Winter School 2025 - Practical course in advanced microscopy

This advanced microscopy course is intended for PhD students and post-graduates with prior experience in microscopy. The goal of the course is to teach and train fundamental knowledge and skills in a specific microscopic technique. Subsequently, students should be able to apply the knowledge in their own present and future projects. Practical work consists of five different modules each covering a specific topic (please see description below). Each practical module lasts the entire duration of the course. Students will participate in one module only and use state-​of-the-art instrumentation. Theoretical sessions will cover basic and advanced knowledge about all the different techniques and topics.

The course is jointly organised by the Center for Microscopy and Image Analysis (ZMB, University of Zurich) and the Scientific Center for Optical and Electron Microscopy (ScopeM, ETHZ)

The course is held 20- 24 January 2025.

Registration deadline: 17. November 2024

500 CHF.

Note: The course fees do not cover lunch, except dinner on Thursday evening and beverages and snacks provided during breaks.

The Winter School is valued with 2-3 ECTS credits (depending on the institution).

In this module, you will learn how to select the most appropriate advanced light microscopy technique such as confocal laser scanning, multiphoton or super resolution (STED) microscopy for your project. Lectures will teach you to understand resolution, multicolor fluorescence acquisition, aberrations and deconvolution in light microscopy. Practical trainings to apply the concepts from lectures will be on advanced widefield, confocal laser scanning, multiphoton, and super resolution microscopes with cell and tissue samples for multicolor and 3D imaging.

Contact: Dr. Jana Döhner, Center for Microscopy and Image Analysis, University of Zurich
(jana.doehner@zmb.uzh.ch, +41 44 635 06 24)

Learning outcomes:

By the end of the module, you should be able to

• know, how to correctly operate advanced microscopes, ranging from widefield imaging over laser scanning systems and multiphoton microscopy, and systems which enhance your resolution by overcoming the diffraction limit.
• decide which microscopic system suits best my research needs.
• understand and apply the underlying basics and main concepts of microscopy (resolution vs magnification, refraction, optics, NA e.g.) to your future imaging procedure.

Practical activities:

• Operate widefield microscopes to acquire images of fixed samples, as well as live cells (short and long term acquisition)
• Operate laser scanning- and multiphoton microscopes to acquire images of thin and thick samples 
• Operate super resolution confocal laser scanning systems to image fixed samples in order to improve the resolution 
• Measure/image possible aberrations on a defined microscope system 

Prerequisites:

• Have at least practical basic experiences with standard widefield fluorescence microscopy
• Have an immediate need to apply the trained skills/one of the techniques for your research question at least within the a year after the course

In this module, you will learn how to identify the most appropriate super-resolution light microscopy technique to address your specific needs and sample. Numerous super-resolution or “quasi” super-resolution methods will be covered including

• Structured Illumination Microscopy (SIM)
• Stimulated emission depletion (STED) microscopy
• Single Molecule Localization (SMLM) 

and “quasi” super-resolution methods like 

• Airy-scan 
• re-scan
• Super Resolution via Optical Re-assignment (SORA)
• Super-Resolution Radial Fluctuations (SRRF) 

Lectures will teach you to understand resolution and the various concepts of resolution enhancement that serve as theoretical basis for the different super-resolution methods. In the practical sessions you will work with the various super-resolution instruments of ScopeM and learn about all the advantages/disadvantages of the different methods, both in terms of imaging and sample preparation possibilities. 

Contact: Dr. Gabor Csucs, ScopeM, ETHZ
(gabor.csucs@scopem.ethz.ch, +41 44 633 62 21)

Learning outcomes:

• Have a thorough understanding of   the capabilities and limitations of the various super-resolution light microscopy methods.
• Establish a solid background enabling you to select which super-resolution technique and instrument suit best your research needs.
• Understand and apply the main concepts/principles of super-resolution microscopy (resolution vs magnification, refraction, optics, NA e.g.) to your future imaging procedure.

Practical activities:

• Hands-on super-resolution microscopy techniques available at ScopeM: SIM, STED, SMLM, Airy-scan, re-scan, SORA.
• In addition to the selected samples provided in the course, opportunity to work with your own samples (the sample preparation should be done before the course - please contact the module responsible in advance to discuss necessary preparation).

Prerequisites:

• Have some practical experience with standard fluorescence microscopy (wide-field or confocal).
• Have an immediate need to apply the trained skills/one of the techniques in your research project within a year.

In this module, you will learn how to prepare biological specimens for 2D and 3D electron microscopy techniques such as serial-section transmission and scanning electron microscopy, focused ion beam and serial block-face scanning electron microscopy. To preserve the sample ultra-structure, chemical and cryo-fixation techniques (high-pressure freezing, freeze-substitution) will be used. Subsequently, resin-embedded samples will be sectioned by ultramicrotomy and imaged by transmission, scanning and focused ion beam scanning electron microscopy. The cellular ultra-structure in the resulting micrographs will be reviewed in context to preparation. Samples will be provided.

Contact: Dr. Andres Kaech, Center for Microscopy and Image Analysis, University of Zurich
(andres.kaech@zmb.uzh.ch, +41 44 634 26 65)

Learning outcomes:

• Participants are able to process biological samples for imaging the subcellular ultra-structure by electron microscopy.
• Participants know the advantages and disadvantages of different preparation steps and procedures.
• Participants know the advantages and disadvantages of the different 2D/3D electron microscopy techniques and are able to select the optimal approach for their research.

Practical activities:

• Room temperature fixation and cryo fixation of biological specimens, dehydration, resin embedding, ultrathin sectioning (ultramicrotomy), contrast enhancement
• Imaging sections in the transmission electron microscope.

Prerequisites:

• Have a need to apply the trained skills/one of the techniques for your research question at least within a year after the course.

Correlative Light and Electron Microscopy (CLEM) combines the best of two worlds: large-scale imaging using light microscopy and high-resolution electron microscopy. This module covers the basics of methodology and preparation techniques needed to perform light and electron microscopic investigations on identical sample areas. Starting with imaging of living cells, we will in the following prepare these samples for EM, and acquire focused-ion beam SEM (FIB-SEM) and serial blockface SEM (SBF-SEM) data of the exact same cells. You will learn to relocate a previously imaged region of interest in the EM, collect correlative 3D data sets and get an understanding of the different image characteristics. The practical work will be rounded off by image processing sessions, covering the basics of post-processing of volume data, aligning the acquired LM and EM data in 3D, visualizing the correlation, and creation of 3D models. Samples will be provided.

Contact: Dr. Miriam Lucas, ScopeM, ETHZ
(miriam.lucas@scopem.ethz.ch, +41 44 633 4424)

Learning outcomes:

• Get familiar with the workflow for different CLEM-approaches
• Understand the requirements for CLEM experiments with respect to sample preparation and choice of microscopy techniques, and be able to design own experiments
• Visualize, process evaluate, and interpret different 3D microscopic data types.

Practical activities:

• Acquisition of light microscopic data of living cells with light microscopy, and selection of regions of interest for optimal correlation with electron microscopy data
• Preparation of the previously imaged cell culture samples for EM
• Relocation of the region of interest in the electron microscope and stack acquisition,  assisted by ScopeM staff
• Inspection, evaluation and processing of the resulting 3D data and visualization of the correlation using Fiji and Amira 3D software
• Discussion of relevant problems concerning preparation acquisition and data interpretation 

Prerequisites:

• Knowledge of theory for light and electron microscopy
• Practical experience in LM, and ideally with basic SEM operation 

The third dimension is of utmost importance for understanding the complex structural context of specimen, in biology as well as in material sciences. This module provides an overview and compares scanning electron microscopy (SEM)-based techniques for volume imaging: (1) focused ion beam-SEM (FIB-SEM), (2) serial blockface SEM (SBF-SEM) and (3) array tomography. For the first two methods, a fresh blockface is created in situ in the SEM employing either a focused ion beam, or a diamond knife respectively, and volume data is acquired by alternating cutting and imaging of the fresh blockface. For array tomography on the other hand, ribbons of sections (i.e. serial sections) are imaged. These are prepared using conventional ultramicrotomy and mounted onto a conductive support for SEM. We will acquire 3D data of the same sample type using all three techniques, and cover the methodology and sample preparation techniques for the respective methods. The module will be rounded off by image processing sessions covering the basics of post-processing of volume data, visualization and 3D modelling of structures of interest. Samples will be provided.

Contact: Dr. Miriam Lucas, ScopeM, ETHZ
(miriam.lucas@scopem.ethz.ch, +41 44 633 4424)

Learning outcomes:

• Become familiar with 3D electron microscopy imaging
• Understand the advantages and disadvantages of the presented 3D imaging techniques and be able to select the optimum approach for your research
• Understand sample requirements and respective preparation techniques for volume SEM
• Visualize, process, evaluate and interpret different 3D microscopic data types 

Practical activities:

• Tips and tricks for serial sectioning
• Choosing a relevant region of interest for image stack acquisition
• Acquisition of 3D image stacks assisted by ScopeM-staff
• Inspection and evaluation of the resulting 3D data, and comparison of the presented imaging techniques
• Processing and visualization of the resulting 3D data using Fiji Amira 3D software
• Discussion of relevant problems concerning preparation acquisition and data interpretation 

Prerequisites:

• Knowledge of electron microscopy theory
• Ideally practical experience with basic SEM operation