The influence of movement on the localization precision of sub‐resolution particles in fluorescence microscopy

Abstract

Obtaining sub‐resolution particle positions in fluorescence microscopy images is essential for single particle tracking and high‐resolution localization microscopy. While the localization precision of stationary single molecules or particles is well understood, the influence of particle motion during image acquisition has been largely neglected. Here, we address this issue and provide a theoretical description on how particle motion influences the centroid localization precision, both in case of 2‐D and 3‐D diffusion. In addition, a novel method is proposed, based on dual‐channel imaging, for the experimental determination of the localization precision of moving particles. For typical single particle tracking experiments, we show that the localization precision is approximately two‐fold worse than expected from the stationary theory. Strikingly, we find that the most popular localization method, based on the fitting of a Gaussian distribution, breaks down for lateral diffusion. Instead, the centroid localization method is found to perform well under all conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Chromosomal locus tracking with proper accounting of static and dynamic errors

“…(5) results from the fact that the effective PSF of a moving object is effectively larger than that of a stationary one. This term was found previously via a different derivation [23] and typically only results in a small inflation of σ . This term settles a standing discrepancy in the literature in that a similar expression was also previously reported in [22], but mistakenly without the factor of 3 in the denominator.…”

“…This treatment of σ is analogous to that done in [23] for the case of pure Brownian motion. Note also that we must multiply the rest of Eq.…”

“…(1) has been known for some time, though recent papers have pointed out that the σ that appears in Eq. (1) is not the same as the localization error of an immobile particle σ 0 , but instead includes an additional correction due to the spreading of photons over a greater area of the detector for a moving particle [22,23]. We discuss this in more detail in the next section.…”

Spot-On: robust model-based analysis of single-particle tracking experiments

“…Nevertheless, Figure Second, Spot-On models the localization error as the static mean localization error and this 410 feature can be used to infer the actual localization error from the data. However, the 411 localization error is affected both by the position of the particle with respect to the focal plane 412 (Lindén et al, 2017) and by motion blur (Deschout et al, 2012). Even though a high signal-413 to-background ratio and fast framerate/stroboscopic illumination help to mitigate these 414 disparities, it is likely that the localization error of fast moving particles will be higher than 415 the bound/slow-moving particles.…”

Particle tracking in drug and gene delivery research: State-of-the-art applications and methods

“…In addition, particle motion increases static error, because motion blur reduces localization precision. In fact, this can be important, as motion blur reduces localization precision by roughly two-fold under typical experimental conditions, as compared to stationary particles [141]. …”