Program
The course will introduce the participant to the various techniques of light microscopy (Monday, Tuesday, and Wednesday), ultrastructural/electron microscopy (Thursday) and image processing and analysis (Friday). The aim is to provide an overview of current microscopy methods, and to bring beginners quickly to an advanced level. After a thorough explanation of the theoretical essentials, various microscopy techniques used in modern biomedical laboratories will be demonstrated. The participants will be divided into 4-5 groups to allow maximum time at the various microscopes for interacting with experienced teachers.
Preliminary program
Monday, 17 October 2022
| 08:15 – 08:45 | Registration |
| 08:45 – 09:00 | Introductory word |
| 09:00 – 09:30 | Image formation in light microscopy | Ivan Novotný Spatial frequency, light diffraction, PSF – point spread function, resolution |
| 09:30 – 10:15 | Light microscopy instrumentation | Pavel Krist Components of microscope: light sources, objectives, fluorescent filters, cubes, principles of detection |
| Coffee break | |
| 10:30 – 11:15 | Contrast-enhancing techniques in optical microscopy | Martin Čapek Köhler illumination, BF/DF, Phase and Nomarski contrast |
| 11:15 – 11:45 | Object visualization in fluorescence microscopy | Jan Valečka Methods of biological sample visualization in fluorescence microscopy: immune labeling, expression and practical approaches |
| Lunch | |
| 12:45 – 13:30 | Confocal scanning microscope: principles and new trends | Ivan Novotný Confocal microscope construction and image formation, pinhole – optical sectioning and resolution |
| 13:30 – 14:00 | Spinning disc confocal microscope: principles, advantages | Michaela Blažíková Image formation in spinning disc microscope, pinhole size and distance, microlenses |
| Coffee break | |
| 14:30 – 17:30 | Practical part (4 x 45 min) Confocal Spinning disc (Andor Dragonfly, room 0.171) | Michaela Blažíková Confocal Microscope (Leica STED, room 0.174) | Ivan Novotný Fluorescence microscope (Leica DM6000, room 0.172) | Jiří Černý Adjusting of Köhler illumination and Phase contrast + DIC (room 0.173) | PRAGOLAB |
Tuesday, 18 October 2022
| 09:00 – 09:30 | Visualization of cell nucleus in super-resolution microscopy | Peter Hoboth Scientific lecture |
| 09:30 – 10:15 | Fluorophores | Jan Sýkora Principles of fluorescence, types of fluorophores |
| Coffee break | |
| 10:30 – 11:15 | Super-resolution approaches in light microscopy | Ivan Novotný Principles and tricks, SMLM, SIM and STED |
| 11:15 – 11:45 | Sample preparation for super-resolution microscopy | Ivan Novotný Size and thickness of the sample, refractive index mismatch and spherical aberration minimizing, mounting & sealing |
| Pizza lunch & business presentations | Carl Zeiss | Pavel Krist KRD | Jiří Vašák Pragolab | TBA |
| 13:15 – 14:00 | Computative high resolution methods| Michaela Blažíková Adaptive deconvolution, optical reassignment, SRRF… |
| Coffee break | |
| 14:30 – 17:30 | Practical part (4 x 45 min) SIM (OMX, room 0.174) | Michaela Blažíková STED (Leica STED, room 0.174) | Ivan Novotný Lightning (Leica SP8 – room 0.172) | Davide Basello Lattice SIM / PALM (Elyra7, room 0.174) | ZEISS |
Wednesday, 19 October 2022
| 09:00 – 09:30 | Adventure in variety of live-cell imaging | Davide Basello Scientific lecture |
| 09:30 – 10:00 | Introduction to live cell imaging | Ivan Novotný Essential equipment: plastic and consumables, incubation, gas, autofocus systems, objectives |
| Coffee break | |
| 10:15 – 10:45 | Time-resolved live cell imaging I. | Michaela Blažíková FRAP, FCS |
| 10:45 – 11:15 | Time-resolved live cell imaging II. | Michaela Blažíková FLIM-FRET |
| Lunch | |
| 12:00 – 12:45 | Light Sheet microscopy | Jiří Černý Principles of spatially illuminated microscopy, dual-side and multi view acquisition and data processing |
| 12:45 – 13:30 | Quantitative phase imaging | Martin Čapek Principles of the method, advantages of quantitative phase image in segmentation and analysis |
| Coffee break | |
| 14:00 – 17:00 | 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 Quantitative Phase Imaging (Telight Q-Phase, room 0.175) | Martin Čapek Light Sheet (Zeiss Z.1, room 0.173) | Jiří Černý |
| 17:00 | Closing remarks |
Thursday, 20 October 2022
| 09:00 – 09:45 | Image formation in transmission electron microscope | Oldřich Benada Electron microscopy basics, properties of electrons, resolution, wavelength of accelerated electrons, the electrons in an electromagnetic field. electron-electron interaction and analytical electron microscopy, transmission electron microscope – design, image creation, interference, image acquisition in the TEM |
| 09:45 – 10:30 | Scanning electron microscopy | Oldřich Benada Scanning electron microscope – the construction, signals and image recording (secondary and back-scattered electron imaging), Scanning Transmission Electron Microscopy, SEM image interpretation |
| Coffee break | |
| 10:45 – 11:30 | Sample preparation for TEM | Jana Nebesářová 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 |
| 11:30 – 12:15 | Sample preparation for SEM | Jana Nebesářová 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), strategies for biological applications; analytical morphomics (morphology, identification, content, functionality) |
| Lunch | |
| 13:15 – 14:00 | Advanced electron microscopy techniques | Vlada Filimoněnko Ultrastructural immunolabeling (imunogold), volume electron microscopy, cryo electron microscopy, analytical techniques |
| 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) | Erik Vlčák EM immunolabeling: detection, clustering and colocalization in new online software tool Pattern (lecture room 0.195) | Vlada Filimoněnko |
Friday, 21 October 2022
| 09:00 – 09:45 | Introduction to image deconvolution | Ivan Novotný Principles of image deconvolution, technical aspect of required image quality, contribution of PSF and noise, expected results |
| 09:45 – 10:30 | 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 |
| Coffee break | |
| 10:45 – 11:30 | 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 |
| 11:30 – 12:15 | Introduction to image processing | Jiří Janáček 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 |
| Lunch | |
| 13:15 – 14:00 | Image analysis and visualization in 3D | Jiří Janáček 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. |
| 14:00 – 14:45 | Preparation of digital images for publication | Oldřich Benada 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? |
| Coffee break | |
| 15:00 – 15:30 | Recap + Evaluation |