Tomasz Kalwarczyk | Searcharxiv

arXiv2026

FcsIT: An Open-Source, Cross-Platform Tool for Correlation and Analysis of Fluorescence Correlation Spectroscopy Data

FcsIT is a platform-independent, open-source tool for calculating the correlation and fitting fluorescence correlation spectroscopy data. The software is written in Python and uses a powerful Dear PyGUI engine for its interface. It provides reading and correlating the TTTR data, as well as TCSPC filtering of the photon time-trace data. The circular-block bootstrap method applied to the calculation of correlation data and its variance results in data quality comparable to that obtained with commercially available software. An intuitive fitting interface provides efficient analysis of large datasets and includes nine predefined mathematical models for fitting correlation curves. Moreover, it allows users to add their own models in a user-friendly manner. Validation of the FcsIT tool against simulated FCS data and real FCS experiments confirms its usability and potential appeal to a wide variety of FCS users.

q-bio.QM↗

arXiv2024

smICA: Open-Source Software for Quantitative, Lifetime-Resolved Mapping of Absolute Fluorophore Concentrations in Living Cells

Advanced microscopy techniques are essential in biomedical research for visualising and tracking biomolecules within living cells and their compartments. Conventional fluorescence microscopy methods, however, often struggle with accurately measuring the absolute concentrations of fluorescent probes in living cells. To overcome these limitations, we introduce an open-source analysis tool, smICA (Single-Molecule Image to Concentration Analyser). The smICA method offers quantitative mapping of absolute fluorophore concentrations, lifetime-resolved filtering methods of the signal, intensity-based cell segmentation, and requires only a few photons per pixel. Our approach also reduces the time required to determine the mean concentration per cell compared to the standard FCS measurement performed in multiple posts. To highlight the robustness of the method, we validated it against standard fluorescence correlation spectroscopy (FCS) measurements by performing in vitro (polymers in aqueous solution) and in vivo (polymers and EGFP in living cells) experiments. Finally, we present exemplary studies on the time evolution of fluorescently labelled mRNA concentration in living cells. The presented methodology, along with the software, is a promising tool for quantitative single-cell studies, including, but not limited to, protein expression, biomolecule degradation (such as proteins and mRNA), and monitoring enzymatic reactions.

q-bio.QM↗