A Practical Guide to Fluorescence Temporal and Spatial Correlation Spectroscopy

Image Correlation ◽

Future Research ◽

Molecular Flow ◽

Basic Principles ◽

Image Correlation Spectroscopy ◽

Properties Of Materials ◽

Temporal And Spatial ◽

Interactive Exercises

ABSTRACT The aim of this article is to introduce the basic principles behind the widely used microscopy tool: fluorescence fluctuation correlation spectroscopy (FFCS). We present the fundamentals behind single spot acquisition (FCS) and its extension to spatiotemporal sampling, which is implemented through image correlation spectroscopy (ICS). The article is an educational guide that introduces theoretic concepts of FCS and some of the ICS techniques, followed by interactive exercises in MATLAB. There, the learner can simulate data time series and the application of various FFCS techniques, as well as learn how to measure diffusion coefficients, molecular flow, and concentration of particles. Additionally, each section is followed by a short exercise to reinforce learning concepts by simulating different scenarios, seek verification of outcomes, and make comparisons. Furthermore, we invite the learner throughout the article to consult the literature for different extensions of FFCS techniques that allow measurements of different physicochemical properties of materials. Upon completion of the modules, we anticipate the learner will gain a good understanding in the field of FFCS that will encourage further exploration and adoption of the FFCS tools in future research and educational practices.

Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

AJP Cell Physiology ◽

10.1152/ajpcell.00313.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1302-C1315 ◽

Cited By ~ 47

Author(s):

Marko Vendelin ◽

Rikke Birkedal

Keyword(s):

Diffusion Coefficient ◽

Diffusion Coefficients ◽

Anisotropic Diffusion ◽

Image Correlation ◽

Rat Cardiomyocytes ◽

Intracellular Diffusion ◽

Average Diffusion ◽

Image Correlation Spectroscopy ◽

Kinetics Of

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 μm2/s in the longitudinal and 52 ± 16 μm2/s in the transverse directions ( n = 8, mean ± SD). Those values are ∼2 (longitudinal) and ∼3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Journal of Visualized Experiments ◽

10.3791/51074 ◽

2014 ◽

Cited By ~ 5

Author(s):

Eva C. Arnspang ◽

Jennifer S. Koffman ◽

Saw Marlar ◽

Paul W. Wiseman ◽

Lene N. Nejsum

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Diffusion Coefficients ◽

Image Correlation ◽

Plasma Membrane Proteins ◽

Image Correlation Spectroscopy ◽

Space Image

Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking

Scientific Reports ◽

10.1038/s41598-020-76272-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sara Makaremi ◽

Markus Rose ◽

Suman Ranjit ◽

Michelle A. Digman ◽

Dawn M. E. Bowdish ◽

...

Keyword(s):

Plasma Membrane ◽

Particle Tracking ◽

Diffusion Coefficients ◽

Basal Membrane ◽

Single Particle Tracking ◽

Image Correlation ◽

Live Cells ◽

Raster Image ◽

Abstract The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.

Exploring oligomeric state of the serotonin1A receptor utilizing photobleaching image correlation spectroscopy: implications for receptor function

Faraday Discussions ◽

10.1039/c7fd00192d ◽

2018 ◽

Vol 207 ◽

pp. 409-421 ◽

Cited By ~ 9

Author(s):

Hirak Chakraborty ◽

Md. Jafurulla ◽

Andrew H. A. Clayton ◽

Amitabha Chattopadhyay

Keyword(s):

Image Correlation ◽

Membrane Cholesterol ◽

Receptor Function ◽

Oligomeric State ◽

Serotonin1a Receptor ◽

Photobleaching image correlation spectroscopy (pbICS) reveals that membrane cholesterol modulates the oligomeric state of the serotonin1A receptor.

Arbitrary-Region Image Correlation Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2015.11.983 ◽

2016 ◽

Vol 110 (3) ◽

pp. 176a

Author(s):

Jelle Hendrix ◽

Tomas Dekens ◽

Don C. Lamb

Keyword(s):

Image Correlation ◽

Arbitrary Region ◽

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

10.1101/240903 ◽

2017 ◽

Cited By ~ 3

Author(s):

Ali Isbilir ◽

Jan Möller ◽

Andreas Bock ◽

Ulrike Zabel ◽

Paolo Annibale ◽

...

Keyword(s):

Single Molecule ◽

Metabotropic Glutamate Receptors ◽

Fluorescent Protein ◽

Methodological Approach ◽

Membrane Receptors ◽

Image Correlation ◽

Intact Cells ◽

Class A ◽

AbstractG protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated.Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization.From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86.Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.