Diffusion–Photodynamics Coupling in Fluorescence Correlation Spectroscopy Studies of Photoswitchable Green Fluorescent Proteins: An Analytical and Simulative Study

2011 ◽  
Vol 115 (34) ◽  
pp. 10311-10321 ◽  
Author(s):  
S. C. Daglio ◽  
N. Banterle ◽  
L. D’Alfonso ◽  
M. Collini ◽  
G. Chirico
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Proteins ◽  
Correlation Spectroscopy ◽  
Green Fluorescent Proteins ◽  
Fluorescence Correlation ◽  
Spectroscopy Studies ◽  
Green Fluorescent

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 Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy

2004 ◽  
Vol 86 (1) ◽  
pp. 384-394 ◽  
Author(s):  
Andreas Schenk ◽  
Sergey Ivanchenko ◽  
Carlheinz Röcker ◽  
Jörg Wiedenmann ◽  
G. Ulrich Nienhaus
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Proteins ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Red Fluorescent Proteins

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

scholarly journals Imaging fluorescence correlation spectroscopy studies of dye diffusion in self-assembled organic nanotubes

2016 ◽  
Vol 18 (25) ◽  
pp. 16766-16774 ◽  
Author(s):  
Hao Xu ◽  
Shinobu Nagasaka ◽  
Naohiro Kameta ◽  
Mitsutoshi Masuda ◽  
Takashi Ito ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Dye Diffusion ◽  
Self Assembled ◽  
Spectroscopy Studies ◽  
Organic Nanotubes

Imaging fluorescence correlation spectroscopy is used to elucidate the rate and mechanism for diffusion of charged molecules within charged, self-assembled organic nanotubes.

Properties of Baculovirus Particles Displaying GFP Analyzed by Fluorescence Correlation Spectroscopy

2002 ◽  
Vol 383 (12) ◽  
pp. 1941-1946 ◽  
Author(s):  
J. Toivola ◽  
K. Ojala ◽  
P.O. Michel ◽  
M. Vuento ◽  
C. Oker-Blom
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Mean Value ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Fluorescence Correlation

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

Octadecylrhodamine B as a Specific Micelle-Binding Fluorescent Tag for Fluorescence Correlation Spectroscopy Studies of Amphiphilic Water-Soluble Block Copolymer Micelles. Spectroscopic Behavior in Aqueous Media

2003 ◽  
Vol 68 (11) ◽  
pp. 2105-2119 ◽  
Author(s):  
Jana Humpolíčková ◽  
Karel Procházka ◽  
Martin Hof
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Polymeric Micelles ◽  
Aqueous Media ◽  
Correlation Spectroscopy ◽  
Water Soluble ◽  
Time Resolved ◽  
Fluorescence Correlation ◽  
Block Copolymer Micelles ◽  
Spectroscopy Studies ◽  
Fluorescent Tag

Fluorescence behavior of octadecylrhodamine B (ORB) and the association-dependent changes in UV-VIS absorption and fluorescence spectra in aqueous solutions were studied by UV-VIS absorption spectroscopy and steady-state and time-resolved fluorometry. Spectral changes are interpreted in terms of the hydrophobically driven formation of H- and J-dimers and higher aggregates. The knowledge gained serves as a basis for the interpretation of results of the fluorescence correlation spectroscopy study of polymeric micelles labeled with ORB.

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