The five-day theoretical course with practical demonstrations and exercises, is intensively devoted to modern methodologies of light and electron microscopy. The program of the course is every year updated to reflect new trends in the light microscopy field. The course covers general methods and fundamental approaches in microscopy and also cutting edge techniques like super-resolution light microscopy, fast confocal spinning disc acquisition or lightsheet microscopy and imaging of whole living organisms.
In the theoretical part, the course will cover fundaments of light microscopy, image creation, and principles of confocal microscopy, tricks of main superresolution methods and many practical aspects like live cell imaging techniques and approaches, and also microscopy sample preparation recommendations.
During the practical part of the course, participants will see demonstration of general Koehler illumination adjustment, widefield fluorescence microscope, fluorescence macroscope, confocal scanning and also spinning disc microscopes, Lightsheet microscope and also advanced superresolution microscopes – STED, SIM and Quantitative phase microscope. The course also deals with the processing of the image data, however, priority is to acquire practical skills in microscopy techniques (image analysis is the main topic of the course Processing and analysis of microscopic images in biomedicine ). After completing the course, the participant will be able to determine what is appropriate microscopic technique used to answer the research questions, including the preparation and data processing for publication. Detailed methodological guidance and technical training are part of the more specialized courses. 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 will be taught in English.
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.
The course will be taught in English.
Place of the event
Institute of Molecular Genetics AS ČR, Vídeňská 1083, Prague 4 - Krč
Prof. Pavel Hozák (Institute of Molecular Genetics, ASCR, Prague)
Dr. Lucie Kubínová (Institute of Physiology, ASCR, Prague)
Ing. Jana Nebesářová (Institute of Parasitology, České
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) - biomathematics
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.
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)“.