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Laboratory course in fluorescence spectroscopy with biological and biochemical applications. The course covers introductory lecture about fluorescence methods and introductions into individual practical works covering: spectra measurements, fluorescence quenching, polarized fluorescence, FRET, fluorescence of proteins, dynamics of biological membranes, physiological characteristics of cells measured by fluorescence techniques, digital image processing.
Last update: Lišková Petra, RNDr., Ph.D. (27.09.2024)
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The course includes an introductory lecture, presentation is available for course participants through Moodle.
Joseph R. Lakowicz (2006): Principles of Fluorescence Spectroscopy, 3rd edition, Springer Last update: Fišer Radovan, doc. RNDr., Ph.D. (24.10.2019)
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To gain the credits, one must be present at the introductory lecture, pass the entrance test operated through the e-learning platform Moodle http://dl2.cuni.cz/course/view.php?id=242 (with minimal success rate 60%). It is also necessary to hand over complete reports in Excel file. Excel file must be uploaded as a task in Moodle to the date determined at the end of the course. Last update: Fišer Radovan, doc. RNDr., Ph.D. (24.10.2019)
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Introductory lecture, entrance test, introductions to individual practical tasks, independent measurements on fluorometer, evaluation of experimental data, preparation of protocols.
1. Introductory lecture: Introduction to fluorescence spectroscopy, Jablonski diagram, absorption, emission spectra, its variations, fluorescence lifetime, probes, fluorescence quenching, FRET, polarization of fluorescence, time-resolved fluorescence, spectroscopic tools and fluorometers.
2. Practical tasks: a) Fluorometer calibration, excitation and emission spectra, fluorescence intensity, polarity of environment. Artifacts (inner filter effect, background correction, Raman scattering). Design of experiments and data evaluation. b) Tryptophan and tyrosine as intrinsic fluorophores, their emission spectra alone and in proteins, spectral shift. c) Polarized fluorescence, fluorescence anisotropy, membrane fluidity and phase transition. d) Time-resolved measurement: determination of concentration of Cl- ions (quenching). e) Evaluation of the data from the confocal microscope: GP of Laurdanu (membrane fluidity); ratiometric fluorescence measurements (changes in calcium concentration in the cells).
Software used: MS Excel or Gnumeric (GNU) spreadsheet, Fityk (data anlysis and fitting, non-linear regression; http://fityk.nieto.pl/), WCIF ImageJ (microscopic image processing; http://fiji.sc/Fiji, http://imagej.net/Welcome). Last update: Fišer Radovan, doc. RNDr., Ph.D. (24.10.2019)
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A successful graduate of the course Fluorescence Spectroscopy in Biology: - describes the basic physical principles of fluorescence, explains the Jablonski diagram and the relationship between excitation and emission spectra - explains the concept of the lifetime of the excited state and its connection with the quantum yield and quenching processes - distinguishes between the types of fluorescence quenching (static, dynamic, self-quenching, etc.) and explains their influence on the measured intensity and lifetime - explains the principle of FRET, the conditions for its occurrence and information about distances and interactions in biomolecular systems that can be derived from FRET detection - describes the principle of polarized fluorescence and anisotropy and their relationship to molecular rotation and microviscosity of the environment - explains the principle of time-resolved fluorescence and the difference between steady-state and pulsed mode measurements - measures the excitation and emission spectra of fluorescent probes and natural fluorophores (Trp, Tyr) and interprets changes in spectra depending on the environment (pH, polarity, binding to proteins or membranes) - performs common fluorescence experiments "in a cuvette", designs basic experiments on a confocal microscope with regard to signal optimization and artifact minimization - recognizes and critically evaluates the most common artifacts of fluorescence measurements (inner filter, Raman scattering, background autofluorescence, light scattering and polarization artifacts) and suggests appropriate corrections - analyzes experimental data of time-resolved fluorescence, determines relevant parameters (lifetimes, component amplitudes) and assesses the quality of the fitted model - describes the design and function of basic types of spectrofluorimeters and confocal microscopes used in fluorescence spectroscopy - critically evaluates the accuracy and reproducibility of own measurements, identifies sources of errors and suggests their minimization - assesses the suitability of selected fluorescence methods and probes for solving a specific biological problem and justifies their choices based on theoretical and practical knowledge - interprets results in the context of current literature and is able to formulate limitations and possible alternative interpretations of data - works safely in a laboratory environment, follows the principles of working with laser and UV sources, biological material and chemicals - effectively communicates experimental results in written (protocols, short reports) and oral form (presentations), including a clear description of methods, error analysis and conclusions Last update: Fišer Radovan, doc. RNDr., Ph.D. (06.02.2026)
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The course precedes the lecture. To be familiar with therms of biochemistry is highly recommended. It is necessary to have skills to work with either MS Excel or Gnumeric or OpenOffice Calc or other. For the processing of the results during the course and for passing the course, own laptop capable of wireless connection (Wifi) is required. Last update: Fišer Radovan, doc. RNDr., Ph.D. (24.10.2019)
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