PAAQ: Paired Alternating AcQuisitions for virtual high frame rate multichannel cardiac fluorescence microscopy.

In vivo fluorescence microscopy is a powerful tool to image the beating heart in its early development stages. A high acquisition frame rate is necessary to study its fast contractions, but the limited fluorescence intensity requires sensitive cameras that are often too slow. Moreover, the problem is even more complex when imaging distinct tissues in the same sample using different fluorophores. We present Paired Alternating AcQuisitions, a method to image cyclic processes in multiple channels, which requires only a single (possibly slow) camera. We generate variable temporal illumination patterns in each frame, alternating between channel-specific illuminations (fluorescence) in odd frames and a motion-encoding brightfield pattern as a common reference in even frames. Starting from the image pairs, we find the position of each reference frame in the cardiac cycle through a combination of image-based sorting and regularized curve fitting. Thanks to these estimated reference positions, we assemble multichannel videos whose frame rate is virtually increased. We characterize our method on synthetic and experimental images collected in zebrafish embryos, showing quantitative and visual improvements in the reconstructed videos over existing nongated sorting-based alternatives. Using a 15 Hz camera, we showcase a reconstructed video containing two fluorescence channels at 100 fps.