Abstract
Continuous fluorescence microphotolysis (CFM) was adapted to flux measurements in single cells. The principle of the method is simple: Cells are equilibrated with a fluorescent solute, an individual cell is continuously irradiated by a laser beam focussed down to approximately the diameter of the cell, and fluorescence originating from the irradiated cell is monitored. In this procedure irradiation irreversibly photolyzes chromophores in the cell while fresh chromophores enter the cell by membrane transport (flux). The resulting fluorescence decay can be analyzed for the rate constants of both membrane transport and photolysis. As an experimental test of the new method the band-3 mediated transport of the fluorescent anion N-(7-nitrobenzofuranzan-4-yl)-taurine (NBD-taurine) across the erythrocyte membrane was measured. For various experimental conditions good agreement between values obtained by CFM and by fluorescence microphotolysis (FM) was observed. By measurements on single ghosts it was furthermore found that photolysis of NBD-taurine is first-order with respect to the power of irradiation. On this basis a stepped-intensity procedure was worked out that facilitates data evaluation in flux measurements. Also, by analysing the relations between CFM and FM flux measurements a method was devised by which FM data can be corrected for (inevitable) photolysis.
Similar content being viewed by others
References
Agard DA (1984) Optical sectioning microscopy: Cellular architecture in three dimensions. Annu Rev Biophys Bioeng 13:191–219
Axelrod D (1983) Lateral motion of membrane proteins and biological function. J Membr Biol 75:1–10
Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurements by analysis of fluorescence photobleaching recovery experiments. Biophys J 16:1055–1069
Brünger A, Peters R, Schulten K (1985) Continuous fluorescence microphotolysis to observe lateral diffusion in membranes. Theoretical methods and applications. J Chem Phys 82:7147–7161
Cherry RJ (1979) Rotational and lateral diffusion of membrane proteins. Biochim Biophys Acta 559:289–327
Davoust J, Devaux PF, Leger L (1982) Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules. EMBO J 1:1233–1238
Edidin M, Zagyanski Y, Lardner TJ (1976) Measurement of membrane lateral diffusion in single cells. Science 191:466–468
Eidelman O, Cabantchik ZI (1980) A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence. Anal Biochem 106:335–341
Jacobson K, Wu E, Poste G (1976) Measurement of the translational mobility of concanavalin A in glycerol-saline solutions and on the cell surface by fluorescence recovery after photobleaching. Biochim Biophys Acta 443:215–222
Koppel DE (1979) Fluorescence redistribution after photobleaching: a new multipont analysis of membrane translational dynamics. Biophys J 28:281–292
Koppel DE, Axelrod D, Schlessinger J, Elson EL, Webb WW (1976) Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J 16:1315–1329
Lang I, Peters R, (1984) Nuclear envelope permeability: A sensitive indicator of pore complex integrity. In: Bolis CL, Helmreich EJM, Passow H (eds) Information and energy transduction in biological membranes. Alan R. Liss, New York, pp 377–386
Lanni F, Ware BR (1981) Intensity dependence of fluorophore photobleaching by a stepped-intensity slow-bleach experiment. Photochem Photobiol 34:279–281
Lanni F, Ware BR (1982) Modulation detection of fluorescence photobleaching recovery. Rev Sci Instrum 53:905–908
Peters R (1981) Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis. Cell Biol Int Rep 5:733–760
Peters R (1983) Nuclear envelope permeability measured by fluorescence microphotolysis of single liver cell nuclei. J. Biol Chem 258:11427–11429
Peters R (1984a) Flux measurement in single cells by fluorescence microphotolysis. Eur Biophys J 11:43–50
Peters R (1984b) Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis. EMBO J 3:1831–1836
Peters R, Passow H (1984) Anion transport in single erythrocyte ghosts measured by fluorescence microphotolysis. Biochim Biophys Acta 777:334–338
Peters R, Richter HP (1981) Translational diffusion in the plasma membrane of sea urchin eggs. Dev Biol 86:285–293
Peters R, Peters J, Tews KH, Bähr W (1974) A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 367:282–294
Peters R, Brünger A, Schulten K (1981) Continuous fluorescence microphotolysis: A sensitive method for the study of translational diffusion in single cells. Proc Natl Acad Sci USA 78:962–966
Schneider MB, Webb WW (1981) Measurement of submicron laser beam radii. Appl Opt 20:1382–1388
Smith BA, McConnell HM (1978) Determination of molecular motion in membranes using periodic pattern photobleaching. Proc Natl Acad Sci USA 75:2759–2763
Smith LM, Weis RM, McConnel HM (1981) Measurement of rotational diffusion of membrane components by fluorescence photobleaching. Biophys J 36:73–91
Thompson NL, Burghardt TP, Axelrod D (1981) Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy. Biophys J 33:435–454
Ware BR (1984) Fluorescence photobleaching recovery. Am Lab (Boston) 16:16–28
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Scholz, M., Schulten, K. & Peters, R. Single-cell flux measurement by continuous fluorescence microphotolysis. Eur Biophys J 13, 37–44 (1985). https://doi.org/10.1007/BF00266308
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00266308