1

Quenching

Non-radiative energy transfer from excited species to other molecules

S0 S1kA

kF

knr

+ Q

kq

2

Quantum Yield and Quenching

S0 S1kA

kF

knr

+ Q

kq

10

1

SnrFSAS nk k - nk

dt

dnQqnkQqnk

nrF

F

pA,

pF,F k k

k

Qqnk

nrF

ASS k k

kn n 0

1

Show that quantum yield in the presence of a quencher is:

FA,p = kAnS0V

FF,p = kFnS1V Qqnk

nrF

F

pA,

pF,F k k

k

3

Dynamic Quenching/Collisional QuenchingRequires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration.

QqnrF

QqnrF

f

of nK

kk

nkkk

1

Since fluorescence emission is directly proportional to quantum yield:

QqnKF

F10

Stern-Volmer Equation

4

Static QuenchingLumophore 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 QuenchingResult 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 FiltersBarrier FiltersDichromatic 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 froma Hg lamp and measure fluorescence emitted at 470 ± 15nm. 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

Fluorescence Microscopy Objectives

Image intensity is a function of the objective numericalaperture and magnification:

2

4

)(

)( mag

NAI obj

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 photodiodeSpatial 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://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.html

http://web.uvic.ca/ail/techniques/epi-fluor.jpg

11

Special Fluorescence Techniques

TIRF

http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

LIF

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