General Info

EMBL Imaging Centre in collaboration with Heidelberg University is offering a 4-day summer school diving into basics of focused ion beam – scanning electron microscopy methods under cryogenic (cryo-FIB-SEM), which allows to study biological samples in their native environment at high-resolution.

Content of the course:

•  Theoretical background and practicals delivered by experts in the field

•  Sample preparation, lamella preparation and cryo-electron tomography

•  Open equipment day to explore further

External invited speaker:

Florent Waltz

University of Basel

Organisers:

Anna Steyer

Mattei Team

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Simone Mattei

Mattei Team

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Petr Chlanda

BioQuant – Heidelberg University

Contact: Anna Steyer anna.steyer@embl.de

To participate to the summer school please register by clicking on the following button. Participation is limited to 9 participants. Deadline for registration is April 1st 2024.

Selected participants will be announced on April 8th 2024.

Sponsor:

Lecture/Practical Content

Lectures:

1. Florent Waltz (external speaker, university of Basel): Exploring the architecture of the cell at high-resolution. The lecture will dive into different molecular machines at high-resolution using cryo-FIB-SEM as well as cryo-tomography.
2. Petr Chlanda (University of Heidelberg): Cryo-CLEM: Structural analysis of virus-infected cells. This lecture will demonstrate how the combination of cryo-LM and cryo-EM can be used to perform structural analysis on pathogens.
3. Georg Wolff (EMBL): Plunging vs. high-pressure: How to prepare your FIB-SEM sample? General basics of sample preparation methods covering the vitrification part of small specimen using a plunge freezer or larger specimen using high-pressure freezing.
4. Zhengyi Yang (EMBL): Cryo-Tomography: Tools and concepts in cryo electron tomography. The lecture will dive into the basic principles of cryo-tomography, what can be achieved with it and a general overview of pipelines, including software that could be used.
5. Anna Steyer (EMBL): Breaking the ice: Basic principals of the FIB-SEM in cryo. General basics of the Focused ion beam – scanning electron microscope hardware and software related will be given as well as practical aspects of lamella preparation.

Practicals:

Practical 1: Sample preparation (Plunge freezing and high-pressure freezing)

High-resolution electron microscopy requires well vitrified specimen. Depending on the thickness of the sample this can be either accomplished by plunge freezing for thinner specimen, up to a few µm or high-pressure freezing up to ~200 µm.

Transmission electron microscopy (TEM) grids are plasma cleaned to make them less hydrophobic, allowing an even spread of sample. Two different plunge freezers allowing one-side and two-sided blotting used for cells or purified proteins are used to vitrify cells/protein suspensions. A high-pressure freezer is used to freeze thicker specimen e.g. C elegans.

The participants will have the opportunity to use:

• Vitrobot Mark 4 (Thermo Fischer Scientific) for freezing protein suspensions
• GP2 (Leica Microsystems) for freezing adherent cells
• EM ICE (Leica Microsystems) high-pressure freezer to vitrify C. elegans in metal carrier

Practical 2: Lamella preparation: Serial-FIB/AutoTEM/iFLM

After sample vitrification the TEM grid is introduced into the FIB-SEM and the region of interest is identified. Different milling steps with decreasing beam currents are applied to gradually remove cell material (starting from 1 nA down to 30 pA). Understanding the geometry and cutting approaches while practicing on a real sample will give the best understanding of the challenges, throughput and the possibilities imposed by this method.

The participants will have the opportunity to use:

• ThermoScientific Aquilos to prepare lamellae using SerialFIB
• ThermoScientific Aquilos 2 with an integrated fluorescence light microscope (iFLM) to prepare lamellae
• Zeiss Crossbeam 550 to prepare lamellae using SerialFIB

Practical 3: Cryo-Tomography setup

The TEM grid is introduced into the transmission electron microscope and an overview image of the full grid is taken. After identifying the regions with the lamellae, medium magnification montages are taken to acquire images of the lamella being able to identify and target regions of interest for high-resolution tilt series.

The participants will have the opportunity to use:

• ThermoScientific Titan Krios

Practical 4: Open equipment practical

This practical will allow everybody to spend some more time practice operating an instrument of their choice.

The participants will have the opportunity to use all the equipment used during duration of the course.

Programme

Practical Information

The course is limited to 9 participants. For selection purposes, please note that your application will not be considered without CV and a letter of motivation, stating your background and how this course would be beneficial for your future work.

Selected participants will be announced on April 8th 2024.

There is no registration fee.

Contact: Anna Steyer anna.steyer@embl.de