Course Topics

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

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 formation, interference, image acquisition in the TEM. Comparison of photographic and digital recording of the microscope, CCD cameras. Scanning electron microscope – the construction, signals and image recording (secondary and back-scattered electron imaging), Scanning Transmission Electron Microscopy, SEM image interpretation. 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. 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). Ultrastructural immunolabeling (immunogold), volume electron microscopy, cryo electron microscopy, analytical techniques.

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.