Large-scale volumetric two-photon calcium imaging enables cellular-resolution mesoscopic mapping of marmoset cortical areas

Primate neocortex is organized into functionally defined domains. Understanding how the functional organization within each domain emerges from single-cell tuning properties has been hampered by the lack of imaging tools that can capture large-scale activity at different depths of the scattering primate brain at cellular resolution and physiological timescales. Here, we demonstrate mesoscopic and volumetric cellular-resolution calcium imaging of entire cortical areas in the marmoset brain at multi-Hertz rates by combining fluorescence lifetime-limited temporally multiplexed two-photon recording based on Light Beads Microscopy (LBM) with optimized calcium indicator targeting. Using this approach, we perform volumetric recording of neuronal population activity within cortical volumes spanning up to 4 × 4 × 0.3 mm ³ capturing the simultaneous activity of over 96,000 cells, including more than 46,000 stimulus-responsive neurons. In auditory cortex, we found fine-grain heterogeneous tuning of individual neurons to frequencies within broader tonotopic gradients. In the middle temporal area (MT) we found direction tuning along functional continua rather than discrete categories, encompassing uni-, bi-, and pandirectional tuning, where cells with similar tuning tend to cluster. Tuning direction follows a columnar organization with a lateral periodicity of ∼500 µm and low variability across depth. Columns are organized by a pinwheel structure. Noise correlations between cells, revealing functional interactions, decay isotropically and monotonically with distance but remain significant even at lateral distances exceeding 3 mm and thus the scale of multiple columns. Together, these discoveries reveal the need for neurotechnologies bridging scales from single cells to multi-columnar systems. The technological advancements open the door for future large-scale, cellular-resolution investigations of functional organization and population dynamics across entire cortical areas in the primate brain.