Correlative Light and Electron Microscopy combines precise targeting of fluorescent proteins in resin sections with fine ultrastructural analysis by electron tomography (from Kukulski et al., J. Cell Biol., 2011).
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 – and for a large variety of biological samples ranging from macromolecules to tissues.
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, 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 conventional methods for sample preparation, we offer access to a set of high-pressure freezing machines that are routinely used to vitrify biological samples. Strong expertise has been developed in yeast cells, adherent cultured cells, Drosophila embryos, nematodes, zebrafish embryos, and mouse tissues. A new microwave-assisted sample processor for chemical fixation greatly reduces time spent preparing the samples (from days to hours). Our electron tomography equipment includes a transmission electron microscope (a FEI TF30 300kV microscope with a field emission gun and Eagle FEI 4K camera) and computing set-up with programs for 3D reconstruction and cellular modelling. Specitfalised EM engineers have expertise in tomography data acquisition and processing.
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: We are in the process of implementing FIB-SEM technology that will allow, among other applications, automated serial image acquisitions. The technology presents new opportunities for the understanding of the cellular fine architecture as it offers high-resolution 3D imaging (5 nm, isotropic) of volumes containing up to several cells. A Crossbeam Auriga 60 from Zeiss has been installed in spring 2014.
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
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
Romero-Brey, I., Merz, A., Chiramel, A., Lee, J.Y., Chlanda, P., Haselman, U., Santarella-Mellwig, R., Habermann, A., Hoppe, S., Kallis, S., Walther, P., Antony, C., Krijnse-Locker, J. & Bartenschlager, R.
PLoS Pathog. 2012, 8(12):e1003056. doi: 10.1371/journal.ppat.1003056. Epub 2012 Dec 6. Abstract
Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography.
Kukulski, W., Schorb, M., Kaksonen, M. & Briggs, J.A.
Cell. 2012 Aug 3, 150(3):508-20. doi: 10.1016/j.cell.2012.05.046. Abstract
Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A.
J Cell Biol. 2011 Jan 10, 192(1):111-9. doi: 10.1083/jcb.201009037. Epub 2011 Jan 3. Abstract
- Chemical fixation, high pressure freezing of cells and multi-cellular specimens.
- Resin embedding.
- Ultramicrotomy and cryo-ultramicrotomy (Tokuyasu technique).
- Sample preparation for single particle analysis.
- Immuno-labelling and TEM imaging.
- Electron tomography.
- FIB-SEM, whole-cell 3D imaging
- Correlative light and electron microscopy.
- Image analysis and 3D cellular modelling.
Teaching and training
- 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.