Spatial Fourier analysis of video photobleaching measurements. Principles and optimization

Tsay, Tsong-Tseh; Jacobson, Ken

doi:10.1016/s0006-3495(91)82061-6

Cited by 85 publications

(103 citation statements)

References 21 publications

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“…Long-range diffusion coefficients parallel and perpendicular to the barriers were calculated from images capturing the recovery process using a 2-dimensional Fourier transform method 20,21 . Briefly, if C(x,y,t) represents the 2-dimensional concentration profile of a diffusing species (i.e., an image of a fluorescent marker) at some time, t, and Ĉ (u, v, t) is the Fourier transform of this data, then u,v,0) represents the transform of data collected at time t=0, and D x and D y are long-range diffusion coefficients along two primary, independent axes.…”

Section: Anisotropic Diffusion Of Nanopatterned Lipid Bilayersmentioning

confidence: 99%

“…The initially round photobleach spot becomes elongated, illustrating the anisotropic properties of this surface and faster long-range diffusion in the vertical direction. At later timepoints, no evidence of the initial photobleach pattern remained, indicating that there is essentially no immobile fraction of TR-DHPE lipids.Long-range diffusion coefficients parallel and perpendicular to the barriers were calculated from images capturing the recovery process using a 2-dimensional Fourier transform method 20,21 . Briefly, if C(x,y,t) represents the 2-dimensional concentration profile of a diffusing species (i.e., an image of a fluorescent marker) at some time, t, and Ĉ (u, v, t) is the Fourier transform of this data, then u,v,0) represents the transform of data collected at time t=0, and D x and D y are long-range diffusion coefficients along two primary, independent axes.…”

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confidence: 99%

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Non-Brownian Diffusion of Membrane Molecules in Nanopatterned Supported Lipid Bilayers

et al. 2008

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Molecules associated with the outer surface of living cells exhibit complex, non-Brownian patterns of diffusion. In this report, supported lipid bilayers were patterned with nanoscale barriers to capture key aspects of this anomalous diffusion in a controllable format. First, long-range diffusion coefficients of membrane-associated molecules were significantly reduced by the presence of the barriers, while short-range diffusion was unaffected. Second, this modulation was more pronounced for large molecular complexes than for individual lipids. Surprisingly, the quantitative effect of these barriers on long-range lipid diffusion could be accurately simulated using a simple, continuum-based model of diffusion on a nanostructured surface; we thus describe a metamaterial that captures the properties of the outer membrane of living cells.The outer surface of cells presents a complex, nanostructured, yet fluid environment that controls the movement of signaling proteins. The lateral movement of many membrane biomolecules, including transmembrane or tethered proteins as well as lipids themselves, can be interpreted as being free and isotropic within compartments of the cell membrane measuring tens to hundreds of nanometers in scale [1][2][3][4][5][6] . These compartments are delineated by semipermeable barriers that arise from interactions between the plasma membrane, underlying cytoskeleton, and associated proteins [6][7][8] . Fluctuations in these structures allow biomolecules to occasionally cross between compartments, allowing long-range, but comparatively slow, transport over the cell surface. More formally, transport along the membrane is an anomalous, non-Brownian process that can be characterized by two diffusion coefficients, one that describes short-range motion within an individual compartment and a second, smaller, effective diffusion coefficient that is associated with long-range motion over many barriers. The extent to which these values differ is dependent on the spacing and properties of the barriers as well as the diffusing molecule. Emerging models suggest significant impacts of this behavior on cell signaling 2, 9, 10 , but experimental systems for testing these hypotheses are not widely available. In this report, we capture this anomalous diffusion by nanopatterning supported lipid bilayers with barriers to lipid diffusion using a geometry that captures the semipermeable nature of those posed to be present in living cells. As is posed by models of these interactions, we aim to gain control over long-range diffusion, while maintaining local, isotropic diffusion associated with a membrane in the absence of such barriers. We demonstrate that these nanopatterned barriers give rise to different short-and long-range diffusion coefficients of lipids and membrane-associated proteins, and provide a quantitative model of this diffusion that suggests specific aspects of membrane structure at the sub-micrometer level. The basic substrate supported lipid bilayer system consists of a phospholipid membrane...

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Section: Anisotropic Diffusion Of Nanopatterned Lipid Bilayersmentioning

confidence: 99%

mentioning

confidence: 99%

Non-Brownian Diffusion of Membrane Molecules in Nanopatterned Supported Lipid Bilayers

et al. 2008

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“…The average intensity measured immediately after a subsequent, extended photobleach period was used to determine background intensity and subtracted from values used for this calculation. The diffusion coefficient for hEFG was calculated by following the time evolution of a nonuniform distribution of Cy5-labeled hEFG by electrophoresis, 29 as detailed in the Results and Discussion.…”

Section: Substrate Preparationmentioning

confidence: 99%

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

et al. 2005

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Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm 2 /s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

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“…Diffusivities were calculated by fitting the fractional recovery of the bleached area against frequency-scaled time, where fractional recovery was measured by the decay of the spatial Fourier transforms of the images. 23,24 Bound fraction was taken as proportional to the amount of fluorescence that did not recover. Stable background fluorescence intensity measurements confirmed that this permanent bleaching was not due to bleaching of the sample from imaging.…”

Section: Image Processing and Calculation Of The Diffusion Coefficientmentioning

confidence: 99%

Composition and transport properties of human ankle and knee cartilage

Fetter

Leddy

Guilak

et al. 2005

Journal Orthopaedic Research

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ABSTRACT:The incidence of osteoarthritis is significantly higher in the knee as compared to the ankle, suggesting that differences in the properties of cartilage from these joints may contribute to the development of osteoarthritis. As an avascular tissue, articular cartilage depends primarily upon diffusion for molecular transport. The goal of this study was to determine if differences in the structure and composition between ankle and knee cartilage were also reflected as differences in solute transport properties. The diffusion coefficient and partition coefficient of a 70-kDa dextran molecule were measured in human ankle and knee articular cartilage using fluorescence recovery after photobleaching (FRAP) and were compared to the proteoglycan, collagen, water, and DNA contents within each zone. The mean partition coefficient was significantly lower in the ankle compared to the knee (0.010 AE 0.002 vs. 0.022 AE 0.003, p < 0.01), but no differences in the diffusion coefficients were observed (34.6 AE 0.9 mm 2 s À1 vs. 35.4 AE 2.4 mm 2 s À1 , p ¼ 0.70). Ankle cartilage exhibited higher proteoglycan content as well as a trend toward lower water content, suggesting that ankle cartilage has a smaller effective pore size than knee cartilage. These findings suggest that differences in the composition of ankle and knee cartilage contribute to a difference in the partition coefficient. The results of this study provide further support for the hypothesis that the transport properties of cartilage may play a role in the differences in the incidence of osteoarthritis in these joints by altering the effective concentration of growth factors and cytokines to which chondrocytes are exposed. ß

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Spatial Fourier analysis of video photobleaching measurements. Principles and optimization

Cited by 85 publications

References 21 publications

Non-Brownian Diffusion of Membrane Molecules in Nanopatterned Supported Lipid Bilayers

Non-Brownian Diffusion of Membrane Molecules in Nanopatterned Supported Lipid Bilayers

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

Composition and transport properties of human ankle and knee cartilage

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