PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy
| Autor: | Ava Handley, Roger Pocock, Alex de Marco, James C. Whisstock, Gediminas Gervinskas, Sergey Gorelick, Monica Pia Caggiano, Genevieve Buckley, Travis K. Johnson |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Electron Microscope Tomography Microscope Ion beam Structural Biology and Molecular Biophysics viruses S. cerevisiae law.invention targeted cryo-FIB 0302 clinical medicine law Microscopy Biology (General) in situ structural biology Neurons D. melanogaster General Neuroscience General Medicine CLEM Tools and Resources Drosophila melanogaster C. elegans Medicine Correlative Materials science QH301-705.5 Science Nanotechnology macromolecular substances General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Optical microscope cryo-FIB Animals correlative microscopy Caenorhabditis elegans cryo-LM Lamella (cell biology) General Immunology and Microbiology Cryoelectron Microscopy Molecular biophysics cryo-ET 030104 developmental biology Multiprotein Complexes cryo-CLEM Electron microscope cryo-light microscopy 030217 neurology & neurosurgery |
| Zdroj: | eLife, Vol 8 (2019) eLife |
| DOI: | 10.5281/zenodo.3260173 |
| Popis: | Cryo-Electron Tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible. |
| Databáze: | OpenAIRE |
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