Summary Recording the activity of large populations of neurons is an important step towards understanding the emergent function of neural circuits. Here we present a simple holographic method to simultaneously perform two-photon calcium imaging of neuronal populations across multiple areas and layers of mouse cortex in vivo. We use prior knowledge of neuronal locations, activity sparsity and a constrained nonnegative matrix factorization algorithm to extract signals from neurons imaged simultaneously and located in different focal planes or fields of view. Our laser multiplexing approach is simple and fast and could be used as a general method to image the activity of neural circuits in three dimensions across multiple areas in the brain.