title: Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal resolution.
abstract: Stochastic optical fluctuation imaging (SOFI) is a super-resolution fluorescence imaging technique that makes use of stochastic fluctuations in the emission of the fluorophores. During a SOFI measurement multiple fluorescence images are acquired from the sample, followed by the calculation of the spatiotemporal cumulants of the intensities observed at each position. Compared to other techniques, SOFI works well under conditions of low signal-to-noise, high background, or high emitter densities. However, it can be difficult to unambiguously determine the reliability of images produced by any superresolution imaging technique. In this work we present a strategy that enables the estimation of the variance or uncertainty associated with each pixel in the SOFI image. In addition to estimating the image quality or reliability, we show that this can be used to optimize the signal-to-noise ratio (SNR) of SOFI images by including multiple pixel combinations in the cumulant calculation. We present an algorithm to perform this optimization, which automatically takes all relevant instrumental, sample, and probe parameters into account. Depending on the optical magnification of the system, this strategy can be used to improve the SNR of a SOFI image by 40% to 90%. This gain in information is entirely free, in the sense that it does not require additional efforts or complications. Alternatively our approach can be applied to reduce the number of fluorescence images to meet a particular quality level by about 30% to 50%, strongly improving the temporal resolution of SOFI imaging.
authors.affiliation: Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Vandenberg
authors.affiliation: Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium.
authors.forename: Sam
authors.initials: S
authors.lastname: Duwé
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; École Polytechnique Fédérale de Lausanne, Laboratoire d'Optique Biomédicale, 1015 Lausanne, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Leutenegger
authors.affiliation: Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium.
authors.forename: Benjamien
authors.initials: B
authors.lastname: Moeyaert
authors.affiliation: Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium; Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Krajnik
authors.affiliation: École Polytechnique Fédérale de Lausanne, Laboratoire d'Optique Biomédicale, 1015 Lausanne, Switzerland.
authors.forename: Theo
authors.initials: T
authors.lastname: Lasser
authors.affiliation: Department of Chemistry, KULeuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Dedecker
keywords: (100.6640) Superresolution; (170.2520) Fluorescence microscopy