Latest publications

10 August 2020

NCBI pubmed
  • FIB-SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20.
    Related Articles

    FIB-SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20.

    J Microsc. 2019 02;273(2):91-104

    Authors: Porrati F, Grewe D, Seybert A, Frangakis AS, Eltsov M

    Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high-pressure freezing and freeze-substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze-substitution with per-mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time-efficient FIB-SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM-TEM workflows to comprehensively analyse biological structures. LAY DESCRIPTION: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three-dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy-based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub-cellular and sub-organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl-embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.

    PMID: 30417390 [PubMed - indexed for MEDLINE]

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    Grahammer F, Wigge C, Schell C, Kretz O, Patrakka J, Schneider S, Klose M, Arnold SJ, Habermann A, Bräuniger R, Rinschen MM, Völker L, Bregenzer A, Rubbenstroth D, Boerries M, Kerjaschki D, Miner JH, Walz G, Benzing T, Fornoni A, Frangakis AS, Huber TB. JCI Insight. 2016 Jun 16;1(9). pii: e86177.
    PubMed PMID: 27430022 


  • Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.
    Mühleip AW, Joos F, Wigge C, Frangakis AS, Kühlbrandt W, Davies KM.
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    PubMed PMID: 27402755 


  • Three-dimensional CTF correction improves the resolution of electron tomograms.
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  • Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography.
    Eltsov M, Dubé N, Yu Z, Pasakarnis L, Haselmann-Weiss U, Brunner D, Frangakis AS.
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    McEwan DG, Richter B, Claudi B, Wigge C, Wild P, Farhan H, McGourty K, Coxon FP, Franz-Wachtel M, Perdu B, Akutsu M, Habermann A, Kirchof A, Helfrich MH, Odgren PR, Van Hul W, Frangakis AS, Rajalingam K, Macek B, Holden DW, Bumann D, Dikic I.
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    Kunz M, Frangakis AS.
    J Struct Biol. 2014 Nov;188(2):107-15. doi: 10.1016/j.jsb.2014.09.010. Epub 2014 Oct 2.
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  • M-free: scoring the reference bias in sub-tomogram averaging and template matching
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    Perkovic M, Kunz M, Endesfelder U, Bunse S, Wigge C, Yu Z, Hodirnau VV, Scheffer MP, Seybert A, Malkusch S, Schuman J Struct Biol. 2014 May;186(2):205-13. doi: 10.1016/j.jsb.2014.03.018. Epub 2014 Mar 31.
    PubMed PMID: 24698954


  • CENP-A arrays are more condensed than canonical arrays at low ionic strength.
    Geiss CP, Keramisanou D, Sekulic N, Scheffer MP, Black BE, Frangakis AS.
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  • Three-dimensional visualization of the molecular architecture of cell-cell junctions in situ by cryo-electron tomography of vitreous sections.
    Al-Amoudi A, Frangakis AS.
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  • 3D Ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography.
    Hummel E, Guttmann P, Werner S, Tarek B, Schneider G, Kunz M, Frangakis AS, Westermann B.
    PLoS One. 2012;7(12):e53293. doi: 10.1371/journal.pone.0053293. Epub 2012 Dec 31.
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  • Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures.
    Saibil HR, Seybert A, Habermann A, Winkler J, Eltsov M, Perkovic M, Castaño-Diez D, Scheffer MP, Haselmann U, Chlanda P, Lindquist S, Tyedmers J, Frangakis AS.

    Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):14906-11. doi: 10.1073/pnas.1211976109. Epub 2012 Aug 27.
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  • Room temperature L1₀ phase transformation in binary CoPt nanostructures prepared by focused-electron-beam-induced deposition.
    Porrati F, Begun E, Winhold M, Schwalb ChH, Sachser R, Frangakis AS, Huth M.
    Nanotechnology. 2012 May 11;23(18):185702. doi: 10.1088/0957-4484/23/18/185702. Epub 2012 Apr 13.
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  • Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure.
    Nishino Y, Eltsov M, Joti Y, Ito K, Takata H, Takahashi Y, Hihara S, Frangakis AS, Imamoto N, Ishikawa T, Maeshima K.
    EMBO J. 2012 Apr 4;31(7):1644-53. doi: 10.1038/emboj.2012.35. Epub 2012 Feb 17.
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  • Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs.
    Hergenreider E, Heydt S, Tréguer K, Boettger T, Horrevoets AJ, Zeiher AM, Scheffer MP, Frangakis AS, Yin X, Mayr M, Braun T, Urbich C, Boon RA, Dimmeler S.

    Nat Cell Biol. 2012 Feb 12;14(3):249-56. doi: 10.1038/ncb2441.
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  • Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro.
    Scheffer MP, Eltsov M, Bednar J, Frangakis AS.

    J Struct Biol. 2012 May;178(2):207-14. doi: 10.1016/j.jsb.2011.11.020. Epub 2011 Nov 22.
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  • Binary Pt-Si nanostructures prepared by focused electron-beam-induced deposition.
    Winhold M, Schwalb CH, Porrati F, Sachser R, Frangakis AS, Kämpken B, Terfort A, Auner N, Huth M.
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  • Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei.
    Scheffer MP, Eltsov M, Frangakis AS.
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  • The three-dimensional molecular structure of the desmosomal plaque.
    Al-Amoudi A, Castaño-Diez D, Devos DP, Russell RB, Johnson GT, Frangakis AS.
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  • Classification of electron sub-tomograms with neural networks and its application to template-matching.
    Yu Z, Frangakis AS.
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  • Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography.
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  • Visualization of macromolecular structures.
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  • Alignator: a GPU powered software package for robust fiducial-less alignment of cryo tilt-series.
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  • Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing.
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    PubMed PMID: 19965468


  • Cytoplasmic relaxation of active Eph controls ephrin shedding by ADAM10.
    Janes PW, Wimmer-Kleikamp SH, Frangakis AS, Treble K, Griesshaber B, Sabet O, Grabenbauer M, Ting AY, Saftig P, Bastiaens PI, Lackmann M.
    PLoS Biol. 2009 Oct;7(10):e1000215. doi: 10.1371/journal.pbio.1000215. Epub 2009 Oct 13.
    PubMed PMID: 19823572


  • Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ.
    Eltsov M, Maclellan KM, Maeshima K, Frangakis AS, Dubochet J.
    Proc Natl Acad Sci U S A. 2008 Dec 16;105(50):19732-7. doi: 10.1073/pnas.0810057105. Epub 2008 Dec 8.
    PubMed PMID: 19064912


    • Performance evaluation of image processing algorithms on the GPU.
      Castaño-Díez D, Moser D, Schoenegger A, Pruggnaller S, Frangakis AS.
      J Struct Biol. 2008 Oct;164(1):153-60. doi: 10.1016/j.jsb.2008.07.006. Epub 2008 Jul 24.
      PubMed PMID: 18692140


  • A visualization and segmentation toolbox for electron microscopy.
    Pruggnaller S, Mayr M, Frangakis AS.
    J Struct Biol. 2008 Oct;164(1):161-5. doi: 10.1016/j.jsb.2008.05.003. Epub 2008 May 17.
    PubMed PMID: 18691905


  • Structural studies on desmosomes.
    Al-Amoudi A, Frangakis AS.
    Biochem Soc Trans. 2008 Apr;36(Pt 2):181-7. doi: 10.1042/BST0360181. Review.
    PubMed PMID: 18363559


  • Reprint of "Fiducial-less alignment of cryo-sections" [J. Struct. Biol. 159 (2007) 413-423].
    Castaño-Díez D, Al-Amoudi A, Glynn AM, Seybert A, Frangakis AS.
    J Struct Biol. 2008 Mar;161(3):249-59. doi: 10.1016/S1047-8477(08)00060-9.
    PubMed PMID: 18342740


  • The molecular architecture of cadherins in native epidermal desmosomes.
    Al-Amoudi A, Díez DC, Betts MJ, Frangakis AS.
    Nature. 2007 Dec 6;450(7171):832-7.
    PubMed PMID: 18064004


  • Classification of cryo-electron sub-tomograms using constrained correlation.
    Förster F, Pruggnaller S, Seybert A, Frangakis AS.
    J Struct Biol. 2008 Mar;161(3):276-86. Epub 2007 Jul 26.
    PubMed PMID: 17720536


  • Fiducial-less alignment of cryo-sections.
    Castaño-Díez D, Al-Amoudi A, Glynn AM, Seybert A, Frangakis AS
    J Struct Biol. 2007 Sep;159(3):413-23. Epub 2007 Jun 9.
    PubMed PMID: 17651988



  • Plastic-embedded protein crystals.
    Ravelli RB, Haselmann-Weiss U, McGeehan JE, McCarthy AA, Marquez JA, Antony C, Frangakis AS, Stranzl G.
    J Synchrotron Radiat. 2007 Jan;14(Pt 1):128-32. Epub 2006 Dec 15.
    PubMed PMID: 17211079


  • Visualization of cell microtubules in their native state.
    Bouchet-Marquis C, Zuber B, Glynn AM, Eltsov M, Grabenbauer M, Goldie KN, Thomas D, Frangakis AS, Dubochet J, Chrétien D.
    Biol Cell. 2007 Jan;99(1):45-53.
    PubMed PMID: 17049046


  • Implementation and performance evaluation of reconstruction algorithms on graphics processors.
    Castaño Díez D, Mueller H, Frangakis AS. 
    J Struct Biol. 2007 Jan;157(1):288-95. Epub 2006 Sep 1.
    PubMed PMID: 17029985


  • Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography.
    Briegel A, Dias DP, Li Z, Jensen RB, Frangakis AS, Jensen GJ.
    Mol Microbiol. 2006 Oct;62(1):5-14.
    PubMed PMID: 16987173


  • Structural analysis of Mycoplasma pneumoniae by cryo-electron tomography.
    Seybert A, Herrmann R, Frangakis AS.
    J Struct Biol. 2006 Nov;156(2):342-54. Epub 2006 May 7.
    PubMed PMID: 16875842


  • Tilt-series and electron microscope alignment for the correction of the non-perpendicularity of beam and tilt-axis.
    Díez DC, Seybert A, Frangakis AS.
    J Struct Biol. 2006 May;154(2):195-205. Epub 2006 Jan 26. 
    PubMed PMID: 16503168


  • Cryo-electron tomography reveals the cytoskeletal structure of Spiroplasma melliferum.
    Kürner J, Frangakis AS, Baumeister W.
    Science. 2005 Jan 21;307(5708):436-8.
    PubMed PMID: 15662018


  • Computational exploration of structural information from cryo-electron tomograms.
    Frangakis AS, Förster F.
    Curr Opin Struct Biol. 2004 Jun;14(3):325-31. Review.
    PubMed PMID: 15193312


  • Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.
    Medalia O, Weber I, Frangakis AS, Nicastro D, Gerisch G, Baumeister W.
    Science. 2002 Nov 8;298(5596):1209-13.
    PubMed PMID: 112424373


  • Identification of macromolecular complexes in cryoelectron tomograms of phantom cells.
    Frangakis AS, Böhm J, Förster F, Nickell S, Nicastro D, Typke D, Hegerl R, Baumeister W.
    Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14153-8. Epub 2002 Oct 21.
    PubMed PMID: 12391313


  • Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis.
    Frangakis AS, Hegerl R.
    J Struct Biol. 2002 Apr-May;138(1-2):105-13.
    PubMed PMID: 12160706


  • Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion.
    Frangakis AS, Hegerl R.
    J Struct Biol. 2001 Sep;135(3):239-50.
    PubMed PMID: 11722164


  • FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study.
    Böhm J, Lambert O, Frangakis AS, Letellier L, Baumeister W, Rigaud JL.
    Curr Biol. 2001 Aug 7;11(15):1168-75.
    PubMed PMID: 11516947


  • Wavelet transform filtering and nonlinear anisotropic diffusion assessed for signal reconstruction performance on multidimensional biomedical data.
    Frangakis AS, Stoschek A, Hegerl R.
    IEEE Trans Biomed Eng. 2001 Feb;48(2):213-22.
    PubMed PMID: 11296877


  • Toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms.
    Bohm J, Frangakis AS, Hegerl R, Nickell S, Typke D, Baumeister W.
    Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14245-50.
    PubMed PMID: 11087814


  • Cryo-electron tomography of neurospora mitochondria.
    Nicastro D, Frangakis AS, Typke D, Baumeister W. 
    J Struct Biol. 2000 Feb;129(1):48-56.
    PubMed PMID: 10675296