Abstract
A technique is described and demonstrated for measuring the orientation distribution of fluorescent molecules in a two-dimensional system. A laser beam is totally internally reflected at the interface between a glass slide and an aqueous solution, which creates a thin layer of evanescent illumination that excites fluorescent molecules near the interface. Molecules with absorption dipoles at different tilts from the normal to the interface are preferentially excited when the laser polarization is rotated. Approximately one-half of the emitted fluorescence is collected with an inverted microscope using a high-aperture objective. The fluorescence vs. polarization curve yields the value of an order parameter that is related to the orientation distribution of absorption dipoles. This technique is applied to phospholipid monolayers made at an air/water interface and transferred to hydrophobic glass microscope slides. Dipalmitoylphosphatidylcholine monolayers were doped with 2 mol% phosphatidylethanolamine labeled with the fluorescent moiety nitrobenzoxadiazole, either on an acyl chain or on the head group. The measured value of the order parameter for the head-labeled probe decreases as a function of the surface pressure at which the monolayer is transferred to the slide, as the surface pressure increases from 10 to 40 dyne/cm. The measured value of the order parameter for the chain-labeled probe is high for all coating pressures. These results can be interpreted in terms of probe partitioning into coexistent fluid and solid domains. Dimyristoylphosphatidylcholine monolayers were doped with 2 mol% chain-labeled phosphatidylethanolamine, either free or covalently conjugated to a small peptide. In these monolayers, the measured value of the order parameter is high at all pressures. The technique presented here may also prove useful for measuring the orientation distribution of proteins bound to or embedded in a planar model membrane.
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Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Axelrod D., Burghardt T. P., Thompson N. L. Total internal reflection fluorescence. Annu Rev Biophys Bioeng. 1984;13:247–268. doi: 10.1146/annurev.bb.13.060184.001335. [DOI] [PubMed] [Google Scholar]
- Balakrishnan K., Hsu F. J., Cooper A. D., McConnell H. M. Lipid hapten containing membrane targets can trigger specific immunoglobulin E-dependent degranulation of rat basophil leukemia cells. J Biol Chem. 1982 Jun 10;257(11):6427–6433. [PubMed] [Google Scholar]
- Borejdo J., Assulin O., Ando T., Putnam S. Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore. J Mol Biol. 1982 Jul 5;158(3):391–414. doi: 10.1016/0022-2836(82)90205-4. [DOI] [PubMed] [Google Scholar]
- Brian A. A., McConnell H. M. Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6159–6163. doi: 10.1073/pnas.81.19.6159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burghardt T. P., Ando T., Borejdo J. Evidence for cross-bridge order in contraction of glycerinated skeletal muscle. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7515–7519. doi: 10.1073/pnas.80.24.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burghardt T. P., Axelrod D. Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics. Biophys J. 1981 Mar;33(3):455–467. doi: 10.1016/S0006-3495(81)84906-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burghardt T. P., Thompson N. L. Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection. Biophys J. 1984 Dec;46(6):729–737. doi: 10.1016/S0006-3495(84)84071-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burton J., Cody R. J., Jr, Herd J. A., Haber E. Specific inhibition of renin by an angiotensinogen analog: studies in sodium depletion and renin-dependent hypertension. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5476–5479. doi: 10.1073/pnas.77.9.5476. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burton J., Poulsen K., Haber E. Competitive inhibitors of renin. Inhibitors effective at physiological pH. Biochemistry. 1975 Aug 26;14(17):3892–3898. doi: 10.1021/bi00688a024. [DOI] [PubMed] [Google Scholar]
- Cartwright G. S., Smith L. M., Heinzelmann E. W., Ruebush M. J., Parce J. W., McConnell H. M. H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1506–1510. doi: 10.1073/pnas.79.5.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hafeman D. G., Seul M., Cliffe C. M., 2nd, McConnell H. M. Superoxide enhances photobleaching during cellular immune attack against fluorescent lipid monolayer membranes. Biochim Biophys Acta. 1984 Apr 25;772(1):20–28. doi: 10.1016/0005-2736(84)90513-3. [DOI] [PubMed] [Google Scholar]
- Hafeman D. G., Smith L. M., Fearon D. T., McConnell H. M. Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils. J Cell Biol. 1982 Jul;94(1):224–227. doi: 10.1083/jcb.94.1.224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hafeman D. G., von Tscharner V., McConnell H. M. Specific antibody-dependent interactions between macrophages and lipid haptens in planar lipid monolayers. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4552–4556. doi: 10.1073/pnas.78.7.4552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McConnell H. M., Tamm L. K., Weis R. M. Periodic structures in lipid monolayer phase transitions. Proc Natl Acad Sci U S A. 1984 May;81(10):3249–3253. doi: 10.1073/pnas.81.10.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakanishi M., Brian A. A., McConnell H. M. Binding of cytotoxic T-lymphocytes to supported lipid monolayers containing trypsinized H-2Kk. Mol Immunol. 1983 Nov;20(11):1227–1231. doi: 10.1016/0161-5890(83)90147-5. [DOI] [PubMed] [Google Scholar]
- Peters R., Beck K. Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7183–7187. doi: 10.1073/pnas.80.23.7183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seul M., Eisenberger P., McConnell H. M. X-ray diffraction by phospholipid monolayers on single-crystal silicon substrates. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5795–5797. doi: 10.1073/pnas.80.18.5795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson N. L., Axelrod D. Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy. Biophys J. 1983 Jul;43(1):103–114. doi: 10.1016/S0006-3495(83)84328-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson N. L., Brian A. A., McConnell H. M. Covalent linkage of a synthetic peptide to a fluorescent phospholipid and its incorporation into supported phospholipid monolayers. Biochim Biophys Acta. 1984 Apr 25;772(1):10–19. doi: 10.1016/0005-2736(84)90512-1. [DOI] [PubMed] [Google Scholar]
- Uzgiris E. E., Kornberg R. D. Two-dimensional crystallization technique for imaging macromolecules, with application to antigen--antibody--complement complexes. Nature. 1983 Jan 13;301(5896):125–129. doi: 10.1038/301125a0. [DOI] [PubMed] [Google Scholar]
- Weis R. M., Balakrishnan K., Smith B. A., McConnell H. M. Stimulation of fluorescence in a small contact region between rat basophil leukemia cells and planar lipid membrane targets by coherent evanescent radiation. J Biol Chem. 1982 Jun 10;257(11):6440–6445. [PubMed] [Google Scholar]
- von Tscharner V., McConnell H. M. Physical properties of lipid monolayers on alkylated planar glass surfaces. Biophys J. 1981 Nov;36(2):421–427. doi: 10.1016/S0006-3495(81)84741-8. [DOI] [PMC free article] [PubMed] [Google Scholar]