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

Electron microscopy basics, properties of electrons, resolution, wavelength of accelerated electrons, the electrons in an electromagnetic field. Electron-electron interaction and analytical electron microscopy (EELS, EDS), transmission electron microscope – design, image formation, interference, image acquisition in the TEM. Comparison of photographic and digital recording of the microscope, CCD and CMOS cameras, direct electron detectors. Scanning electron microscope – the construction, signals and image recording (secondary and back-scattered electron imaging), Scanning Transmission Electron Microscopy (STEM), 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, critical-point drying (CPD) method, metal coating, freeze fracturing, etching and freeze-drying. Sample preparation for cryo-SEM, low vacuum SEM; correlative light-electron microscopy (CLEM) in cryo and room temperature conditions, strategies for biological applications; analytical morphomics (morphology, identification, content, functionality). Ultrastructural immunolabeling (immunogold) on-section and in the volume. Volume electron microscopy: serial imaging – array tomography, SBF-SEM, FIB-SEM; electron tomography. Cryo electron microscopy, single particle analysis (SPA), cryo-electron tomography, cryo-FIB lamella.

IV. Image processing

1) Digitizing the image

Essentials (pixel size and spatial resolution, levels of grey and contrast), The image parameters (contrast, noise), histogram. Spatial calibration. Data file formats (binary, grayscale, RGB, HSV, Lab) and compression (lossy, lossless).

2) Measurement of geometric characteristics of digital image

Interactive methods: position, length, profiles, histograms in ROI.

Filtration and image processing. Segmentation by thresholding. Objects separation by watershed.

Use of Crofton formulas for circumference measurements in 2D, area, perimeter, Feret averages, number, Euler characteristic. The effect of anisotropy of the object and of noise on the measurement accuracy.

3) Image analysis and visualization in 3D

Data Sources: CLSM, CT, 3D TEM and MRI. Dimensional calibration. Filtering and segmentation data. Use of Crofton formula for measuring the surface and the length in the 3D measurement. Visualization: volume and surface rendering. Visualization cues – movies, lighting, texture, stereopsis, fog, depth color coding etc.

V. Preparation of digital images for publication

Thorough review of both technical and ethical aspects of publication of scientific images.

Image formats – lossless vs. lossy. Image interpretation – the human eye is not a perfect device. Primary image interpretation – mainly based on previous experiences. Image Handling and Processing – essential hardware calibration, software image processing -What is allowed and prohibited? Preparation of digital images for publications – a 300 DPI nightmare. Scale bars, arrows, and lettering – bitmaps vs. vector graphic. Black&White vs. Color Images – hardware gamma adjustment, primal color management in PC, color blindness problem – color maps. Color in scanning electron microscopy – Does it matter?