Intravital 2-photon microscopy enabling 6D single cell RNA seq in immunocompetent glioblastoma xenografts A variety of in vitro and ex vivo methods have been used to study mammalian cells deciphering signaling pathways and validating new drug targets. However, properties of cell isolated from native environment are unavoidably altered. Histology provides only static information on molecular and cellular processes. Medical imaging paradigms such as CT, PET and MRI do not allow dynamic visualization of cells at microscopic resolution in living animals. Recent progress in two-photon (2p) imaging and labeling tools has ushered in a new era of intravital imaging, which not only enables the longitudinal visualization of cells at high spatial and temporal resolution, but also sheds light on the function of cells through a variety of indicators. Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor in adults. There is a pressing need of high resolution intravital functional imaging tools to interrogate GBM tumor heterogeneity, which is the key determinant of GBM recurrence and resistance to therapy. This proposal aims to address this pressing need by establishing an in vivo functional single cell tracking platform with the capacity of molecular profiling. With extensive research experience in 2p imaging and labeling tools, the PI has developed a cell positioning system (CPS) for precise, longitudinal functional single cell tracking in live animals. In this proposal, we combine long-term functional single cell tracking platform with single cell RNA sequencing (scRNA seq). Compared with conventional scRNA seq, two more dimensions are added: time (t) and cell function (f), constituting the 6D scRNA seq (x, y, z, color, t, and f). 6D scRNA seq will be used to delineate the molecular features of GSCs whose cellular behaviors have been collected during 2p imaging. If successful, this 6D seq platform will significantly advance the field of single-cell sequencing and provide an enabling technology that promises a more in-depth understanding of the pathogenesis and therapy of brain cancers or other diseases. Our platform also allows comprehensive in vivo evaluation of tumor responses to chemotherapy as a drug in vivo screening platform toward establishing individualized targeted therapy for GBM and beyond.