyocytes under Static Stretch

“…Although the contrast of our SHG confocal does not allow us to distinguish among layers of myofibrils, its resolution (0.55 μm lateral 37 and 1.2 μm axial 64 ) does allow us to observe sarcomere-addition sites relative to the entire cell body. Our experiments show that myofibrillar splitting occurs in the central region of myofibrillar bundles, where the myofibrils, upon splitting, are less likely to bind or directly connect to the focal adhesion assemblies that mechanically link the extracellular matrix and the cell skeleton.…”

“…Cells were cultured for 5 days in the microgrooves on the PDMS (biochip) membrane that was affixed to the cell stretcher and housed in a standard cell incubator (5% CO 2 and 37 °C) and then, the entire device including the cell culture was placed in an on-stage incubator mounted on the imaging stage of the customized 2 P/SHG confocal microscope. The setup of the 2 P/SHG microscope was described previously 37 . The 2 P channel was utilized to image mitochondrial distribution (for these experiments, neonatal ventricular CMs were fluorescently labeled with MitoTracker Red (Life Technologies, US)), and the SHG channel was used to image sarcomeric patterns.…”

“…Consequently, it can be used to image a specific molecule that has the required molecular structure, such as a sarcomeric myosin filament, without cell staining and thus, to achieve live cell imaging. Using our custom-built two photon (2P)/SHG confocal microscope 37 , we have been studying dynamic sarcomeric addition during myofibrillar remodeling in single, living neonatal CMs under acute, uniaxial, static, sustained stretch. Here we report, for the first time, live-cell observations of various modes of dynamic sarcomeric addition and how these real-time images compare to the static images from hypertrophic hearts reported in the literature.…”

Analysis of time-course drug response in rat cardiomyocytes cultured on a pattern of islands

“…6,7 However, fluorescent indicators may cause photodamage. 8 These studies were focused on the physiological processes of cardiomyocytes and did not reveal information on the beating properties of cardiomyocytes.…”

“…27,28 Thus a stage-top incubator that consists of a sealed chamber is essential for the accurate analysis of cardiomyocyte activities. 8,29,30 However, because SECM requires strict control of the microelectrode position at a resolution in the order of nanometer, it is difficult to perform SECM measurements in a sealed chamber. Therefore, at present only a temperature controlled-plate has been used for SECM experiments on living cells.…”