Abstract Multiplexed spatial profiling of protein markers in cells and tissues is critical to basic research and clinical applications. Unfortunately, we currently lack tools that can rapidly and routinely profile a large number of proteins in situ in large tissues with subcellular resolution in a time and cost-effective fashion. Existing tools for in situ protein analysis including immunohistochemistry and immunofluorescence suffer from low multiplexing because of limited separation of spectral channels. Recent single-cell sequencing methods lack the critical spatial context needed to understand complex heterogeneous samples. Other spatial proteomics methods that are based on serial labeling and imaging, indirect indexed mass spectrometry or sequencing are complicated, time-consuming and expensive. This proposed project will develop a new spatial proteomics technology termed as Phasor S- FLIM that enables direct, simultaneous, high-plex spatial profiling of protein markers in large and thick tissues with just one-round of staining and imaging. Our Phasor S-FLIM system, for the first time, allows true parallel, simultaneous lifetime and spectral detection with phasor analysis to obtain fast, unbiased, high-precision lifetime and spectral data that can be processed in real time. In the proposed work, we will adapt and further develop Phasor S-FLIM for high-plex spatial proteomics applications, including (a) implementation of Pulsed Interleaved Excitation (PIE) dual excitation with 2-photon lasers and sensitive multi-channel GaAsP PMT arrays, enabling exciting and detecting a broad range of fluorophores (Aim 1), (b) development of a novel fluorophore-quencher labeling strategy to generate a large repertoire of probes with orthogonal lifetime and spectrum signatures for high-plex target encoding (Aim 2), and (c) characterization, validation and benchmarking of Phasor S-FLIM for multiplexed spatial protein analysis using broadly relevant biological and clinical tissue models (Aim 3). Once developed, we expect the Phasor S-FLIM can detect at least 30 different protein targets through direct, one round of staining and imaging, in thick (>0.5 mm) tissues, with subcellular resolution (200 nm), and in high imaging throughput (1 x 1 mm2 plane in <15 min), which is currently not possible with existing methods. Upon successful completion of the proposed work, we will have established a working prototype ready to quickly serve the scientific community to address a broad range of biological and clinical questions that are previously impossible or impractical. Our technology can potentially shift current practice in interrogating protein and cellular processes as well as the complexity and systems in biology and disease with high resolution, throughput and scale.