What is Fluorescence?

What is Fluorescence?

http://elchem.kaist.ac.kr/vt/chem-ed/spec/molec/mol-fluo.htm

A type of electronic spectroscopy, that is,it involves excitation of electrons—energy absorption to an excited state— followed by emission of a smaller energy as radiation.

Fluorescence andPhosphorescence are two types of Luminescence.

What is Fluorescence?

Link to Simulation spectrometer: http://terpconnect.umd.edu/~toh/models/Fluorescence.html

S refers to ‘singlet’ and T to ‘triplet’ states. The S0 state is the ground state and the subscript numbers, S1 and S2 , identify individual excited states.

What is Fluorescence?

Xe lamp

Sample cell

photodetector

Emissionmonochromator

excitationmonochromator

Why is Fluorescence useful in DNA (and other biochemical) Studies?

-Fluorescence generally is much more sensitive to the environment of the chromophore than is light absorption. - Fluorescence is a useful technique for following the binding of ligands or conformational changes. - The sensitivity of fluorescence is a consequence of the relatively long time a molecule stays in an excited singlet state before de-excitation.

- Absorption is a process that is over in 10-15 sec, a time scale where the molecule and its environment are effectively static.

- In Fluorescence , during the 10-9 to 10-8 sec that a singlet remains excited, all kinds of processes can occur, including protonation or deprotonation reactions, solvent-cage relaxation, local conformational changes, and any processes coupled to translational or rotational motion.

What is the connection between Fluorescence and DNA Photocleavage?

Ru(bpy)2(L)2+

Ground state

{Ru(bpy)2(L)2+}*Excited state

Ru(bpy)2(L)2+

Ground state

{Ru(bpy)2(L)2+}*Excited state

+ hn

What is the connection between Fluorescence and DNA Photocleavage?

Ru(bpy)2(L)2+

Ground state

{Ru(bpy)2(L)2+}*Excited stateMLCT,d e- p* orbital

1O2 singlet state, S

3O2 triplet state, T

“Ru(bpy)2(L)2+“ Relaxed state

O=O

O=O“triplet energy transfer"

What is the connection between Fluorescence and DNA Photocleavage?

Ru(bpy)2(L)2+

Ground state

{Ru(bpy)2(L)2+}*

3O2

“Ru(bpy)2(L)2+“ Relaxed state

1O2

+ hn

ATTACGGCATCG

ATTACoxGGCATCG

What is the connection between Fluorescence and DNA Photocleavage? Oxidized Guanine bases

3O2

1O2

ATTACGGCATCG

ATTACoxGGCATCG