Multiplexed Dynamic Assessment of Cellular Composition and Function In Vivo

About This Grant

Project summary Understanding the dynamic change of cell-type composition, functional evolution, migration, and interaction of single cells within complex tissue or in vivo environments is essential for advancing our knowledge of developmental, pathological, and therapeutic processes. Yet current single-cell analytical methods such as flow cytometry, histological staining, sequencing, and mass cytometry are inherently destructive and provide only static views of dynamic cellular processes. While intravital microscopy with multiphoton technology enables nondestructive real-time imaging of single-cell behavior in three- dimension (3D) tissue environment, its multiplexing capacity is constrained by reliance on a narrow range of spectrally distinct colors. As a result, we remain unable to continuously track live cells and their functional evolution in physiologically relevant environments. My research program addresses this critical gap by developing Spatiotemporal Multiplexed Antigen Revealing Technology (SMART), an innovative platform for multiplexed, longitudinal imaging of live cells. SMART enables repeated cycles of labeling and erasing of fluorescent signals on cell surface, allowing continuous tracking of dozens of extracellular markers on the same cells over time. By reconstructing these signals across cycles, SMART provides a dynamic view of cell phenotypes, migration, differentiation, and communication that current methods cannot achieve. Over the next five years, my laboratory will develop and establish SMART as a versatile and reliable tool through applications in PBMCs (peripheral blood mononuclear cells) phenotyping, immune cell migration and function, stem cell differentiation, and neuron–astrocyte interactions. These studies will demonstrate SMART’s ability to uncover how cellular states evolve and how microenvironmental interactions shape biological outcomes. Looking ahead, my long-term vision is to extend SMART into living organisms, enabling noninvasive, real-time, multiplexed imaging of cellular dynamics in vivo. This advance would transform our ability to study immune responses, stem cell fate, and neural development directly in their native environments, providing insights that cannot be obtained with current technology. Ultimately, SMART has the potential to redefine how biomedical research observes and understands cellular behavior across diverse biological systems.