scholarly journals A Comprehensive Review of Fluorescence Correlation Spectroscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Lan Yu ◽  
Yunze Lei ◽  
Ying Ma ◽  
Min Liu ◽  
Juanjuan Zheng ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Fluorescence Correlation Spectroscopy ◽  
Pair Correlation ◽  
Time Correlation ◽  
Correlation Spectroscopy ◽  
Local Concentration ◽  
Fluorescence Correlation ◽  
Pros And Cons

Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. By time-correlation of the fluorescence fluctuations induced by molecules diffusing through a focused light, FCS can quantitatively evaluate the concentration, diffusion coefficient, and interaction of the molecules in vitro or in vivo. In this review, the basic principle and implementation of FCS are introduced. Then, the advances of FCS variants are reviewed, covering dual-color FCCS, multi-focus FCS, pair correlation function (pCF), scanning FCS, focus-reduced FCS, SPIM-FCS, and inverse-FCS. Besides, the applications of FCS are demonstrated with the measurement of local concentration, hydrodynamic radius, diffusion coefficient, and the interaction of different molecules. Lastly, a discussion is given by summarizing the pros and cons of different FCS techniques, as well as the outlooks and perspectives of FCS.

scholarly journals Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy

2021 ◽  
Vol 22 (6) ◽  
pp. 2969
Author(s):  
Aurélie Crepin ◽  
Edel Cunill-Semanat ◽  
Eliška Kuthanová Trsková ◽  
Erica Belgio ◽  
Radek Kaňa
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Higher Plants ◽  
Fluorescence Emission ◽  
Correlation Spectroscopy ◽  
Photochemical Quenching ◽  
High Ph ◽  
Fluorescence Correlation ◽  
Non Photochemical Quenching

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

scholarly journals Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo

2020 ◽  
Vol 154 (5) ◽  
pp. 507-519 ◽  
Author(s):  
Michael L. Dawes ◽  
Christian Soeller ◽  
Steffen Scholpp
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Full Range ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Biophysical Studies ◽  
Living Organisms ◽  
And Function ◽  
Compare And Contrast

AbstractCell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

scholarly journals An Alternative Framework for Fluorescence Correlation Spectroscopy

2018 ◽  
Author(s):  
Sina Jazani ◽  
Ioannis Sgouralis ◽  
Omer M. Shafraz ◽  
Marcia Levitus ◽  
Sanjeevi Sivasankar ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Light Exposure ◽  
Correlation Spectroscopy ◽  
High Signal ◽  
Fluorescence Correlation ◽  
Fluorescence Confocal Microscopy ◽  
Long Time

ABSTRACTFluorescence correlation spectroscopy (FCS), is a flexible and widely used tool routinely exploited forin vivoandin vitroapplications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted fromµ-s long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy based approaches, to probe processes several orders of magnitude faster in time and permits a reduction of phototoxic effects on living samples induced by long periods of light exposure.

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy

2000 ◽  
Vol 113 (22) ◽  
pp. 3921-3930 ◽  
Author(s):  
R.H. Kohler ◽  
P. Schwille ◽  
W.W. Webb ◽  
M.R. Hanson
Keyword(s):  
Active Transport ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Long Distance ◽  
Fluorescence Correlation ◽  
Photon Excitation ◽  
Green Fluorescent

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

scholarly journals In vivo fluorescence correlation spectroscopy analyses of FMBP ‐1, a silkworm transcription factor

FEBS Open Bio ◽  
2016 ◽  
Vol 6 (2) ◽  
pp. 106-125 ◽  
Author(s):  
Motosuke Tsutsumi ◽  
Hideki Muto ◽  
Shohei Myoba ◽  
Mai Kimoto ◽  
Akira Kitamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
In Vivo Fluorescence

Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy

2004 ◽  
Vol 385 (1) ◽  
pp. 87-93 ◽  
Author(s):  
J. Toivola ◽  
P. O. Michel ◽  
L. Gilbert ◽  
T. Lahtinen ◽  
V. Marjomäki ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Serum Sample ◽  
Acute Phase ◽  
Fluorescence Correlation Spectroscopy ◽  
Parvovirus B19 ◽  
Correlation Spectroscopy ◽  
Human Parvovirus B19 ◽  
Fluorescence Correlation ◽  
Average Diffusion Coefficient ◽  
Average Diffusion

AbstractFluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and pastimmunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7x10exp-7 cm(2)s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5x10exp-8 cm(2)s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a pastimmunity serum sample gave an average diffusion coefficient of 3.5x10exp-8 cm(2)s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7x10exp-7 to 1.6x10exp-7 cm(2)s(-1), which is much smaller than that observed with acute phase or pastimmunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and pastimmunity sera. FCS measurement of VLPimmune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

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