Practical hands-on training on 3D correlative light and electron microscopy combining confocal fluorescence imaging and scanning electron microscopy with focused ion beam milling (FIB-SEM).
Light, particulary fluorescence, microscopy offers live-cell compatibility and flexibility in labelling by specific molecular probes. In this way, insight into the function of cells at their native state can be obtained. Electron microscopy, on the hand, provides unmatched spatial resolution. Correlative light and electron microscopy (CLEM) combines the unique information from both techniques in a single image.During the course you will try hands-on a complete CLEM workflow from sample preparation to image processing. The hands-on sessions will be conducted using the state-of-the-art equipment available at IMCF, including high end fluorescence confocal microscopes and a scanning electron microscope (SEM) with focused ion beam (FIB) milling.
Poslední úprava: Benda Aleš, Mgr., Ph.D. (31.05.2020)
Sylabus - angličtina
Lectures:
Introduction to 3D & CLEM principles and workflows
Fluorescence Microscopy Under Cryogenic Conditions – Cryo-CLEM
(Cryo)-Electron Tomography and its application in (cryo)-CLEM
Volume-EM: principles, sample preparation and CLEM applications
A practical guide to multimodal data correlation
3D-CLEM Applications
Hands on sessions:
Sample preparation and 3D Fluorescence microscopy
Cryo-fixation (Plunge freezing and High-pressure freezing)
Cryo-Fluorescence microscopy
Sample processing & ultramicrotomy for EM
Electron Tomography
Electron microscopy (SEM, FIB-SEM)
Cryo-Transmission Electron Microscopy
Poslední úprava: Benda Aleš, Mgr., Ph.D. (06.02.2026)
Výsledky učení - angličtina
Knowledge and Understanding
Describes the fundamental principles of correlative light and electron microscopy (CLEM) and explains how 3D‑CLEM integrates fluorescence and volume‑EM modalities.
Explains the theoretical basis, capabilities, and limitations of super‑resolution fluorescence microscopy, cryo‑fluorescence imaging, electron tomography, and FIB‑SEM volume imaging.
Identifies the key steps in 3D‑CLEM workflows, including sample preparation, cryo‑fixation, ultramicrotomy, and imaging strategies for correlating light and electron microscopy data.
Practical and Technical Skills
Performs supervised sample preparation procedures for 3D fluorescence imaging, cryo‑fixation (plunge and high‑pressure freezing), and EM sample processing.
Operates state‑of‑the‑art imaging systems (super‑resolution FM, cryo‑FM, SEM/FIB‑SEM, TEM/ET) to acquire 3D datasets suitable for correlative analysis.
Applies appropriate imaging parameters, detector settings, and acquisition workflows to ensure high‑quality fluorescence and electron microscopy data for downstream correlation.
Data Analysis and Interpretation
Processes FM and EM datasets using dedicated 3D‑imaging software, including reconstruction, segmentation, volume rendering, and alignment of multimodal datasets.
Analyzes spatial relationships, ultrastructural features, and co‑localization patterns within correlated 3D volumes to address defined biological questions.
Evaluates common artefacts, technical limitations, and sources of error in 3D‑CLEM workflows and proposes strategies to mitigate them.
Critical Thinking and Integration
Assesses the suitability of different 3D‑CLEM approaches and sample‑processing protocols for specific experimental models and research objectives.
Integrates multimodal imaging data to formulate evidence‑based interpretations of cellular structures, molecular localization, or ultrastructural organization.
Communication and Research Competence
Communicates methodological choices, analytical results, and troubleshooting strategies clearly and professionally in discussions and collaborative problem‑solving sessions.
Poslední úprava: Benda Aleš, Mgr., Ph.D. (06.02.2026)
