This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kiessling, V. Articles by Tamm, L. K. Search for Related Content PubMed PubMed Citation Articles by Kiessling, V. Articles by Tamm, L. K.

Measuring Distances in Supported Bilayers by Fluorescence Interference-Contrast Microscopy: Polymer Supports and SNARE Proteins

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908-0736 USA

Correspondence: Address reprint requests to Lukas K. Tamm, Dept. of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 800736, Charlottesville, VA 22908-0736. Tel.: 434-982-3578; Fax: 434-982-1616; E-mail: lkt2e{at}virginia.edu.

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 ± 1.0 nm. The cleft distance was increased to 3.9 ± 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 ± 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.

This article has been cited by other articles:

				C. W. Harland, D. Rabuka, C. R. Bertozzi, and R. Parthasarathy The Mycobacterium tuberculosis Virulence Factor Trehalose Dimycolate Imparts Desiccation Resistance to Model Mycobacterial Membranes Biophys. J., June 15, 2008; 94(12): 4718 - 4724. [Abstract] [Full Text] [PDF]

				G. Oncins, L. Picas, J. Hernandez-Borrell, S. Garcia-Manyes, and F. Sanz Thermal Response of Langmuir-Blodgett Films of Dipalmitoylphosphatidylcholine Studied by Atomic Force Microscopy and Force Spectroscopy Biophys. J., October 15, 2007; 93(8): 2713 - 2725. [Abstract] [Full Text] [PDF]

				S. Garg, J. Ruhe, K. Ludtke, R. Jordan, and C. A. Naumann Domain Registration in Raft-Mimicking Lipid Mixtures Studied Using Polymer-Tethered Lipid Bilayers Biophys. J., February 15, 2007; 92(4): 1263 - 1270. [Abstract] [Full Text] [PDF]

				M. C. Howland, A. W. Szmodis, B. Sanii, and A. N. Parikh Characterization of Physical Properties of Supported Phospholipid Membranes Using Imaging Ellipsometry at Optical Wavelengths Biophys. J., February 15, 2007; 92(4): 1306 - 1317. [Abstract] [Full Text] [PDF]

				V. Kiessling, J. M. Crane, and L. K. Tamm Transbilayer Effects of Raft-Like Lipid Domains in Asymmetric Planar Bilayers Measured by Single Molecule Tracking Biophys. J., November 1, 2006; 91(9): 3313 - 3326. [Abstract] [Full Text] [PDF]

				K. Tawa and K. Morigaki Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy Biophys. J., October 1, 2005; 89(4): 2750 - 2758. [Abstract] [Full Text] [PDF]

				T. Liu, W. C. Tucker, A. Bhalla, E. R. Chapman, and J. C. Weisshaar SNARE-Driven, 25-Millisecond Vesicle Fusion In Vitro Biophys. J., October 1, 2005; 89(4): 2458 - 2472. [Abstract] [Full Text] [PDF]

				C. M. Ajo-Franklin, P. V. Ganesan, and S. G. Boxer Variable Incidence Angle Fluorescence Interference Contrast Microscopy for Z-Imaging Single Objects Biophys. J., October 1, 2005; 89(4): 2759 - 2769. [Abstract] [Full Text] [PDF]

				M. E. Bowen, K. Weninger, A. T. Brunger, and S. Chu Single Molecule Observation of Liposome-Bilayer Fusion Thermally Induced by Soluble N-Ethyl Maleimide Sensitive-Factor Attachment Protein Receptors (SNAREs) Biophys. J., November 1, 2004; 87(5): 3569 - 3584. [Abstract] [Full Text] [PDF]

				B. L. Stottrup, S. L. Veatch, and S. L. Keller Nonequilibrium Behavior in Supported Lipid Membranes Containing Cholesterol Biophys. J., May 1, 2004; 86(5): 2942 - 2950. [Abstract] [Full Text] [PDF]

				J. M. Crane and L. K. Tamm Role of Cholesterol in the Formation and Nature of Lipid Rafts in Planar and Spherical Model Membranes Biophys. J., May 1, 2004; 86(5): 2965 - 2979. [Abstract] [Full Text] [PDF]

				R. Richter, A. Mukhopadhyay, and A. Brisson Pathways of Lipid Vesicle Deposition on Solid Surfaces: A Combined QCM-D and AFM Study Biophys. J., November 1, 2003; 85(5): 3035 - 3047. [Abstract] [Full Text] [PDF]