Imaging distinct neuronal populations with a dual channel miniscope

Miniature fluorescence microscopes (miniscopes) are one of the most powerful and versatile tools for recording large scale neural activity in freely moving rodents with single cell resolution. Recent advances in the design of genetically encoded calcium indicators (GECIs) allow to target distinct neuronal populations with non-overlapping emission spectral profiles. However, conventional miniscopes are limited to a single excitation, single focal plane imaging, which does not allow to compensate for chromatic aberration and image from two spectrally distinct calcium indicators. In this paper we present an open-source dual channel miniscope capable of simultaneous imaging of genetically or functionally distinct neuronal populations. Chromatic aberrations are corrected using an electrowetting lens (EWL), which allows fast focal plane change between frames. To demonstrate the capabilities of the dual channel miniscope, we labeled layer specific excitatory neurons or inhibitory interneurons in the medial prefrontal cortex (mPFC) with a red fluorescence protein, and simultaneously imaged neural activity of distinct neuronal populations of freely moving mice via a green GECI.