Myofibrillogenesis in live neonatal cardiomyocytes observed with hybrid two-photon excitation fluorescence-second harmonic generation microscopy

Liu, Honghai; Wan, Qin; Shao, Yufeng; Ma, Zhen; Ye, Tong; Borg, T. K.; Gao, Bruce Z.

doi:10.1117/1.3662457

Cited by 15 publications

(23 citation statements)

References 23 publications

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“…Because the SHG signal from cardiomyocytes arises from the coiled-rod region of myosin filaments [16], dynamic assembly of myosin filaments onto a myofibril may be studied without protein labeling [17][18][19]. Through the combination of two-photon excited fluorescence (TPEF) and SHG (TPEF-SHG), additional structural information can be obtained from a cardiomyocyte [20,21]; this microscopic technique is ideal for tracking how specific sarcomeric proteins are assembled onto myofibrils during myofibrillogenesis.…”

Section: Introductionmentioning

confidence: 99%

“…Through the combination of two-photon excited fluorescence (TPEF) and SHG (TPEF-SHG), additional structural information can be obtained from a cardiomyocyte [20,21]; this microscopic technique is ideal for tracking how specific sarcomeric proteins are assembled onto myofibrils during myofibrillogenesis. Since myofibrillogenesis is a process that spans hours to days, we have developed a hybrid TPEF-SHG imaging system with an on-stage incubator that provides normal physiological conditions to isolated cardiomyocytes [17]. We have developed a hybrid TPEF-SHG polarization-imaging system with an on-stage incubator that provides normal physiological conditions to isolated cardiomyocytes during imaging of myofibrillogenesis [17,22].…”

Section: Introductionmentioning

confidence: 99%

“…Figure 1 shows the entire system of our custom-built TPEF-SHG imaging system. The design of the light path of the system can be found in our previous publication 17 . The temperature inside the on-stage incubator was maintained at 37ºC, and a mixture of CO 2 (5%) and air (95%) was passed through a humidifying module and pumped into the incubator.…”

Section: Introductionmentioning

confidence: 99%

“…Since myofibrillogenesis is a process that spans hours to days, we have developed a hybrid TPEF-SHG imaging system with an on-stage incubator that provides normal physiological conditions to isolated cardiomyocytes [17]. We have developed a hybrid TPEF-SHG polarization-imaging system with an on-stage incubator that provides normal physiological conditions to isolated cardiomyocytes during imaging of myofibrillogenesis [17,22]. Here, we report the dynamic process of new-sarcomere addition in neonatal cardiomyocytes during 10 hours of on-stage incubation.…”

Section: Introductionmentioning

confidence: 99%

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Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

et al. 2012

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Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length during maturation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

et al. 2012

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“…However, during the early stages of cell culture in which sarcomeric additions occur, neonatal cardiomyocytes are extremely sensitive to photodamage. The only solution to this problem has been to reduce the power of the incident laser; 1-5 the tradeoff is greatly compromised image quality. Even with this precaution, internal cellular process within cardiomyocytes may still be affected.…”

Section: Introductionmentioning

confidence: 99%

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

et al. 2014

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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.

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Region-Specific Microstructure in the Neonatal Ventricles of a Porcine Model

et al. 2018

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The neonate transitions from placenta-derived oxygen, to supply from the pulmonary system, moments after birth. This requires a series of structural developments to divert more blood through the right heart and onto the lungs, with the tissue quickly remodelling to the changing ventricular workload. In some cases, however, the heart structure does not fully develop causing poor circulation and inefficient oxygenation, which is associated with an increase in mortality and morbidity. This study focuses on developing an enhanced knowledge of the 1-day old heart, quantifying the region-specific microstructural parameters of the tissue. This will enable more accurate mathematical and computational simulations of the young heart. Hearts were dissected from 12, 1-day-old deceased Yorkshire piglets (mass: 2.1-2.4 kg, length: 0.38-0.51 m), acquired from a breeding farm. Evans blue dye was used to label the heart equator and to demarcate the left and right ventricle free walls. Two hearts were used for three-dimensional diffusion-tensor magnetic resonance imaging, to quantify the fractional anisotropy (FA). The remaining hearts were used for two-photon excited fluorescence and second-harmonic generation microscopy, to quantify the cardiomyocyte and collagen fibril structures within the anterior and posterior aspects of the right and left ventricles. FA varied significantly across both ventricles, with the greatest in the equatorial region, followed by the base and apex. The FA in each right ventricular region was statistically greater than that in the left. Cardiomyocyte and collagen fibre rotation was greatest in the anterior wall of both ventricles, with less dispersion when compared to the posterior walls. In defining these key parameters, this study provides a valuable insight into the 1-day-old heart that will provide a valuable platform for further investigation the normal and abnormal heart using mathematical and computational models.

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Myofibrillogenesis in live neonatal cardiomyocytes observed with hybrid two-photon excitation fluorescence-second harmonic generation microscopy

Cited by 15 publications

References 23 publications

Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

Region-Specific Microstructure in the Neonatal Ventricles of a Porcine Model

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