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: Immungold labeling is a routine technique offered by the facility. In this work, performed by Charlotta Funaya, Erg11-HA in asi1∆ cells is labelled with antibodies and identified by 10 nm gold particles (from Foresti et al., Science, 2014).
The facility provides advanced expertise in electron microscopy, from sample preparation to image analysis, for a large 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: We offer access to a set of high-pressure freezing machines that 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). Our electron tomography equipment includes a transmission electron microscope (a FEI TECNAI F30 300kV microscope with a field emission gun and Gatan OneView 4K camera) and computing workstations set-up with programs 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 automated serial imaging in scanning electron microscopy (ASI-SEM), such as 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.
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
ENDOCYTOSIS. Endocytic sites mature by continuous bending and remodeling of the clathrin coat.
Avinoam O, Schorb M, Beese CJ, Briggs JA, Kaksonen M.
Science 2015, 348(6241):1369-1372. doi: 10.1126/science.aaa9555 | Abstract
Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1.
Francis MK, Holst MR, Vidal-Quadras M, Henriksson S, Santarella-Mellwig R, Sandblad L, Lundmark R.
J Cell Sci 2015, 128:4183-4195. doi: 10.1242/jcs.174417 | Abstract
An arp2/3 nucleated f-actin shell fragments nuclear membranes at nuclear envelope breakdown in starfish oocytes.
Mori M, Somogyi K, Kondo H, Monnier N, Falk HJ, Machado P, Bathe M, Nédélec F, Lénárt P.
Curr. Biol. 2014, 24(12):1421-1428. doi: 10.1016/j.cub.2014.05.019 | Abstract
Quality control of inner nuclear membrane proteins by the Asi complex.
Foresti O, Rodriguez-Vaello V, Funaya C, Carvalho P.
Science. 2014, 346(6210):751-5. doi: 10.1126/science.1255638. Epub 2014 Sep 18. Abstract
Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.
Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M & Briggs JA.
J Cell Biol. 2011 Jan 10, 192(1):111-9. doi: 10.1083/jcb.201009037. Epub 2011 Jan 3. Abstract
- 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.
The Electron Microscopy Core Facility collaborates closely with its technology partners.