scholarly journals Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging

2018 ◽  
Author(s):  
Falk Schneider ◽  
Dominic Waithe ◽  
Silvia Galiani ◽  
Jorge Bernadino de la Serna ◽  
Erdinc Sezgin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Temporal Organization ◽  
Live Cells ◽  
Apparent Diffusion Coefficients ◽  
Diffusion Dynamics ◽  
Spatio Temporal

AbstractThe diffusion dynamics in the cellular plasma membrane provides crucial insights into the molecular interactions, organization and bioactivity. Fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (STED-FCS) measures such dynamics with high spatial and temporal resolution and reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method; line interleaved excitation scanning STED-FCS (LIESS-FCS) which discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatio-temporal organization of GPI-anchored proteins in the plasma membrane of live cells which interestingly show multiple diffusion modes at different spatial positions.

Super-resolution optical microscopy of lipid plasma membrane dynamics

2015 ◽  
Vol 57 ◽  
pp. 69-80 ◽  
Author(s):  
Christian Eggeling
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Molecular Diffusion ◽  
Super Resolution ◽  
Optical Microscope ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Diffusion Dynamics ◽  
Lipid Protein Interactions

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.

scholarly journals Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

2019 ◽  
Author(s):  
Nirmalya Bag ◽  
David A. Holowka ◽  
Barbara A. Baird
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Mast Cells ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Resting Cells ◽  
Functional Protein ◽  
Diffusion Properties

ABSTRACTA myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Herein, we quantified the diffusion properties of a panel of structurally distinct lipid-anchored and transmembrane (TM) probes in RBL mast cells by multiplexed Imaging Fluorescence Correlation Spectroscopy. We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences as small as 10% in diffusion coefficients, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid-anchored probes can be explained by their dynamic partitioning into ordered proteo-lipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion by functional protein modules in both lipid-anchored and TM probes reflect additional complexity in steady-state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represent the dynamic resting steady-state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.

scholarly journals Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

2017 ◽  
Author(s):  
Dominic Waithe ◽  
Falk Schneider ◽  
Jakub Chojnacki ◽  
Mathias Clausen ◽  
Dilip Shrestha ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spatial Heterogeneity ◽  
Fluorescence Correlation Spectroscopy ◽  
Large Scale ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
The Other ◽  
Statistical Accuracy ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics

AbstractScanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

scholarly journals Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

2019 ◽  
Author(s):  
Aurélien Barbotin ◽  
Silvia Galiani ◽  
Iztok Urbančič ◽  
Christian Eggeling ◽  
Martin Booth
Keyword(s):  
Adaptive Optics ◽  
Molecular Diffusion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Correction Method ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Extreme Sensitivity

Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (3D) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.

scholarly journals G1/S transcription factors assemble in discrete clusters that increase in number as cells grow

2019 ◽  
Author(s):  
Labe Black ◽  
Sylvain Tollis ◽  
Guo Fu ◽  
Jean-Bernard Fiche ◽  
Savanna Dorsey ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Super Resolution ◽  
Temporal Organization ◽  
Stochastic Simulations ◽  
Live Cells ◽  
Number Of Clusters ◽  
Localization Microscopy ◽  
Spatio Temporal ◽  
Cluster Behavior ◽  
Target Promoters

AbstractThe spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing ∼8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor

2016 ◽  
Vol 52 (90) ◽  
pp. 13269-13272 ◽  
Author(s):  
Michael R. Dent ◽  
Ismael López-Duarte ◽  
Callum J. Dickson ◽  
Phoom Chairatana ◽  
Harry L. Anderson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Plasma Membranes ◽  
Live Cell ◽  
Phase Behaviour ◽  
Live Cells ◽  
Molecular Rotor ◽  
Membrane Phase ◽  
Artificial Lipid Bilayers ◽  
Cell Plasma

A thiophene-based molecular rotor was used to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.

scholarly journals Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

2009 ◽  
Vol 424 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Ilya Levental ◽  
Fitzroy J. Byfield ◽  
Pramit Chowdhury ◽  
Feng Gai ◽  
Tobias Baumgart ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Lipid Phase ◽  
Plasma Membrane Vesicles ◽  
Liquid Ordered ◽  
Lipid Phase Separation

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Sign in / Sign up

Export Citation Format

Share Document