Skip to main content
SpringerLink
Log in

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Adams SR, Campbell RE, Gross LA, Martin BR, Walkup GK, Yao Y, Llopis J, Tsien RY (2002) New biarsenical ligands and tetracysteine motifs for protein labelling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 124:6063–6076

    Article  PubMed  CAS  Google Scholar 

  • Acton S, Scherer PE, Lodish HF, Krieger M (1994) Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem 269:21003–21009

    PubMed  CAS  Google Scholar 

  • Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M (1996) Identification of scavenger receptor SR-BI as high density lipoprotein receptor. Science 271:518–520

    Article  PubMed  CAS  Google Scholar 

  • Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RG, Xu S, Krieger M (1997) Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272:13242–13249

    Article  PubMed  CAS  Google Scholar 

  • Bascom RA, Srinivasan S, Nussbaum RL (1999) Identification and characterization of Golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain. J Biol Chem 274:2953–2962

    Article  PubMed  CAS  Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805

    Article  PubMed  CAS  Google Scholar 

  • Dantuma NP, Pijnenburg MA, Diederen JH, Van der Horst DJ (1998) Electron microscopic visualization of receptor-mediated endocytosis of DiI-labeled lipoproteins by diaminobenzidine photoconversion. J Histochem Cytochem 46:1085–1090

    PubMed  CAS  Google Scholar 

  • Deerinck TJ, Martone ME, Lev-Ram V, Green DP, Tsien RY, Spector DL, Huang S, Ellisman MH (1994) Fluorescence photooxidation with Eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J Cell Biol 126:901–910

    Article  PubMed  CAS  Google Scholar 

  • De-Miguel FF, Muller KJ, Adams WB, Nicholls JG (2002) Axotomy of single fluorescent nerve fibres in developing mammalian spinal cord by photoconversion of diaminobenzidine. J Neurosci Methods 117:73–79

    Article  PubMed  CAS  Google Scholar 

  • Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M (2003) The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation. J Cell Biol 160:201–212

    Article  PubMed  CAS  Google Scholar 

  • Eckhardt ERM, Cai L, Sun B, Webb NR, Van der Westhuyzen DR (2004) High density lipoprotein uptake by scavenger receptor SR-BII. J Biol Chem 279:14372–14381

    Article  PubMed  CAS  Google Scholar 

  • Fahimi HD, Baumgart E (1999) Current cytochemical techniques for the investigation of peroxisomes: a review. J Histochem Cytochem 47:1219–1232

    PubMed  CAS  Google Scholar 

  • Farhan H, Reiterer V, Korkhov VM, Schmid JA, Freissmuth M, Sitte HH (2007) Concentrative export from the endoplasmic reticulum of the γ-aminobutyric acid transporter 1 requires binding to SEC24D. J Biol Chem 282:7679–7689

    Article  PubMed  CAS  Google Scholar 

  • Fomina AF, Deerinck TJ, Ellisman MH, Cahalan MD (2003) Regulation of membrane trafficking and subcellular organisation of endocytic compartments revealed with FM1–43 in resting and activated human T cells. Exp Cell Res 291:150–166

    Article  PubMed  CAS  Google Scholar 

  • Gaietta GM, Deerinck TJ, Adams SR, Bouwer J, Tour O, Laird DW, Sosinsky GE, Tsien RY, Ellisman MH (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296:503–507

    Article  PubMed  CAS  Google Scholar 

  • Gaietta GM, Giepmans BN, Deerinck TJ, Smith WB, Ngan L, Llopis J, Adams SR, Tsien RY, Ellisman MH (2006) Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy. Proc Natl Acad Sci USA 103:17777–17782

    Article  PubMed  CAS  Google Scholar 

  • Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224

    Article  PubMed  CAS  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 2:857–862

    Article  PubMed  CAS  Google Scholar 

  • Graham RC Jr, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302

    PubMed  CAS  Google Scholar 

  • Greenbaum L, Rothmann C, Lavie R, Malik Z (2000) Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen. J Biol Chem 381:1251–1258

    Article  CAS  Google Scholar 

  • Guastella J, Nelson N, Nelson H, Czyzyk L, Keynan S, Miedel MC, Davidson N, Lester HA, Kanner BI (1990) Cloning and expression of a rat brain GABA transporter. Science 249:1303–1306

    Article  PubMed  CAS  Google Scholar 

  • Harata N, Ryan TA, Smith SJ, Buchanan J, Tsien RW (2001) Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1–43 photoconversion. Proc Natl Acad Sci 98:12748–12753

    Article  PubMed  CAS  Google Scholar 

  • Hillson DA, Lambert N, Freedman RB (1984) Formation and isomerization of disulfide bonds in proteins: protein disulfide-isomerase. Method Enzymol 107:281–194

    Article  CAS  Google Scholar 

  • Höglund P, Adzic D, Scicluna SJ, Lindblom J, Fredriksson R (2005) The repertoire of solute carriers of family 6: identification of new human and rodent genes. Biochem Biophys Res Commun 336:175–189

    Article  PubMed  CAS  Google Scholar 

  • Huitema K, Van den Dikkenberg J, Brouwers JF, Holthuis JC (2004) Identification of a family of animal sphingomyelin synthases. EMBO J 23:33–44

    Article  PubMed  CAS  Google Scholar 

  • Jyoti K, Simon J, Simon SM (2004) Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol 14:497–504

    Article  CAS  Google Scholar 

  • Kacza J, Härtig W, Seeger J (1997) Oxygen-enriched photoconversion of fluorescent dyes by means of a closed conversion chamber. J Neurosci Methods 71:225–232

    Article  PubMed  CAS  Google Scholar 

  • Kingsley DM, Krieger M (1984) Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity. Proc Natl Acad Sci USA 81:5454–5458

    Article  PubMed  CAS  Google Scholar 

  • Krieger M (2001) Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. J Clin Invest 108:793–797

    PubMed  CAS  Google Scholar 

  • Ladinsky MS, Kremer JR, Furcinitti PS, McIntosh JR, Howell KE (1994) HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat. J Cell Biol 127:29–38

    Article  PubMed  CAS  Google Scholar 

  • Ladinsky MS, Mastronarde DN, McIntosh JR, Howell KE, Staehelin LA (1999) Golgi structures in three dimensions: functional insights from the normal rat kidney cell. J Cell Biol 144:1135–1149

    Article  PubMed  CAS  Google Scholar 

  • Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91

    Article  PubMed  CAS  Google Scholar 

  • Lübke J (1993) Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product. Microsc Res Tech 1:2–14

    Article  Google Scholar 

  • Maranto AR (1982) Neuronal mapping: a photooxidation reaction makes lucifer yellow useful for electron microscopy. Science 217:953–955

    Article  PubMed  CAS  Google Scholar 

  • Martin OC, Pagano RE (1994) Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms. J Cell Biol 125:769–781

    Article  PubMed  CAS  Google Scholar 

  • Merrill AH, Jones DD (1990) An update of enzymology and regulation of sphingomyelin metabolism. Biochim Biophys Acta 1044:1–12

    PubMed  CAS  Google Scholar 

  • Monosov EZ, Wenzel TJ, Lüers GH, Heyman JA, Subramani S (1996) Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells. J Histochem Cytochem 44:581–589

    PubMed  CAS  Google Scholar 

  • Morris JI, Varandani PT (1988) Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase/oxidoreductase). Biochim Biophys Acta 949:169–180

    PubMed  CAS  Google Scholar 

  • Munro S, Pelham HR (1987) A c-terminal signal prevents secretion of luminal ER proteins. Cell 48:899–907

    Article  PubMed  CAS  Google Scholar 

  • Ni M, Lee AS (2007) ER chaperones in mammalian development and human diseases. FEBS Lett 581:3641–3651

    Article  PubMed  CAS  Google Scholar 

  • Noiva R, Lennarz WJ (1992) Protein disulfide isomerase: a multifunctional protein resident in the lumen of the endoplasmic reticulum. J Biol Chem 267:3553–3556

    PubMed  CAS  Google Scholar 

  • Pagano RE, Sepanski MA, Martin OC (1989) Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J Cell Biol 109:2067–2079

    Article  PubMed  CAS  Google Scholar 

  • Pagano RE, Martin OC, Kang HC, Haugland RP (1991) A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol 113:1267–1279

    Article  PubMed  CAS  Google Scholar 

  • Pagler TA, Rhode S, Neuhofer A, Laggner H, Strobl W, Hinterndorfer C, Volf I, Pavelka M, Eckhardt ER, Van der Westhuyzen DR, Schütz GJ, Stangl H (2006) SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux. J Biol Chem 281:11193–11204

    Article  PubMed  CAS  Google Scholar 

  • Pavelka M, Ellinger A (1989) Pre-embedding labeling techniques applicable to intracellular binding sites. In: Plattner H Electron microscopy of subcellular dynamics, CDC Press, Boca Raton, pp 199–218

  • Pavelka M, Ellinger A, Debbage P, Loewe C, Vetterlein M, Roth J (1998) Endocytic routes to the Golgi apparatus. Histochem Cell Biol 109:555–570

    Article  PubMed  CAS  Google Scholar 

  • Pavelka M, Neumüller J, Ellinger A (2008) Retrograde traffic in the biosynthetic-secretory route. Histochem Cell Biol 129:277–288

    Article  PubMed  CAS  Google Scholar 

  • Rajfur Z, Roy P, Otey C, Romer L, Jacobson K (2002) Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat Cell Biol 4:286–293

    Article  PubMed  CAS  Google Scholar 

  • Sandell JH, Masland RH (1988) Photoconversion of some fluorescent markers to a diaminobenzidine product. J Histochem Cytochem 36:555–559

    PubMed  CAS  Google Scholar 

  • Satoh A, Wang Y, Malsam J, Beard MB, Warren G (2003) Golgin-84 is a rab1 binding partner involved in Golgi structures. Traffic 4:153–160

    PubMed  CAS  Google Scholar 

  • Schmid JA, Scholze P, Kudlacek O, Freissmuth M, Singer EA, Sitte HH (2001) Oligomerization of human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells. J Biol Chem 276:3805–3810

    Article  PubMed  CAS  Google Scholar 

  • Singleton CD, Casagrande VA (1996) A reliable and sensitive methode for fluorescent photoconversion. J Neurosci Methods 64:47–54

    Article  PubMed  CAS  Google Scholar 

  • Surrey T, Elowitz MB, Wolf PE, Yang F, Nedelec F, Shokat K, Leibler S (1998) Chromophore-assisted light inactivation and self-organization of microtubules and motors. Proc Natl Acad Sci USA 95:4293–4298

    Article  PubMed  CAS  Google Scholar 

  • Tour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY (2003) Genetically targeted chromophore-assisted light inactivation. Nat Biotech 21:1505–1508

    Article  CAS  Google Scholar 

  • Tsien RY (1998) The green fluorescent protein. Ann Rev Biochem 67:509–544

    Article  PubMed  CAS  Google Scholar 

  • Vetterlein M, Ellinger A, Neumüller J, Pavelka M (2002) Golgi apparatus and TGN during endocytosis. Histochem Cell Biol 117:143–150

    Article  PubMed  CAS  Google Scholar 

  • Vetterlein M, Niapir M, Ellinger A, Neumüller J, Pavelka M (2003) Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin. Histochem Cell Biol 120:121–128

    Article  PubMed  CAS  Google Scholar 

  • Voelker DR, Kennedy EP (1982) Cellular and enzymic synthesis of sphingomyelin. Biochemistry 21:2753–2759

    Article  PubMed  CAS  Google Scholar 

  • Von Bartheld CS, Cunningham DE, Rubel EW (1990) Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion of light and electron microscopic analysis. J Histochem Cytochem 38:725–733

    Google Scholar 

  • Yuste R (2005) Fluorescence microscopy today. Nat Methods 2:902–904

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the valuable technical assistance of Mrs.Elfriede Scherzer, and Mrs.Beatrix Mallinger, and like to thank Mr.Ulrich Kaindl for the excellent work done in connection with the 3D-modelling, and the preparation of the figures. Parts of the work were supported by the Austrian Science Foundation (FWF) grant P20116-B11, and SFB 35.

Author information

Authors and Affiliations

  1. Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University Vienna, Schwarzspanierstrasse 17, 1090, Vienna, Austria

    Claudia Meißlitzer-Ruppitsch, Monika Vetterlein, Josef Neumüller, Margit Pavelka & Adolf Ellinger

  2. Institute of Medical Chemistry, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria

    Herbert Stangl

  3. Institute of Pharmacology, Center for Biomolecular Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria

    Susanne Maier & Michael Freissmuth

Authors
  1. Claudia Meißlitzer-Ruppitsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Monika Vetterlein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herbert Stangl
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Susanne Maier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Josef Neumüller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Freissmuth
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Margit Pavelka
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Adolf Ellinger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Adolf Ellinger.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meißlitzer-Ruppitsch, C., Vetterlein, M., Stangl, H. et al. Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion. Histochem Cell Biol 130, 407–419 (2008). https://doi.org/10.1007/s00418-008-0429-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-008-0429-4

Keywords

Search

Navigation