In-vivo co-registering of functional calcium activity with nine other virally expressed fluorophores through an implanted GRIN lens

Head-mounted miniscopes offer an interesting tradeoff: at the expense of image quality and versatility, they enable the correlation of animal behavior with neuronal activity without limiting free movement. Combining this with the ability to use additional fluorescent markers in-vivo, would allow us to gather further information on the observed neurons, such as genetic traits, specific cell-types, or connections to other brain regions. Unfortunately, current miniscopes cannot discriminate more than one additional fluorophore, and sacrificing the animals for post-experiment brain fixation and ex-vivo imaging is cumbersome, risks failure, and severely complicates the final co-registration and identification of the functional neurons.Instead, we show how to use confocal microscopy to create a high-resolution, multicolor map of the visible brain area by imaging through the implanted GRIN lens of the live, head-fixed animal. Even though GRIN lenses induce a multitude of chromatic and non-chromatic image distortions, they are identical for both imaging modalities, which greatly simplifies co-registration, and are not prohibitive for sophisticated confocal microscopes. Strong chromatic axial shifts are readily compensated, and despite severe GRIN-induced astigmatism and focal plane curvature, chromatic distortions in the optical plane are negligible, thereby allowing co-registration of all color channels in x,y,z with high confidence. Excitation multiplexing and multispectral lambda-detection further allow the differentiation of fluorophores based on both their emission and excitation spectra, increasing the number and spectral proximity of possible fluorophores.Using this method, we were able to discriminate nine spectrally distinct fluorescent proteins alongside GCaMP expressed in the mPFC of a live, head-fixed mouse.