Multi-Spectral Widefield Microscopy of the Beating Heart Through Post-Acquisition Synchronization and Unmixing

Multi-spectral imaging allows distinguishing biological structures. For cardiac microscopy, available devices are either too slow or require illumination intensities that are detrimental to the sample. We present a method for spectral super-resolution imaging of samples whose motion is quasi-periodic by sequentially acquiring movies in wavelength ranges with filters of overlapping bands. Following an initial calibration procedure, we synchronize and unmix the movies to produce multi-spectral sequences. We characterized our approach to retrieve the transmittance of a colored microscopic target whose motion we controlled, observing measurements within of 10% that of a reference spectrometer. We further illustrate our approach to observe the beating embryonic zebrafish heart, demonstrating new possibilities for studying its development.