Characteristics

The five-day theoretical course with practical demonstrations is devoted to modern light and electron microscopy, and the program is updated every year to reflect the newest trends. The course covers the theoretical background of microscopy as well as fundamental techniques in microscopy, and continues quickly to cutting-edge methods like super-resolution light microscopy, light-sheet microscopy, imaging of whole living organisms, and cryo-electron microscopy.

In the theoretical part, the course will cover the principles of light and electron microscopy, image creation, biological sample preparation recommendations, and will provide a guidance through the principal image processing procedures. The practical part of the course consists of demonstrations in smaller groups at the instruments, covering a wide range of microscopy methods, including Koehler illumination adjustment, widefield fluorescence microscopy, fluorescence macroscopy, confocal scanning and spinning disc microscopy, quantitative phase microscopy, light-sheet microscopy, advanced superresolution microscopy (STED, SIM), transmission electron microscopy, scanning electron microscopy, and cryo-methods of EM sample preparation. The course is taught by experts and scientists from the field and by specialists from leading microscopy companies.

After completing the course, the participant will be able to determine which microscopic technique should be used in order to answer the research question, including the sample preparation and data processing for publication. However, do not expect a detailed technical training on the microscopes or advanced training on sample preparation, super-resolution techniques or electron microscopy, these are part of the more specialized courses which we highly recommend: http://microscopytraining.eu/

The course is primarily intended for PhD students and young researchers in the biomedical fields. A number of doctoral committees counts this course towards the fulfillment of student's study obligations.

The course is an introduction to both light and electron microscopy with solid theoretical background extended with many practical presentations. The lectures and also practical sessions are taught by experts and scientists from the field or by product specialists from leading microscopy companies.







Language
The course will be taught in English.





Place of the event
Institute of Molecular Genetics AS ČR, Vídeňská 1083, Prague 4 - Krč




Scientific supervisors
Prof. Pavel Hozák (Institute of Molecular Genetics, ASCR, Prague) - course organizer
Dr. Lucie Kubínová (Institute of Physiology, ASCR, Prague)
Ing. Jana Nebesářová (Institute of Parasitology, České Budějovice)




Coordinators of course modules

Ing. Ivan Novotný (Institute of Molecular Genetics, ASCR, Prague) - light microscopy
Mgr. Vlada Philimonenko (Institute of Molecular Genetics, ASCR, Prague) - electron microscopy

Dr. Jiří Janáček (Institute of Physiology, ASCR, Prague) - image processing





The course content


I. Microscopy essentials
Propagation of light and electrons, optical systems, waves, reflection, diffraction, interference, and polarization.
 
II. Light microscopy
The microscope and its components, image formation, microscopy in both transmitted and fluorescent light, Kohler illumination, optical aberrations, objective lens types, phase contrast, interference contrast, polarization, fluorescence microscopy, laser confocal microscopy, two-photon confocal microscopy, super-resolution microscopy, study of dynamic processes in living cells, immunofluorescence.
 
III. Electron microscopy
Characteristics of electrons, resolution, wavelength of accelerated electrons, the electrons in a magnetic field. Scanning electron microscope: the construction, the detection of secondary and backscattered electron imaging, formation of X-ray radiation and its use for qualitative and quantitative microanalysis, preparation of biological specimens (fixation, dehydration, drying preparations - method of critical point, freezing methods) SEM image digitization. Transmission electron microscope: design, image formation, interference, preparation of biological samples (chemical methods - fixation, dehydration, infiltration, embedding, preparation of ultrathin sections, contrasting; physical methods - low-temperature processes, microwaves), digitizing the image in TEM. Ultrastructural immunolabeling (imunogold). Comparison of photographic and digital recording of the microscope, CCD cameras. Correlative microscopy. Use of digitization and the internet in a virtual electron microscopy.
 
IV. Image processing
1) Scanning and digitizing the image
Essentials (resolution, levels of grey, frame repetition frequency), the advantages and disadvantages of digital processing, basic ideological scheme of digitizing the image. Types of cameras (analog versus digital) and their important characteristics. The types of capture cards in the PC, so. "frame grabbers", the basic principles of operation. Usability and accessibility SW. Specific examples of configurations (potential suppliers) and solving some typical problems. The image parameters (contrast, noise), histogram. Densitometric calibration. Data file formats (binary, grayscale, RGB, HSV, Lab) and compression (lossy, lossless). Filtration and image processing.
 
2) The basic methods of segmentation
Detection areas: thresholding and growth areas, edge detection: Operators highlight contours (Sobel, LoG, DoG), active contours.

3) Measurement of geometric characteristics of digital image
Interactive methods: position, length, profiles, histograms in ROI. Use of Crofton formulas for circumference measurements in 2D. Interactive stereological methods - STESYS system and automatic - area, perimeter, Feret averages, number, Euler characteristic. The effect of anisotropy of the object and noise measurement accuracy.

4) Image analysis and visualization in 3D
Data Sources CLSM and MRI, dimensional calibration. Filtering and segmentation data. Use of Croftonov formula for measuring the surface and the length in the 3D measurement. Interactive stereological methods: Fakir probe and Slicer, automatic volume, number, surface and length in 3D. Visualization: volume and surface rendering.  

V. Stereology and morphometry
Traditional morphometric methods: measuring length, area, perimeter and number. Introduction to stereology. Sampling in stereology, Cavalieri's principle for measuring volume point method. Examples of stereological methods for measurement of volume, surface, length and number. Methods for measuring lengths and three-dimensional surface structures of thin sections: a method of vertical sections, orientator. Methods based on focusing the thick cuts: dissector principle for calculating three-dimensional particles (eg. cells), methods for measurement of spatial curves (eg. capillary) and three-dimensional surface structure.
 





Sponsors

   

      

The course is organized by the Microscopy Centre - Core Facility for Light Microscopy & Core Facility for Electron Microscopy, which are supported from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project “National Infrastructure for Biological and Medical Imaging (Czech-BioImaging – LM2015062)“.