scholarly journals Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells

2015 ◽  
Vol 2 (6) ◽  
pp. 140454 ◽  
Author(s):  
D. J. Nieves ◽  
Y. Li ◽  
D. G. Fernig ◽  
R. Lévy
Keyword(s):  
Gold Nanoparticles ◽  
Single Molecule ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Live Cells ◽  
Glycerol Water ◽  
Raster Image ◽  
Diffusion Speed ◽  
Image Correlation Spectroscopy ◽  
Representative Area

Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic.

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

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 ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Live Cells ◽  
Raster Image ◽  
Image Correlation Spectroscopy

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.

scholarly journals Raster image correlation spectroscopy in live cells

2010 ◽  
Vol 5 (11) ◽  
pp. 1761-1774 ◽  
Author(s):  
Molly J Rossow ◽  
Jennifer M Sasaki ◽  
Michelle A Digman ◽  
Enrico Gratton
Keyword(s):  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Live Cells ◽  
Raster Image ◽  
Image Correlation Spectroscopy

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

2017 ◽  
Author(s):  
Ali Isbilir ◽  
Jan Möller ◽  
Andreas Bock ◽  
Ulrike Zabel ◽  
Paolo Annibale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Fluorescent Protein ◽  
Methodological Approach ◽  
Correlation Spectroscopy ◽  
Membrane Receptors ◽  
Image Correlation ◽  
Intact Cells ◽  
Class A ◽  
Image Correlation Spectroscopy

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.

Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds

2011 ◽  
Vol 7 (12) ◽  
pp. 4195-4203 ◽  
Author(s):  
S.C.P. Norris ◽  
J. Humpolíčková ◽  
E. Amler ◽  
M. Huranová ◽  
M. Buzgo ◽  
...  
Keyword(s):  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Raster Image ◽  
Nanofiber Scaffolds ◽  
Image Correlation Spectroscopy

scholarly journals Measuring Lateral Diffusion of Receptors On Plasma Membrane of Macrophages Using Raster Image Correlation Spectroscopy

2018 ◽  
Vol 24 (S1) ◽  
pp. 1356-1357
Author(s):  
S. Makaremi ◽  
S. Ranjit ◽  
M.A. Digman ◽  
E. Gratton ◽  
D. M.E. Bowdish ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lateral Diffusion ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Raster Image ◽  
Image Correlation Spectroscopy

scholarly journals Fluctuations as a source of information in fluorescence microscopy

2008 ◽  
Vol 6 (suppl_1) ◽  
Author(s):  
Zdeněk Petrášek ◽  
Petra Schwille
Keyword(s):  
Single Molecule ◽  
Single Point ◽  
Quantitative Information ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Photon Detection ◽  
Fluorescence Correlation ◽  
Additional Information ◽  
Image Correlation Spectroscopy ◽  
Spatio Temporal

Fluctuations in fluorescence spectroscopy and microscopy have traditionally been regarded as noise—they lower the resolution and contrast and do not permit high acquisition rates. However, fluctuations can also be used to gain additional information about a system. This fact has been exploited in single-point microscopic techniques, such as fluorescence correlation spectroscopy and analysis of single molecule trajectories, and also in the imaging field, e.g. in spatio-temporal image correlation spectroscopy. Here, we discuss how fluctuations are used to obtain more quantitative information from the data than that given by average values, while minimizing the effects of noise due to stochastic photon detection.

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