------------------- GENERAL INFORMATION ------------------- Related publication (journal paper, book, etc.): Subdiffraction fluorescence lifetime imaging with Flipper-TR, Methods and Applications in Fluorescence (2026). DOI: 10.1088/2050-6120/aeb69f Date(s) of data collection: Collection of raw experimental data happened in days: 13/01/2026, 23/01/2026, 04/02/2026, 17/02/2026, 24/03/26, 30/06/26. Geographic location of data collection: Advanced Optical Microscopy Unit in CNB-CSIC (C. Darwin, 3, Fuencarral-El Pardo, 28049 Madrid) General description: The data are organised into five different folders. Four of the folders correspond to the figures in the paper and contain the raw data used to generate the figures, as well as the fitting values. The fifth folder, called “software,” contains the programs used to perform the fittings. -------------------------- SHARING/ACCESS INFORMATION -------------------------- Licenses/restrictions placed on the data, or limitations of reuse: - Links/relationships to previous or related data sets: None -------------------- DATA & FILE OVERVIEW -------------------- File list: Inside folder Fig1: Fig1_left_conf_profile.csv Fig1_left_fit_results.csv Fig1_left_STED_profile.csv Fig1_readme.docx Fig1_right_conf_profile.csv Fig1_right_fit_results.csv Fig1_right_STED_profile.csv Fig1_STED.tiff Inside folder Fig2: Fig2(a)_conf_from0ps_decay.csv Fig2(a)_STED_from0ps_decay.csv Fig2(b)_saturationcurve.csv Fig2(c)_fit.csv Fig2(c)_STED_from2900ps_decay.csv Fig2_fittings.tiff Fig2_readme.docx Inside folder Fig3: Fig3(b)_histogramdata_confocal.csv Fig3(b)_histogramdata_STED.csv Fig3(b)_histogramfit.csv Fig3(c)_conf_decay.csv Fig3(c)_decayfit.csv Fig3(c)_STED_decay.csv Fig3_comparison.tiff Fig3_readme.docx Inside folder Fig4: Fig4(b)_16lines_profile.csv Fig4(b)_4lines_profile.csv Fig4(b)_8lines_profile.csv Fig4(b)_fitresults.csv Fig4(c)_histogramdata_16lines.csv Fig4(c)_histogramdata_4lines.csv Fig4(c)_histogramdata_8lines.csv Fig4(c)_histogramfit.csv Fig4_lines.tiff Fig4_readme.docx Inside folder software: erf_gaussian_fitting.py example_hitogramfitting.ipynb example_profilesfitting.ipynb Fig1_left_conf_profile.csv Fig1_left_fit_results.csv Fig1_left_STED_profile.csv Fig3(b)_confocal_histofit.csv Fig3(b)_confocal_histofit.svg Fig3(b)_histogramdata_confocal.csv profiles.txt software_readme.docx step_lorentzian_fitting.py tools_histogram.py tools_profiles.py Relationship between files: the relationship between files as well as the contents of the files are explained inside each folders '*_readme.docx' Formats: All data files are in '*.csv' format Version: First uploaded version -------------------------- METHODOLOGICAL INFORMATION -------------------------- Description of methods used for *collection/generation* of data: For a thorough description of the microscopy methods, materials and samples used please go to the Methods section of the paper. The data here presented come from STED, confocal, STED-FLIM and confocal-FLIM images of B. subtilis bacteria labelled with probe Flipper-TR. Profile data are generated from raw intensity images using Fiji (https://imagej.net/software/fiji/). Decay data are generated from FLIM images using FLIMfit 5.1.1 (https://flimfit.org/downloads/5.1.1/) Histogram data are generated from lifetime images calculated using FLIMfit 5.1.1 with Fiji. Methods for *processing* the data: The fitting methods for profile and histogram data, and how to use them are described in the software folder 'software_readme.docx' Fitting of fluorescence decay was performed using FLIMfit 5.1.1 Software- or Instrument-specific information needed to *access/open/interpret* the data, including software and hardware version numbers: Phyton, Jupyter Environmental/experimental conditions: Confocal laser scanning microscopy (CLSM) and STED-FLIM images were acquired using a commercial Leica Stellaris 8 STED 3X microscope (Leica Microsystems) equipped with a 93x glycerol immersion objective with N.A.= 1.3. A pulsed white light laser (80 MHz, resulting in a 12.5 ns observation window) was used for excitation at 488 nm and a pulsed laser at 775 nm (50% laser power, 80MW/cm2 peak power density at the sample) for depletion. A phase element right before the objective lens was used to ensure circular polarization at the imaging plane. All images were acquired using 16 bits weight and 1024 x 1024 pixels with a pixel size of 20.36 nm x 20.36 nm. The emission was collected between 500 and 700 nm with a HyDX detector set to photon counting mode that discards the information containing more than 1 photon/count. Images were recorded using 0.6 Airy Unit which results in an optical section of 0.608 µm. The scan was run in unidirectional mode with a speed of 170 Hz with a maximum of 16 line repetitions and 1 frame repetition with a pixel dwell time of 4.48 µs. Images were collected using Leica Application Suite LAS X FLIM/FCS software. 2 µM Flipper-TR was added to the bacterium sample to promote fluorophore exchange and minimize photobleaching. Images were recorded at room temperature. -------------------------- DATA-SPECIFIC INFORMATION -------------------------- Variable list, defining any abbreviations, units of measure, codes or symbols used: Distance_(microns) Gray_Value: value of intensity from the intensity image Profile type: can be either ext (meaning it crosses the boundary from the outside to the inside of the bacterium in whichever direction) or int (crosses a membrane boundary from the inside to the inside of the bacterium) Image type: whether the profile comes from an STED image: STED or a confocal image: conf Direction: in the case of an ext profile, whether the direction is from the outside to the inside of the bacterium (1) or vice versa (-1) Param: each of the parameters of the fitting model, more details about this in 'Fig1_readme.docx' Cut-off time: The time at which we start the decay fitting. All time bins with lower values are excluded from the fitting. Fit decay: value of the fitted model at a certain time bin