Chapter 10: Fluorescence

Question 1

What is a Jablonski diagram?

Answer 1

A visualisation of different energy levels and the processes by which electrons move between them and connect them.

Question 2

Describe the process of fluorescent light emission

Answer 2

1. Electrons absorb a photon with a specific amount of energy, hv, so are excited from the ground state, s_0, to an excited state, s_1.
2. Internal non-radioactive conversion occurs so the electron reaches the lowest s_1 state.
   3a. Energy can be emitted by fluorescence where a new photon with a difference amount of energy is emitted directly.
   3b. Energy can be emitted by inter-system crossing where orbital coupling occurs and allows the electron to enter into an excited triplet state before relaxing into the ground state by phosphorescence.

Question 3

How long does electron excitation take?

Answer 3

~ 10^-15 s

Question 4

Excited electrons usually absorb photons within the __ range.

Answer 4

UV

Question 5

How long does internal non-radioactive conversion take?

Answer 5

~ 10^-12 s

Question 6

How long does fluorescence take?

Answer 6

~ 10^-8 s

Question 7

Define fluorophores

Answer 7

Molecules which show fluorescence. They often have a specific molecular structure that allows them to do this.

Question 8

Define green fluorescent protein (GFP)

Answer 8

A protein found in a species of jellyfish that is cylindrical in shape (barrel shaped). A molecular portion of the protein in the centre of the barrel can be excited and emit fluorescent light.

Question 9

How many amino acids are in GFP?

Answer 9

238 amino acids

Question 10

What are the dimensions of GFP?

Answer 10

42 x 24 Å

Question 11

How has GFP been significant on molecular biophysics?

Answer 11

It’s structure has been studied and now the DNA required to produce this protein can now be introduced into a difference cell using genetic engineering. This means it can be used as a genetic marker and adapted so that it has different emission spectra.

Question 12

Define Föster Resonance Energy Transfer (FRET)

Answer 12

A technique used to measure distances on the molecular scale using fluorescent proteins. It is especially important in studies of molecular dynamics.

Question 13

What are the most commonly used colour pairs for FRET?

Answer 13

Blue (BFP) and Green (GFP)

Question 14

Outline the process of FRET

Answer 14

1. A fluorophore donor is excited by incident light of a specific wavelength.
2. The excitation energy in transferred onto an acceptor fluorophore molecule via a non-radiative process (not mediated by a photon) where it is emitted at a much longer wavelength.
3. The emitted light is then monitored to measure distances on the nm scale, as the energy transfer depends on distance.

Question 15

What type of interaction does this process reply on?

Answer 15

Dipole-dipole interactions between the donor and acceptor molecules.

Question 16

What is one requirement for this technique to occur successfully?

Answer 16

The donor emission and acceptor absorption spectrums must overlap.

Question 17

During FRET, the intensity of the emission depends on the ________ of the energy transfer. This in turn depends on ________ __________.

Answer 17

Efficiency
Separation distance

Question 18

Give the equation of the Föster-type energy transfer

Answer 18

E_T = efficiency
R_0 = fluorophore-specific constant (2-6nm)
R = distance between donor and acceptor

Question 19

What does the value of the fluorophore-specific constant depend on??

Answer 19

• Spectral overal
• Refractive index of the medium
• Relative dipole orientation
• Quantum yield of the donor (photons emitted/ photons absorbed)

Question 20

Give the equation for the fluorophore-specific constant

Answer 20

Question 21

Why is FRET usually used for relative distances?

Answer 21

Because the calculation depends on the relative dipole orientation.

Question 22

How can the results of FRET be interpreted quantitatively? (EXTRA: what method is used for this to be done?)

Answer 22

At least one dipole must rotate freely (to have an equal probability of having an rotation.

EXTRA: using a flexible linker