Simulations of camera-based single-molecule fluorescence experiments

PLoS One. 2018 Apr 13;13(4):e0195277. doi: 10.1371/journal.pone.0195277. eCollection 2018.

Abstract

Single-molecule microscopy has become a widely used technique in (bio)physics and (bio)chemistry. A popular implementation is single-molecule Förster Resonance Energy Transfer (smFRET), for which total internal reflection fluorescence microscopy is frequently combined with camera-based detection of surface-immobilized molecules. Camera-based smFRET experiments generate large and complex datasets and several methods for video processing and analysis have been reported. As these algorithms often address similar aspects in video analysis, there is a growing need for standardized comparison. Here, we present a Matlab-based software (MASH-FRET) that allows for the simulation of camera-based smFRET videos, yielding standardized data sets suitable for benchmarking video processing algorithms. The software permits to vary parameters that are relevant in cameras-based smFRET, such as video quality, and the properties of the system under study. Experimental noise is modeled taking into account photon statistics and camera noise. Finally, we survey how video test sets should be designed to evaluate currently available data analysis strategies in camera-based sm fluorescence experiments. We complement our study by pre-optimizing and evaluating spot detection algorithms using our simulated video test sets.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Fluorescence Resonance Energy Transfer*
  • Markov Chains
  • Software*
  • Statistics as Topic
  • Video Recording

Grants and funding

Financial support by the European Research Council (ERC Starting Grant MIRNA 259092, to RKOS, http://erc.europa.eu/), the University of Zurich (to RKOS and Forschungskredit Grants FK-14-096 and FK-15-095, to RB; FK-13-108, to DK; FK-13-091, to MCASH; FK-57010302, to SLBK, http://www.researchers.uzh.ch/en/funding.html), as well as from the Swiss National Science Foundation, and the Swiss State Secretariat for Education and Research (COST Action CM1105, to RKOS) is gratefully acknowledged. This work was also partially accomplished within the project localizeIT (funding code 03IPT608X, to MR, http://localize-it.de/) funded by the Federal Ministry of Education and Research (BMBF, Germany, https://www.bmbf.de/) in the program of Entrepreneurial Regions InnoProfile-Transfer (https://www.unternehmen-region.de/de/1849.php). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.