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Quenching

Quenching. Non- radiative energy transfer from excited species to other molecules. Quantum Yield and Quenching. Show that quantum yield in the presence of a quencher is:. F A,p = k A n S0 V. F F,p = k F n S1 V. Dynamic Quenching/ Collisional Quenching

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Quenching

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  1. Quenching Non-radiative energy transfer from excited species to other molecules

  2. Quantum Yield and Quenching Show that quantum yield in the presence of a quencher is: FA,p = kAnS0V FF,p = kFnS1V

  3. Dynamic Quenching/Collisional Quenching Requires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration. Stern-Volmer Equation Since fluorescence emission is directly proportional to quantum yield:

  4. Static Quenching Lumophore in ground state and quencher form dark complex. Luminescence is only observed from unbound lumophore. Luminescence lifetime not affected by static quenching. Dopamine Sensor!

  5. Long-Range Quenching/Förster Quenching Result of dipole-dipole coupling between donor (lumophore) and acceptor (quencher). Rate of energy transfer drops with R-6. Used to assess distances in proteins (good for 2-10 nm). Förster/Fluorescence Resonance Energy Transfer Single DNA molecules with molecular Beacons

  6. Fluorescence Microscopy Need 3 filters: Exciter Filters Barrier Filters Dichromatic Beamsplitters http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html

  7. Are you getting the concept? You plan to excite catecholamine with the 406 nm line from a Hg lamp and measure fluorescence emitted at 470 ± 15 nm. Choose the filter cube you would buy to do this. Sketch the transmission profiles for the three optics. http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html

  8. FluorescenceMicroscopy Objectives Image intensity is a function of the objective numerical aperture and magnification: Fabricated with low fluorescence glass/quartz with anti- reflection coatings http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

  9. Fluorescence Microscopy Detectors No spatial resolution required: PMT or photodiode Spatial resolution required: CCD http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html

  10. Epi-Fluorescence Microscopy • Light Source - Mercury or xenon lamp (external to reduce thermal effects) • Dichroic mirror reflects one range of wavelengths and allows another range to pass. • Barrier filter eliminates all but fluorescent light. http://web.uvic.ca/ail/techniques/epi-fluor.jpg http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.html

  11. Special Fluorescence Techniques TIRF LIF http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

  12. Photoactivated Localization Microscopy http://www.hhmi.org/bulletin/nov2006/upfront/image.html Left: Viewing a mitochondrion using conventional diffraction-limited microscopy offers a resolution (200 nanometers) barely sufficient to visualize the mitochondrial internal membranes. Right: Viewing the same mitochondrion by imaging sparsely activated fluorescent molecules one at a time—using PALM—provides much better resolution (20 nanometers), producing a detailed picture of the mitochondrion’s internal membranes. http://www.hhmi.org/news/palm20060810.html

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