Demixing fluorescence time traces transmitted by multimode fibers

Fiber photometry is a significantly less invasive method compared to other deep brain imaging microendoscopy approaches due to the use of thin multimode fibers (MMF diameter $<$ 500 $\mu$m). Nevertheless, the transmitted signals get scrambled upon propagation within the MMF, thus limiting the technique's potential in resolving temporal readouts with cellular resolution. Here, we demonstrate how to separate the time trace signals of several fluorescent sources probed by a thin ($\approx$ 200 $\mu$m) MMF with typical implantable length in a mouse brain. We disentangled several spatio-temporal fluorescence signals by using a general unconstrained non-negative matrix factorization (NMF) algorithm directly on the raw video data. Furthermore, we show that commercial and low-cost open-source miniscopes display enough sensitivity to image the same fluorescence patterns seen in our proof of principle experiment, suggesting that a whole new avenue for novel minimally invasive deep brain studies with multimode fibers in freely-behaving mice is possible.