Raster Image Correlation Spectroscopy Performance Evaluation
Artikel i vetenskaplig tidskrift, 2019

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 mu m(2) s(-1), a typical value for intracellular measurements, similar to 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 x 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-mM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical leastsquares 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.

Författare

Marco Longfils

Chalmers, Matematiska vetenskaper, Tillämpad matematik och statistik

Göteborgs universitet

Nick Smisdom

Universiteit Hasselt

KU Leuven

Marcel Ameloot

Universiteit Hasselt

Mats Rudemo

Chalmers, Matematiska vetenskaper, Tillämpad matematik och statistik

Göteborgs universitet

Veerle Lemmens

Universiteit Hasselt

KU Leuven

Guillermo Solis Fernandez

RISE Research Institutes of Sweden

Magnus Roding

RISE Research Institutes of Sweden

Niklas Loren

RISE Research Institutes of Sweden

Chalmers, Fysik, Eva Olsson Group

Jelle Hendrix

Universiteit Hasselt

Aila Särkkä

Göteborgs universitet

Chalmers, Matematiska vetenskaper, Tillämpad matematik och statistik

Biophysical Journal

0006-3495 (ISSN) 1542-0086 (eISSN)

Vol. 117 10 1900-1914

Ämneskategorier

Biofysik

Datorseende och robotik (autonoma system)

DOI

10.1016/j.bpj.2019.09.045

PubMed

31668746

Mer information

Senast uppdaterat

2019-12-06