Biology subjects

Milstein, J. N.

Publications and source records attributed to Milstein, J. N..

10 recordsLinked to original sources

bioRxiv2023

Extending the Range of Rupture Force Measurements with Axial Optical Tweezers

Axial optical tweezers provide a natural geometry for performing biomechanical assays such as rupture force measurements of protein binding. Axial traps, however, are typically weaker than their lateral counterparts requiring high laser power to maintain a well calibrated, linear restoring force. Here we show how to extend the spatial range over which well calibrated forces can be applied by considering aberration effects and extend the range of applied forces by accounting for the nonlinear response that appears when an optically trapped bead is moved far from the trap center. These refinements to the force calibration can be used to apply higher axial forces at reduced laser powers deeper into a sample. To illustrate the method, we reproduce both the linear extension regime and overstretching transition observed in dsDNA at significantly reduced laser powers. http://dx.doi.org/10.1364/ao.XX.XXXXXX

biophysics↗

bioRxiv2023

Spatial exclusion leads to tug-of-war ecological dynamics between competing species within microchannels

Competition is ubiquitous in microbial communities, shaping both their spatial and temporal structure and composition. Many classic minimal models, such as the Moran model, have been employed in ecology and evolutionary biology to understand the role of fixation and invasion in the maintenance of a population. Informed by recent experimental studies of cellular competition in confined spaces, we extend the Moran model to explicitly incorporate spatial exclusion through mechanical interactions among cells within a one-dimensional, open microchannel. The results of our spatial exclusion model differ significantly from those of its classical counterpart. The fixation/extinction probability of a species sharply depends on the species initial relative abundance, and the mean time to fixation is greatly accelerated, scaling logarithmically, rather than algebraically, with the system size. In non-neutral cases, spatial exclusion tends to attenuate the effects of fitness differences on the probability of fixation, and the fixation times increase as the relative fitness differences between species increase. Successful fixation by invasive species, whether through mutation or immigration, are also less probable on average than in the Moran model. Surprisingly, in the spatial exclusion model, successful fixations occur on average more rapidly in longer channels. The mean time to fixation heuristically arises from the boundary between populations performing either quasi-neutral diffusion, near a semi-stable fixed point, or quasi-deterministic avalanche dynamics away from the fixed point. These results, which can be tested in microfluidic monolayer devices, have implications for the maintenance of species diversity in dense bacterial ecosystems where spatial exclusion is central to the competition, such as in organized biofilms or intestinal crypts. The results may be broadly applied to any system displaying tug-of-war type dynamics with a region of quasi-neutral diffusion centered around regions of deterministic population collapse. Author summaryCompetition for territory between different species has far reaching consequences for the diversity and fate of bacterial communities. In this study, we theoretically and computationally study the competitive dynamics of two bacterial populations competing for space in confined environments. The model we develop extends classical models that have served as paradigms for understanding competitive dynamics but did not explicitly include spatial exclusion. We find that spatial effects drastically change the probability of one species successfully outcompeting the other and accelerates the mean time it takes for a species to exclude the other from the environment. In comparison to the predictions of population models that neglect spatial exclusion, species with higher selective advantages are less heavily favoured to outcompete their rival species. Moreover, spatial exclusion influences the success of an invasive species taking over a densely populated community. Compared to classical well-mixed models, there is a reduction in the effectiveness of an invaders fitness advantage at improving the chances of taking over the population. Our results show that spatial exclusion has rich and unexpected repercussions on species dominance and the long-time composition of populations. These must be considered when trying to understand complex bacterial ecosystems such as biofilms and intestinal flora.

ecology↗

bioRxiv2021

Quantitative Single-Molecule Imaging with Statistical Machine Learning

Single-molecule localization microscopy (SMLM) is a super-resolution technique capable of rendering nanometer scale images of cellular structures. Recently, much effort has gone into developing SMLM into a quantitative method capable of determining the abundance and stoichiometry of macromolecular complexes. These methods often require knowledge of the complex photophysical properties of photoswitchable flourophores. We previously developed a simpler method built upon the observation that most photswitchable fluorophores emit an exponentially distributed number of blinks before photobleaching, but its utility was limited by the need to calibrate for the blinking distribution. Here we extend this method by incorporating a machine learning technique known as Expectation-Maximization (EM) and apply it to a statistical mixture model of monomers, dimers and trimers. We show that the protomer fractions and the underlying single-fluorophore blinking distributions can be inferred, simultaneously, from SMLM datasets, obviating the need for an additional calibration and greatly expanding the applicability of this technique. To illustrate the utility of our approach, we benchmark the method on both simulated datasets and experimental datasets assembled from dSTORM images of Alexa-647 labeled DNA nanostructures.

biophysics↗

bioRxiv2021

Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy

In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photo-switching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.

biophysics↗

bioRxiv2020

An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

We demonstrate an ultra-stable, highly dense single-molecule assay ideal for observing protein-DNA interactions. Stable click Tethered Particle Motion (scTPM) leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, has a high antifouling resistance, is stable at elevated temperatures to at least 45 {degrees}C, and is compatible with Mg2+, an important ionic component of many regulatory protein-DNA interactions. Prepared samples remain stable, with little degradation, for > 6 months in physiological buffers. These improvements enabled us to study previously inaccessible sequence and temperature dependent effects on DNA binding by the bacterial protein H-NS, a global transcriptional regulator found in E. Coli. This greatly improved assay can directly be translated to accelerate existing tethered particle based, single-molecule biosensing applications.

biophysics↗

bioRxiv2019

Pinhole Optical Tweezers: Extending the Fluorescence Lifetime in the Presence of an Optical Trap by Wavefront Engineering

We present a new method for combining optical tweezers with single-molecule fluorescence in an engineered geometry we have coined a pinhole optical trap. By utilizing an appropriately constructed Laguerre-Gaussian (LG) or donut beam, and applying force along the axis of the trapping laser, one can maintain a low-intensity region of near-infrared (IR) light directly below the optical trap in which a biomolecule may be probed by both force spectroscopy and fluorescence. We show that within this region of low IR light intensity, the photobleaching lifetime of Alexa-647, an organic dye that is particularly sensitive to the high intensity trap light, can be significantly extended. This approach enables us to spatially separate the trap light from the fluorescence illumination without the need to physically separate, by many micrometers, the optical trap from the biological sample.

biophysics↗

bioRxiv2019

FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) yields an image resolution 1-2 orders of magnitude below that of conventional light microscopy, resolving fine details on intracellular structure and macromolecular organization. The massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Density based clustering algorithms, such as DBSCAN, can provide spatial statistics on protein/nucleic acid aggregation or dispersion while explicitly identifying macromolecular clusters. The performance of DBSCAN, however, is typically dependent upon an arbitrary, or at least highly subjective, parametric tuning of the algorithm. Moreover, DBSCAN can be computationally expensive, which makes it arduous to evaluate on large image stacks. This is all the more important in 3-dimensions where there exist limited alternatives for quantifying clustering in SMLM data, and where a 2-dimensional analysis of true 3-dimensional data may give rise to image artefacts. We have developed an open-source software package in Python for both identifying and quantifying spatial clustering in 3-dimensional SMLM datasets. FOCAL3D is an extension of our previously developed, 2-dimensional, grid based clustering algorithm FOCAL. FOCAL3D provides a highly efficient way to spatially cluster SMLM datasets, scaling linearly with the number of localizations, and the algorithmic parameters may be systematically optimized so that the resulting analysis is insensitive to variation over a range of parameter choices. We initially validate the performance and parametric insensitivity of FOCAL3D on simulated datasets, then apply the algorithm to 3-dimensional, astigmatic dSTORM images of the nuclear pore complex in human osteosarcoma cells.\n\nThe data and software package are available at: http://www.utm.utoronto.ca/milsteinlab/software/

bioinformatics↗

bioRxiv2019

Nanoscopic Stoichiometry and Single-Molecule Counting

Single-molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here we present a method for molecular counting with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. We provide a practical guide to molecular counting, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLMs utility for optical -omics analysis.

biophysics↗

bioRxiv2017

Polarization effects and the calibration of a donut beam axial optical tweezers for single-molecule force spectroscopy

Advances in light shaping techniques are leading to new tools for optical trapping and micromanipulation. For example, optical tweezers made from Laguerre-Gaussian or donut beams display an increased axial trap strength and can impart angular momentum to rotate a specimen. However, their application to precision, biophysical measurements remains limited as their are a number of challenges to applying this tool to optical force spectroscopy. One notable complication, not present when trapping with a Gaussian beam, is that the polarization of the trap light can significantly affect the tweezers strength as well as the precise location of the trap. In this article, we provide a practical implementation of a donut beam optical tweezers for applying axial forces. We show how to precisely calibrate the height of the optical trap above the coverslip surface while accounting for focal shifts in the trap position that arise due to radiation pressure, mismatches in the index of refraction, and polarization induced intensity variations.

biophysics↗

bioRxiv2016

Resolving Biology Beyond the Diffraction Limit with Single-Molecule Localization Microscopy

Optical imaging provides a window into the microscopic world, but the level of observable detail is ultimately limited by the wavelength of light being employed. By harnessing the physics of photoswitchable dyes and fluorescent proteins, single-molecule localization microscopy (SMLM) provides a window into the nano-world of biology. This mini-review article provides a short overview of SMLM and discusses some of its prospects for the future.

biophysics↗