Microscopy and instrumental methods in biology 2025
This course gives an overview of modern measurement and imaging techniques used in biophysics, molecular and cell biology. Its main goal is to teach students about the capabilities and limitations of each of the most popular techniques and explain how to select the simplest and the most effective approach to answer a particular experimental question. Practicals will include a complete workflow of processing real experimental datasets from fluorescence and circular dichroism experiments, planning chromatography experiments, and advanced work with fluorescence microscopy images using ImageJ.
All classes will be given in Ukrainian.
Electromagnetic waves. Wavelength, color. Light absorption, scattering, refraction. The dipole moment of molecules and absorption wavelength. Spectrophotometers. Natural chromophores. Absorption-based methods.
Principles of fluorescence. Jablonsky diagram. Fluorescence quantum yield. Fluorophores. Brightness. Solvatochromism. Fluorometer.
Tryptophan and other natural fluorophores. Intercalating dyes. Cys-, Lys- reactive dyes. Tags. Click reactions. UAA. GFP.
FRET. Fluorescence lifetime. Fluorescence anisotropy.
HTS methods. Assay development ► Homework: Processing plate reader data: determination of inhibitor IC50 from kinetic data.
Transmission microscopy, phase contrast. Fluorescence microscopy. Principal schemes of microscopes. Lasers. Filters. Dichroic mirrors. Channels. Digital image collection. Image resolution, micrones, and pixels. Confocal microscopy. Z-slices.
Colocalization. FRET and detection of interactions in microscopy. FLIM. TIRF. Diffraction limit. Superresolution, STORM. PALM. Two-photon microscopy. FRAP.
Basics of work with eucaryotic cell lines. Commonly used cell lines. Passages. Transfection.
Introduction of fluorescent proteins. Small molecule dyes. Channel crosstalk and selection of fluorophores. Membrane trackers, staining of nuclei. Photodegradation during measurements. Light intensity and damage to cells.
ImageJ/Fiji. ► Homework: image processing in Fiji
Electrophoresis of proteins and oligonucleotides. Analytical ultracentrifugation. DLS. FCS. FCCS.
Polarized light. CD spectroscopy to determine protein structure. IR spectroscopy.
Spin. 13C and 15N protein labeling. NMR for protein structure analysis. Solid state NMR. MRI imaging. ESR and free radicals.
Protein crystallization. SAXS.
Basics of statistics and errors. Linear and non-linear regression. Data visualization.
Principle and scheme of microscopes. XY and Z resolution. Sample preparation. Scanning speed and sample damage. Application for protein unfolding.
Principle. Steady-state. Association and dissociation kinetics.
HPLC principle, preparative and analytical applications. Types of columns. Ion exchange chromatography. Size-exclusion.
Mass-spectrometry. LC-MS. ESI, MALDI, and other ionization methods. Types of mass detectors. Fragmentation.
What is that? Sample preparation.
Immunocytochemistry/immunohistochemistry, flow cytometry, in situ hybridization.
PCR, microarray, Western blotting, ELISA, two-dimensional gel electrophoresis, mass spectrometry.
First-generation sequencing, second- or next-generation sequencing, third-generation sequencing, digital PCR, liquid biopsy).
Principle. Resolution. Modes. Sample preparation.
Kd, pH, buffers. ► Homework: A set of problems to train simple calculations.
Virtual lab work ‘inhibition of enzymatic reaction’.
► Homework: Calculation of protein affinity to membrane based on Trp emission changes.
►Homework: applied problems on CD and SDS-PAGE.
► Homework: analysis of distances between residues in protein based on PDB structure.
