Instruct-ERIC hands-on workshop on: Advanced cryo-FIB-SEM methods (09.-13.11.2026)

General

EMBL Imaging Centre is offering a 5-day course diving into advanced operations of focused ion beam – scanning electron microscopy methods under cryogenic conditions (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

•  Advanced sample preparation using waffle method, advanced lamella preparation, correlative workflows and lift-out procedures.

•  Open equipment day to explore further

The course is limited to 12 participants. Deadline for registration is July 1^st 2026.

There is no registration fee.

Selected participants from an Instruct member state can receive a travel grant up to 550€.

Speakers/Trainers

Anna Steyer

EMBL Imaging Centre

Zhengyi Yang

EMBL Imaging Centre

Georg Wolff

EMBL Imaging Centre

Evgenia Zagoriy

MSB Unit, EMBL Heidelberg

Invited speaker:

Calina Glynn

Rosalind Franklin Institute

Lectures/Practical Content

1. Calina Glynn: Future directions with the FIB – What can the Plasma-FIB bring?
   This lecture will cover the use and the benefit of using a plasma-FIB for larger volumes and higher throughput.
2. Evgenia Zagoriy: Cellular sample preparation for the FIB. How to prepare your sample in the best possible way for FIB milling and how to target and correlate.
3. Zhengyi Yang: Automated lamella preparation on grid and lift-out procedures: How to use software to automate lamella preparation and most recent developments regarding lift-out.
4. Georg Wolff: How to prepare your FIB-SEM sample using the Waffle method?
   General basics of this sample preparation method for on-grid lamella preparation will be given, covering the vitrification part and specifics about milling lamellae later on in the FIB-SEM.
5. Anna Steyer: Cryo-volume imaging – the method for difficult specimen?
   In this talk the focus will be on the pros and cons about cryo volume imaging, giving different example projects.

Practical 1: Sample preparation by Waffle method (HPF and cryo-LM)

Standard cellular lamella production by cryo-FIB milling poses major challenges when preparing small, unicellular samples (e.g. bacteria or protists) that often result too thin to offer proper lateral support to the generated lamella. At the same time, thick multi-cellular specimen (e.g. 3D organoids, C. elegans) cannot be properly preserved by simple plunge-freezing preparation, where the limited heat transfer results in crystalline ice formation. Both challenges have been recently overcome by introducing a new method, termed “waffle method” (Kelley et al. 2022, Nature), allowing on-grid lamella preparation after high-pressure freezing.

The waffle method starts with the preparation of the high-pressure freezing (HPF) carrier and glow discharging of the transmission electron microscopy (TEM) grids. For the actual freezing by high-pressure freezing, a sandwich of HPF carriers surrounding a TEM grid where the sample was applied is assembled and frozen. After samples vitrification, the TEM grid is gently separated from the carrier and can now be checked by cryo-LM. Different objectives (5x, 10x and 100x) in combination with reflected light and fluorescence microscopy will reveal the structure of interest and its location on the grid.

The participants will have the opportunity to use:

• Leica EM ICE high-pressure freezer to vitrify the waffle carriers
• Zeiss LSM900 with Linkam cryo-stage for cryo-confocal imaging on the vitrified specimen.

Practical 2: Specifics of on grid cryo- lamella preparation for waffle samples

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 from different angles to remove material (starting from 16 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 2 with an integrated fluorescence light microscope (iFLM) to prepare lamellae on grids prepared with the waffle method

Practical 3: Automating lamella preparation and targeting using Serial-FIB and AutoTEM

Vitrified cells on a TEM will be introduced into the FIB-SEM to target regions of interest (SerialEM and AutoTEM toolbox). Multiple positions will be set-up to be milled automatically with the focused ion beam to get lamellae suitable for tomographic data acquisition.

The participants will have the opportunity to use:

• ThermoScientific Aquilos with Serial-FIB and the AutoTEM software toolkit to set up lamella preparation and polishing milling multiple lamellae automatically

Practical 4: Lift-out of waffled samples

Being able to visualize fully hydrated multicellular samples as close as possible to native conditions at highest resolution has seen an increasing interest by the community, made possible by improvements in efficacy and throughput in lift-out methods. Samples will be introduced into the FIB-SEM and prepared for lift-out, including ROI preparation, needle attachment, lift-out and slab deposition. As a last step lamella are thinned down in an automated process.

The participants will have the opportunity to use:

• Cryo-FIB-SEM for lift-out workflow

Sponsor

Selection info

Selected participants will be announced on July 14^th 2026. For selection purposes, please note that your application will not be considered without CV and a letter of motivation, stating your scientific background, available instrumentation at your home institute and how this course would be beneficial for your future work.

The participants are selected based on EMBL’s course and conferences inclusive criteria (which can be found on the course and conference homepage) and are summarized below:

• Selection of candidates is based on there being a good match between the learning objectives of the course and the stated needs of the candidate, as well as accessibility to the instruments/workflows to the candidate after the course.
• Depending on the applicants, the course will not welcome more than 25% of participants from the same institution’s host country. Same institutions have to be avoided in order to ensure a broader geographical distribution of the knowledge learned during the course.
• Aim for gender balance when selecting the final participants (at least 30% of the minority gender). 
• Applications from organisers, speakers, or trainer laboratories are not prioritised.