scholarly journals Tweezepy: A Python package for calibrating forces in single-molecule video-tracking experiments

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0262028
Author(s):  
Ian L. Morgan ◽  
Omar A. Saleh
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Three Dimensional ◽  
Likelihood Estimation ◽  
Video Tracking ◽  
Thermal Motion ◽  
Parameter Estimates ◽  
Comprehensive Overview ◽  
Calibration Methods ◽  
Python Package

Single-molecule force spectroscopy (SMFS) instruments (e.g., magnetic and optical tweezers) often use video tracking to measure the three-dimensional position of micron-scale beads under an applied force. The force in these experiments is calibrated by comparing the bead trajectory to a thermal motion-based model with the drag coefficient, γ, and trap spring constant, κ, as parameters. Estimating accurate parameters is complicated by systematic biases from spectral distortions, the camera exposure time, parasitic noise, and least-squares fitting methods. However, while robust calibration methods exist that correct for these biases, they are not always used because they can be complex to implement computationally. To address this barrier, we present Tweezepy: a Python package for calibrating forces in SMFS video-tracking experiments. Tweezepy uses maximum likelihood estimation (MLE) to estimate parameters and their uncertainties from a single bead trajectory via the power spectral density (PSD) and Allan variance (AV). It is well-documented, fast, easy to use, and accounts for most common sources of biases in SMFS video-tracking experiments. Here, we provide a comprehensive overview of Tweezepy’s calibration scheme, including a review of the theory underlying thermal motion-based parameter estimates, a discussion of the PSD, AV, and MLE, and an explanation of their implementation.

scholarly journals LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional single microtubule imaging

2018 ◽  
Author(s):  
Steve Simmert ◽  
Mohammad Kazem Abdosamadi ◽  
Gero Hermsdorf ◽  
Erik Schäffer
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Light Emitting Diode ◽  
Optical Design ◽  
Three Dimensional ◽  
Interference Microscopy ◽  
Light Emitting ◽  
Versatile Tool ◽  
Microscopy Techniques ◽  
3D Profile

AbstractOptical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be resolved with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-resolution 3D microscopy.OCIS codes(180.3170) Interference microscopy; (120.4570) Optical design of instruments (350.4855); Optical tweezers or optical manipulation.

A Cautionary Note on Incremental Fit Indices Reported by LISREL

Methodology ◽  
2005 ◽  
Vol 1 (2) ◽  
pp. 81-85 ◽  
Author(s):  
Stefan C. Schmukle ◽  
Jochen Hardt
Keyword(s):  
Factor Analysis ◽  
Maximum Likelihood ◽  
Structural Equation ◽  
Structural Equation Models ◽  
Null Model ◽  
Likelihood Estimation ◽  
Parameter Estimates ◽  
Fit Indices ◽  
Cautionary Note ◽  
Confirmatory Factor

Abstract. Incremental fit indices (IFIs) are regularly used when assessing the fit of structural equation models. IFIs are based on the comparison of the fit of a target model with that of a null model. For maximum-likelihood estimation, IFIs are usually computed by using the χ2 statistics of the maximum-likelihood fitting function (ML-χ2). However, LISREL recently changed the computation of IFIs. Since version 8.52, IFIs reported by LISREL are based on the χ2 statistics of the reweighted least squares fitting function (RLS-χ2). Although both functions lead to the same maximum-likelihood parameter estimates, the two χ2 statistics reach different values. Because these differences are especially large for null models, IFIs are affected in particular. Consequently, RLS-χ2 based IFIs in combination with conventional cut-off values explored for ML-χ2 based IFIs may lead to a wrong acceptance of models. We demonstrate this point by a confirmatory factor analysis in a sample of 2449 subjects.

Missing Data - Better "Not to Have Them", but What If You Do? (Part 1)

Marketing ZFP ◽  
2019 ◽  
Vol 41 (4) ◽  
pp. 21-32
Author(s):  
Dirk Temme ◽  
Sarah Jensen
Keyword(s):  
Missing Data ◽  
Statistical Power ◽  
Missing Values ◽  
Graphical Representation ◽  
Marketing Research ◽  
Likelihood Estimation ◽  
Parameter Estimates ◽  
Full Information Maximum Likelihood ◽  
Definition Of ◽  
Traditional Approaches

Missing values are ubiquitous in empirical marketing research. If missing data are not dealt with properly, this can lead to a loss of statistical power and distorted parameter estimates. While traditional approaches for handling missing data (e.g., listwise deletion) are still widely used, researchers can nowadays choose among various advanced techniques such as multiple imputation analysis or full-information maximum likelihood estimation. Due to the available software, using these modern missing data methods does not pose a major obstacle. Still, their application requires a sound understanding of the prerequisites and limitations of these methods as well as a deeper understanding of the processes that have led to missing values in an empirical study. This article is Part 1 and first introduces Rubin’s classical definition of missing data mechanisms and an alternative, variable-based taxonomy, which provides a graphical representation. Secondly, a selection of visualization tools available in different R packages for the description and exploration of missing data structures is presented.

scholarly journals Membrane fusion studied by colloidal probes

2021 ◽  
Vol 50 (2) ◽  
pp. 223-237 ◽  
Author(s):  
Hannes Witt ◽  
Filip Savić ◽  
Sarah Verbeek ◽  
Jörn Dietz ◽  
Gesa Tarantola ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Optical Tweezers ◽  
Three Dimensional ◽  
Biological Processes ◽  
Supported Membranes ◽  
Supported Bilayers ◽  
Force Microscopy ◽  
Advantages And Disadvantages ◽  
Atomic Force ◽  
Colloidal Probes

AbstractMembrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.

scholarly journals Optical tweezers in single-molecule biophysics

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Carlos J. Bustamante ◽  
Yann R. Chemla ◽  
Shixin Liu ◽  
Michelle D. Wang
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Molecule Biophysics

scholarly journals Assembly of multicomponent structures from hundreds of micron-scale building blocks using optical tweezers

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Jeffrey E. Melzer ◽  
Euan McLeod
Keyword(s):  
Optical Tweezers ◽  
Three Dimensional ◽  
Building Blocks ◽  
Optical Tweezer ◽  
Device Fabrication ◽  
Positional Accuracy ◽  
Component Structure ◽  
Material Development ◽  
Critical Process Parameters ◽  
Optical Positioning

AbstractThe fabrication of three-dimensional (3D) microscale structures is critical for many applications, including strong and lightweight material development, medical device fabrication, microrobotics, and photonic applications. While 3D microfabrication has seen progress over the past decades, complex multicomponent integration with small or hierarchical feature sizes is still a challenge. In this study, an optical positioning and linking (OPAL) platform based on optical tweezers is used to precisely fabricate 3D microstructures from two types of micron-scale building blocks linked by biochemical interactions. A computer-controlled interface with rapid on-the-fly automated recalibration routines maintains accuracy even after placing many building blocks. OPAL achieves a 60-nm positional accuracy by optimizing the molecular functionalization and laser power. A two-component structure consisting of 448 1-µm building blocks is assembled, representing the largest number of building blocks used to date in 3D optical tweezer microassembly. Although optical tweezers have previously been used for microfabrication, those results were generally restricted to single-material structures composed of a relatively small number of larger-sized building blocks, with little discussion of critical process parameters. It is anticipated that OPAL will enable the assembly, augmentation, and repair of microstructures composed of specialty micro/nanomaterial building blocks to be used in new photonic, microfluidic, and biomedical devices.

scholarly journals Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

2021 ◽  
Vol 7 (6) ◽  
pp. eabe3902
Author(s):  
Martin Rieu ◽  
Thibault Vieille ◽  
Gaël Radou ◽  
Raphaël Jeanneret ◽  
Nadia Ruiz-Gutierrez ◽  
...  
Keyword(s):  
High Precision ◽  
High Throughput ◽  
Single Molecule ◽  
Particle Tracking ◽  
Focal Plane ◽  
Tracking System ◽  
Three Dimensional ◽  
3D Tracking ◽  
Single Molecule Force Spectroscopy ◽  
A New Technique

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

scholarly journals Weakening of resistance force by cell–ECM interactions regulate cell migration directionality and pattern formation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masaya Hagiwara ◽  
Hisataka Maruyama ◽  
Masakazu Akiyama ◽  
Isabel Koh ◽  
Fumihito Arai
Keyword(s):  
Cell Migration ◽  
Pattern Formation ◽  
Epithelial Cells ◽  
Optical Tweezers ◽  
Three Dimensional ◽  
Lung Epithelial Cells ◽  
Resistance Force ◽  
Collective Cell Migration ◽  
Physical Forces ◽  
Cellular Movement

AbstractCollective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell–cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

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