Quantitative analysis of variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) of cell/substrate contacts

Burmeister, Jeffrey S.; Truskey, George A.; Reichert, William M.

doi:10.1111/j.1365-2818.1994.tb03426.x

Cited by 79 publications

(59 citation statements)

References 20 publications

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“…For a given refractive index material, the penetration depth can be modulated by varying the incidence angle i (Fig. 2); this method is VIA-TIRFM (15)(16)(17).…”

mentioning

confidence: 99%

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Ajo‐Franklin

Kam

Boxer

2001

Proc. Natl. Acad. Sci. U.S.A.

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Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO 2 on lithium niobate (LiNbO3, n ‫؍‬ 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead͞ protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

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“…For a given refractive index material, the penetration depth can be modulated by varying the incidence angle i (Fig. 2); this method is VIA-TIRFM (15)(16)(17).…”

mentioning

confidence: 99%

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Ajo‐Franklin

Kam

Boxer

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Calculations of this separation from variable-angle total internal reflection fluorescence microscopy measurements averaged 24+13 nm [20]. In a study made using tandem scanning confocal microscopy, calculated separations between the plasmalemma and the substrate ranged from 10 to 50 nm and increased radially from the center of focal contacts [7].…”

Section: Interpreting Height Profiles Of Ventral Plasma Membranesmentioning

confidence: 99%

Cytoplasmic topography of focal contacts

et al. 1996

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To investigate the structure of focal contacts, the cytoplasmic faces of fibroblast membranes were examined in solution by scanning force and immunofluorescence microscopy. Focal contacts were Identified in scanning force topographs by correlation with fluorescence images. Finer details were resolved in topographs of the focal contacts than in fluorescence micrographs. Increased separation of ventral plasma membranes from the substrate correlated with the duration of cell culture. The cytoplasmic projections of the focal contacts also Increased with the cell culture period. These changes accompanied lateral spreading of flbroblasts during a period of several hours after seeding cells in culture medium.

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“…The TIRF flow cell and its alignment were as described previously. 11,13 From TIRFM images, the contact area was determined as a function of distance from the point of closest approach, using a value of ⌬ o equal to 15 nm.…”

Section: Total Internal Reflection Fluorescence Microscopy (Tirfm)mentioning

confidence: 99%

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers

Burmeister

McKinney

Reichert

et al. 1999

J. Biomed. Mater. Res.

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The objective of this study was to examine the effect of substrate hydrophobicity on cell-substrate contact area and the affinity between adsorbed fibronectin (Fn) and its receptor. Homo- and copolymer films of hydrophobic ethyl methacrylate (EMA) and hydrophilic hydroxyethyl methacrylate (HEMA) were spun-cast onto glass slides. Bovine aortic endothelial cells (BAEC) were plated for 2 h in serum-free medium onto polymers preadsorbed with Fn. Cells were fixed, labeled, and examined by total internal reflection fluorescence microscopy (TIRFM) to determine the topography of the basal surface as a function of distance from the substrate. Phase contrast microscopy was used to examine the total projected area of adherent cells. The cumulative contact area was greatest on cells attached to surfaces prepared from 0% HEMA and lowest on surfaces with the highest HEMA content. An equilibrium adhesion model used these data together with the critical force for detachment and the Fn density (Burmeister et al., J Biomed Mater Res 1996;30:13-22) to determine the affinity between Fn and its receptor and the bond strength. The affinity and force per bond decreased with increasing HEMA content. These results indicate that differences in the strength of endothelial cell adhesion to polymers are influenced by the conformation of the adsorbed adhesion proteins.

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Quantitative analysis of variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) of cell/substrate contacts

Cited by 79 publications

References 20 publications

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Cytoplasmic topography of focal contacts

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers

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