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

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Single 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.
Original languageEnglish
JournalJournal of Microscopy
Volume252
Issue number2
Pages (from-to)169-188
ISSN0022-2720
DOIs
Publication statusPublished - 2013

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Noise
Signal-To-Noise Ratio
Lighting
Fibroblasts
Fluorescence
Skin
human NPC2 protein

Keywords

  • Cholesterol
  • Diffusion
  • Image denoising
  • Niemann-Pick disease
  • Photobleaching
  • Transport

Cite this

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title = "A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility",
abstract = "Single 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.",
keywords = "Cholesterol, Diffusion, Image denoising, Niemann-Pick disease, Photobleaching, Transport",
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A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility. / Lund, F. W.; Wustner, D.

In: Journal of Microscopy, Vol. 252, No. 2, 2013, p. 169-188.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

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AU - Wustner, D.

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AB - Single 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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