scholarly journals Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

2018 ◽  
Author(s):  
J. Rocha ◽  
J. Corbitt ◽  
T. Yan ◽  
C. Richardson ◽  
A. Gahlmann
Keyword(s):  
Data Analysis ◽  
Single Molecule ◽  
Time Window ◽  
Analysis Framework ◽  
Cellular Functions ◽  
Single Molecule Tracking ◽  
State Switching ◽  
Single Protein ◽  
Simplifying Assumptions ◽  
Analytical Approaches

AbstractThe trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in 2D or 3D single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion (TAD) analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.

scholarly journals A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

2017 ◽  
Author(s):  
Rajarshi P Ghosh ◽  
J Matthew Franklin ◽  
Will E. Draper ◽  
Quanming Shi ◽  
Jan T. Liphardt
Keyword(s):  
Single Molecule ◽  
Precision Measurement ◽  
Single Molecules ◽  
Living Cells ◽  
Background Suppression ◽  
Molecular Heterogeneity ◽  
Single Molecule Tracking ◽  
High Brightness ◽  
Cellular Processes ◽  
State Switching

AbstractCellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

scholarly journals Single molecule tracking and analysis framework including theory-predicted parameter settings

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Timo Kuhn ◽  
Johannes Hettich ◽  
Rubina Davtyan ◽  
J. Christof M. Gebhardt
Keyword(s):  
Single Molecule ◽  
Spatial Information ◽  
Reaction Rates ◽  
Data Sets ◽  
Analysis Framework ◽  
Tracking Errors ◽  
Experimental Conditions ◽  
Single Molecule Tracking ◽  
External Software ◽  
User Friendly

AbstractImaging, tracking and analyzing individual biomolecules in living systems is a powerful technology to obtain quantitative kinetic and spatial information such as reaction rates, diffusion coefficients and localization maps. Common tracking tools often operate on single movies and require additional manual steps to analyze whole data sets or to compare different experimental conditions. We report a fast and comprehensive single molecule tracking and analysis framework (TrackIt) to simultaneously process several multi-movie data sets. A user-friendly GUI offers convenient tracking visualization, multiple state-of-the-art analysis procedures, display of results, and data im- and export at different levels to utilize external software tools. We applied our framework to quantify dissociation rates of a transcription factor in the nucleus and found that tracking errors, similar to fluorophore photobleaching, have to be considered for reliable analysis. Accordingly, we developed an algorithm, which accounts for both tracking losses and suggests optimized tracking parameters when evaluating reaction rates. Our versatile and extensible framework facilitates quantitative analysis of single molecule experiments at different experimental conditions.

scholarly journals Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

2015 ◽  
Vol 112 (3) ◽  
pp. E267-E276 ◽  
Author(s):  
Sulagna Das ◽  
Taofei Yin ◽  
Qingfen Yang ◽  
Jingqiao Zhang ◽  
Yi I. Wu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Membrane Lipids ◽  
Exchange Process ◽  
Single Particle Tracking ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Membrane Translocation ◽  
Cellular Functions ◽  
Single Molecule Tracking ◽  
Spatial Regulation

Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.

scholarly journals Single molecule tracking and analysis framework including theory-predicted parameter settings

2020 ◽  
Author(s):  
Timo Kuhn ◽  
Johannes Hettich ◽  
J. Christof M. Gebhardt
Keyword(s):  
Single Molecule ◽  
Spatial Information ◽  
Reaction Rates ◽  
Data Sets ◽  
Analysis Framework ◽  
Tracking Errors ◽  
Experimental Conditions ◽  
Single Molecule Tracking ◽  
External Software ◽  
User Friendly

AbstractImaging, tracking and analyzing individual biomolecules in living systems is a powerful technology to obtain quantitative kinetic and spatial information such as reaction rates, diffusion coefficients and localization maps. Common tracking tools often operate on single movies and require additional manual steps to analyze whole data sets or to compare different experimental conditions. We report a fast and comprehensive single molecule tracking and analysis framework (TrackIt) to simultaneously process several multi-movie data sets. A user-friendly GUI offers convenient tracking visualization, multiple state-of-the-art analysis procedures, display of results, and data im- and export at different levels to utilize external software tools. We applied our framework to quantify dissociation rates of a transcription factor in the nucleus and found that tracking errors, similar to fluorophore photobleaching, have to be considered for reliable analysis. Accordingly, we developed an algorithm, which accounts for both tracking losses and suggests optimized tracking parameters when evaluating reaction rates. Our versatile and extensible framework facilitates quantitative analysis of single molecule experiments at different experimental conditions.

Design and Analysis for Quantitative Research in Music Education

2018 ◽  
Author(s):  
Peter Miksza ◽  
Kenneth Elpus
Keyword(s):  
Education Research ◽  
Data Analysis ◽  
Music Education ◽  
General Education ◽  
Research Design ◽  
Quantitative Research ◽  
Traditional Design ◽  
Design Type ◽  
To Come ◽  
Analytical Approaches

This book is an introduction to quantitative research design and data analysis presented in the context of music education scholarship. The book aims for readers to come away with a familiarity of prototypical research design possibilities as well as a fundamental understanding of data analysis techniques necessary for carrying out scientific inquiry. The book includes examples that demonstrate how the methodological and statistical concepts presented throughout can be applied to pertinent issues in music education. For the majority of Part I, the strategy is to present traditional design categories side by side with explanations of general analytical approaches for dealing with data yielded from each respective design type. Part II consists of chapters devoted to methodological and analytical approaches that have become common in related fields (e.g., psychology, sociology, general education research, educational policy) but are as yet not frequently exploited by music education researchers. Ultimately, this work is motivated by a desire to help scholars acquire the means to actualize their research curiosities and to contribute to the advancement of rigor in music education research throughout the profession at large.

Polarization-resolved single-molecule tracking reveal strange dynamics of individual fluorescent tracers through a deep rubbery polymer network

2021 ◽  
Author(s):  
Jaladhar Mahato ◽  
Sukanya Bhattacharya ◽  
Dharmendar Kumar Sharma ◽  
Arindam Chowdhury
Keyword(s):  
Single Molecule ◽  
Local Structure ◽  
Biological Systems ◽  
Polymer Network ◽  
Soft Materials ◽  
Complex Environments ◽  
Fluorescent Tracers ◽  
Single Molecule Tracking ◽  
Structure And Dynamics

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However,...

scholarly journals Synthesis and Photostability of Unimolecular Submersible Nanomachines: Toward Single-Molecule Tracking in Solution

2016 ◽  
Vol 18 (10) ◽  
pp. 2343-2346 ◽  
Author(s):  
Víctor García-López ◽  
Jonathan Jeffet ◽  
Shunsuke Kuwahara ◽  
Angel A. Martí ◽  
Yuval Ebenstein ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Tracking
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