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Open AccessArticle

Temporal Filtering to Improve Single Molecule Identification in High Background Samples

1
Institute of Applied Physics, TU Wien, Getreidemarkt 9, A-1060 Vienna, Austria
2
Department of Microbiology, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria
3
Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstrasse 1a, A-1060 Vienna, Austria
*
Author to whom correspondence should be addressed.
Current Address: Institute of Food Technology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Molecules 2018, 23(12), 3338; https://doi.org/10.3390/molecules23123338
Received: 30 October 2018 / Revised: 6 December 2018 / Accepted: 13 December 2018 / Published: 17 December 2018
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable. View Full-Text
Keywords: single molecule microscopy; super-resolution microscopy; Fourier filter; background fluorescence; image processing single molecule microscopy; super-resolution microscopy; Fourier filter; background fluorescence; image processing
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Reismann, A.W.A.F.; Atanasova, L.; Schrangl, L.; Zeilinger, S.; Schütz, G.J. Temporal Filtering to Improve Single Molecule Identification in High Background Samples. Molecules 2018, 23, 3338.

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