The goal of this study is to explore the potential of pulsed and continuous wave light optogenetic stimulation on mouse embryonic cardiodynamics. Toward this goal, we engineered mouse embryos expressing the light-sensitive protein Channelrhodopsin-2 (ChR2) ubiquitously through the embryo. The embryos were dissected live and the optogenetic light stimulation of ChR2 at 473-nm was performed under imaging guidance. The pulsed stimulation allowed for a range of cardiodynamic behaviors and was overall found to have a milder effect on embryo viability, while the continuous wave stimulation provided an advantage in the faster mapping of optogenetic cardiac responses.
Cilia beat frequency (CBF) is an essential measure of fallopian tube function. In the present study, we adapted a previously developed functional optical coherence tomography (OCT) method, to spatially map and quantify CBF in one human sample ex vivo. Time lapsed image sets at different locations were acquired using OCT (n=8) and 50x magnification brightfield (BF) microscopy (n=2) as ground truth. A sliding window Fourier analysis was performed on OCT and BF image sets to quantify CBF on a pixel-by-pixel basis. Parameters were optimized to maximize contrast of cilia, and were uniformly applied for all OCT image sets. CBF color maps were created and were qualitatively compared to unprocessed OCT intensity image sets to evaluate the spatial mapping accuracy of CBF values. Line plots of amplitude vs. frequency at 1 second intervals, and pixel peak frequency histograms of whole image sets, were created to visualize the dominant CBFs. An analysis of variance was used to compare CBFs as measured with OCT and BF microscopy. Our results revealed qualitatively accurate spatial mapping of non-zero CBF values to pixels in ciliated areas, which were visibly appreciable on unprocessed intensity image sets. There was no significant difference between the dominant CBFs as measured with OCT and ground truth BF microscopy (3.2 ± 1.6Hz, 3.2 ± 0, p=0.97). Bulk sample movement was a significant source of temporal variability and amplified high-frequency noise.
The mouse embryo is an established model for investigation of regulatory mechanisms controlling cardiac development and congenital heart defects in humans. Optogenetics, originally developed for neuroscience research, has recently been shown to control embryonic mouse heartbeat with pulsed light in a range of stimulation frequencies. The aim of this study was to explore continuous wave optogenetic light stimulation for activation of contractions through regions of the heart. This study suggests a potential to use continuous wave optogenetic stimulation for quick identification and investigation of developmental origin of cardiac cell populations in addition to previously established manipulation of embryo cardiodynamics.
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