Quantitative theory for the diffusive dynamics of liquid condensates

Key processes of biological condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date, a physics-based, quantitative framework for the dynamics of labeled condensate components is lacking. Here we derive the corresponding dynamic equations, building on the physics of phase separation, and quantitatively validate the related framework via experiments. We show that by using our framework we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Interestingly, our theory can also be used to determine a relationship between the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This enables us to investigate the effect of salt addition on partitioning and bypasses recently described quenching artifacts in the dense phase. Our approach opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities, and quantifying rates of biochemical reactions in liquid condensates.

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Quantitative Theory for the Diffusive Dynamics of Liquid Condensates

10.1101/2021.03.08.434288 ◽

2021 ◽

Author(s):

Lars Hubatsch ◽

Louise M. Jawerth ◽

Celina Love ◽

Jonathan Bauermann ◽

T.-Y. Dora Tang ◽

...

Keyword(s):

Temporal Analysis ◽

Dense Phase ◽

Time Resolved ◽

Fluorescence Measurements ◽

Accuracy And Precision ◽

Diffusive Dynamics ◽

Quantitative Understanding ◽

Spatio Temporal ◽

Molecule Dynamics ◽

Material Exchange

AbstractTo unravel the biological functions of membraneless liquid condensates it is crucial to develop a quantitative understanding of the physics underlying their dynamics. Key processes within such condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date we lack a physics-based quantitative framework for the dynamics of labeled condensate components. Here, we derive the corresponding theory, building on the physics of phase separation, and quantitatively validate this framework via experiments. We show that using our theory we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Strikingly, our theory can also be used to determine the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This bypasses recently described quenching artefacts in the dense phase, which can underestimate partition coefficients by orders of magnitude. Our experimentally verified theory opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities and quantifying rates of biochemical reactions in liquid condensates.

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MitoWave: Spatio-temporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion

10.1101/2020.05.21.108670 ◽

2020 ◽

Author(s):

D. Ashok ◽

B. O’Rourke

Keyword(s):

Membrane Potential ◽

Ventricular Myocytes ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Temporal Analysis ◽

Time Resolved ◽

Glass Coverslip ◽

Spatio Temporal ◽

Microscopy Data

AbstractMitochondria exhibit non-stationary unstable membrane potential (ΔΨm) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in response to I/R stress in an unbiased and reproducible manner. We designed a simple ImageJ-MATLAB-based workflow called ‘MitoWave’ to unravel dynamic mitochondrial ΔΨm changes that occur during ischemia and reperfusion. MitoWave employs MATLAB’s wavelet transform toolbox. In-vitro Ischemia was effected by placing a glass coverslip for 60 minutes on a monolayer of neonatal mouse ventricular myocytes (NMVMs). Removal of the coverslip allowed for reperfusion. ΔΨm response to I/R was recorded on a confocal microscope using TMRM as the indicator. As proof-of-principle, we used MitoWave analysis on ten invitro I/R experiments. Visual observations corroborated quantitative MitoWave analysis results in classifying the ten I/R experiments into five outcomes that were observed based on the oscillatory state of ΔΨm throughout the reperfusion time period. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion shows significant differences between five different outcomes (p< 0.001). Features such as time-points of ΔΨm depolarization during I/R, area of mitochondrial clusters and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. We found that mitochondria from NMVMs subjected to I/R oscillate in the frequency range of 8.6-45mHz, with a mean of 8.73±4.35mHz. Oscillating clusters had smaller areas ranging from 49.78±40.64 μm2 while non-oscillating clusters had larger areas 65.97±42.07μm2. A negative correlation between frequency and mitochondrial cluster area was seen. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a way to quantify complex time-resolved mitochondrial behavior. It provides an easy to follow workflow to automate microscopy analysis and allows for unbiased, reproducible quantitation of complex nonstationary cellular phenomena.Statement of SignificanceUnderstanding mitochondrial instability in Ischemia Reperfusion injury is key to determining efficacy of interventions. The MitoWave analysis is a powerful yet simple tool that enables even beginner MATALAB-Image J users to automate analysis of time-series from microscopy data. While we used it to detect ΔΨm changes during I/R, it can be adapted to detect any such spatio-temporal changes. It standardizes the quantitative analysis of complex biological signals, opens the door to in-depth screening of the genes, proteins and mechanisms underlying metabolic recovery after ischemia-reperfusion.

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On the Dynamics of Active Flow Control Over a Separated Airfoil Using Leading Edge Unsteady Blowing

Volume 1: Symposia, Parts A and B ◽

10.1115/fedsm2006-98097 ◽

2006 ◽

Author(s):

John J. Charonko ◽

Pavlos P. Vlachos

Keyword(s):

Flow Control ◽

Control Strategies ◽

Flow Behavior ◽

Active Flow Control ◽

Leading Edge ◽

Temporal Analysis ◽

Time Resolved ◽

Momentum Coefficient ◽

Pulsed Flow ◽

Spatio Temporal

Flow control has become universally accepted as an important technology that can potentially be implemented in future air and naval vehicles. It has been shown that flow control strategies can be effective in increasing lift, reducing or increasing drag, delaying or controlling separation, or even reattaching separated flows. However, there is still a need for better understanding of the physics that govern these processes. In order to address this, Time-Resolved Digital Particle Image Velocimetry (TRDPIV) was used in this experiment to quantitatively image the velocity field around a NACA-0015 airfoil at an angle of attack of 25 degrees and a Reynolds number of about 38,000. A slot was located approximately 0.1 chord lengths behind the leading edge and was used for pulsed flow injection at the natural shedding frequency of the wing. Ten cases with varying momentum ratios and pulse duty cycles were tested. A spatio-temporal analysis of the resulting flow fields was conducted. Reattachment and flow turning were observed, and the important features of the flow and their interactions with the wing are described. Analysis showed that the cycle-averaged momentum coefficient may govern flow behavior more than its peak value, and that the primary influence of flow control may be limited to the area within the pre-blowing separation region.

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Spatio-temporal Analysis of Electroconvection in Nematics

Zeitschrift für Naturforschung A ◽

10.1515/zna-1998-3-404 ◽

1998 ◽

Vol 53 (3-4) ◽

pp. 117-126

Author(s):

Heidrun Amm ◽

Maren Grigutsch ◽

Ralf Stannarius

Keyword(s):

Temporal Analysis ◽

Time Averaging ◽

Time Resolved ◽

Low Contrast ◽

Optical Images ◽

Optical Pattern ◽

Spatio Temporal ◽

Director Dynamics ◽

Convection Rolls ◽

First Time

Abstract We investigate electroconvection in nematic liquid crystals by means of optical microscopy. Time resolved optical images are used to study the director dynamics. For the first time we present instant images in the dielectric regime. A numerical simulation of the optical transmission patterns is performed on the basis of Fermat's principle. In the instant images of dielectric rolls, the periodicity of the observed optical pattern is equal to the wavelength λ0 of the convection rolls. The well known low contrast 'stationary' optical texture observed in conventional experiments results from time averaging of these instant images; its wavelength is λ0/2.

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