Figure 1: On section CLEM: the signal from fluorescent proteins is used to target the EM analysis to specific subcellular timepoints during the endocytic process (adapted from Avinoam, et al. 2015).
Figure 2: Live-cell correlative light and FIB-SEM captures specific stages of anaphase in dividing HeLa cells, revealing the formation of new nuclear envelope and the integration of nuclear pores. Adapted with permission from Otsuka et al. 2018
The facility provides advanced expertise in electron microscopy, from sample preparation to image analysis, for a wide variety of biological samples.
The EMCF activities cover a large spectrum of EM techniques with a major focus on sample preparation, immuno-localisation of proteins, ultrastructural analysis in 2D and 3D, correlative light and electron microscopy and data processing. Staff in the facility can help you to define optimal experimental conditions for your project – we have experience spanning virtually the full spectrum of biological specimens, with high-level resources for both research and training.
Major projects and accomplishments
Advanced equipment: Besides the EM imaging, we offer access to a high-pressure freezing machine. These instruments are routinely used to vitrify biological samples. Specimens can then be dehydrated, stabilised and embedded in resins in specific freeze-substitution units. Strong expertise has been developed for yeast cells, adherent cultured cells, Drosophila embryos, nematodes, zebrafish embryos, and mouse tissues. A microwave-assisted sample processor, used for chemical fixation, dehydration and embedding, greatly reduces time spent preparing the samples (from days to hours). For Tomography, two transmission electron microscopes (a FEI TECNAI F30 300kV and a 200kV JEOL JEM 2100Plus) are available. EMCF services include access to EMBL IT's central storage and compute facilities for 3D reconstruction and cellular modelling.
The Electron microscopist ‘savoir faire’: We are deeply involved in method development and training. A recent example in correlative light and electron microscopy (CLEM) is the implementation of a technique developed by the Briggs and Kaksonen groups, which tracks the signal of fluorescent proteins in resin sections with high precision.
The future in perspective: Since 2014, the facility has started to provide services in focused ion beam SEM (FIB-SEM). This technique complements our portfolio of 3D imaging applications (serial section, electron tomography) by providing new opportunities for understanding the cellular fine architecture of multi-cellular specimens. The facility is also offering support for the recently developed cryo-CLEM techniques (see Schorb and Briggs 2014, 2016), which will nicely bridge structural and cell biology.
Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.
Cortese M, Lee JY, Cerikan B, Neufeldt CJ, Oorschot VMJ, Köhrer S, Hennies J, Schieber NL, Ronchi P, Mizzon G, Romero-Brey I, Santarella-Mellwig R, Schorb M, Boermel M, Mocaer K, Beckwith MS, Templin RM, Gross V, Pape C, Tischer C, Frankish J, Horvat NK, Laketa V, Stanifer M, Boulant S, Ruggieri A, Chatel-Chaix L, Schwab Y, Bartenschlager R.
Cell Host Microbe. 2020 Nov 17:S1931-3128(20)30620-X. doi: 10.1016/j.chom.2020.11.003
In-cell architecture of the nuclear pore and snapshots of its turnover.
Allegretti M, Zimmerli CE, Rantos V, Wilfling F, Ronchi P, Fung HKH, Lee CW, Hagen W, Turoňová B, Karius K, Börmel M, Zhang X, Müller CW, Schwab Y, Mahamid J, Pfander B, Kosinski J, Beck M.
Nature. 2020 Oct;586(7831):796-800. doi: 10.1038/s41586-020-2670-5
Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.
Hampoelz B, Schwarz A, Ronchi P, Bragulat-Teixidor H, Tischer C, Gaspar I, Ephrussi A, Schwab Y, Beck M.
Cell. 2019 Oct 17;179(3):671-686.e17. doi: 10.1016/j.cell.2019.09.022
Software tools for automated transmission electron microscopy.
Schorb M, Haberbosch I, Hagen WJH, Schwab Y, Mastronarde DN.
Nature Methods. 2019 Jun. doi: 10.1038/s41592-019-0396-9.
Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings.
Otsuka S, Steyer AM, Schorb M, Hériché JK, Hossain MJ, Sethi S, Kueblbeck M, Schwab Y, Beck M, Ellenberg J.
Nat Struct Mol Biol. 2018 Jan;25(1):21-28. doi: 10.1038/s41594-017-0001-9. | Abstract
Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.
Hampoelz B, Mackmull MT, Machado P, Ronchi P, Bui KH, Schieber N, Santarella-Mellwig R, Necakov A, Andrés-Pons A, Philippe JM, Lecuit T, Schwab Y, Beck M.
Cell 2016, 166(3):664-78. doi: 10.1016/j.cell.2016.06.015 | Abstract
ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.
Curwin AJ, Brouwers N, Alonso Y Adell M, Teis D, Turacchio G, Parashuraman S, Ronchi P, Malhotra V.
Elife 2016, 5. pii: e16299. doi: 10.7554/eLife.16299 | Abstract
Single-particle and tomography cryo-EM for structural biology projects is offered through the cryo-EM service platform.
- Negative staining.
- Chemical fixation, high pressure freezing of cells and multi-cellular specimens.
- Resin embedding.
- Ultramicrotomy (including serial sectioning).
- Cryo-ultramicrotomy (for the Tokuyasu technique and for CEMOVIS).
- 3D EM imaging (TEM tomography, FIB-SEM).
- Correlative light and electron microscopy (live cell imaging CLEM, on section CLEM, cryo-CLEM, 3D CLEM).
- Image analysis and 3D cellular modelling.
- Training of the users on all the accessible techniques.
- Organising courses and lectures on EM methods in cell biology.