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Poslední úprava: Mgr. Aleš Benda, Ph.D. (19.06.2017)
The course covers traditional methods of single molecule observation using TIRF based wide-field microscopy and confocal microscopy combined with spectral and excited state lifetime resolved detection. The single molecule manipulation, including force measurement and confocal imaging of the manipulated molecules, is performed using the optical tweezers. The methodologies covered include single particle tracking and various modifications of fluorescence correlation spectroscopy. |
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Poslední úprava: Mgr. Aleš Benda, Ph.D. (19.06.2017)
Day 1 9:00 – 16:00 Theory - Introduction to single molecule techniques (TIRFm single molecule visualization, optical tweezers, FCS) - Introduction to molecular motors and diffusible microtubule-associated proteins - Introduction to data analysis
Day 2-5: 9:00 – 14:00 Experimental sections – each group of 4 one section per day 14:00 – 17:00 Data analysis (tracking of single molecules, calculation of velocity, mean square displacement and diffusion constant, force time-traces analysis, autocorrelation analysis, etc…)
Section 1 – TIRF 1 - Imaging of single kinesin motors walking along microtubules (at different ATP concentrations -> different velocity)
Section 2 – TIRF 2 - Measurement of forces exerted by single kinesin molecule (visualizing single steps, measuring stall force, describing force-velocity relationship)
Section 3 - optical tweezers - Imaging of single diffusible microtubule crosslinkers (at different salt concentrations -> different diffusion constant)
Section 4 - FCS - Measuring concentrations and diffusion coefficients of various molecules in solution at different conditions (temperature, viscosity), quantifying molecular interaction using fluorescence cross-correlation analysis, counting and sorting single molecules. |