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Cryo-focused ion beam scanning electron microscope to prepare cells for visualising their molecular architecture by electron cryo-tomography

Applicant Kukulski Wanda
Number 198524
Funding scheme R'EQUIP
Research institution Institut für Biochemie und Molekulare Medizin Universität Bern
Institution of higher education University of Berne - BE
Main discipline Cellular Biology, Cytology
Start/End 01.11.2021 - 31.10.2022
Approved amount 525'000.00
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All Disciplines (2)

Discipline
Cellular Biology, Cytology
Molecular Biology

Keywords (5)

in situ structural biology; cyo-electron tomography; cryo-correlative microscopy; cryo-FIB SEM; cellular imaging

Lay Summary (German)

Lead
Lebewesen bestehen aus Zellen, welche einen komplexen Aufbau haben. Die räumliche Organisation des Zellinneren dient der Zellfunktion: Organellen bilden Kompartimente mit unterschiedlichen Aufgaben, und das Zytoskelett verleiht Stabilität und Form. Auch auf der Ebene von Molekülen sind Zellen organisiert, so führen akute Zellaktivitäten zu einer lokalen Anhäufung oder Umverteilung spezifischer Proteine. Die molekulare Architektur ist wichtig für das Verständnis der Vorgänge in Zellen, aber sie ist wenig erforscht.
Lay summary

Das Zellinnere wird seit Jahrzenten mit Elektronenmikroskopen abgebildet. Allerdings war die Interpretation stets durch die Präparation limitiert. Ein wichtiger Fortschritt ist die Kryo-Elektronenmikroskopie von Zellen. Dabei werden Zellen so rasch gefroren, dass Wasser nicht zu Eis kristallisiert. Die Zellen sind in einem nahezu unveränderten Momentzustand, und molekulare Strukturen sind bis in kleinste Details erhalten. Allerdings sind Zellen meist zu ‘dick’ um direkt im Elektronenmikroskop abgebildet zu werden. Es müssen dünne Zellschnitte hergestellt werden. Dies geschieht mit einem fokussierten Ionenstrahl im gekühlten Rasterelektronenmikroskop (cryo-FIB SEM). Dabei ‘fräst’ der Ionenstrahl den Grossteil der Zelle präzis weg, so dass eine dünne Scheibe übrigbleibt, welche sich mittels Kryo-Elektronentomographie abbilden lässt. Die resultierenden 3D Bilder liefern beispiellose Einblicke ins Zellinnere. Mit Bildverarbeitung kann man die Auflösung einzelner Strukturen weiter erhöhen. So lassen sich Proteinkomplexe in ihrer natürlichen Umgebung untersuchen, was Rückschlüsse auf die Mechanismen ihrer Aktivität erlaubt. Das cryo-FIB SEM wird daher dazu beitragen, neue Erkenntnisse zu den molekularen Vorgängen in gesunden und pathologischen Zellen zu gewinnen.

Direct link to Lay Summary Last update: 26.08.2021

Responsible applicant and co-applicants

Project partner

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Abstract

Electron cryo-tomography (cryo-ET) can visualise the native architecture inside cells at molecu-lar resolution. It allows to see individual protein molecules within their endogenous environ-ment and hence to study their architecture in situ. The resolution can be increased further by processing the data using subtomogram averaging, building on the methodology for single par-ticle cryo-EM that has revolutionised structural biology. It is widely believed that cellular cryo-ET coupled to in situ structural biology is the next revolution in the cryo-EM field. Like cryo-EM samples, cells are vitrified for cryo-ET by rapid plunge-freezing in liquid ethane. However, for the most part cells are too thick for the electron beam to pass. To perform cryo-ET it is there-fore necessary to produce thin sections through cells. In recent years, milling with a focused ion beam (FIB) in a scanning electron microscope (SEM) has proven ideal for thinning cells whilst maintaining near-native preservation. A dedicated instrument is referred to as cryo-FIB-SEM or cryo-dual beam microscope. It is used to ablate large parts of individual cells, leaving so-called lamellae, which are ~200 nm sections through the cell. After thinning, the sample is transferred to a cryo-transmission electron microscope (TEM) for collection of cryo-ET data. The resulting 3D volumes have the potential to provide unprecedented views into diverse cellu-lar interiors and reveal the arrangement of macromolecules. At present, the University of Bern has a growing community of experts in cellular cryo-ET, however lacks the necessary state-of-the-art equipment. With this proposal we wish to present the need for a cryo-FIB-SEM at the University of Bern, which will be part of the infrastructure for cellular cryo-ET and will serve a broad community of researchers at the University of Bern. Accordingly, the research proposed here by the applicants and project partners reflects diverse applications, from in situ determi-nation of target protein structures, to the visualisation of complex organisation of subcelullar compartments, and the development of novel workflows for making medically relevant experi-mental models accessible by cryo-ET. We are convinced that enabling cryo-FIB-SEM-based cellular cryo-ET will boost new interdisciplinary research directions at University of Bern, and will contribute critically to increasing fundamental biomedical knowledge.
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