Monday, October 13, 2025

08:15 – 08:45	Registration
08:45 – 09:00	Introductory word
09:00 – 09:30	From a living cell to microscopical sample | Pavel Hozák Important aspects of sample preparation – fixation. How to preserve fine structures and epitopes for later visualization Explore how to prepare a living cell to become a high-quality microscopic sample. This session is focused on the crucial steps if fixation, a process that preserves fragile cell structures and key epitopes, so they remain visible in acquisition. You’ll learn differences in fixation variants to preserve fine details and maintaining the sample’s integrity for best results.
09:30 – 10:00	Image formation in light microscopy | Ivan Novotný Spatial frequency, light diffraction, PSF – point spread function, resolution This lecture uncovers how an image is formed in light microscopy, focusing on essential concepts like spatial frequencies and light diffraction. Get familiar with the Point Spread Function (PSF) and how it impacts resolution, determining intensities and contrasts in your images. By understanding these principles, you’ll be able to maximize the quality of your microscopy work.
Coffee break
10:30 – 11:00	Light microscopy instrumentation | Pavel Krist Components of microscope: light sources, objectives, fluorescent filters, cubes, principles of detection This session covers everything from light sources and objectives to specialized filters and cubes. You’ll also learn the principles behind detection techniques, equipping you with a solid information background for proper selection of microscopy tools in your research.
11:00 – 11:30	Contrast-enhancing techniques in optical microscopy | Martin Čapek Köhler illumination, BF/DF, Phase, Nomarski contrast and quantitative phase Discover various methods for enhancing contrast in optical microscopy for effective visualization of your object of interest. Lecture includes Köhler illumination, Bright Field/Dark Field techniques, Phase contrast, and Nomarski contrast; explains also quantitative phase imaging, a valuable tool for analyzing transparent samples like living cells without dyes or stains.
Lunch
12:30 – 13:00	Confocal scanning microscope: principles and new trends | Ivan Novotný Confocal microscope construction and image formation, pinhole – optical sectioning and resolution Learn the basics of confocal microscopy, principles of illumination, detection and image formation, including the pinhole feature that increase image resolution and allows 3D visualization by optical sectioning. This lecture also discusses innovations in confocal microscopy technology and approaches, offering insights into last trends and application options in modern biology.
13:00 – 13:30	Spinning disc confocal microscope: principles, advantages | Michaela Blažíková Image formation in spinning disc microscope, pinhole size and distance, microlenses Get an introduction to spinning disc confocal microscopy, a technique that allows optical sectioning at high speed. You’ll learn how the arrangement of pinholes and microlenses improve signal intensities in images, making it ideal for capturing dynamic processes in live cells or acquiring large structures. This session highlights the practical advantages and specialized uses of this advanced tool.
13:30 – 14:00	Image acquisition by two-photon microscopy | Daniel Hadraba 2-photon microscopy basics – 2-photon excitation demands – 2-photon excitation (dis)advantages – 2-photon excitation hardware and systems Explore the fundamentals of two-photon microscopy, a technique for deep tissue imaging. This session covers the basics of two-photon excitation, including its essential requirements, benefits, and limitations. You’ll also learn about the specialized hardware and systems involved, gaining insight into how two-photon microscopy enables imaging in complex biological samples.
Coffee break
14:30 – 17:30	Practical part (4 x 45 min) Confocal Spinning disc (Andor Dragonfly, room 0.171) | Jan Valečka Confocal Microscope (Leica Stellaris 8, room 0.172) | Ivan Novotný Fluorescence microscope (Leica DM6000, room 0.172) | Helena Chmelová Adjusting of Köhler illumination and Phase contrast + DIC (room 0.173) + QPI| PRAGOLAB/Martin Čapek

Tuesday, October 14, 2025

09:00 – 09:30	Fluorophores | Jan Sykora Principles of fluorescence, types of fluorophores Lecture goes into the principles of fluorescence and the usage of variety of fluorophores available for biological imaging. This session explains how fluorophores work, the differences between types, and how to choose the right ones for your experiments to achieve accurate visualization of biological structures within images.
09:30 – 10:00	Object visualization in fluorescence microscopy | Jan Valečka Methods of biological sample visualization in fluorescence microscopy: immune labeling, expression, and practical approaches Explore key methods for visualizing biological samples in fluorescence microscopy. This lecture covers immune labeling, protein expression, and other practical techniques, helping you understand which approach best suits your research for clear, informative images.
Coffee break
10:30 – 11:00	Super-resolution approaches in light microscopy | Ivan Novotný Principles and tricks, SMLM, SIM and STED Discover advanced super-resolution microscopy techniques, including Single Molecule Localization Microscopy (SMLM), Structured Illumination Microscopy (SIM), and Stimulated Emission Depletion (STED). Learn about the principles and tricks behind these methods to push the boundaries of conventional resolution limits.
11:00 – 11:30	Computative high resolution methods| Michaela Blažíková Adaptive deconvolution, optical reassignment, SRRF… Explore the advantages of computational methods to enhance resolution and image quality, covering techniques like adaptive deconvolution, optical reassignment, and Super-Resolution Radial Fluctuations (SRRF). This session will introduce you to advanced image-acquisition/processing tools, allowing you to improve image quality in high-resolution studies.
11:30 – 12:00	Sample preparation for super-resolution microscopy | Ivan Novotný Size and thickness of the sample, refractive index mismatch and spherical aberration minimizing, mounting & sealing Correct sample preparation is crucial not only for super-resolution microscopy. This session highlights how factors like sample size, thickness, and refractive index mismatches affect image quality. You’ll also learn tips for reducing spherical aberration effect and techniques for mounting and sealing samples to get the best results.
Lunch
13:00 – 13:30	Super-resolution in live-cell imaging | Ivan Novotný Tips and tricks for SR imaging of living structures, how to prolong viability, minimize photobleaching and remove motion artifacts. Suitable methods and applications Unlock the potential of super-resolution (SR) imaging for live-cell studies with practical tips and tricks. This session provides strategies for extending cell viability during imaging, minimizing photobleaching, and reducing motion artifacts. You’ll also learn about the most suitable SR methods and applications for studying living structures in detail, making it ideal for capturing dynamic cellular processes.
Coffee break
14:00 – 17:00	Practical part (4 x 45 min) SIM (OMX, room 0.174) | Helena Chmelová STED (Leica STED, room 0.174) | Ivan Novotný Lightning (Leica Stellaris 8 – room 0.172) | Michaela Blažíková STORM / PALM (Elyra7, room 0.174) | ZEISS/TBA

Wednesday, October 15, 2025

09:00 – 09:30	Introduction to live cell imaging | Ivan Novotný Essential equipment: plastic and consumables, incubation, gas, autofocus systems, objectives Get a complex overview of live cell imaging essentials. This session covers the key equipment and consumables needed for successful live-cell imaging, including incubation setups, gas control, autofocus systems, and specialized objectives. Learn how to create an optimal environment to keep cells alive and healthy during observation.
09:30 – 10:00	Time-resolved live cell imaging I. | Michaela Blažíková FRAP, FCS Focus into time-resolved imaging techniques such as FRAP (Fluorescence Recovery After Photobleaching), photoactivation and FCS (Fluorescence Correlation Spectroscopy). These methods allow you to study and reveal the dynamics and interactions of molecules in living cells over time, providing information about their intracellular functions.
Coffee break
10:30 – 11:00	Time-resolved live cell imaging II. | Michaela Blažíková FLIM-FRET Explore FLIM-FRET, a powerful tool for studying molecular interactions using Fluorescence Lifetime Imaging Microscopy (FLIM) to measure Förster Resonance Energy Transfer (FRET). This approach enables precise analysis of molecular interactions, providing essential insights into complex cellular processes.
11:00 – 11:30	Light Sheet microscopy | Jiří Černý Principles of spatially illuminated microscopy, dual-side and multi view acquisition and data processing Learn about Light Sheet Microscopy, a technique that illuminates samples from the side, allowing for rapid, gentle imaging of 3D structures. This session explains principles of dual-side and multi-view acquisition and how to process the resulting data, providing a tips for visualizing complex biological samples.
11:30 – 12:00	Optical projection tomography | Martin Čapek Visualization of big 3D specimens, sample volume reconstruction from physical slices, principles of computed and optical tomography, transmission and fluorescence modes, optical clearing Focus on Optical Projection Tomography (OPT), a technique designed for visualizing large 3D specimens. This session covers the fundamentals of reconstructing sample volumes from physical slices and explains the principles behind both computed and optical tomography. Learn how to use transmission and fluorescence modes, as well as optical clearing techniques, to acquire detailed images of larger biological samples, making OPT ideal for complex structural studies.
Banquette lunch in the lecture room
12:30 – 13:30	Course Partners Presentations and Discussion | Pavel Hozák Join us for a series of short presentations from our course partners, each lasting about 10 minutes. This session offers an excellent opportunity to learn about the latest tools, technologies, and resources available to enhance your research. Following the presentations, engage in an open discussion with the partners to ask questions, share ideas, and explore potential collaborations.
13:30 – 14:00	Automated microscopy and analysis | Jan Valečka Principles of automated high-content screening, advantages of high-content imaging and bias-free robust analysis Explore the world of automated high-content microscopy, which combines imaging with unbiased, robust analysis. This lecture explains how automated systems enable high-content screening and reduce human error, offering reliable, reproducible data for complex biological studies.
Coffee break
14:30 – 17:30	Practical part (4 x 45 min) Time-resolved: FRAP and FLIM (Leica Stellaris 8, room 0.172) | Michaela Blažíková Live cell imaging (Andor Dragonfly, room 0.171) |Ivan Novotný High-content microscopy (Evident ScanR, room 0.175) | Jan Valečka Light Sheet (Zeiss Z.1, room 0.173) | Jiří Černý
17:30	Closing remarks

Thursday, October 16, 2025

09:00 – 09:45	Image formation in transmission electron microscope | Oldřich Benada Basic principles of electron microscopy, electron properties, construction and image acquisition in transmission electron microscopes Get a complex overview of electron microscopy essentials and understand the specifics of ultrastructural imaging. Learn when the electron microscopy is the method of choice and how to understand what is behind the image from transmission electron microscope.
09:45 – 10:30	Scanning electron microscopy | Oldřich Benada Scanning electron microscope – the construction, signals and image recording, Scanning Transmission Electron Microscopy (STEM), SEM image interpretation Learn about scanning electron microscopy as a unique tool not only to reveal fine details of various surfaces, but also to obtain multiple kinds of information about the sample. Specific way of image formation in SEM will be explained to help you interpret the data obtained in SEM.
Coffee break
10:45 – 11:30	Sample preparation for TEM | Dominik Pinkas Physical and chemical principles of sample preparation for electron microscopy; chemical methods – fixation, dehydration, infiltration, embedding, preparation of ultrathin sections, contrasting; physical methods – low-temperature processes, microwaves The intrinsic properties of live samples are not compatible with the conditions inside the electron microscope, making direct observation impossible. To overcome these challenges, complex specialized methods of sample preparation should be used. This lecture presents overview of available methods of sample preparation for transmission electron microscopy and explains how to choose a proper one.
11:30 – 12:15	Sample preparation for SEM | Markéta Dalecká Fixation, dehydration, CPD method, metal coating, freeze fracturing, etching and freeze drying. Sample preparation for cryo-SEM, low vacuum SEM; correlative light-electron microscopy (CLEM-SEM) While some of the factors important for sample preparation procedure are common between transmission and scanning electron microscopy, the SEM has its specifics. In this session, you will learn how to prepare the samples for imaging of it’s surface, without losing the sample shape, how to overcome the problem of sample charging by electrons during observation, and how to combine information form light microscope and SEM.
Lunch
13:15 – 14:00	Advanced electron microscopy techniques | Vlada Filimoněnko Ultrastructural immunolabeling (imunogold), volume electron microscopy, cryo electron microscopy, analytical techniques, CLEM-TEM Get an overview of various advanced application-oriented approaches in electron microscopy. You will learn how to detect specific molecular targets, how to obtain volume information, and which technique to choose according to desired resolution and size of the area of interest. Detection of specific elements in the sample can help you to analyze the distribution of natural or exogenous compounds or particles in the sample. Cryo-electron microscopy deals with close-to-native frozen-hydrated samples and allows imaging cellular structures or building high-resolution models of isolated molecular complexes. Correlation of light microscopy and TEM imaging allows to study dynamic or rare events and target specific structures.
Coffee break
14:30 – 17:30	Practical part (4 x 45 min) Transmission electron microscopy (JEM-1400FLASH, room 01.152) | Dominik Pinkas Scanning electron microscopy (building “U”) | Oldřich Benada High-pressure freezing, freeze substitution and ultramicrotomy (EM CF labs – rooms 01.155.1., 01.155.2) | Markéta Dalecká Cryo FIB-SEM applications in life sciences (Amber Cryo, room 01.189.2) | Martin Uher, TESCAN

Friday, October 17, 2025

09:00 – 09:30	Introduction to image deconvolution | Ivan Novotný Principles of image deconvolution, technical aspect of required image quality, contribution of PSF and noise, expected results Get a fundamental understanding of image deconvolution process, a technique used to image restoration, contrast and resolution increasing. This session covers the core principles, the importance of image quality, and how factors like the Point Spread Function (PSF) and noise influence results.
09:30 – 10:00	Practical examples of image deconvolution | Ivan Novotný Practical demonstration of Huygens deconvolution software, process settings, image input parameters, results evaluation. A practical demonstration of deconvolution using Huygens software. This session covers setting up the deconvolution process, adjusting image input parameters, and evaluating results to optimize image clarity and detail. Additionally, you’ll be introduced to batch processing and template preparation for efficient workflow management in deconvolution tasks.
Coffee break
10:45 – 11:15	Introduction to image processing | Jiří Janáček Formats of image files, dimensionality, multipage tiff image. FiJi ImageJ basics. Digital coding of multispectral images – formats of image files. Dimensional calibration. ImageJ (FiJi) – widely used freeware for scientific image processing. Basic manipulation with images. Regions of interest. Overlays – scale bar and annotation. Macro language and plugins
11:15 – 12:00	Image analysis and visualization in 3D | Jiří Janáček Image dimensionalities quantification, 3D visualization, view modalities, lookup tables. Quantitative information in images – geometric characteristics of real biological samples and how to estimate them. Visualization of 3D data from biomedical modalities – surface rendering, maximum intensity projection and volume rendering. Visualization cues – movies, lighting, texture, stereopsis, fog, depth color coding etc.
Lunch
13:00 – 13:45	Preparation of digital images for publication | Oldřich Benada Image formats, interpretation, handling and processing, publication preparation, scale bars and graphics, color management, color in electron microscopy. Image formats – lossless vs. lossy. Image interpretation – the human eye is not a perfect device. Primary image interpretation – mainly based on previous experiences. Image Handling and Processing – essential hardware calibration, software image processing -What is allowed and prohibited? Preparation of digital images for publications – a 300 DPI nightmare. Scale bars, arrows, and lettering – bitmaps vs. vector graphic. Black&White vs. Color Images – hardware gamma adjustment, primal color management in PC, color blindness problem – color maps. Color in scanning electron microscopy – Does it matter?
13:45 – 14:30	From imaging to publication of results | Martin Gregor Workflow, image acquisition, processing, analysis, result presentation, statistical analysis Workflow from acquisition of microscopy images through image processing and analysis to comprehensive presentation of results. Brief introduction into statistical analysis of the results.
Coffee break
15:00 – 15:30	Recap + Evaluation | Pavel Hozák A summary of key topics covered throughout the course, followed by an evaluation session. This is an opportunity to review important concepts, ask final questions, and provide feedback on the course experience.