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This dataset contains raw and processed data supporting the development and validation of phasor-based spectral flow cytometry (phSFC). This framework extends spectral phasor analysis, traditionally used in hyperspectral microscopy, to high-throughput flow cytometry, enabling model-free visualization and quantification of complex fluorescence signals. Dataset Contents The record is organized by experimental model and acquisition modality: Multilamellar Vesicles (MLVs): Data of DOPC, DPPC, and Cholesterol mixtures labeled with LAURDAN. Used for cross-modal validation and establishing reference phasor coordinates for membrane order. Vero Cell Culture: Data from live cells treated with methyl-β-cyclodextrin (MβCD) to induce cholesterol depletion. Ex Vivo Leukocytes: Data from a mouse model of lung inflammation (LPS-induced). Contains multi-color spectral flow cytometry data (LAURDAN + CD11c + CD45) from bronchoalveolar lavage (BAL). Metadata & Gating: Supplementary tables defining the spectral channel configurations for the Cytek Aurora (V1–V16) and the Zeiss LSM 880 lambda mode settings. Data Organization & File Formats To ensure reproducibility and transparency, the files are categorized as follows: Spectral Flow Cytometry (.fcs): * Ungated Folder: Raw acquisition files directly from the Cytek Aurora. Pre-gated Folder: Files containing specific populations (e.g., singlets, LAURDAN+ events, or specific cell types) exported after the gating workflows described in the Methods. Hyperspectral Microscopy (.lsm): * Raw Data: Original hyperspectral stacks. Masks Folder: Contains binary masks (stored as image files) used to restrict analysis to specific regions of interest (ROIs), such as vesicle membranes or cellular boundaries. Technical Specifications Spectral Flow Cytometry: Acquired on a Cytek Aurora (3-laser). Spectral range analyzed: 428–812 nm (16 channels, Violet excitation). Hyperspectral Microscopy: Acquired on a Zeiss LSM 880 in Lambda Mode (63×/1.4 NA). Spectral range: 423–693 nm (28 channels, 10 nm spacing). Processing: Initial gating performed in FlowJo. Downstream spectral phasor transformation and component analysis performed using the PhasorPy library in Python.