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 – 08:45Registration
08:45 – 09:00Introductory word
09:00 – 09:30Image formation in light microscopy | Ivan Novotný
Spatial frequency, light diffraction, PSF – point spread function, resolution
09:30 – 10:15Light microscopy instrumentation | Pavel Krist
Components of microscope: light sources, objectives, fluorescent filters, cubes, principles of detection
Coffee break
10:30 – 11:15Contrast-enhancing techniques in optical microscopy | Martin Čapek
Köhler illumination, BF/DF, Phase and Nomarski contrast
11:15 – 11:45Object visualization in fluorescence microscopy | Jan Valečka
Methods of biological sample visualization in fluorescence microscopy: immune labeling, expression and practical approaches
12:45 – 13:30Confocal scanning microscope: principles and new trends | Ivan Novotný
Confocal microscope construction and image formation, pinhole – optical sectioning and resolution
13:30 – 14:00Spinning disc confocal microscope: principles, advantages | Michaela Blažíková
Image formation in spinning disc microscope, pinhole size and distance, microlenses
Coffee break
14:30 – 17:30Practical 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 – 09:30Visualization of cell nucleus in super-resolution microscopy | Peter Hoboth
Scientific lecture
09:30 – 10:15Fluorophores | Jan Sýkora
Principles of fluorescence, types of fluorophores
Coffee break
10:30 – 11:15Super-resolution approaches in light microscopy | Ivan Novotný
Principles and tricks, SMLM, SIM and STED
11:15 – 11:45Sample 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 presentationsCarl Zeiss | Pavel Krist
KRD | Jiří Vašák
Pragolab | TBA
13:15 – 14:00Computative high resolution methods| Michaela Blažíková
Adaptive deconvolution, optical reassignment, SRRF…
Coffee break
14:30 – 17:30Practical 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 – 09:30Adventure in variety of live-cell imaging | Davide Basello
Scientific lecture
09:30 – 10:00Introduction to live cell imaging | Ivan Novotný
Essential equipment: plastic and consumables, incubation, gas, autofocus systems, objectives
Coffee break
10:15 – 10:45Time-resolved live cell imaging I. | Michaela Blažíková
10:45 – 11:15Time-resolved live cell imaging II. | Michaela Blažíková
12:00 – 12:45Light Sheet microscopy | Jiří Černý
Principles of spatially illuminated microscopy, dual-side and multi view acquisition and data processing
12:45 – 13:30Quantitative phase imaging | Martin Čapek
Principles of the method, advantages of quantitative phase image in segmentation and analysis
Coffee break
14:00 – 17:00Practical 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
17:00Closing remarks

Thursday, 20 October 2022

09:00 – 09:45Image 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 – 10:30Scanning 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 – 11:30Sample 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 – 12:15Sample 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)
13:15 – 14:00Advanced electron microscopy techniques | Vlada Filimoněnko
Ultrastructural immunolabeling (imunogold), volume electron microscopy, cryo electron microscopy, analytical techniques
Coffee break
14:30 – 17:30Practical 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 – 09:45Introduction to image deconvolution | Ivan Novotný
Principles of image deconvolution, technical aspect of required image quality, contribution of PSF and noise, expected results
09:45 – 10:30Optical 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 – 11:30Image 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 – 12:15Introduction 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
13:15 – 14:00Image 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 – 14:45Preparation 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 – 15:30Recap + Evaluation