Laminar optical tomography (LOT) is a new technique that combines the advantages of diffuse optical tomography image reconstruction and a microscopy-based setup to allow noncontact imaging with 100–200‐µm resolution effective over depths of 0–2.5 mm. LOT is being developed primarily for multispectral imaging of rat cortex, for which resolving functional dynamics in various layers of the brain’s cortex (to depths of 1500 µm) is of increasing interest to neurophysiologists. System design and image reconstruction techniques are described, along with simulation and phantom results that demonstrate the characteristics and limitations of system accuracy and resolution.