Abstract
Unraveling the pathways of intracellular cholesterol transport is of great importance for biomedicine, since disturbed cholesterol trafficking is involved in many metabolic diseases. Most fluorescent probes for cholesterol, however, have physico-chemical properties deviating from the natural sterol. Intrinsically fluorescent sterols like dehydroergosterol (DHE) and the related cholestatrienol (CTL) have great potential for analysis of sterol trafficking due to their close resemblance of ergosterol and cholesterol, respectively. Excitation and emission of both sterols are in the ultraviolet (UV), which, together with high bleaching propensity and low brightness, make fluorescence imaging of DHE and CTL challenging. Here, we present an overview of how UV-sensitive wide field (UV-WF) and multiphoton (MP) microscopy can be applied to image both sterols in living cells and tissues. In addition, we show, for the first time, how applying advanced image denoising can dramatically enhance the signal-to-noise ratio in MP image sequences of DHE. This allowed us to track DHE-containing vesicles and surface protrusions in cells over prolonged time. We also discuss the properties of BODIPY-tagged cholesterol (BChol) compared to DHE and cholesterol and present an overview of fluorescence imaging techniques for analyzing cellular sterol dynamics.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ariola FS, Li Z, Cornejo C, Bittman R, Heikal AA (2009) Membrane fluidity and lipid order in ternary giant unilamellar vesicles using a new bodipy-cholesterol derivative. Biophys J 96:2696–2708
Arthur JR, Heinecke KA, Seyfried TN (2011) Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain. J Lipid Res 52:1345–1351
Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069
Baumgart T, Hunt G, Farkas ER, Webb WW, Feigenson GW (2007) Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta 1768:2182–2194
Beattie ME, Veatch SL, Stottrup BL, Keller SL (2005) Sterol structure determines miscibility versus melting transitions in lipid vesicles. Biophys J 89:1760–1768
Beaudouin J, Mora-Bermúdez F, Klee T, Daigle N, Ellenberg J (2006) Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins. Biophys J 90:1878–1894
Beh CT, McMaster CR, Kozminski KG, Menon AK (2012) A detour for yeast oxysterol-binding proteins. J Biol Chem 287:11481–11488
Behnke O, Tranum-Jensen J, van Deurs B (1984) Filipin as a cholesterol probe. I. Morphology of filipin-cholesterol interaction in lipid model systems. Eur J Cell Biol 35:189–199
Behnke O, Tranum-Jensen J, van Deurs B (1984) Filipin as a cholesterol probe. II. Filipin-cholesterol interaction in red blood cell membranes. Eur J Cell Biol 35:200–215
Bergy ME, Eble TE (1968) The filipin complex. Biochemistry 7:653–659
Bittman R (1978) Sterol-polyene antibiotic complexation: probe of membrane structure. Lipids 13:686–691
Bittman R, Chen WC, Blau L (1974) Stopped-flow kinetic and equilibrium studies of filipin III binding to sterols. Biochemistry 13:1374–1379
Blau L, Bittman R (1978) Cholesterol distribution between the two halves of the lipid bilayer of human erythrocyte ghost membranes. J Biol Chem 253:8366–8388
Borroni V, Barrantes FJ (2011) Cholesterol modulates the rate and mechanism of acetylcholine receptor internalization. J Biol Chem 286:17122–17132
Braga J, Desterro JM, Carmo-Fonseca M (2004) Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol Biol Cell 15:4749–4760
Brandt S (1999) Datenanalyse. Spektrum Verlag, Heidelberg, Berlin
Bridgman PC, Nakajima Y (1983) Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine clusters. J Cell Biol 96:363–372
Brown AC, Towles KB, Wrenn SP (2007) Measuring raft size as a function of membrane composition in PC-based systems: Part 1–binary systems. Langmuir 23:11180–11187
Burger K, Gimpl G, Fahrenholz F (2000) Regulation of receptor function by cholesterol. Cell Mol Life Sci 57:1577–1592
Castanho MA, Coutinho A, Prieto MJ (1992) Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol. J Biol Chem 267:204–209
Cedar O, Ryhage R (1964) The structure of filipin. Acta Chem Scand 18:558–560
Cheruku SR, Xu Z, Dutia R, Lobel P, Storch J (2006) Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport. J Biol Chem 281:31594–31604
Coxey RA, Pentchev PG, Campbell G, Blanchette-Mackie EJ (1993) Differential accumulation of cholesterol in Golgi compartments of normal and Niemann-Pick type C fibroblasts incubated with LDL: a cytochemical freeze-fracture study. J Lipid Res 34:1165–1176
de Saint-Jean M, Delfosse V, Douguet D, Chicanne G, Payrastre B, Bourguet W, Antonny B, Drin G (2011) Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers. J Cell Biol 195:965–978
Du H, Duanmu M, Witte D, Grabowski GA (1998) Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage. Hum Mol Genet 7:1347–1354
Elson EL (2011) Fluorescence correlation spectroscopy: past, present, future. Biophys J 101:2855–2870
Ewers H, Smith AE, Sbalzarini IF, Lilie H, Koumoutsakos P, Helenius A (2005) Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes. Proc Natl Acad Sci USA 102:15110–15115
Frolov A, Petrescu A, Atshaves BP, So PT, Gratton E, Serrero G, Schroeder F (2000) High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. J Biol Chem 275:12769–12780
Garvik O, Benediktsen P, Ipsen JH, Simonsen AC, Wüstner D (2008) The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes. Chem Phys Lipids 159:114–118
Garvik O, Benediktson P, Simonsen AC, Ipsen JH, Wüstner D (2009) The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes. Chem Physics Lipids 159:114–118
Ge L, Qi W, Miao HH, Cao J, Qu YX, Li BL, Song BL (2008) The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1. Cell Metab 7:508–519
Georgiev AG, Sullivan DP, Kersting MC, Dittman JS, Beh CT, Menon AK (2011) Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM. Traffic 12:1341–1355
Gerstbrein B, Stamatas G, Kollias N, Driscoll M (2005) In vivo spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans. Aging Cell 4:127–137
Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR (1998) An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells. J Cell Biol 142:923–936
Gimpl G (2010) Cholesterol-protein interaction: methods and cholesterol reporter molecules. Subcell Biochem 51:1–45
Gimpl G, Gehrig-Burger K (2007) Cholesterol reporter molecules. Biosci Rep 27:335–358
Gonzalez RC, Woods RE (2002) Digital Image Processing. Chapter 11. Prentice Hall, New Jersey, pp 675–683
Greenspan P, Mayer EP, Fowler SD (1985) Nile Red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965–973
Grosheva I, Haka AS, Qin C, Pierini LM, Maxfield FR (2009) Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization. Arterioscler Thromb Vasc Biol 29:1615–1621
Haka AS, Grosheva I, Chiang E, Buxbaum AR, Baird BA, Pierini LM, Maxfield FR (2009) Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins. Mol Biol Cell 20:4932–4940
Hao M, Bogan JS (2009) Cholesterol regulates glucose-stimulated insulin secretion through phosphatidylinositol 4,5-bisphosphate. J Biol Chem 284:29489–29498
Hao M, Head WS, Gunawardana SC, Hasty AH, Piston DW (2007) Direct effect of cholesterol on insulin secretion: a novel mechanism for pancreatic beta-cell dysfunction. Diabetes 56:2328–2338
Hao M, Lin SX, Karylowski OJ, Wüstner D, McGraw TE, Maxfield FR (2002) Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J Biol Chem 277:609–617
Hao M, Mukherjee S, Maxfield FR (2001) Cholesterol depletion induces large scale domain segregation in living cell membranes. Proc Natl Acad Sci USA 98:13072–13077
Hartwig Petersen N, Færgeman NJ, Yu L, Wüstner D (2008) Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells. J Lipid Res 49:2023–2037
Hebert B, Costantino S, Wiseman PW (2005) Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. Biophys J 88:3601–3614
Henriksen J, Rowat AC, Brief E, Hsueh YW, Thewalt JL, Zuckermann MJ, Ipsen JH (2006) Universal behavior of membranes with sterols. Biophys J 90:1639–1649
Hoekstra M, Van Eck M, Korporaal SJ (2012) Genetic studies in mice and humans reveal new physiological roles for the high-density lipoprotein receptor scavenger receptor class B type I. Curr Opin Lipidol 23:127–132
Hofsass C, Lindahl E, Edholm O (2003) Molecular dynamics simulations of phospholipid bilayers with cholesterol. Biophys J 84:2192–2206
Horvat S, McWhir J, Rozman D (2011) Defects in cholesterol synthesis genes in mouse and in humans: lessons for drug development and safer treatments. Drug Metab Rev 43:69–90
IV Humphries WH, Fay NC, Payne CK (2010) Intracellular degradation of low-density lipoprotein probed with two-color fluorescence microscopy. Integr Biol (Camb) 2:536–544
Hölttä-Vuori M, Uronen RL, Repakova J, Salonen E, Vattulainen I, Panula P, Li Z, Bittman R, Ikonen E (2008) BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms. Traffic 9:1839–1849
Ipsen JH, Karlstrom G, Mouritsen OG, Wennerstrom H, Zuckermann MJ (1987) Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta 905:162–172
Jensen MO, Mouritsen OG (2004) Lipids do influence protein function-the hydrophobic matching hypothesis revisited. Biochim Biophys Acta 1666:205–226
Jeon JH, Tejedor V, Burov S, Barkai E, Selhuber-Unkel C, Berg-Sørensen K, Oddershede L, Metzler R (2011) In vivo anomalous diffusion and weak ergodicity breaking of lipid granules. Phys Rev Lett 106:048103
Jia L, Betters JL, Yu L (2011) Niemann-Pick C1-like 1 (NPC1L1) protein in intestinal and hepatic cholesterol transport. Annu Rev Physiol 73:239–259
John K, Kubelt J, Muller P, Wustner D, Herrmann A (2002) Rapid transbilayer movement of the fluorescent sterol dehydroergosterol in lipid membranes. Biophys J 83:1525–1534
Kolin DL, Wiseman PW (2007) Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells. Cell Biochem Biophys 49:141–164
Kolovou G, Anagnostopoulou K, Mikhailidis DP, Cokkinos DV (2008) Apolipoprotein E knockout models. Curr Pharn Des 14:338–351
Lagane B, Mazères S, Le Grimellec C, Cézanne L, Lopez A (2002) Lateral distribution of cholesterol in membranes probed by means of a pyrene-labelled cholesterol: effects of acyl chain unsaturation. Biophys Chem 95:7–22
Le Guyader L, Le Roux C, Mazères S, Gaspard-Iloughmane H, Gornitzka H, Millot C, Mingotaud C, Lopez A (2007) Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe. Biophys J 93:4462–4473
Le Lay S, Ferre P, Dugail I (2004) Adipocyte cholesterol balance in obesity. Biochem Soc Trans 32:103–106
Lindahl E, Edholm O (2000) Spatial and energetic-entropic decomposition of surface tension in lipid bilayers from molecular dynamics simulations. J Chem Phys 113:3882–3893
Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol (suppl 7):S7–14
Listenberger LL, Brown DA (2007) Fluorescent detection of lipid droplets and associated proteins. Curr Protoc Cell Biol 24:Unit 24.2
Liu JP, Tang Y, Zhou S, Toh BH, McLean C, Li H (2010) Cholesterol involvement in the pathogenesis of neurodegenerative diseases. Mol Cell Neurosci 43:33–42
Liu R, Lu P, Chu JW, Sharom FJ (2009) Characterization of fluorescent sterol binding to purified human NPC1. J Biol Chem 284:1840–1852
Loura LMS, Fedorov A, Prieto M (2001) Exclusion of a cholesterol analog from the cholesterol-rich phase in model membranes. Biochim Biophys Acta 1511:236–243
Luisier F, Vonesch C, Blu T, Unser M (2009) Fast Haar-wavelet denoising of multidimensional fluorescence microscopy data. Proceedings of the sixth IEEE international symposium on biomedical imaging: From nano to macro, Boston MA, USA, June 28–July 1, pp. 310–313
Luisier F, Vonesch C, Blu T, Unser M (2010) Fast interscale wavelet denoising of Poisson-corrupted images. Signal Processing 90:415–427
Lund FW, Lomholt MA, Solanko LM, Wüstner D (2012) Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in Chinese hamster ovary cells (Submitted for publication)
Masters BR, So PTC (2008) Classical and quantum theory of one-photon and multiphoton fluorescence spectroscopy. In: Masters BR, So PTC (eds) Handbook of biomedical nonlinear optical microscopy. Oxford University Press, Oxford, pp 91–152
Matyash V, Geier C, Henske A, Mukherjee S, Hirsh D, Thiele C, Grant B, Maxfield FR, Kurzchalia TV (2001) Distribution and transport of cholesterol in Caenorhabditis elegans. Mol Biol Cell 12:1725–1736
Maxfield FR, Menon AK (2006) Intracellular sterol transport and distribution. Curr Opin Cell Biol 18:379–385
Maxfield FR, Wüstner D (2002) Intracellular cholesterol transport. J Clin Invest 110:891–898
Maxfield FR, Wüstner D (2012) Analysis of cholesterol trafficking with fluorescent probes. Methods Cell Biol 108:367–393
McGookey DJ, Anderson RW (1983) Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Biol 97:1156–1168
McIntosh AL, Atshaves BP, Huang H, Gallegos AM, Kier AB, Schroeder F (2008) Fluorescence techniques using dehydroergosterol to study cholesterol trafficking. Lipids 43:1185–1208
McIntosh AL, Gallegos AM, Atshaves BP, Storey SM, Kannoju D, Schroeder F (2003) Fluorescence and multiphoton imaging resolve unique structural forms of sterol in membranes of living cells. J Biol Chem 278:6384–6403
Mesmin B, Maxfield FR (2009) Intracellular sterol dynamics. Biochim Biophys Acta 1791:636–645
Mesmin B, Pipalia NH, Lund FW, Ramlall TF, Sokolov A, Eliezer D, Maxfield FR (2011) STARD4 abundance regulates sterol transport and sensing. Mol Biol Cell 22:4004–4015
Miao L, Nielsen M, Thewalt J, Ipsen JH, Bloom M, Zuckermann M, Mouritsen OG (2002) From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers. Biophys J 82:1429–1444
Mondal M, Mesmin B, Mukherjee S, Maxfield FR (2009) Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells. Mol Biol Cell 20:581–588
Mouritsen OG, Zuckermann MJ (2004) What's so special about cholesterol? Lipids 39:1101–1113
Mueller V, Ringemann C, Honigmann A, Schwarzmann G, Medda R, Leutenegger M, Polyakova S, Belov VN, Hell SW, Eggeling C (2011) STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. Biophys J 101:1651–1660
Mukherjee S, Zha X, Tabas I, Maxfield FR (1998) Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys J 75:1915–1925
Müller F, Mazza D, Stasevich TJ, McNally JG (2010) FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know? Curr Opin Cell Biol 22:403–411
Müller P, Herrmann A (2002) Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes. Biophys J 82:1418–1428
Nagao T, Qin C, Grosheva I, Maxfield FR, Pierini LM (2007) Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation. Arterioscler Thromb Vasc Biol 27:1596–1602
Neufeld EB, Cooney AM, Pitha J, Dawidowicz EA, Dwyer NK, Pentchev PG, Blanchette-Mackie EJ (1996) Intracellular trafficking of cholesterol monitored with a cyclodextrin. J Biol Chem 271:21604–21613
Norman AW, Demel RA, de Kruyff B, van Deenen LLM (1972) Studies on the biological properties of polyene antibiotics. J Biol Chem 247:1918–1929
Petersen NO, Höddelius PL, Wiseman PW, Seger O, Magnusson KE (1993) Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. Biophys J 65:1135–1146
Pipalia NH, Hao M, Mukherjee S, Maxfield FR (2006) Sterol, protein, and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect. Traffic 8:130–141
Pipalia NH, Huang A, Ralph H, Rujoi M, Maxfield FR (2006) Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells. J Lipid Res 47:284–301
Prattes S, Horl G, Hammer A, Blaschitz A, Graier WF, Sattler W, Zechner R, Steyrer E (2000) Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures. J Cell Sci 113:2977–2989
Puri V, Watanabe R, Singh RD, Dominguez M, Brown JC, Wheatley CL, Marks DL, Pagano RE (2001) Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J Cell Biol 154:535–547
Qian H, Sheetz MP, Elson EL (1991) Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J 60:910–921
Qin C, Nagao T, Grosheva I, Maxfield FR, Pierini LM (2006) Elevated plasma membrane cholesterol content alters macrophage signaling and function. Arterioscler Thromb Vasc Biol 26:372–378
Rodal SK, Skretting G, Garred O, Vilhardt F, van Deurs B, Sandvig K (1999) Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10:961–974
Rychnovsky SD, Richardson TI (1995) Relative and absolute configuration of filipin III. Angew Chem Int Ed Engl 34:1227–1230
Sage D, Neumann FR, Hediger F, Gasser SM, Unser M (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans Image Processing 14:1372–1383
Saxton MJ (1983) Lateral diffusion in an archipelago. Biophys J 64:1766–1780
Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399
Sbalzarini IF, Hayer A, Helenius A, Koumoutsakos P (2006) Simulations of (an)isotropic diffusion on curved biological surfaces. Biophys J 90:878–885
Sbalzarini IF, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 151:182–195
Scheidt HA, Müller P, Herrmann A, Huster D (2003) The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol. J Biol Chem 278:45563–45569
Schrader M, Bahlmann K, Hell SW (1997) Three-photon-excitation microscopy: theory, experiment, and applications. Optik 104:116–124
Schroeder F, Barenholz Y, Gratton E, Thompson TE (1987) A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles. Biochemistry 26:2441–2448
Schroeder F, Holland JF, Bieber LL (1971) Fluorometric evidence for the binding of cholesterol to the filipin complex. J Antibiot (Tokyo) 24:846–849
Schroeder F, Nemecz G, Gratton E, Barenholz Y, Thompson TE (1988) Fluorescence properties of cholestatrienol in phosphatidylcholine bilayer vesicles. Biophys Chem 32:57–72
Schütz GJ, Schindler H, Schmidt T (1997) Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys J 73:1073–1080
Severs NJ, Simons HL (1983) Failure of filipin to detect cholesterol-rich domains in smooth muscle plasma membrane. Nature 303:637–638
Shaw JE, Epand RF, Epand RM, Li Z, Bittman R, Yip CM (2006) Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization. Biophys J 90:2170–2178
Shrivastava S, Haldar S, Gimpl G, Chattopadhyay A (2009) Orientation and dynamics of a novel fluorescent cholesterol analogue in membranes of varying phase. J Phys Chem B 113:4475–4481
Steck TL, Ye J, Lange Y (2002) Probing red cell membrane cholesterol movement with cyclodextrin. Biophys J 83:2118–2125
Steer CJ, Bisher M, Blumenthal R, Steven AC (1984) Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat. J Cell Biol 99:315–319
Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, McGraw TE (1999) Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Nat Acad Sci U S A 96:6775–6780
Swedlow JR, Platani M (2002) Live cell imaging using wide-field microscopy and deconvolution. Cell Struct Funct 27:335–341
Tabas I, Rosoff WJ, Boykow GC (1988) Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate. J Biol Chem 263:1266–1272
Tolić-Nørrelykke IM, Munteanu EL, Thon G, Oddershede L, Berg-Sørensen K (2004) Anomalous diffusion in living yeast cells. Phys Rev Lett 93:078102
Veatch SL, Keller SL (2002) Organization in lipid membranes containing cholesterol. Phys Rev Lett 89:268101
Veatch SL, Polozov IV, Gawrisch K, Keller SL (2004) Liquid domains in vesicles investigated by NMR and fluorescence microscopy. Biophys J 86:2910–2922
Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8:571–573
Vist MR, Davis JH (1990) Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2 H nuclear magnetic resonance and differential scanning calorimetry. Biochemistry 29:451–464
Volpon L, Lancelin J-M (2000) Solution NMR structures of the polyene macrolide antibiotic filipin III. FEBS Lett 478:137–140
Weiss M (2004) Challenges and artefacts in quantitative photobleaching experiments. Traffic 5:662–671
Whitfield GB, Brock TD, Ammann A, Gottlieb D, Carter HF (1955) Filipin, an antifungal antibiotic: isolation and properties. J Am Chem Soc 77:4799–4801
Wiseman PW, Petersen NO (1999) Image correlation spectroscopy: II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells. Biophys J 76:963–977
Wiseman PW, Squier JA, Ellisman MH, Wilson KR (2000) Two-photon image correlation spectroscopy and image cross-correlation spectroscopy. J Microsc 200:14–25
Wolf DE (2007) Fundamentals of fluorescence and fluorescence microscopy. Methods Cell Biol 81:64–91
Wrenn SP, Kaler EW, Lee SP (1999) A fluorescence energy transfer study of lecithin-cholesterol vesicles in the presence of phospholipase C. J Lipid Res 40:1483–1494
Wüstner D (2007) Fluorescent sterols as tools in membrane biophysics and cell biology. Chem Phys Lipids 146:1–25
Wüstner D (2007) Plasma membrane sterol distribution resembles the surface topography of living cells. Mol Biol Cell 18:211–228
Wüstner D (2008) Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages. Chem Phys Lipids 154:129–136
Wüstner D (2009) Intracellular cholesterol transport. In: Ehnholm C (ed) Cellular lipid metabolism. Springer Press, Heidelberg, Germany, pp 157–190
Wüstner D (2012) Following intracellular cholesterol transport by linear and non-linear optical microscopy of intrinsically fluorescent sterols. Curr Pharm Biotechnol 13:303–318
Wüstner D, Brewer JR, Bagatolli LA, Sage D (2011) Potential of ultraviolet widefield imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes. Microsc Res Tech 74:92–108
Wüstner D, Færgeman NJ (2008) Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets. Cytometry A 73:727–744
Wüstner D, Færgeman NJ (2008) Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells. Histochem Cell Biol 130:891–908
Wüstner D, Herrmann A, Hao M, Maxfield FR (2002) Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells. J Biol Chem 277:30325–30336
Wüstner D, Landt Larsen A, Færgeman NJ, Brewer JR, Sage D (2010) Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate based image segmentation. Traffic 11:440–454
Wüstner D, Mondal M, Huang A, Maxfield FR (2004) Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells. J Lipid Res 45:427–437
Wüstner D, Mondal M, Tabas I, Maxfield FR (2005) Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells. Traffic 6:396–412
Wüstner D, Sage D (2010) Multicolor bleach-rate imaging enlightens in vivo sterol transport. Commun Integr Biol 3:1–4
Wüstner D, Solanko LM, Sokol E, Lund FW, Garvik O, Li Z, Bittman R, Korte T, Herrmann A (2011) Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol. Chem Phys Lipids 164:221–235
Xu C, Zipfel WR (2008) Multiphoton excitation of fluorescent probes. In: Masters BR, So PTC (eds) Handbook of biomedical nonlinear optical microscopy. Oxford University Press, Oxford, pp 311–333
Xu XX, Tabas I (1991) Lipoproteins activate acyl-coenzyme A:cholesterol acyltransferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level. J Biol Chem 266:17040–17048
Xu Z, Farver W, Kodukula S, Storch J (2008) Regulation of sterol transport between membranes and NPC2. Biochemistry 47:11134–11143
Yu L, Bharadwaj S, Brown JM, Ma Y, Du W, Davis MA, Michaely P, Liu P, Willingham MC, Rudel LL (2006) Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake. J Biol Chem 281:6616–6624
Zhang W, McIntosh AL, Xu H, Wu D, Gruninger T, Atshaves B, Liu JC, Schroeder F (2005) Structural analysis of sterol distributions in the plasma membrane of living cells. Biochemistry 44:2864–2884
Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377
Zuckermann MJ, Ipsen JH, Miao L, Mouritsen OG, Nielsen M, Polson J, Thewalt J, Vattulainen I, Zhu H (2004) Modeling lipid-sterol bilayers: applications to structural evolution, lateral diffusion, and rafts. Methods Enzymol 383:198–229
Acknowledgements
DW acknowledges funding by grants of the Lundbeck Foundation, the Novo Nordisk Foundation, the Danish Research Agency Forskningsstyrelsen, Forskningsrådet for Natur og Univers (FNU) and the Danish Research Agency Forskningsstyrelsen, Forskningsrådet for Sundhed og sygdom (FSS). We are grateful to Florian Luisier (Statistics and Information Sciences Laboratory, Harvard University, Cambridge, USA) and Daniel Sage (Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland) for their helpful comments on image denoising using the PURE-LET approach and on an early version of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Wüstner, D., Lund, F.W., Solanko, L.M. (2012). Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution. In: Mély, Y., Duportail, G. (eds) Fluorescent Methods to Study Biological Membranes. Springer Series on Fluorescence, vol 13. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2012_57
Download citation
DOI: https://doi.org/10.1007/4243_2012_57
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-33127-5
Online ISBN: 978-3-642-33128-2
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)