Measuring molecular motions inside single cells with improved analysis of single-particle trajectories

Rowland, David J.; Biteen, Julie S.

doi:10.1016/j.cplett.2017.02.052

Cited by 16 publications

(20 citation statements)

References 34 publications

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“…Both single-molecule localization data sets were then analyzed with the same single-molecule tracking algorithm: trajectories were determined ( Fig. 3 c) by optimizing all possible pairings of molecules between consecutive frames using the Hungarian algorithm (30)(31)(32). Measured diffusion coefficients for PolC-PAmCherry in the high-background cells matched our previously reported low-background measurements (29) ( Fig.…”

Section: Validating Small-labs With Live-cell Singlemolecule Trackingsupporting

confidence: 65%

SMALL-LABS: Measuring Single-Molecule Intensity and Position in Obscuring Backgrounds

Isaacoff

Lee

et al. 2019

Biophysical Journal

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Single-molecule and super-resolution imaging relies on successful, sensitive, and accurate detection of the emission from fluorescent molecules. Yet, despite the widespread adoption of super-resolution microscopies, single-molecule data processing algorithms can fail to provide accurate measurements of the brightness and position of molecules in the presence of backgrounds that fluctuate significantly over time and space. Thus, samples or experiments that include obscuring backgrounds can severely, or even completely, hinder this process. To date, no general data analysis approach to this problem has been introduced that is capable of removing obscuring backgrounds for a wide variety of experimental modalities. To address this need, we present the Single-Molecule Accurate LocaLization by LocAl Background Subtraction (SMALL-LABS) algorithm, which can be incorporated into existing single-molecule and super-resolution analysis packages to accurately locate and measure the intensity of single molecules, regardless of the shape or brightness of the background. Accurate background subtraction is enabled by separating the foreground from the background based on differences in the temporal variations of the foreground and the background (i.e., fluorophore blinking, bleaching, or moving). We detail the function of SMALL-LABS here, and we validate the SMALL-LABS algorithm on simulated data as well as real data from single-molecule imaging in living cells.

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Section: Validating Small-labs With Live-cell Singlemolecule Trackingsupporting

confidence: 65%

SMALL-LABS: Measuring Single-Molecule Intensity and Position in Obscuring Backgrounds

Isaacoff

Lee

et al. 2019

Biophysical Journal

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“…A promising way to increase the precision of diffusion constant estimation is to quantify the localization error and include this in the model for the MLE or CVE [38,43]. When multiple underlying diffusional states are present, the model becomes more complex, and other approaches have been developed for this task, which are often based on Bayesian statistics [44][45][46][47]. Considering these recent developments, motion analysis of multistate diffusion seems to come within reach, but there is no golden standard yet, and the optimal analysis method still has to be determined on a case-by-case basis.…”

Section: Overcoming the Diffraction Limit: Single-molecule Localizatimentioning

confidence: 99%

Single-molecule observation of diffusion and catalysis in nanoporous solids

Maris

Meirer

et al. 2021

Adsorption

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Nanoporous solids, including microporous, mesoporous and hierarchically structured porous materials, are of scientific and technological interest because of their high surface-to-volume ratio and ability to impose shape- and size-selectivity on molecules diffusing through them. Enormous efforts have been put in the mechanistic understanding of diffusion–reaction relationships of nanoporous solids, with the ultimate goal of developing materials with improved catalytic performance. Single-molecule localization microscopy can be used to explore the pore space via the trajectories of individual molecules. This ensemble-free perspective directly reveals heterogeneities in diffusion and diffusion-related reactivity of individual molecules, which would have been obscured in bulk measurements. In this article, we review developments in the spatial and temporal characterization of nanoporous solids using single-molecule localization microscopy. We illustrate various aspects of this approach, and showcase how it can be used to follow molecular diffusion and reaction behaviors in nanoporous solids.

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“…Recorded Swi6-PAmCherry single-molecule positions were detected and localized with 2D Gaussian fitting with home-built MATLAB software as previously described, and connected into trajectories using the Hungarian algorithm (Munkres, 1957; Rowland and Biteen, 2017). These single-molecule trajectory datasets were analyzed by a non-parametric Bayesian framework to reveal heterogeneous dynamics (Karslake et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization

Biswas

Karslake

Chen

et al. 2021

Preprint

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HP1 proteins bind with low affinity but high specificity to sites of histone H3 lysine 9 methylation (H3K9me) in the genome. HP1 binding to H3K9me compartmentalizes the genome into transcriptionally inactive heterochromatin and actively transcribed euchromatin. A characteristic feature of HP1 proteins is their dynamic and rapid turnover from sites of heterochromatin formation. How low-affinity H3K9me recognition enables HP1 proteins to rapidly and efficiently traverse a complex and crowded chromatin landscape on the millisecond timescale remains a paradox. Here, we visualize the real-time motions of an HP1 homolog, the fission yeast protein Swi6, in its native chromatin environment. By analyzing the motions of Swi6 with high spatial and temporal resolution, we map individual mobility states that are directly linked to discrete biochemical intermediates. We find that nucleic acid binding titrates Swi6 away from sites of heterochromatin formation, whereas increasing the valency of chromodomain-mediated H3K9me recognition promotes specific chromatin localization. We propose that Swi6 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for protein turnover. Our high-resolution biophysical studies provide a comprehensive framework for in vivo biochemistry and reveal how the competing biochemical properties of Swi6 affect H3K9me recognition in living cells.

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Measuring molecular motions inside single cells with improved analysis of single-particle trajectories

Cited by 16 publications

References 34 publications

SMALL-LABS: Measuring Single-Molecule Intensity and Position in Obscuring Backgrounds

SMALL-LABS: Measuring Single-Molecule Intensity and Position in Obscuring Backgrounds

Single-molecule observation of diffusion and catalysis in nanoporous solids

HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization

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