Dataset for phSFC framework: Spectral Flow Cytometry and Hyperspectral Confocal Microscopy Files

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 fluorescent signals. Archives all_data.zip: the complete dataset (all raw, pre-gated and technical benchmark files, for spectral flow cytometry and hyperspectral microscopy), organized as described below. source_data_fig_1.zip: Source Data for Figure 1. source_data_fig_2.zip: Source Data for Figure 2. source_data_extended_data_fig_1.zip: Source Data for Extended Data Figure 1. The three Source Data archives contain copies of the files from all_data.zip that are needed to reproduce the corresponding figure. Each one is organized in folders named after the figure panel they feed, so each figure can be reproduced without downloading the full dataset. Files used by more than one figure (for example, the MLV and BAL pre-gated files) are included in each archive that uses them. Dataset Contents The record is organized by experimental model, technical validation benchmarks, 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. Includes an OLS Unmixed subfolder containing raw data unmixed via the ordinary least-squares algorithm. 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). Flow Rate Analysis: Whole blood cells stained with Hoechst and CD45-BV510, acquired across variable throughput regimes (Low, Medium, and Fast sample flow rates) to benchmark phasor coordinate stability. Gain Analysis: Unstained Vero cells acquired under systematic detector gain attenuation (-20%, -40%, -60%, and -80%) to evaluate robustness against signal dynamic range shifts. Data Organization & File Formats To ensure reproducibility and transparency, the files in all_data.zip are categorized as follows: Spectral Flow Cytometry (.fcs): Ungated Folder: Raw acquisition files directly exported from the Cytek Aurora. Pre-gated Folder: Files containing specific populations (e.g., singlets, LAURDAN+ events, or specific cell subsets) exported following the gating workflows described in the Methods. Technical Benchmarks: Dedicated folders for Flow rate analysis, Gain analysis, and OLS Unmixed files. Hyperspectral Microscopy (.lsm): Raw Data: Original hyperspectral stacks. Masks Folder: Binary masks (stored as image files) used to restrict analysis to specific regions of interest (ROIs), such as vesicle membranes or cellular boundaries. The Source Data archives contain only spectral flow cytometry files (.fcs), taken from the Pre-gated folders, the Flow rate analysis and Gain analysis folders, and, for Figure 2d, one file from the Ungated folder. Technical Specifications Spectral Flow Cytometry: Acquired on a Cytek Aurora (3-laser configuration). 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 & Analysis: Initial gating and traditional unmixing performed in FlowJo / SpectroFlo. Downstream spectral phasor transformations, component calculations, and visualization performed using the PhasorPy library in Python.