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This repository contains image datasets associated with the manuscript “PSF-Driven Spatio-Temporal Blending in Fluorescence Lifetime Imaging Microscopy and Its Mitigation via Mean-Shift Super-Resolution–Based Masking.” The dataset is organized into two groups of FLIM images obtained from standardized biological samples. (a) Two-color confocal FLIM images of U2OS cells (GattaQuant® reference slide) The first dataset consists of time-resolved confocal fluorescence lifetime images of U2OS cells obtained from a commercially available reference slide (GattaQuant®). The sample contains three fluorescently labeled cellular structures: nuclear DNA stained with DAPI, mitochondria labeled via TOM20 immunostaining using Alexa Fluor 532, and microtubules labeled through α-tubulin immunostaining with Alexa Fluor 555. The samples were mounted in ProLong Diamond antifade medium and sealed prior to imaging. Time-domain FLIM data were acquired using a custom-built confocal microscope platform (iRT-FLIM-001, Intek Scientific) previously described in the literature. Excitation was provided by a pulsed 515 nm laser source (Prima, PicoQuant) operating at a repetition rate of 25 MHz. Fluorescence emission was collected through a 60× objective lens (UPLXAPO60, Olympus) using a pinhole diameter of 0.5 Airy units, resulting in a pixel sampling of 54 nm. Emitted photons in the spectral range of 550–614 nm were isolated using a bandpass filter (FF01-582/64-25, Semrock) suitable for detecting Alexa Fluor 532 and Alexa Fluor 555 signals and were recorded using a photomultiplier tube detector (H10720-01, Hamamatsu Photonics). For each image pixel, a full fluorescence decay profile was recorded within a nanosecond-scale temporal window. Phasor coordinates (G and S) were calculated using a modulation frequency of 50 MHz corresponding to the second harmonic of the excitation repetition rate. Additional details regarding the instrumentation and acquisition strategy are available in: Hwang, W., Raymond, T., McPartland, T. et al. Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high-dimensional spatial biology in 3D. Communications Biology 7, 1012 (2024). https://doi.org/10.1038/s42003-024-06702-8 After image acquisition, temporal analysis was performed The images of this set are: 2CGQ_U2OS_50MHz_Intensity_px54.tif 2CGQ_U2OS_50MHz_G.csv 2CGQ_U2OS_50MHz_S.csv (b) Three-color confocal FLIM images of U2OS cells (GattaQuant® reference slide) The second dataset corresponds to three-color confocal FLIM images acquired from the same reference slide described above. In this case, an additional detection channel was used to capture the fluorescence lifetime signal from the DAPI-labeled nuclei alongside the Alexa Fluor–labeled mitochondrial and microtubule markers. These measurements were performed using a Leica TCS SP8 STED 3X microscope equipped with a FALCON-FLIM module, which employs photon-counting hybrid detectors combined with galvanometric scanning and a 63× high–numerical aperture oil-immersion objective. The excitation source operated at a repetition frequency of 78 MHz for lifetime acquisition. The images of this set are: 3CGQ_U2OS_78MHz_FLIM.ptu ATTO488_200nM_LaserPower10_10LineRep_10FrameAccum_80MHz.ptu (this is the lifetime reference for temporal analysis by phasors)