Electron Microscopy (EM) in Practice - From Sample Preparation to 3D-(Immuno)CLEM

The idea for this event originated during a collaborative discussion at the successful COMULIS Globe meeting in Stellenbosch in January 2025. The goal was to promote and implement advanced techniques—such as Tokuyasu cryosectioning, immunolabelling, Correlative Light and Electron Microscopy (CLEM), and 3D imaging—across laboratories in South Africa.  The event featured a two-day Symposium with talks presented by Dr. Muriel Mari (Aarhus University, DK), Viola Oorschot (EMBL, DE), Dr Frederic Leroux (DiATOME, CH) and keynote addresses by Profs Judith Klumperman (UMC Utrecht, NL) and Jemima Burden (UCL, UK). Presentations from industry partners—including Separations (Leica), Carl Zeiss, and Angstrom/ALS (JEOL/Nikon)— provided insight into the latest tools and technologies. Nearly 50 delegates from over 6 research institutions from across SA and Namibia attended, reflecting strong regional interest and engagement.

Following the symposium, selected participants joined an 8-day hands-on workshop supervised by Muriel Mari, Viola Oorschot and Frederic Leroux.  Attendance was limited to 12 to ensure each participant could actively practice the techniques and gain true technical independence. Impressively, over 80% of attendees for the entire event were from designated groups, and more than 60% were female, demonstrating a strong commitment to diversity and representativity.

While the Symposium aimed to provide the foundational knowledge of the EM techniques covered, the workshop focused on equipping participants with practical skills and the confidence to apply advanced microscopy techniques in their own laboratories. The content covered included:

Conventional EM:

·        Sample processing from fixation to embedding

·        Manual and targetted trimming

·        Glass knife-making

·        Ultramicrotomy and pick-up of semi- and ultra-thin sections (Leica Enuity)

Tokuyasu Cryosectioning:

·        Fixation, gelatine embedding, cryoprotection, freezing, and storage

·     Trimming combined with cryo-ultramicrotomy and collection of thick and ultrathin sections (Leica Enuity)

Participants then applied these samples for immuno-gold and fluorescence labelling, followed by imaging with fluorescence microscopy and transmission electron microscopy (TEM) to demonstrate CLEM on Tokuyasu cryo-sections.

Advanced 3D Techniques:

·        Correlative Light and Electron Microscopy (CLEM) using a ZEISS CLSM 880 with Airyscan, and TEM JEOL 2100F

·        Array tomography

·        Acquisition of ±60° tilt series using SerialEM on a JEOL 1400 Flash TEM

·        Reconstruction of image stacks with IMOD software

The final day allowed delegates to consolidate learning, revisit challenging techniques, and receive personalized guidance where needed.

A Google survey conducted after the Symposium and workshop revealed overwhelming support from the participants (i.e. > agree / strongly agree)

·       A strong need for additional events emphasising upon hands-on practice and extra one-to-one time with trainers.

·       High relevance of the content for their ongoing research

·       Confidence in their ability implementing these techniques in their laboratories

These results confirm that the event successfully addressed a critical skills gap in South African microscopy and highlight the importance of repeating it in the future.

This event would not have been possible without the dedication and support of:

Key contributors

·       Dr. Muriel Mari, Ms Viola Oorschot and Dr. Frederic Leroux for their tireless efforts, patience and passion

·       Drs James Wesley-Smith and Antoinette Lensink for their crucial role in raising funds and dedicating time and energy to establishing this workshop at the University of Pretoria

·       Profs J. Klumperman and Jemima Burden for their outstanding online presentations

·       Ms. Charity Maepa for managing event logistics and preparing the hosting laboratory

Institutional Support

·       National Research Foundation (NRF) for funding Dr. Antoinette Lensink’s Equipment Related Travel and Training Grant (ERTTG) proposal, which supported event costs and participant accommodation

·       University of Pretoria for hosting the event

·       Aarhus University and EMBL for enabling travel for Prof. Mari and Ms. Oorschot

Industry Support:

·        Helmut Gnaegi, Frederic Leroux, and DiATOME Pty for the loan of diamond knives

·        Leica Microsystems (via Separations) for the loan of an Enuity cryo-ultramicrotome

·        Carl Zeiss, JEOL, Angstrom/ALS, and the Microscopy Society of Southern Africa (MSSA) Trust for financial contributions

·        EMPOWER Microscopy for coordinating this event.

Organising Committee & Support Staff:

·        M. Mari, V. Oorschot, J. Wesley-Smith, A. Lensink, C. Maepa

·        Laboratory for Microscopy and Microanalysis staff: Nonkululeko Phili, for preparing and maintaining the laboratory, and Mohlokomedi Bopape, for assisting with equipment logistics, including handling liquid nitrogen (LN₂).

·        Mr. Michael Kleynhans and UP Dept. of Physiology for accommodating the Symposium and IT support

Finally, our gratitude and congratulations to workshop participants who approached an intensive program of novel techniques with enthusiasm and a positive attitude, making the event a resounding success.