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J Cell Biol. 1986 October 1; 103(4): 1221–1234.
PMCID: PMC2114341

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells

Abstract

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa- 1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Arndt-Jovin DJ, Robert-Nicoud M, Kaufman SJ, Jovin TM. Fluorescence digital imaging microscopy in cell biology. Science. 1985 Oct 18;230(4723):247–256. [PubMed]
  • Becker JS, Oliver JM, Berlin RD. Fluorescence techniques for following interactions of microtubule subunits and membranes. Nature. 1975 Mar 13;254(5496):152–154. [PubMed]
  • Benson DM, Bryan J, Plant AL, Gotto AM, Jr, Smith LC. Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells. J Cell Biol. 1985 Apr;100(4):1309–1323. [PMC free article] [PubMed]
  • BLIGH EG, DYER WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. [PubMed]
  • Ciechanover A, Schwartz AL, Dautry-Varsat A, Lodish HF. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed]
  • Ellens H, Bentz J, Szoka FC. H+- and Ca2+-induced fusion and destabilization of liposomes. Biochemistry. 1985 Jun 18;24(13):3099–3106. [PubMed]
  • Fung BK, Stryer L. Surface density determination in membranes by fluorescence energy transfer. Biochemistry. 1978 Nov 28;17(24):5241–5248. [PubMed]
  • Gibson GA, Loew LM. Application of Forster resonance energy transfer to interactions between cell or lipid vesicle surfaces. Biochem Biophys Res Commun. 1979 May 14;88(1):141–146. [PubMed]
  • Herman BA, Fernandez SM. Dynamics and topographical distribution of surface glycoproteins during myoblast fusion: a resonance energy transfer study. Biochemistry. 1982 Jul 6;21(14):3275–3283. [PubMed]
  • Johnson DA, Voet JG, Taylor P. Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor. J Biol Chem. 1984 May 10;259(9):5717–5725. [PubMed]
  • Johnson LV, Walsh ML, Bockus BJ, Chen LB. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol. 1981 Mar;88(3):526–535. [PMC free article] [PubMed]
  • Kapitza HG, McGregor G, Jacobson KA. Direct measurement of lateral transport in membranes by using time-resolved spatial photometry. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4122–4126. [PubMed]
  • Keller PM, Person S, Snipes W. A fluorescence enhancement assay of cell fusion. J Cell Sci. 1977 Dec;28:167–177. [PubMed]
  • Kremer JM, Esker MW, Pathmamanoharan C, Wiersema PH. Vesicles of variable diameter prepared by a modified injection method. Biochemistry. 1977 Aug 23;16(17):3932–3935. [PubMed]
  • Nichols JW, Pagano RE. Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. Biochemistry. 1982 Apr 13;21(8):1720–1726. [PubMed]
  • Pagano RE, Longmuir KJ. Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J Biol Chem. 1985 Feb 10;260(3):1909–1916. [PubMed]
  • Pagano RE, Longmuir KJ, Martin OC. Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts. J Biol Chem. 1983 Feb 10;258(3):2034–2040. [PubMed]
  • Pagano RE, Longmuir KJ, Martin OC, Struck DK. Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts. J Cell Biol. 1981 Dec;91(3 Pt 1):872–877. [PMC free article] [PubMed]
  • Pardee JD, Simpson PA, Stryer L, Spudich JA. Actin filaments undergo limited subunit exchange in physiological salt conditions. J Cell Biol. 1982 Aug;94(2):316–324. [PMC free article] [PubMed]
  • Plant AL, Benson DM, Smith LC. Cellular uptake and intracellular localization of benzo(a)pyrene by digital fluorescence imaging microscopy. J Cell Biol. 1985 Apr;100(4):1295–1308. [PMC free article] [PubMed]
  • Rouser G, Siakotos AN, Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids. 1966 Jan;1(1):85–86. [PubMed]
  • Sleight RG, Pagano RE. Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus. J Cell Biol. 1984 Aug;99(2):742–751. [PMC free article] [PubMed]
  • Struck DK, Hoekstra D, Pagano RE. Use of resonance energy transfer to monitor membrane fusion. Biochemistry. 1981 Jul 7;20(14):4093–4099. [PubMed]
  • Struck DK, Pagano RE. Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J Biol Chem. 1980 Jun 10;255(11):5404–5410. [PubMed]
  • Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. [PubMed]
  • Stryer L, Haugland RP. Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A. 1967 Aug;58(2):719–726. [PubMed]
  • Tanasugarn L, McNeil P, Reynolds GT, Taylor DL. Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH. J Cell Biol. 1984 Feb;98(2):717–724. [PMC free article] [PubMed]
  • Taylor DL, Reidler J, Spudich JA, Stryer L. Detection of actin assembly by fluorescence energy transfer. J Cell Biol. 1981 May;89(2):362–367. [PMC free article] [PubMed]
  • Terasaki M, Song J, Wong JR, Weiss MJ, Chen LB. Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell. 1984 Aug;38(1):101–108. [PubMed]
  • Thomas DD, Carlsen WF, Stryer L. Fluorescence energy transfer in the rapid-diffusion limit. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5746–5750. [PubMed]
  • Tietze C, Schlesinger P, Stahl P. Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling. J Cell Biol. 1982 Feb;92(2):417–424. [PMC free article] [PubMed]
  • Tycko B, Maxfield FR. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. [PubMed]
  • Uster PS, Deamer DW. Fusion competence of phosphatidylserine-containing liposomes quantitatively measured by a fluorescence resonance energy transfer assay. Arch Biochem Biophys. 1981 Jul;209(2):385–395. [PubMed]
  • Vanderwerf P, Ullman EF. Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels. Biochim Biophys Acta. 1980 Feb 28;596(2):302–314. [PubMed]

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