scholarly journals SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics

2019 ◽  
Author(s):  
J.D. Karslake ◽  
E.D. Donarski ◽  
S.A. Shelby ◽  
L.M. Demey ◽  
V.J. DiRita ◽  
...  
Keyword(s):  
Bayesian Statistics ◽  
Real Time ◽  
Single Molecule ◽  
Single Particle ◽  
Single Molecules ◽  
Living Cells ◽  
Cellular Systems ◽  
Nonparametric Bayesian ◽  
Experimental Systems ◽  
Nonparametric Bayesian Statistics

AbstractSingle-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory (SPT) dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of SPT datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.Statement of SignificanceSuper-resolution microscopy allows researchers access to the motions of individual molecules inside living cells. However, due to experimental constraints and unknown interactions between molecules, rigorous conclusions cannot always be made from the resulting datasets when model fitting is used. SMAUG (Single-Molecule Analysis by Unsupervised Gibbs sampling) is an algorithm that uses Bayesian statistical methods to uncover the underlying behavior masked by noisy datasets. This paper outlines the theory behind the SMAUG approach, discusses its implementation, and then uses simulated data and simple experimental systems to show the efficacy of the SMAUG algorithm. Finally, this paper applies the SMAUG method to two model living cellular systems—one bacterial and one mammalian—and reports the dynamics of important membrane proteins to demonstrate the usefulness of SMAUG to a variety of systems.

scholarly journals SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics

Methods ◽  
2020 ◽  
Author(s):  
Joshua D. Karslake ◽  
Eric D. Donarski ◽  
Sarah A. Shelby ◽  
Lucas M. Demey ◽  
Victor J. DiRita ◽  
...  
Keyword(s):  
Bayesian Statistics ◽  
Single Molecule ◽  
Nonparametric Bayesian ◽  
Nonparametric Bayesian Statistics

scholarly journals Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2826 ◽  
Author(s):  
Shangguo Hou ◽  
Courtney Johnson ◽  
Kevin Welsher
Keyword(s):  
Fluorescence Spectroscopy ◽  
Real Time ◽  
Temporal Resolution ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

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 Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

Open Biology ◽  
2012 ◽  
Vol 2 (6) ◽  
pp. 120090 ◽  
Author(s):  
Tchern Lenn ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
Molecular Basis ◽  
Life Sciences ◽  
Short Review ◽  
Living Cells ◽  
Cellular Systems ◽  
Biological Processes ◽  
Sensitivity Level ◽  
Experimental Approaches ◽  
The Impact

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.

scholarly journals Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

2016 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Polarization ◽  
Single Molecules ◽  
Living Cells ◽  
Molecular Assembly ◽  
Higher Order ◽  
Actin Network ◽  
Molecular Assemblies ◽  
Position And Orientation ◽  
Orientation Of Molecules

AbstractRegulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells, yet remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within, ∼350nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide new insights into how micron-scale ordered assemblies emerge from nanoscale molecules in living cells.Significance StatementIn living cells, the 3D architecture of molecular assemblies such as chromosomes, lipid bilayers, and the cytoskeleton is regulated through the interaction among their component molecules. Monitoring the position and orientation of constituent molecules is important for understanding the mechanisms that govern the structure and function of these assemblies. We have developed an instantaneous fluorescence polarization microscope to track the position and orientation of fluorescently labeled particles, including single molecules, which form micron-scale macromolecular assemblies in living cells. Our imaging approach is broadly applicable to the study of dynamic molecular interactions that underpin the function of micron-scale assemblies in living cells.

Probing Single Molecules in Single Living Cells

2001 ◽  
Vol 7 (S2) ◽  
pp. 28-29
Author(s):  
Tyler A. Byassee ◽  
Warren C. W. Chan ◽  
Shuming Nie
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Single Molecules ◽  
Cancer Cell Line ◽  
Living Cells ◽  
Cervical Cancer Cell Line ◽  
Biological Macromolecules ◽  
Background Fluorescence ◽  
Molecular Events

Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). However, a key remaining question is whether single-molecule methodologies could be developed to study complex molecular processes in living cells. in contrast to clean and well-controlled conditions in-vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules.We demonstrate that fluorescence detection of single molecules can be achieved by tightly focusing a laser beam into a living cell (see Figure 1). The observed background fluorescence is indeed higher than that in-vitro (e.g., pure biological buffer), but this background is continuous and stable, and does not significantly interfere with the measurement of single-molecule photon bursts. Specifically, we report single-molecule results on three types of extrinsic fluorescent molecules in cultured human HeLa cells (a cervical cancer cell line).

scholarly journals Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

2020 ◽  
Author(s):  
Sviatlana Shashkova ◽  
Mikael Andersson ◽  
Stefan Hohmann ◽  
Mark C Leake
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Single Molecule ◽  
Living Cells ◽  
Membrane Channels ◽  
Environmental Perturbations ◽  
Molecular Scale ◽  
Transmembrane Channels ◽  
Import And Export ◽  
Time Changes

AbstractMembrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.

scholarly journals Single-molecule imaging and tracking of molecular dynamics in living cells

2017 ◽  
Vol 4 (5) ◽  
pp. 739-760 ◽  
Author(s):  
Nan Li ◽  
Rong Zhao ◽  
Yahong Sun ◽  
Zi Ye ◽  
Kangmin He ◽  
...  
Keyword(s):  
Single Molecule ◽  
Quantitative Information ◽  
Living Cells ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Biomolecular Interaction ◽  
Ensemble Averaging ◽  
Spatiotemporal Resolution ◽  
Detection And Tracking ◽  
Single Molecule Studies

Abstract Unlike the ensemble-averaging measurements, the single-molecule imaging and tracking (SMIT) in living cells provides the real-time quantitative information about the locations, kinetics, dynamics and interactions of individual molecules in their native environments with high spatiotemporal resolution and minimal perturbation. The past decade has witnessed a transforming development in the methods of SMIT with living cells, including fluorescent probes, labeling strategies, fluorescence microscopy, and detection and tracking algorithms. In this review, we will discuss these aspects with a particular focus on their recent advancements. We will then describe representative single-molecule studies to illustrate how the single-molecule approaches can be applied to monitor biomolecular interaction/reaction dynamics, and extract the molecular mechanistic information for different cellular systems.

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