Chapter 10: Fluorescence Flashcards
What is a Jablonski diagram?
A visualisation of different energy levels and the processes by which electrons move between them and connect them.
Describe the process of fluorescent light emission
- 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.
- 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.
How long does electron excitation take?
~ 10^-15 s
Excited electrons usually absorb photons within the __ range.
UV
How long does internal non-radioactive conversion take?
~ 10^-12 s
How long does fluorescence take?
~ 10^-8 s
Define fluorophores
Molecules which show fluorescence. They often have a specific molecular structure that allows them to do this.
Define green fluorescent protein (GFP)
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.
How many amino acids are in GFP?
238 amino acids
What are the dimensions of GFP?
42 x 24 Å
How has GFP been significant on molecular biophysics?
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.
Define Föster Resonance Energy Transfer (FRET)
A technique used to measure distances on the molecular scale using fluorescent proteins. It is especially important in studies of molecular dynamics.
What are the most commonly used colour pairs for FRET?
Blue (BFP) and Green (GFP)
Outline the process of FRET
- A fluorophore donor is excited by incident light of a specific wavelength.
- 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.
- The emitted light is then monitored to measure distances on the nm scale, as the energy transfer depends on distance.
What type of interaction does this process reply on?
Dipole-dipole interactions between the donor and acceptor molecules.
What is one requirement for this technique to occur successfully?
The donor emission and acceptor absorption spectrums must overlap.
During FRET, the intensity of the emission depends on the ________ of the energy transfer. This in turn depends on ________ __________.
Efficiency
Separation distance
Give the equation of the Föster-type energy transfer
E_T = efficiency
R_0 = fluorophore-specific constant (2-6nm)
R = distance between donor and acceptor
What does the value of the fluorophore-specific constant depend on??
- Spectral overal
- Refractive index of the medium
- Relative dipole orientation
- Quantum yield of the donor (photons emitted/ photons absorbed)
Give the equation for the fluorophore-specific constant
Why is FRET usually used for relative distances?
Because the calculation depends on the relative dipole orientation.
How can the results of FRET be interpreted quantitatively? (EXTRA: what method is used for this to be done?)
At least one dipole must rotate freely (to have an equal probability of having an rotation.
EXTRA: using a flexible linker
