Abstract
Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.
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References
Adler J, Parmryd I (2010) Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytom A 77:733–742
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645
Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc Oxf 224:213–232
Churchman LS, Okten Z, Rock RS, Dawson JF, Spudich JA (2005) Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc Natl Acad Sci USA 102:1419–1423
Endesfelder U, Malkusch S, Flottmann B, Mondry J, Liguzinski P, Verveer PJ, Heilemann M (2011) Chemically induced photoswitching of fluorescent probes—a general concept for super-resolution microscopy. Molecules 16:3106–3118
Fehon RG, McClatchey AI, Bretscher A (2010) Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 11:276–287
French AP, Mills S, Swarup R, Bennett MJ, Pridmore TP (2008) Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat Protoc 3:619–628
Grecco HE, Verveer PJ (2011) FRET in cell biology: still shining in the age of super-resolution? Chemphyschem 12:484–490
Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149:243–251
Heilemann M, Margeat E, Kasper R, Sauer M, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127:3801–3806
Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172–6176
Koyama-Honda I, Ritchie K, Fujiwara T, Iino R, Murakoshi H, Kasai RS, Kusumi A (2005) Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. Biophys J 88:2126–2136
Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF (2004a) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24:4070–4081
Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF (2004b) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24:4070–4081
McClatchey AI, Fehon RG (2009) Merlin and the ERM proteins—regulators of receptor distribution and signaling at the cell cortex. Trends Cell Biol 19:198–206
McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133
Pertsinidis A, Zhang Y, Chu S (2010) Subnanometre single-molecule localization, registration and distance measurements. Nature 466:647–651
Radenovic A, Annibale P, Scarselli M, Kodiyan A (2010) Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long-lived dark state in the red photoconverted form. J Phys Chem Lett 1:1506–1510
Ronneberger O, Baddeley D, Scheipl F, Verveer PJ, Burkhardt H, Cremer C, Fahrmeir L, Cremer T, Joffe B (2008) Spatial quantitative analysis of fluorescently labeled nuclear structures: problems, methods, pitfalls. Chromosome Res 16:523–562
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795
Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336
Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175
Smith CS, Joseph N, Rieger B, Lidke KA (2010) Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7:373–375
Wolter S, Endesfelder U, van de Linde S, Heilemann M, Sauer M (2011) Measuring localization performance of super-resolution algorithms on very active samples. Optics Express 19:7020–7033
Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M, Cornish VW, Sauer M (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719
Zinchuk V, Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4:4–19
Acknowledgments
M.H. is grateful for funding by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315262) and the German Science Foundation (DFG, grant nr. HE 6166/2-1). P.J.V is grateful for funding by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315257) and by the German Science Foundation, as part of the NanoSci-ERA consortium (grant nr. VE 579/1-1).
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S. Malkusch and U. Endesfelder contributed equally.
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Malkusch, S., Endesfelder, U., Mondry, J. et al. Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochem Cell Biol 137, 1–10 (2012). https://doi.org/10.1007/s00418-011-0880-5
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DOI: https://doi.org/10.1007/s00418-011-0880-5