Předmět Fluorescence spectroscopy in biology (MB140C75E)
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Další informace
Sylabus
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).
Literatura
Joseph R. Lakowicz (2006): Principles of Fluorescence Spectroscopy, 3rd edition, Springer
Požadavky
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.
Garant
RNDr. Radovan Fišer, Ph.D.
Vyučující
RNDr. Radovan Fišer, Ph.D.Mgr. Petra Matyska Lišková