Fluorescence Spectroscopy Flashcards
Who first discovered Fluorescence?
George Stokes
What are the spins of electrons in a Singlet State?
Opposite
What are the spins of electrons in a Triplet State?
Same
What are the TWO structural requirements for Fluorescence?
- Rigid structure
- Large energy gap between S1 and S0 states
What is the duration of Fluorescence?
1 - 10ns
Which hydrocarbons are frequently fluorescent?
Aromatic
Do heavy atoms increase or decrease fluorescence?
Decrease
What is Stokes Shift?
Emission spectrum is shifted towards longer wavelength (lower energy)
What is the Mirror Rule?
When the fluorescence spectrum resembles the absorption spectrum
What does the Shape of the fluorescence spectrum indicate?
Emission is independent of wavelength
What are the FIVE characteristics of Fluorescence?
- Excitation Spectrum
- Emission Spectrum
- Quantum Yield
- Decay Time (lifetime)
- Polarisation / Anisotropy
What is the equation for Fluorescence in dilute solutions?
What are the units of Fluorescence?
RFU
TRUE OR FALSE:
Measurement of fluorescence sensitivity down to single molecules is possible?
True
What is the practical sensitivity of fluorescence?
Low nM / pM
Why are long-lived excited states more unstable?
- Excess of energy
- Different pKa, Redox potentials, reactivity
Which TWO amino acids provide a strong UV fluorescence?
Trp and Tyr
Which coenzymes are fluorescent?
- NADH / NADPH
- FAD+
Which coenzymes are non-fluorescent?
- NAD+ / NADP+
- FADH
Are DNA and RNA fluorescent or non-fluorescent?
Non-fluorescent
What is the fluorescent stain is used to identify NAs in live cells?
DAPI
What regions are stained in NAs?
A-T rich regions
What does fluorescence-activated cell sorting (FACS) allow?
Sorting of heterogenous mixture of cells into sub-populations
What are THREE special formats of fluorescence?
- Fluorescence Quenching
- Fluorescence Energy Transfer
- Fluorescence Polarisation
What are the THREE requirements for Forster Resonance Energy Transfer (FRET)?
- Donor - Acceptor distance <100 A
- Orientation of D and A dipoles
- Spectral Overlap
What are the SIX advantages of Fluorescence?
- High Sensitivity & Selectivity
- Versatility
- ‘On/Off\ Switching
- Imaging Capabilities (2D/3D)
- Multi-Colour Detection
- Communication with Human Molecules