This 5-day theoretical course with demonstrations and practical exercises covers the modern methodologies of light and electron microscopy (including principles of biological specimens’ preparation), digital image acquisition and processing, and stereology. In addition, the participants will be introduced to the basics of atomic force microscopy and this technique will be also demonstrated. The course is aimed especially at PhD students and junior researchers in biomedical fields. Many doctoral committees recognize this course as part of the PhD student’s duties. After the course the participants will be able to determine what microscopy technique should be used to answer a particular research question. However, do not expect detailed methodological instructions and technical training - these are provided by more specialized courses. The course will be taught in Czech.

Institute of Molecular Genetics AS CR, Vídeňská 1083, Prague 4 - Krč, Czech Republic

Scientific coordinators
Prof. Pavel Hozák (Institute of Molecular Genetics of AS CR, Prague)
Dr. Lucie Kubínová (Institute of Physiology of AS CR, Prague)
Ing. Jana Nebesářová (Institute of Parasitology of AS CR, České Budějovice)

Course content
I. Theoretical background
Light and electron diffusion, optical systems, waves, reflection, diffraction, interference, polarization.
II. Light microscopy
A microscope and its parts, image formation, Köhler illumination, optical aberrations, types of lenses, phase contrast, interference contrast, polarization, fluorescence microscopy, laser confocal microscopy, two-photon confocal microscopy, superresolution microscopy, study of dynamic processes in living cells, immunofluorescence.
III. Electron microscopy
Characteristics of electrons, resolution ability, wavelength of an accelerated electron, an electron in a magnetic field. A scanning electron microscope: design, detection of secondary and reflected electrons, image creation, X-ray origin and its use for qualitative and quantitative microanalysis, biological specimens’ preparation (fixation, dehydration, drying of specimens – a critical point method, frost preparation methods), SEM image digitalization. A transmission electron microscope: design, image creation, interference effects, biological specimens’ preparation (chemical methods - fixation, dehydration, infiltration, irrigation, preparation of ultra thin sections, contrasting, physical methods – low-temperatures processes, microwaves), TEM image digitalization. Ultra structural immunodetection (immunogold). Comparison of a photographic and a digital record from a microscope, CCD cameras. Correlative microscopy. The use of the digitalization and the Internet in virtual electron microscopy.
IV. Image processing
1) Image scanning and digitalization
Basic terms (resolution, gray scales, repeating scanning frequency), advantages and disadvantages of digital processing, basic ideological scheme of image digitalization. Types of cameras (analogue versus digital) and their significant features. Types of capture cards for PC, so called “frame grabbers”, basic principle of activities. Possible applications and software availability. Concrete examples of configuration (potential suppliers) and resolving of some typical problems. Image parameters: contrast, image noise, histogram. Densitometric calibration. Formats of data files (binary, gray-scale, RGB, HSV, Lab) and compression (dissipative, non-dissipative). Filtering and image processing.
2) Basic methods of segmentation
Areas detection: thresholding and areas growth, edge detection: operators highlighting contours (Sobel, LoG, DoG), active contours.

3) Measurement of geometric features of a digital image
Interactive methods: location, length, profiles, histograms in ROI. Usage of Croft’s formula for measuring circumference in 2D. Interactive stereologic methods – STESYS system and automatic – surface, circumference, Feret diameter, quantity, Euler characteristic. Effect of object anisotropy and noise on measurement accuracy.

4) Image analysis and visualization in 3D
CLSM and MRI data resources, dimensional calibration. Filtering and data segmentation. Usage of Croft’s formula for measuring the surface and length in 3D measurements. Interactive stereologic methods: Fakir probe and Slicer, automatic: capacity, quantity, surface and length in 3D. Visualization: volume and surface rendering.  

V. Stereology and morphometry
Traditional morphometric methods: measurement of length, surface, circumference, and quantity. Introduction to stereology. Sampling in stereology. Cavalieri principle for capacity measurement, dot method. Examples of stereologic methods for measuring capacity, surface, length and quantity. Methods for measuring length and surface of 3D structures from thin sections: a method of vertical sections, orientator. Methods based on sharpening thicker sections: disector principle for counting 3D particles (e.g. cells), methods for length measurement of space curves (e.g. capillaries) and surface of 3D structures.

Course sponsors