High-throughput serial two-photon tomography with BakingTray and BrainSaw

Volumetric imaging is increasingly important in fields such as neuroscience, where quantifying structure and mapping features are inherently three-dimensional problems. Block-face serial two-photon tomography (STPT) has emerged as a powerful approach for high-resolution, whole-organ volumetric imaging. STPT avoids complex sample preparation and balances image quality, speed, and scalability. However, STPT remains niche, in part because of the absence of flexible, general-purpose control software. Here we present BakingTray, an open-source software platform for STPT that extends ScanImage. BakingTray’s modular framework accommodates diverse hardware configurations and allows users to build systems customised to their needs. Twelve such systems, which we call ‘BrainSaws’, have been constructed worldwide since 2016, with more under construction, and have so far contributed to 35 research papers. The three systems at the Sainsbury Wellcome Centre alone have acquired over 7,000 samples in a busy multi-user facility. We demonstrate the platform across its core neuroscience applications: electrode track localisation, brain-wide cell counting, and bulk projection mapping. Beyond these, we map the cell-type-specific synaptic output of inhibitory neurons in the ventral lateral geniculate nucleus and, with whole-mount staining, amyloid plaques brain-wide in a mouse model of Alzheimer’s disease. BakingTray is a mature platform for groups or facilities needing a customised volumetric imaging solution, whether built new or converted cheaply from an existing two-photon microscope.