|
|
|
||
|
The course assumes basic knowledge of light fluorescence microscopy, including the preparation of slides and
image imaging. The course will include the preparation of samples, the selection, and the application of microscopic techniques. The sample studies will also include data management, statistics, and the preparation of a pictorial appendix for publication purposes. The course will be taught in English. Last update: Sacherová Veronika, RNDr., Ph.D. (09.05.2022)
|
|
||
|
J. Lakowicz, Principles of fluorescence spectroscopy, Springer 2007
G. Jung, Fluorescent Proteins I and II, Springer 2011
Kianianmomeni, Optogenetics: Methods and Protocols, Springer 2016 Last update: Sacherová Veronika, RNDr., Ph.D. (09.05.2022)
|
|
||
|
For the final exam please prepare the final project. The project will contain several parts. 1. Define your research question. 2. pick up one microscopy technique and software. 3. prepare a theoretical pipeline of image analysis of your task. Last update: Sacherová Veronika, RNDr., Ph.D. (09.05.2022)
|
|
||
|
Introduction, Research Data Management
Basic principles of detection of molecular probes - optical (fluorescence, luminescence, vibrational spectroscopy), spin, radioactive, electron microscopic, mass-spectrometric, acousto-optical, AFM, NMR, CT
Specifics of applications of molecular probes - in vitro, in fixed cells and tissues, in living cells and tissues, in living organisms; labeling of lipids, proteins, nucleic acids; comparison of approaches detecting individual molecules and sets of molecules
Labeling principles - exogenous (affinity, incl. Immunodetection, FlAsH / ReAsH, covalent (click-chemistry, photoaffinity labeling, chromobodies), nucleic acid hybridization (DNA barcoding, DIANA), endogenous (genetically encoded - natural and unnatural amino acids)
Marking for superresolution optical microscopy - exogenous and genetic marking, relation to the principles of optical superresolution
Fluorescent proteins - spectral and other biophysical properties (structure, reactivity, maturation, photoconversion, immunogenicity, FRET, tolerance of modifications), non-traditional fluorescent proteins
Self-labeling enzymes - dehalogenases (HALOtag), methyltransferases and other alkyl transferases (SNAP-tag, CLIP-tag)
Bioluminescence - luciferins and luciferases, BRET, instrumentation
Raman Marks - Examples and limits of use in linear and nonlinear Raman techniques
Optogenetic approaches - membrane tension modulation, ion concentration, cell localization, molecular interactions
Detection of neuronal activity - changes in membrane tension, changes in calcium concentration, probes detecting neurotransmitters, probes of G protein signaling activity, and other processes
Marking in electron, cryo-electron, and combined microscopy and tomography. Last update: Sacherová Veronika, RNDr., Ph.D. (09.05.2022)
|