The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).
Mélodie C. A. S. Hadzic, Danny Kowerko, Richard Börner, Susann Zelger-Paulus, and Roland K. O. Sigel, "Detailed analysis of complex single molecule FRET data with the software MASH," Proc. SPIE 9711, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 971119 (Presented at SPIE BiOS: February 17, 2016; Published: 6 April 2016); https://doi.org/10.1117/12.2211191.
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