OPTRACE: Optical Imaging–Guided Transplantation and Tracking of Cells in the Mouse Brain

Intracerebral cell transplantation holds promise for treating stroke and neurological disorders, yet challenges in precise delivery and post‐engraftment monitoring impede progress. This work introduces OPTRACE (OPtical imaging‐guided Transplantation and tRAcking of CElls), a two‐step optical framework integrating real‐time visualization during transplantation with longitudinal post‐transplantation in vivo cell tracking. Leveraging cost‐effective translucent glass micropipettes and two innovative predictive mathematical modeling—the retention–depth model (predicts retained fraction versus injection depth) and the hypoperfusion–volume model (predicts hypoperfused fraction versus graft volume)—that this work fits to data (depth–retention R 2 = 0.91; volume–growth R 2 = 0.78)—OPTRACE optimizes delivery parameters to maximize engraftment and minimize hypoxia. A novel pulse‐elevation injection technique further enhances the precision of superficial cortical retention. Following transplantation, multicolor labeling combined with two‐photon fluorescence microscopy permits longitudinal single‐cell tracking, revealing host microglial responses, and altered neuronal calcium signaling at the graft interface. OPTRACE provides micrometer precision, longitudinal dynamics and quantitative insights of cells during and after transplantation, accelerating mechanistic understanding and therapeutic development for regenerative cell therapies.