1. Non-radiative energy transfer from excited species to other molecules
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. Stern-Volmer Equation
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. Dopamine Sensor! 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

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.

8. Fluorescence Microscopy Objectives
Image intensity is a function of the objective numerical
aperture and magnification:
Fabricated with low fluorescence glass/quartz with anti-
reflection coatings

9. Fluorescence Microscopy Detectors
No spatial resolution required: PMT or photodiode
Spatial resolution required: CCD

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.

11. Special Fluorescence Techniques
TIRF
LIF

12. Photoactivated Localization Microscopy
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.