Fig. 2. Experimental pipeline for combining functional imaging during motor behaviors with structural imaging in macaque monkeys.

a Prior to imaging, we performed a neutralizing antibody assay in order to select an appropriate viral serotype tailored to the immune response of each monkey. b Viral constructs were injected into cortex to deliver the calcium reporter gene. c A chamber designed for chronic 2P imaging in premotor cortex and motor cortex was implanted. d Widefield (1P) imaging was used to assess GCaMP expression and establish vascular fiducial markers for navigating to specific sites on the cortex (representative example, single image collected prior to most recording sessions). e A macaque was trained to perform a reaching task to radially arranged targets. f 2P imaging was used to obtain functional signals at single-cell resolution from motor cortex (contrast-enhanced mean-intensity projection from one representative dataset of 36 sessions). g During training trials, a decoder was trained on the imaging data obtained during reaching movements. Subsequently, during test trials, this decoder was run in real time to decode (predict) the reach target from 2P imaging data. h Ex vivo CLARITY was performed to identify cell morphology, projection patterns and cell type (anti-GCaMP antibody labeling green, vasculature white, and results from single imaging session).