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Correlative video-light–electron microscopy: development, impact and perspectives

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Abstract

Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the ‘history,’ or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light–electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular ‘context’ to the fluorescent dynamic structures of interest. CLEM ‘multiplies’ the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.

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Abbreviations

CLEM:

Correlative video-light–electron microscopy

EM:

Electron microscopy

GFP:

Green fluorescent protein

COPI:

Coat protein I

COPII:

Coat protein II

ER:

Endoplasmic reticulum

DAB:

3,3′-Diaminobenzidine

FIB–SEM:

Focused-ion beam–scanning electron microscopy

FRET:

Fluorescence resonance energy transfer

References

  • Baldassarre M, Pompeo A, Beznoussenko G, Castaldi C, Cortellino S, McNiven MA, Luini A, Buccione R (2003) Dynamin participates in focal extracellular matrix degradation by invasive cells. Mol Biol Cell 14(3):1074–1084

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Brown E, Mantell J, Carter D, Tilly G, Verkade P (2009) Studying intracellular transport using high-pressure freezing and correlative light electron microscopy. Semin Cell Dev Biol 20(8):910–919

    Article  CAS  PubMed  Google Scholar 

  • Deschout H, Shivanandan A, Annibale P, Scarselli M, Radenovic A (2014) Progress in quantitative single-molecule localization microscopy. Histochem Cell Biol 142(1):5–17

    CAS  PubMed  Google Scholar 

  • Godman GC, Morgan C, Breitenfeld PM, Rose HM (1960) A correlative study by electron and light microscopy of the development of type 5 adenovirus. II. Light microscopy. J Exp Med 112:383–402

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Grabenbauer M, Geerts WJ, Fernadez-Rodriguez J, Hoenger A, Koster AJ, Nilsson T (2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nat Methods 2(11):857–862

    Article  CAS  PubMed  Google Scholar 

  • Hayat I (1987) Correlative microscopy in biology. Academic Press, London

    Google Scholar 

  • Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD, Lippincott-Schwartz J (1998) Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J Cell Biol 143(6):1485–1503

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Horrocks MH, Palayret M, Klenerman D, Lee SF (2014) The changing point-spread function: single-molecule-based super-resolution imaging. Histochem Cell Biol 141(6):577–585

    CAS  PubMed  Google Scholar 

  • Klein T, Proppert S, Sauer M (2014) Eight years of single-molecule localization microscopy. Histochem Cell Biol 141(6):561–575

    CAS  PubMed  Google Scholar 

  • Kukulski W, Schorb M, Kaksonen M, Briggs JA (2012) Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150(3):508–520

    Article  CAS  PubMed  Google Scholar 

  • Liljedahl M, Maeda Y, Colanzi A, Ayala I, Van Lint J, Malhotra V (2001) Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell 104(3):409–420

    Article  CAS  PubMed  Google Scholar 

  • Lucas MS, Guenthert M, Gasser P, Lucas F, Wepf R (2014) Correlative 3D imaging: CLSM and FIB–SEM tomography using high-pressure frozen, freeze-substituted biological samples. Methods Mol Biol 1117:593–616

    Article  PubMed  Google Scholar 

  • Mellman I, Warren G (2000) The road taken: past and future foundations of membrane traffic. Cell 100(1):99–112

    Article  CAS  PubMed  Google Scholar 

  • Mironov AA, Polishchuk RS, Luini A (2000) Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy. Trends Cell Biol 10(8):349–353

    Article  CAS  PubMed  Google Scholar 

  • Mironov AA, Mironov AA Jr, Beznoussenko GV, Trucco A, Lupetti P, Smith JD, Geerts WJ, Koster AJ, Burger KN, Martone ME, Deerinck TJ, Ellisman MH, Luini A (2003) ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains. Dev Cell 5(4):583–594

    Article  CAS  PubMed  Google Scholar 

  • Muller-Reichert T, Verkade P (2012) Introduction to correlative light and electron microscopy. Methods Cell Biol 111:xvii–xix

    Article  PubMed  Google Scholar 

  • Nakata T, Terada S, Hirokawa N (1998) Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J Cell Biol 140(3):659–674

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Polishchuk RS, Polishchuk EV, Marra P, Alberti S, Buccione R, Luini A, Mironov AA (2000) Correlative light–electron microscopy reveals the tubular–saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. J Cell Biol 148(1):45–58

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Polishchuk EV, Di Pentima A, Luini A, Polishchuk RS (2003) Mechanism of constitutive export from the Golgi: bulk flow via the formation, protrusion, and en bloc cleavage of large trans-Golgi network tubular domains. Mol Biol Cell 14(11):4470–4485

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Powell RD, Halsey CM, Hainfeld JF (1998) Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech 42(1):2–12

    Article  CAS  PubMed  Google Scholar 

  • Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111(2):229–233

    Article  CAS  PubMed  Google Scholar 

  • Rieder CL, Davison EA, Jensen LC, Cassimeris L, Salmon ED (1986) Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol 103(2):581–591

    Article  CAS  PubMed  Google Scholar 

  • Robinson JM, Takizawa T, Pombo A, Cook PR (2001) Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap. J Histochem Cytochem 49(7):803–808

    Article  CAS  PubMed  Google Scholar 

  • Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4):e1001041

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stiebing C, Matthaus C, Krafft C, Keller A-A, Weber K, Lorkowski S, Popp J (2014) Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy. Anal Bioanal Chem, 1–10

  • Svitkina TM, Verkhovsky AB, Borisy GG (1995) Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. J Struct Biol 115(3):290–303

    Article  CAS  PubMed  Google Scholar 

  • Takizawa T, Suzuki K, Robinson JM (1998) Correlative microscopy using FluoroNanogold on ultrathin cryosections. Proof of principle. J Histochem Cytochem 46(10):1097–1102

    Article  CAS  PubMed  Google Scholar 

  • Valente C, Turacchio G, Mariggio S, Pagliuso A, Gaibisso R, Di Tullio G, Santoro M, Formiggini F, Spano S, Piccini D, Polishchuk RS, Colanzi A, Luini A, Corda D (2012) A 14-3-3gamma dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIbeta to regulate post-Golgi carrier formation. Nat Cell Biol 14(4):343–354

    Article  CAS  PubMed  Google Scholar 

  • van Rijnsoever C, Oorschot V, Klumperman J (2008) Correlative light–electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5(11):973–980

    Article  PubMed  Google Scholar 

  • Zumbusch A, Langbein W, Borri P (2013) Nonlinear vibrational microscopy applied to lipid biology. Prog Lipid Res 52(4):615–632

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Daniela Corda for critical reading of the manuscript. We thank AIRC (Italy), MIUR (FaReBio, PON01–00117, PON01–00862, PON03–00025, Progetti EPIGEN and Invecchiamento) (Italy) and FIRC (Italy) for financial support.

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Authors and Affiliations

  1. Institute of Protein Biochemistry, Consiglio Nazionale Delle Ricerche (CNR-IBP), Via P. Castellino 111, 80131, Naples, Italy

    Riccardo Rizzo, Seetharaman Parashuraman & Alberto Luini

  2. Telethon Institute for Genetics and Medicine (TIGEM), Via P. Castellino 111, 80131, Naples, Italy

    Alberto Luini

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  1. Riccardo Rizzo
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  2. Seetharaman Parashuraman
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  3. Alberto Luini
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Correspondence to Alberto Luini.

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Rizzo, R., Parashuraman, S. & Luini, A. Correlative video-light–electron microscopy: development, impact and perspectives. Histochem Cell Biol 142, 133–138 (2014). https://doi.org/10.1007/s00418-014-1249-3

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