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Raster Image Correlation Spectroscopy Performance Evaluation
Chalmers University of Technology, Sweden; University of Gothenburg, Sweden.
KU Leuven, Belgium; Hasselt University, Belgium .
Hasselt University, Belgium .
Chalmers University of Technology, Sweden; University of Gothenburg, Sweden.
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2019 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 117, nr 10, s. 1900-1914Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three- and fivefold. For D = 1 μm2 s−1, a typical value for intracellular measurements, ∼25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (≪50 × 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E = 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM–μM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical least-squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient. 

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Biophysical Society , 2019. Vol. 117, nr 10, s. 1900-1914
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URN: urn:nbn:se:ri:diva-40865DOI: 10.1016/j.bpj.2019.09.045Scopus ID: 2-s2.0-85074365080OAI: oai:DiVA.org:ri-40865DiVA, id: diva2:1376791
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Funding details: KU Leuven, C14/16/053; Funding details: Fonds Wetenschappelijk Onderzoek, FWO; Funding details: Knut och Alice Wallenbergs Stiftelse; Funding text 1: Dr. Kris Janssen (Chemistry, KU Leuven) is thanked for programming the acquisitioning software of the homebuilt microscope. Johan Hofkens (Molecular Imaging and Photonics, KU Leuven) is gratefully acknowledged for the usage of his imaging facilities. Financial support from the Swedish Foundation for Strategic Research and Knut and Alice Wallenberg Foundation is highly appreciated. J.H. acknowledges the KU Leuven for funding ( C14/16/053 ). V.L. acknowledges the UHasselt Bijzonder Onderzoeksfonds fund ( BOF17DOC11 ) for a PhD scholarship. Guillermo Solís Fernández is grateful for a PhD scholarship from the Research Foundation Flanders.

Tillgänglig från: 2019-12-10 Skapad: 2019-12-10 Senast uppdaterad: 2019-12-10Bibliografiskt granskad

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