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BC3001: Structural & Experimental Biochemistry > Fluorescence Spectroscopy > Flashcards

Fluorescence Spectroscopy Flashcards

1
Q

Who first discovered Fluorescence?

A

George Stokes

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2
Q

What are the spins of electrons in a Singlet State?

A

Opposite

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3
Q

What are the spins of electrons in a Triplet State?

A

Same

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4
Q

What are the TWO structural requirements for Fluorescence?

A
  1. Rigid structure
  2. Large energy gap between S1 and S0 states
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5
Q

What is the duration of Fluorescence?

A

1 - 10ns

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6
Q

Which hydrocarbons are frequently fluorescent?

A

Aromatic

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7
Q

Do heavy atoms increase or decrease fluorescence?

A

Decrease

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8
Q

What is Stokes Shift?

A

Emission spectrum is shifted towards longer wavelength (lower energy)

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9
Q

What is the Mirror Rule?

A

When the fluorescence spectrum resembles the absorption spectrum

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10
Q

What does the Shape of the fluorescence spectrum indicate?

A

Emission is independent of wavelength

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11
Q

What are the FIVE characteristics of Fluorescence?

A
  1. Excitation Spectrum
  2. Emission Spectrum
  3. Quantum Yield
  4. Decay Time (lifetime)
  5. Polarisation / Anisotropy
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12
Q

What is the equation for Fluorescence in dilute solutions?

A
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13
Q

What are the units of Fluorescence?

A

RFU

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14
Q

TRUE OR FALSE:
Measurement of fluorescence sensitivity down to single molecules is possible?

A

True

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15
Q

What is the practical sensitivity of fluorescence?

A

Low nM / pM

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16
Q

Why are long-lived excited states more unstable?

A
  • Excess of energy
  • Different pKa, Redox potentials, reactivity
17
Q

Which TWO amino acids provide a strong UV fluorescence?

A

Trp and Tyr

18
Q

Which coenzymes are fluorescent?

A
  • NADH / NADPH
  • FAD+
19
Q

Which coenzymes are non-fluorescent?

A
  • NAD+ / NADP+
  • FADH
20
Q

Are DNA and RNA fluorescent or non-fluorescent?

A

Non-fluorescent

21
Q

What is the fluorescent stain is used to identify NAs in live cells?

A

DAPI

22
Q

What regions are stained in NAs?

A

A-T rich regions

23
Q

What does fluorescence-activated cell sorting (FACS) allow?

A

Sorting of heterogenous mixture of cells into sub-populations

24
Q

What are THREE special formats of fluorescence?

A
  1. Fluorescence Quenching
  2. Fluorescence Energy Transfer
  3. Fluorescence Polarisation
25
Q

What are the THREE requirements for Forster Resonance Energy Transfer (FRET)?

A
  1. Donor - Acceptor distance <100 A
  2. Orientation of D and A dipoles
  3. Spectral Overlap
26
Q

What are the SIX advantages of Fluorescence?

A
  1. High Sensitivity & Selectivity
  2. Versatility
  3. ‘On/Off\ Switching
  4. Imaging Capabilities (2D/3D)
  5. Multi-Colour Detection
  6. Communication with Human Molecules

BC3001: Structural & Experimental Biochemistry (13 decks)

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