This dataset supports findings of the research article “smICA: Open-Source Software for Quantitative, Lifetime-Resolved Mapping of Absolute Fluorophore Concentrations in Living Cells”. The article introduces smICA (Single-Molecule Image-to-Concentration Analyser), an open-source method and software package for converting time-resolved fluorescence microscopy images into spatial maps of absolute fluorophore concentration. The method combines time-resolved fluorescence imaging measurements, lifetime-based signal filtration, image segmentation, and photon-to-concentration calculations. It was validated against fluorescence correlation spectroscopy (FCS) measurements using fluorescently labelled dextrans and EGFP in living cells. Dextrans were also investigated in aqueous solutions. The dataset also supports an exemplary analysis of the time evolution of fluorescently labelled mRNA concentration in living cells.
The source measurements include time-tagged, time-resolved (TTTR) fluorescence microscopy data stored in PicoQuant PTU format and corresponding FCS measurements. The microscopy data were processed with smICA v2.2.0 (https://tkmist.github.io/smICA/). Depending on the analysis presented in the article, the raw photon data were analysed without any temporal filtering, with time gating based on the time-correlated single-photon counting (TCSPC) histogram of fluorescence decay times, or with statistical background filtering based on the TCSPC histogram pattern. Calibration files used for smICA analysis were prepared from calibration measurements in aqueous solutions of standard dyes for FCS calibration, as well as from brightness measurements utilised with FCS in the target sample. Those files provide molecular brightness and effective focal-volume parameters. ROI definitions are text files that provide the cellular or solution regions of interest used in the calculations. Phot2Conc processing within smICA converts the calibrated fluorescence signal into molecule-number and absolute-concentration results. The deposited data and scripts reproduce the analyses and outputs presented in Figures 5–8 of the article and the corresponding supplementary figures.
The repository contains four types of deposited files. The README.md file provides detailed instructions for extraction, software, processing, and reproduction, written in Markdown format. The files article_data.zip, together with all accompanying parts article_data.z01–article_data.z08, form a single multipart archive containing the selected raw PTU and FCS inputs, calibration files, ROI definitions, and intermediate analysis results. All multipart archive files must be downloaded and kept together; extraction should start with article_data.zip, as described in the README.md file. ARTICLE_WORKFLOW_SCRIPTS.zip contains readable Linux and Windows workflows that automate the complete analysis through the smICA TCP interface, from PTU extraction and filtering through calibration loading, ROI analysis, concentration calculation, result export, and preparation of figure data. This workflow was used to perform all analyses and provide all data required to prepare all figures of the manuscript. Depending on the computing power, repeating the workflow may take around 10 h or more.
FIGURES.zip is a self-contained package containing prepared CSV and NPZ plotting datasets extracted from the workflow results, plotting scripts, metadata, and reference outputs.
The repository provides two levels of data reproduction. The first level provides reconstruction of Figures 5–8 and the supplementary data. This level requires only the FIGURES.zip file. Repeating the smICA analysis is not necessary in this case. The second level requires reprocessing the data from the deposited complete multipart article_data archive, the workflow scripts from the ARTICLE_WORKFLOW_SCRIPTS.zip archive, and a compatible installation of smICA v2.2.0. Note that the directory names created during archive extraction should be preserved because the workflows resolve their inputs relative to these locations.
Further details on archive extraction, data inventory, calibration selection, filtering variants, software requirements, processing order, and figure reconstruction are available in the README.md file.
(2026-10-29)
