Course Topics – Syllabus

I. Microscopy essentials

Propagation of light and electrons, optical systems, waves, reflection, diffraction, interference, and polarization.

II. Light microscopy

The Light microscopy part of the course provides an introduction to light microscopy, covering its basic principles, techniques, and equipment. Starting with concepts: image formation spatial frequency, light diffraction, and point spread function (PSF) closing with an understanding of resolution in microscopy. The components of the microscope, including light sources, objectives, fluorescent filters, and detection mechanisms, are explained in detail.

The course highlights various microscopy techniques, with a significant focus on fluorescence microscopy. Detailed sessions cover the principles and construction of confocal microscope, properties of spinning disc microscope, including aspects like pinhole size and function of microlenses. The fundament of super-resolution methods like STED, SIM, and SMLM will be explained and extended with computational high-resolution methods such as adaptive deconvolution, optical reassignment, and SRRF.

A dedicated section explores live-cell imaging techniques, emphasizing both imaging methods pronouncing a needed equipment from WF basics up to Lightsheet, and functional techniques like FRAP/FCS, FLIM, and FRET.

A significant part of the course also explains principles of microscopic sample preparation, from fixation to visualization of objects under the microscope.

Structured for optimal learning, half the course time is dedicated to theory, while the other half is reserved for practical demonstrations. This balance ensures that participants receive real experience to complement their theoretical knowledge.

III. Electron microscopy

The Electron Microscopy section of the course provides an in-depth exploration of electron microscopy (EM) techniques, emphasizing both their theoretical foundations and practical applications in biological research.

The section begins with the fundamental physical principles of electron microscopy, such as properties of electrons, focusing the electrons in an electromagnetic field, achievable resolution, and various kinds of signals that can be collected. Analytical techniques such as Electron Energy Loss Spectroscopy (EELS) and Energy Dispersive X-ray Spectroscopy (EDS) will be also introduced. Participants will learn the design of Transmission Electron Microscope (TEM) and principles of image formation including interference effects and image acquisition methods, as well as comparison of photographic and digital recording techniques using CCD and CMOS cameras and direct electron detectors.

In the next part, the basics of scanning electron microscopy (SEM) will be explained, focusing on the construction, signal generation, and image recording in SEM. The principles and applications of Scanning Transmission Electron Microscopy (STEM) and SEM image interpretation are also discussed in detail.

Sample preparation for electron microscopy is a critical aspect. Chemical techniques such as fixation, dehydration, infiltration, embedding, ultrathin sectioning, and contrasting are covered, as well as physical methods such as low temperature processes, microwave fixation, critical point drying (CPD), metal coating, freeze fracturing, and freeze drying. Special emphasis is placed on sample preparation for cryo-SEM and low-vacuum SEM, as well as the specifics of cryo-electron microscopy and correlative light-electron microscopy (CLEM) for biological applications.

The course also covers cutting-edge techniques in volume electron microscopy, including serial imaging methods such as array tomography, serial block-face SEM (SBF-SEM), Focused Ion Beam SEM (FIB-SEM), and electron tomography. In addition, participants will gain insight into advanced techniques such as ultrastructural immunolabeling (immunogold) for both section and volume applications, as well as cryo-electron tomography and single-particle analysis (SPA) for high-resolution structural studies.

Theoretical lectures will be complemented by practical demonstrations of selected techniques to provide a better orientation for the application of electron microscopy techniques in participants’ own research.

IV. Image processing and presentation of results

The image processing part of the course introduces image deconvolution, from basic principles and image requirements to practical applications and examples using Huygens software. Image quality, the contribution of PSF, and noise will be explained. Batch processing will be demonstrated, and the evaluation of the results will be discussed.

In the next part, essential image terminology will be explained, along with image parameters and histograms. Data file formats and compression types will be presented. Basic measurements of geometric characteristics of digital images will be shown, including interactive methods like position, length, profiles, and histograms. This part also includes filtration, segmentation by thresholding, and object separation by watershed. Measurements of circumference in 2D, area, perimeter, Feret averages, and Euler characteristic will be explained.

Image analysis and visualization in 3D will cover data sources such as CLSM, CT, 3D TEM, and MRI. Dimensional calibration, filtering, and segmentation of such data will be presented. Measurements of surface and length in 3D will be explained. The basics of visualization, including volume and surface rendering, will be shown. Visualization cues such as movies, lighting, texture, stereopsis, fog, depth, color coding, etc., will be explained as well.

The last part of the course will be dedicated to the preparation of data for publication, starting from digital images, including both technical and ethical aspects of publishing scientific images. It includes image handling and processing – what is allowed and prohibited, preparation of digital images for publications, scale bars, arrows, and lettering, bitmaps vs. vector graphics. Black & white vs. color images will be discussed as well, together with hardware gamma adjustment, primary color management on PC, and color blindness issues with color maps.

The results of the image processing tasks include not only images but also videos, graphs, and tables with numerical results. The complete workflow from acquiring microscopic images through image processing and analysis to the comprehensive presentation of the results will be presented and discussed. A brief introduction to the statistical analysis of the results will also be included.