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Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.
Alberti S, Gladfelter A, Mittag T (2019) Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176(3): 419−434
Alshareedah I, Moosa MM, Raju M, Potoyan DA, Banerjee PR (2020) Phase transition of RNA-protein complexes into ordered hollow condensates. Proc Natl Acad Sci USA 117(27): 15650−15658
Alshareedah I, Thurston GM, Banerjee PR (2021) Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates. Biophys J 120(7): 1161−1169
Beutel O, Maraspini R, Pombo-Garcia K, Martin-Lemaitre C, Honigmann A (2019) Phase separation of zonula occludens proteins drives formation of tight junctions. Cell 179(4): 923−936.e911
Bracha D, Walls MT, Wei MT, Zhu L, Kurian M, Avalos JL, Toettcher JE, Brangwynne CP (2018) Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds. Cell 175(6): 1467−1480.e1413
Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77(1): 553−567
Chiantia S, Ries J, Schwille P (2009) Fluorescence correlation spectroscopy in membrane structure elucidation. Biochim Biophys Acta 1788(1): 225−233
Chong S, Dugast-Darzacq C, Liu Z, Dong P, Dailey GM, Cattoglio C, Heckert A, Banala S, Lavis L, Darzacq X, Tjian R (2018) Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361(6400): eaar2555. https://doi.org/10.1126/science.aar2555
Cubuk J, Alston JJ, Incicco JJ, Singh S, Stuchell-Brereton MD, Ward MD, Zimmerman MI, Vithani N, Griffith D, Wagoner JA, Bowman GR, Hall KB, Soranno A, Holehouse AS (2021) The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun 12(1): 1936. https://doi.org/10.1038/s41467-021-21953-3
Delarue M, Brittingham GP, Pfeffer S, Surovtsev IV, Pinglay S, Kennedy KJ, Schaffer M, Gutierrez JI, Sang D, Poterewicz G, Chung JK, Plitzko JM, Groves JT, Jacobs-Wagner C, Engel BD, Holt LJ (2018) mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174(2): 338−349.e320
Dertinger T, Pacheco V, von der Hocht I, Hartmann R, Gregor I, Enderlein J (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8(3): 433−443
Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schonle A, Hell SW (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457(7233): 1159−1162
Ehrenberg M, Rigler R (1974) Rotational brownian motion and fluorescence intensify fluctuations. Chem Physics 4: 390−401
Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci USA 91(13): 5740−5747
Elson EL (2011) Fluorescence correlation spectroscopy: past, present, future. Biophys J 101(12): 2855−2870
Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. 1. Conceptual basis and theory. Biopolymers 13(1): 1−27
Erdel F, Rademacher A, Vlijm R, Tunnermann J, Frank L, Weinmann R, Schweigert E, Yserentant K, Hummert J, Bauer C, Schumacher S, Al Alwash A, Normand C, Herten DP, Engelhardt J, Rippe K (2020) Mouse heterochromatin adopts digital compaction states without showing hallmarks of HP1-driven liquid-liquid phase separation. Mol Cell 78(2): 236−249.e237
Felekyan S, Sanabria H, Kalinin S, Kuhnemuth R, Seidel CA (2013) Analyzing Forster resonance energy transfer with fluctuation algorithms. Methods Enzymol 519: 39−85
Fisher RS, Elbaum-Garfinkle S (2020) Tunable multiphase dynamics of arginine and lysine liquid condensates. Nat Commun 11(1): 4628. https://doi.org/10.1038/s41467-020-18224-y
Fujioka Y, Alam JM, Noshiro D, Mouri K, Ando T, Okada Y, May AI, Knorr RL, Suzuki K, Ohsumi Y, Noda NN (2020) Phase separation organizes the site of autophagosome formation. Nature 578(7794): 301−305
Gamari BD, Zhang D, Buckman RE, Milas P, Denker JS, Chen H, Li H, Goldner LS (2014) Inexpensive electronics and software for photon statistics and correlation spectroscopy. Am J Phys 82(7): 708−722
Ghosh A, Karedla N, Thiele JC, Gregor I, Enderlein J (2018) Fluorescence lifetime correlation spectroscopy: basics and applications. Methods 140−141: 32−39
Guillen-Boixet J, Kopach A, Holehouse AS, Wittmann S, Jahnel M, Schlussler R, Kim K, Trussina I, Wang J, Mateju D, Poser I, Maharana S, Ruer-Gruss M, Richter D, Zhang X, Chang YT, Guck J, Honigmann A, Mahamid J, Hyman AA, Pappu RV, Alberti S, Franzmann TM (2020) RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell 181(2): 346−361.e317
Haustein E, Schwille P (2003) Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. Methods 29(2): 153−166
He HT, Marguet D (2011) Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy. Annu Rev Phys Chem 62: 417−436
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(5): 3601−3614
Hellenkamp B, Schmid S, Doroshenko O, Opanasyuk O, Kuhnemuth R, Rezaei Adariani S, Ambrose B, Aznauryan M, Barth A, Birkedal V, Bowen ME, Chen H, Cordes T, Eilert T, Fijen C, Gebhardt C, Gotz M, Gouridis G, Gratton E, Ha T, Hao P, Hanke CA, Hartmann A, Hendrix J, Hildebrandt LL, Hirschfeld V, Hohlbein J, Hua B, Hubner CG, Kallis E, Kapanidis AN, Kim JY, Krainer G, Lamb DC, Lee NK, Lemke EA, Levesque B, Levitus M, McCann JJ, Naredi-Rainer N, Nettels D, Ngo T, Qiu R, Robb NC, Rocker C, Sanabria H, Schlierf M, Schroder T, Schuler B, Seidel H, Streit L, Thurn J, Tinnefeld P, Tyagi S, Vandenberk N, Vera AM, Weninger KR, Wunsch B, Yanez-Orozco IS, Michaelis J, Seidel CAM, Craggs TD, Hugel T (2018) Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat Methods 15(9): 669−676
Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41(3): 697−705
Hess ST, Webb WW (2002) Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy. Biophys J 83(4): 2300−2317
Honigmann A, Mueller V, Ta H, Schoenle A, Sezgin E, Hell SW, Eggeling C (2014) Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells. Nat Commun 5: 5412. https://doi.org/10.1038/ncomms6412
Kask P, Palo K, Ullmann D, Gall K (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci USA 96(24): 13756−13761
Kaur T, Raju M, Alshareedah I, Davis RB, Potoyan DA, Banerjee PR (2021) Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies. Nat Commun 12(1): 872. https://doi.org/10.1038/s41467-021-21089-4
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(3): 141−164
Korlach J, Schwille P, Webb WW, Feigenson GW (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 96(15): 8461−8466
Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys : 65:251−297
Lerner E, Barth A, Hendrix J, Ambrose B, Birkedal V, Blanchard SC, Borner R, Sung Chung H, Cordes T, Craggs TD, Deniz AA, Diao J, Fei J, Gonzalez RL, Gopich IV, Ha T, Hanke CA, Haran G, Hatzakis NS, Hohng S, Hong SC, Hugel T, Ingargiola A, Joo C, Kapanidis AN, Kim HD, Laurence T, Lee NK, Lee TH, Lemke EA, Margeat E, Michaelis J, Michalet X, Myong S, Nettels D, Peulen TO, Ploetz E, Razvag Y, Robb NC, Schuler B, Soleimaninejad H, Tang C, Vafabakhsh R, Lamb DC, Seidel CA, Weiss S (2021) FRET-based dynamic structural biology: challenges, perspectives and an appeal for open-science practices. eLife 10: e60416. https://doi.org/10.7554/eLife.60416
Lerner E, Cordes T, Ingargiola A, Alhadid Y, Chung S, Michalet X, Weiss S (2018) Toward dynamic structural biology: two decades of single-molecule Forster resonance energy transfer. Science 359(6373): eaan1133. https://doi.org/10.1126/science.aan1133
Lyu X, Wang J, Wang J, Yin YS, Zhu Y, Li LL, Huang S, Peng S, Xue B, Liao R, Wang SQ, Long M, Wohland T, Chua BT, Sun Y, Li P, Chen XW, Xu L, Chen FJ, Li P (2021) A gel-like condensation of Cidec generates lipid-permeable plates for lipid droplet fusion. Dev Cell 56(18): 2592−2606.e2597
Macdonald P, Johnson J, Smith E, Chen Y, Mueller JD (2013) Brightness analysis. Methods Enzymol 518: 71−98
Machan R, Kapusta P, Hof M (2014) Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy. Anal Bioanal Chem 406(20): 4797−4813
Magatti D, Ferri F (2003) 25 ns software correlator for photon and fluorescence correlation spectroscopy. Rev Sci Instrum 74: 1135−1144
Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. 2. Experimental realization. Biopolymers 13(1): 29−61
Magde D, Webb WW, Elson E (1972) Thermodynamic fluctuations in a reacting system — Measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29(11): 705. https://doi.org/10.1103/PhysRevLett.29.705
Mahen R, Jeyasekharan AD, Barry NP, Venkitaraman AR (2011) Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis. Proc Natl Acad Sci USA 108(22): 9310−9315
Martin EW, Harmon TS, Hopkins JB, Chakravarthy S, Incicco JJ, Schuck P, Soranno A, Mittag T (2021) A multi-step nucleation process determines the kinetics of prion-like domain phase separation. Nat Commun 12(1): 4513. https://doi.org/10.1038/s41467-021-24727-z
Martin EW, Holehouse AS, Peran I, Farag M, Incicco JJ, Bremer A, Grace CR, Soranno A, Pappu RV, Mittag T (2020) Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. Science 367(6478): 694−699
Meseth U, Wohland T, Rigler R, Vogel H (1999) Resolution of fluorescence correlation measurements. Biophys J 76(3): 1619−1631
Mueller V, Honigmann A, Ringemann C, Medda R, Schwarzmann G, Eggeling C (2013) FCS in STED microscopy: studying the nanoscale of lipid membrane dynamics. Methods Enzymol 519: 1−38
Muller JD (2004) Cumulant analysis in fluorescence fluctuation spectroscopy. Biophys J 86(6): 3981−3992
Palo K, Mets U, Jager S, Kask P, Gall K (2000) Fluorescence intensity multiple distributions analysis: concurrent determination of diffusion times and molecular brightness. Biophys J 79(6): 2858−2866
Peng S, Li W, Yao Y, Xing W, Li P, Chen C (2020) Phase separation at the nanoscale quantified by dcFCCS. Proc Natl Acad Sci USA 117(44): 27124−27131
Perroud TD, Huang B, Zare RN (2005) Effect of bin time on the photon counting histogram for one-photon excitation. Chemphyschem 6(5): 905−912
Petersen NO, Hoddelius PL, Wiseman PW, Seger O, Magnusson KE (1993) Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. Biophys J 65(3): 1135−1146
Sanders DW, Kedersha N, Lee DSW, Strom AR, Drake V, Riback JA, Bracha D, Eeftens JM, Iwanicki A, Wang A, Wei MT, Whitney G, Lyons SM, Anderson P, Jacobs WM, Ivanov P, Brangwynne CP (2020) Competing protein-rna interaction networks control multiphase intracellular organization. Cell 181(2): 306−324.e328
Schaub E (2013) High countrate real-time FCS using F2Cor. Opt Exp 21: 23543−23555
Schuler B (2018) Perspective: chain dynamics of unfolded and intrinsically disordered proteins from nanosecond fluorescence correlation spectroscopy combined with single-molecule FRET. J Chem Phys 149(1): 010901. https://doi.org/10.1063/1.5037683
Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72(4): 1878−1886
Shakya A, King JT (2018a) DNA local-flexibility-dependent assembly of phase-separated liquid droplets. Biophys J 115(10): 1840−1847
Shakya A, King JT (2018b) Non-fickian molecular rransport in protein-DNA droplets. ACS Macro Lett 7: 1220−1225
Shakya A, Park S, Rana N, King JT (2020) Liquid-liquid phase separation of histone proteins in cells: role in chromatin organization. Biophys J 118(3): 753−764
Shimobayashi SF, Ronceray P, Sanders DW, Haataja MP, Brangwynne CP (2021) Nucleation landscape of biomolecular condensates. Nature 599(7885): 503−506
Shin Y, Brangwynne CP (2017) Liquid phase condensation in cell physiology and disease. Science 357(6357): eaaf4382. https://doi.org/10.1126/science.aaf4382
Tetin SY, Ruan Q, Skinner JP (2013) Studying antibody-antigen interactions with fluorescence fluctuation spectroscopy. Methods Enzymol 519: 139−166
Trivedi P, Palomba F, Niedzialkowska E, Digman MA, Gratton E, Stukenberg PT (2019) The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nat Cell Biol 21(9): 1127−1137
Uversky VN (2017) Intrinsically disordered proteins in overcrowded milieu: membrane-less organelles, phase separation, and intrinsic disorder. Curr Opin Struct Biol 44: 18−30
Wang ZL, Kang N, Liang YQ, Xiao Q, Huang SH (2018) Cortector SX100: principles and applications of benchtop fluorescence correlation spectrometer. Prog Biochem Biophys 45: 1166−1177
Wei MT, Elbaum-Garfinkle S, Holehouse AS, Chen CC, Feric M, Arnold CB, Priestley RD, Pappu RV, Brangwynne CP (2017) Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat Chem 9(11): 1118−1125
Weidemann T, Schwille P (2013) Dual-color fluorescence cross-correlation spectroscopy with continuous laser excitation in a confocal setup. Methods Enzymol 518: 43−70
Wen J, Hong L, Krainer G, Yao QQ, Knowles TPJ, Wu S, Perrett S (2021) Conformational expansion of Tau in condensates promotes irreversible aggregation. J Am Chem Soc 143(33): 13056−13064
Wennmalm S, Chmyrov V, Widengren J, Tjernberg L (2015) Highly sensitive FRET-FCS detects amyloid beta-peptide oligomers in solution at physiological concentrations. Anal Chem 87(23): 11700−11705
Wu B, Muller JD (2005) Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy. Biophys J 89(4): 2721−2735
Wu B, Singer RH, Mueller JD (2013) Time-integrated fluorescence cumulant analysis and its application in living cells. Methods Enzymol 518: 99−119
Zhu L, Richardson TM, Wacheul L, Wei MT, Feric M, Whitney G, Lafontaine DLJ, Brangwynne CP (2019) Controlling the material properties and rRNA processing function of the nucleolus using light. Proc Natl Acad Sci USA 116(35): 17330−17335
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