A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

F.W. LUND; D. WÜSTNER

doi:10.1111/jmi.12080

journal article Sep 19, 2013

A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

F.W. LUND D. WÜSTNER

Journal of Microscopy Vol. 252 No. 2 pp. 169-188 · Wiley

View at Publisher Save 10.1111/jmi.12080

Abstract

SummarySingle particle tracking (SPT) is becoming a standard method to extract transport parameters from time‐lapse image sequences of fluorescent vesicles in living cells. Another method to obtain these data is temporal image correlation spectroscopy (TICS), but this method is less often used for measurement of intracellular vesicle transport. Here, we present an extensive comparison of SPT and TICS. First we examine the effect of photobleaching, shading and noise on SPT and TICS analysis using simulated image sequences. To this end, we developed a simple photophysical model, which relates spatially varying illumination intensity to the bleaching propensity and fluorescence intensity of the moving particles. We found that neither SPT nor TICS are affected by photobleaching per se, but the transport parameters obtained by both methods are sensitive to the signal‐to‐noise ratio. In addition, the number of obtained trajectories in SPT is affected by noise. Diffusion constants determined by TICS are significantly overestimated when large immobile fluorescent structures are present in the image sequences, while the opposite is true for SPT. To improve the performance of both techniques, we compare three different methods for image denoising. Appropriate denoising significantly reduced the effect of noise and of immobile structures on both methods. Shape fluctuations of simulated particles had a more pronounced effect on TICS than on SPT analysis. In denoised images of fluorescent beads or cytosolic vesicles containing fluorescent protein NPC2 in human skin fibroblast cells, the transport parameters acquired by SPT and TICS were comparable emphasizing the value of both analysis methods.

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Details

Published: Sep 19, 2013
Vol/Issue: 252(2)
Pages: 169-188
License: View

Authors

F

F.W. LUND

Department of Biochemistry and Molecular Biology, University of Southern Denmark DK‐5230 Odense M Denmark

D

D. WÜSTNER

Department of Biochemistry and Molecular Biology, University of Southern Denmark DK‐5230 Odense M Denmark

Cite This Article

F.W. LUND, D. WÜSTNER (2013). A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility. Journal of Microscopy, 252(2), 169-188. https://doi.org/10.1111/jmi.12080

A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

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