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Last update: doc. RNDr. Radovan Fišer, Ph.D. (08.10.2020)
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. |
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Last update: KONOP (19.04.2007)
Cílem praktického cvičení je seznámit s fluorescencí jako všestrannou metodou použitelnou v biologii. Studenti při samostatné práci s fluorometrem zvládnou základní techniky včetně vyhodnocení výsledků a návrhu experimentů. |
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Last update: doc. RNDr. Radovan Fišer, Ph.D. (24.10.2019)
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 |
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Last update: doc. RNDr. Radovan Fišer, Ph.D. (24.10.2019)
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. |
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Last update: doc. RNDr. Radovan Fišer, Ph.D. (24.10.2019)
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). |
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Last update: doc. RNDr. Radovan Fišer, Ph.D. (24.10.2019)
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. |