scholarly journals Computationally-efficient spatiotemporal correlation analysis super-resolves anomalous diffusion

2020 ◽  
Author(s):  
Shawn Yoshida ◽  
William Schmid ◽  
Nam Vo ◽  
William Calabrase ◽  
Lydia Kisley
Keyword(s):  
Correlation Analysis ◽  
Anomalous Diffusion ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Brownian Diffusion ◽  
Computationally Efficient ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics ◽  
Nanoscale Structure ◽  
User Friendly

AbstractAnomalous diffusion dynamics in confined nanoenvironments govern the macroscale properties and interactions of many biophysical and material systems. Currently, it is difficult to quantitatively link the nanoscale structure of porous media to anomalous diffusion within them. Fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) has been shown to extract nanoscale structure and Brownian diffusion dynamics within gels, liquid crystals, and polymers, but has limitations which hinder its wider application to more diverse, biophysically-relevant datasets. Here, we parallelize the least-squares curve fitting step on a GPU improving computation times by up to a factor of 40, implement anomalous diffusion and two-component Brownian diffusion models, and make fcsSOFI more accessible by packaging it in a user-friendly GUI. We apply fcsSOFI to simulations of the protein fibrinogen diffusing in polyacrylamide of varying matrix densities and super-resolve locations where slower, anomalous diffusion occurs within smaller, confined pores. The improvements to fcsSOFI in speed, scope, and usability will allow for the wider adoption of super-resolution correlation analysis to diverse research topics.

scholarly journals Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

2021 ◽  
Author(s):  
Silvia Galiani ◽  
Katharina Reglinski ◽  
Pablo Carravilla ◽  
Aurelien Barbotin ◽  
Iztok Urbančič ◽  
...  
Keyword(s):  
Molecular Interactions ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Interaction Dynamics ◽  
Diffusion Dynamics ◽  
Interaction Partners ◽  
Target Binding ◽  
Import Receptor

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

scholarly journals FoCuS-point: software for STED fluorescence correlation and time-gated single photon counting

2015 ◽  
Vol 32 (6) ◽  
pp. 958-960 ◽  
Author(s):  
Dominic Waithe ◽  
Mathias P. Clausen ◽  
Erdinc Sezgin ◽  
Christian Eggeling
Keyword(s):  
Single Photon ◽  
Photon Counting ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Single Photon Counting ◽  
Fluorescence Correlation ◽  
Point Data ◽  
User Friendly ◽  
Kinetics Of ◽  
Focus Point

Abstract Motivation: Fluorescence Correlation Spectroscopy (FCS) is a popular tool for measuring molecular mobility and how mobility relates to molecular interaction dynamics and bioactivity in living cells. The FCS technique has been significantly advanced by its combination with super-resolution STED microscopy (STED-FCS). Specifically, the use of gated detection has shown great potential for enhancing STED-FCS, but has also created a demand for software which is efficient and also implements the latest algorithms. Prior to this study, no open software has been available which would allow practical time-gating and correlation of point data derived from STED-FCS experiments. Results: The product of this study is a piece of stand-alone software called FoCuS-point. FoCuS-point utilizes advanced time-correlated single-photon counting (TCSPC) correlation algorithms along with time-gated filtering and innovative data visualization. The software has been designed to be highly user-friendly and is tailored to handle batches of data with tools designed to process files in bulk. FoCuS-point also includes advanced fitting algorithms which allow the parameters of the correlation curves and thus the kinetics of diffusion to be established quickly and efficiently. Availability and implementation: FoCuS-point is written in python and is available through the github repository: https://github.com/dwaithe/FCS_point_correlator. Furthermore, compiled versions of the code are available as executables which can be run directly in Linux, Windows and Mac OSX operating systems. Contact: [email protected]

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 Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

2020 ◽  
Author(s):  
Valentin Dunsing ◽  
Annett Petrich ◽  
Salvatore Chiantia
Keyword(s):  
Correlation Analysis ◽  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Diffusion Dynamics ◽  
Cross Correlation Analysis

AbstractFluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

scholarly journals Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

ACS Nano ◽  
2015 ◽  
Vol 9 (9) ◽  
pp. 9158-9166 ◽  
Author(s):  
Lydia Kisley ◽  
Rachel Brunetti ◽  
Lawrence J. Tauzin ◽  
Bo Shuang ◽  
Xiyu Yi ◽  
...  
Keyword(s):  
Porous Materials ◽  
Fluorescence Correlation Spectroscopy ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

Soft Matter ◽  
2016 ◽  
Vol 12 (18) ◽  
pp. 4190-4203 ◽  
Author(s):  
Daniel S. Banks ◽  
Charmaine Tressler ◽  
Robert D. Peters ◽  
Felix Höfling ◽  
Cécile Fradin
Keyword(s):  
Anomalous Diffusion ◽  
Fluorescence Correlation Spectroscopy ◽  
Polymer Solutions ◽  
Tracer Diffusion ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Wide Range

FCS with a wide range of beam waists was used to measure tracer diffusion in crowded media over five decades in time, thus providing a strong test for different models of anomalous diffusion.

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