Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins
10.1038/srep09583
Crossref journal-article
Springer Science and Business Media LLC
Scientific Reports (297)
Abstract

AbstractWe introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40–50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

Bibliography

Johnson, E., Seiradake, E., Jones, E. Y., Davis, I., Grünewald, K., & Kaufmann, R. (2015). Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Scientific Reports, 5(1).

Authors 6
  1. Errin Johnson (first)
  2. Elena Seiradake (additional)
  3. E. Yvonne Jones (additional)
  4. Ilan Davis (additional)
  5. Kay Grünewald (additional)
  6. Rainer Kaufmann (additional)
References 60 Referenced 85
  1. Sjollema, K. A., Schnell, U., Kuipers, J., Kalicharan, R. & Giepmans, B. N. Correlated Light Microscopy and Electron Microscopy. Method. Cell Biol. 111, 157 (2012). (10.1016/B978-0-12-416026-2.00009-1) / Method. Cell Biol. by KA Sjollema (2012)
  2. Hell, S. W. & Wichmann, J. Breaking the Diffraction Resolution Limit by Stimulated-Emission - Stimulated-Emission-Depletion Fluorescence Microscopy. Opt. Lett. 19, 780–782 (1994). (10.1364/OL.19.000780) / Opt. Lett. by SW Hell (1994)
  3. Heintzmann, R. & Cremer, C. Laterally modulated excitation microscopy: Improvement of resolution by using a diffraction grating. Proc. SPIE 3568, 185–196 (1999). (10.1117/12.336833) / Proc. SPIE by R Heintzmann (1999)
  4. Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000). (10.1046/j.1365-2818.2000.00710.x) / J. Microsc. by MGL Gustafsson (2000)
  5. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006). (10.1126/science.1127344) / Science by E Betzig (2006)
  6. Rust, M. J., Bates, M. & Zhuang, X. W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006). (10.1038/nmeth929) / Nat. Methods by MJ Rust (2006)
  7. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006). (10.1529/biophysj.106.091116) / Biophys. J. by ST Hess (2006)
  8. Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80–84 (2011). (10.1038/nmeth.1537) / Nat. Methods by S Watanabe (2011)
  9. Kopek, B. G., Shtengel, G., Xu, C. S., Clayton, D. A. & Hess, H. F. Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. PNAS 109, 6136–6141 (2012). (10.1073/pnas.1121558109) / PNAS by BG Kopek (2012)
  10. Löschberger, A., Franke, C., Krohne, G., van de Linde, S. & Sauer, M. Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution. J. Cell Sci. 127, 4351–4355 (2014). / J. Cell Sci. by A Löschberger (2014)
  11. Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008). (10.1126/science.1154228) / Science by V Westphal (2008)
  12. Jones, S. A., Shim, S. H., He, J. & Zhuang, X. W. Fast, three-dimensional super-resolution imaging of live cells. Nat. Methods 8, 499–U496 (2011). (10.1038/nmeth.1605) / Nat. Methods by SA Jones (2011)
  13. Schnell, U., Dijk, F., Sjollema, K. A. & Giepmans, B. N. Immunolabeling artifacts and the need for live-cell imaging. Nat. Methods 9, 152–158 (2012). (10.1038/nmeth.1855) / Nat. Methods by U Schnell (2012)
  14. Bleck, C. K. E. et al. Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis. J. Microsc. 237, 23–38 (2010). (10.1111/j.1365-2818.2009.03299.x) / J. Microsc. by CKE Bleck (2010)
  15. Kaufmann, R. et al. Super-Resolution Microscopy using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions. Nano Lett. 14, 4171–4175 (2014). (10.1021/nl501870p) / Nano Lett. by R Kaufmann (2014)
  16. Chang, Y.-W. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11, 737–739 (2014). (10.1038/nmeth.2961) / Nat. Methods by Y-W Chang (2014)
  17. Kaufmann, R., Hagen, C. & Grünewald, K. Fluorescence cryo-microscopy: current challenges and prospects. Curr. Opin. Chem. Biol. 20, 86–91 (2014). (10.1016/j.cbpa.2014.05.007) / Curr. Opin. Chem. Biol. by R Kaufmann (2014)
  18. Wild, P., Schraner, E. M., Adler, H. & Humbel, B. M. Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze‐substitution. Microsc. Res. Techniq. 53, 313–321 (2001). (10.1002/jemt.1098) / Microsc. Res. Techniq. by P Wild (2001)
  19. Peddie, C. J. et al. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy 143, 3–14 (2014). (10.1016/j.ultramic.2014.02.001) / Ultramicroscopy by CJ Peddie (2014)
  20. Schwarz, H. & Humbel, B. M. in Methods in Molecular Biology, Vol. 1117 (ed. Kuo J., ed. ) Ch. 25, 559–592 (Humana Press, 2014). / Methods in Molecular Biology by H Schwarz (2014)
  21. Mutasa, H. Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins. Histochem. J. 21, 249–258 (1989). (10.1007/BF01757177) / Histochem. J. by H Mutasa (1989)
  22. Tokuyasu, K. Immunochemistry on ultrathin frozen sections. Histochem. J. 12, 381–403 (1980). (10.1007/BF01011956) / Histochem. J. by K Tokuyasu (1980)
  23. Cortese, K., Diaspro, A. & Tacchetti, C. Advanced correlative light/electron microscopy: current methods and new developments using Tokuyasu cryosections. J.Histochem.Cytochem. 57, 1103–1112 (2009). (10.1369/jhc.2009.954214) / J.Histochem.Cytochem. by K Cortese (2009)
  24. Fritschy, J. M. Is my antibody‐staining specific? How to deal with pitfalls of immunohistochemistry. Eur. J. Neurosci. 28, 2365–2370 (2008). (10.1111/j.1460-9568.2008.06552.x) / Eur. J. Neurosci. by JM Fritschy (2008)
  25. Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. PNAS 104, 20308–20313 (2007). (10.1073/pnas.0710517105) / PNAS by H Shroff (2007)
  26. Hein, B., Willig, K. I. & Hell, S. W. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. PNAS 105, 14271–14276 (2008). (10.1073/pnas.0807705105) / PNAS by B Hein (2008)
  27. Kopek, B. G., Shtengel, G., Grimm, J. B., Clayton, D. A. & Hess, H. F. Correlative photoactivated localization and scanning electron microscopy. PloS One 8, e77209 (2013). (10.1371/journal.pone.0077209) / PloS One by BG Kopek (2013)
  28. Kukulski, W. et al. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–119 (2011). (10.1083/jcb.201009037) / J. Cell Biol. by W Kukulski (2011)
  29. Fabrowski, P. et al. Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila. Nat. Commun. 4, 2244 (2013). (10.1038/ncomms3244) / Nat. Commun. by P Fabrowski (2013)
  30. Yang, Z., Hu, B., Zhang, Y., Luo, Q. & Gong, H. Development of a plastic embedding method for large-volume and fluorescent-protein-expressing tissues. PloS one 8, e60877 (2013). (10.1371/journal.pone.0060877) / PloS one by Z Yang (2013)
  31. Hell, S. W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009). (10.1038/nmeth.1291) / Nat. Methods by SW Hell (2009)
  32. Lemmer, P. et al. SPDM: light microscopy with single-molecule resolution at the nanoscale. Appl. Phys. B 93, 1–12 (2008). (10.1007/s00340-008-3152-x) / Appl. Phys. B by P Lemmer (2008)
  33. Biteen, J. S. et al. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat. Methods 5, 947–949 (2008). (10.1038/nmeth.1258) / Nat. Methods by JS Biteen (2008)
  34. Fölling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat. Methods 5, 943–945 (2008). (10.1038/nmeth.1257) / Nat. Methods by J Fölling (2008)
  35. Lucas, M. S., Günthert, M., Gasser, P., Lucas, F. & Wepf, R. Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures. Method. Cell Biol. 111, 325–356 (2012). (10.1016/B978-0-12-416026-2.00017-0) / Method. Cell Biol. by MS Lucas (2012)
  36. Nixon, S. J. et al. A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic 10, 131–136 (2009). (10.1111/j.1600-0854.2008.00859.x) / Traffic by SJ Nixon (2009)
  37. Giddings, T. Freeze‐substitution protocols for improved visualization of membranes in high‐pressure frozen samples. J. Microsc. 212, 53–61 (2003). (10.1046/j.1365-2818.2003.01228.x) / J. Microsc. by T Giddings (2003)
  38. Lakadamyali, M., Babcock, H., Bates, M., Zhuang, X. & Lichtman, J. 3D multicolor super-resolution imaging offers improved accuracy in neuron tracing. PloS one 7, e30826 (2012). (10.1371/journal.pone.0030826) / PloS one by M Lakadamyali (2012)
  39. Ding, B., Turgeon, R. & Parthasarathy, M. Substructure of freeze-substituted plasmodesmata. Protoplasma 169, 28–41 (1992). (10.1007/BF01343367) / Protoplasma by B Ding (1992)
  40. Hawes, P., Netherton, C., Mueller, M., Wileman, T. & Monaghan, P. Rapid freeze‐substitution preserves membranes in high‐pressure frozen tissue culture cells. J. Microsc. 226, 182–189 (2007). (10.1111/j.1365-2818.2007.01767.x) / J. Microsc. by P Hawes (2007)
  41. Seiradake, E., Harlos, K., Sutton, G., Aricescu, A. R. & Jones, E. Y. An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly. Nat. Struct. Mol. Biol. 17, 398–402 (2010). (10.1038/nsmb.1782) / Nat. Struct. Mol. Biol. by E Seiradake (2010)
  42. Grüll, F., Kirchgessner, M., Kaufmann, R., Hausmann, M. & Kebschull, U. in International Conference on Field Programmable Logic and Applications, Vol. 21, 1–5 (IEEE, 2011). / International Conference on Field Programmable Logic and Applications by F Grüll (2011)
  43. Kaufmann, R. et al. Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy. PloS one 7, e31128 (2012). (10.1371/journal.pone.0031128) / PloS one by R Kaufmann (2012)
  44. Kaufmann, R., Müller, P., Hildenbrand, G., Hausmann, M. & Cremer, C. Analysis of Her2/neu membrane protein clusters in different types of breast cancer cells using localization microscopy. J. Microsc. 242, 46–54 (2011). (10.1111/j.1365-2818.2010.03436.x) / J. Microsc. by R Kaufmann (2011)
  45. Durisic, N., Cuervo, L. L. & Lakadamyali, M. Quantitative super-resolution microscopy: pitfalls and strategies for image analysis. Curr. Opin. Chem. Biol. 20, 22–28 (2014). (10.1016/j.cbpa.2014.04.005) / Curr. Opin. Chem. Biol. by N Durisic (2014)
  46. Simionescu, N. & Simionescu, M. Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure and evidence for mordanting effect. J. Cell Biol. 70, 608–621 (1976). (10.1083/jcb.70.3.608) / J. Cell Biol. by N Simionescu (1976)
  47. Hayat, M. A. in Stains and cytochemical methods, Vol. 1 (ed. Hayat M. A., ed. ) 336–347 (Springer Science & Business Media, 1993). / Stains and cytochemical methods by MA Hayat (1993)
  48. Weil, T. T. et al. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat. Cell Biol. 14, 1305–1313 (2012). (10.1038/ncb2627) / Nat. Cell Biol. by TT Weil (2012)
  49. Melo, R. C., Morgan, E., Monahan-Earley, R., Dvorak, A. M. & Weller, P. F. Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes. Nat. Prot. 9, 2382–2394 (2014). (10.1038/nprot.2014.163) / Nat. Prot. by RC Melo (2014)
  50. Perkovic, M. et al. Correlative Light-and Electron Microscopy with chemical tags. J. Struct. Biol. 186, 205–213 (2014). (10.1016/j.jsb.2014.03.018) / J. Struct. Biol. by M Perkovic (2014)
  51. Xiong, H. et al. Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging. Nat. Commun. 5, 3992 (2014). (10.1038/ncomms4992) / Nat. Commun. by H Xiong (2014)
  52. Zanacchi, F. C. et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat. Methods 8, 1047–1049 (2011). (10.1038/nmeth.1744) / Nat. Methods by FC Zanacchi (2011)
  53. Hell, S., Reiner, G., Cremer, C. & Stelzer, E. H. Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. J. Microsc. 169, 391–405 (1993). (10.1111/j.1365-2818.1993.tb03315.x) / J. Microsc. by S Hell (1993)
  54. Aricescu, A. R. & Jones, E. Y. Immunoglobulin superfamily cell adhesion molecules: zippers and signals. Curr. Opin. Cell Biol. 19, 543–550 (2007). (10.1016/j.ceb.2007.09.010) / Curr. Opin. Cell Biol. by AR Aricescu (2007)
  55. Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012). (10.1038/nmeth.2171) / Nat. Methods by AJ Lam (2012)
  56. Coschi, C. H. et al. Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive. Gene. Dev. 24, 1351–1363 (2010). (10.1101/gad.1917610) / Gene. Dev. by CH Coschi (2010)
  57. Schaupp, A. et al. The composition of EphB2 clusters determines the strength in the cellular repulsion response. J. Cell Biol. 204, 409–422 (2014). (10.1083/jcb.201305037) / J. Cell Biol. by A Schaupp (2014)
  58. Endesfelder, U., Malkusch, S., Fricke, F. & Heilemann, M. A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment. Histochem. Cell Biol. 141, 629–638 (2014). (10.1007/s00418-014-1192-3) / Histochem. Cell Biol. by U Endesfelder (2014)
  59. Baddeley, D., Cannell, M. B. & Soeller, C. Visualization of localization microscopy data. Microsc. Microanal. 16, 64–72 (2010). (10.1017/S143192760999122X) / Microsc. Microanal. by D Baddeley (2010)
  60. Reynolds, E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963). (10.1083/jcb.17.1.208) / J. Cell Biol. by ES Reynolds (1963)
Dates
Type When
Created 10 years, 4 months ago (March 31, 2015, 5:05 a.m.)
Deposited 2 years, 7 months ago (Jan. 5, 2023, 7:21 p.m.)
Indexed 1 month ago (July 20, 2025, 12:08 a.m.)
Issued 10 years, 4 months ago (March 31, 2015)
Published 10 years, 4 months ago (March 31, 2015)
Published Online 10 years, 4 months ago (March 31, 2015)
Funders 0

None

@article{Johnson_2015, title={Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins}, volume={5}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/srep09583}, DOI={10.1038/srep09583}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Johnson, Errin and Seiradake, Elena and Jones, E. Yvonne and Davis, Ilan and Grünewald, Kay and Kaufmann, Rainer}, year={2015}, month=mar }