OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization

Front Plant Sci. 2024 Apr 19:15:1358935. doi: 10.3389/fpls.2024.1358935. eCollection 2024.

Abstract

Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

Keywords: BRI1; RLP44; cluster analysis; localization; photoconvertible/-activatable fluorophores; plasma membrane; single-particle tracking PALM; super-resolution microscopy.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Our research was supported by the German Research Foundation (DFG) via the CRC grant 1101 (project A09, D02, D03, Z02), the TRR grant 356 (project B01, B02) and individual grants to KH (HA 2146/22, HA 2146/23). We also thank the DFG for grants for scientific equipment (FUGG: INST 37/991-1, INST 37/992-1, INST 37/819-1, INST 37/965-1). We acknowledge support from the Open Access Publication Fund of the University of Tübingen.