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.
Congenital Heart Disease (CHD) is the most common congenital malformation in newborns in the US. Although
knowledge of CHD is limited, altered hemodynamic conditions are suspected as the factor that stimulates cardiovascular
cell response, resulting in the heart morphology remodeling that ultimately causes CHDs. Therefore, one of recent efforts
in CHD study is to develop high-speed imaging tools to correlate the rapidly changing hemodynamic condition and the
morphological adaptations of an embryonic heart in vivo. We have developed a high-speed streak mode OCT that works
at the center wavelength of 830 nm and is capable of providing images (292x220 μm2) of the outflow tract of an
embryonic chick heart at the rate of 1000 Hz. The modality can provide a voxel resolution in the range of 10 μm3, and
the spectral resolution allows a depth range of 1.63 mm. In the study reported here, each of the 4D images of an outflow
tract was recorded for 2 seconds. The recording was conducted every 2 hours (HH17 to HH18), 3 hours (HH14 to HH17),
and 4 hours (HH18 to HH19). Because of the fast scan speed, there is no need for postacquisition processing such as use
of gating techniques to provide a fine 3D structure. In addition, more details of the outflow tract are preserved in the
recorded images. The 4D images can be used in the future to determine the role of blood flow in CHD development.