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Stokes Shift

stokes

The wavelength difference between absorption and emission maxima—enabling separation of excitation from fluorescence.

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Definition
The Stokes shift is the difference between absorption and emission peak wavelengths of a fluorophoreLoading.... Emission always occurs at longer wavelength (lower energy) than absorption because energy is lost to vibrational relaxation in the excited stateLoading.... This shift enables optical separation of excitation light from fluorescence and is a key consideration in donor-acceptor pairLoading... selection.
Emission > Absorption λ
Always red-shifted
Energy loss mechanism
Vibrational relaxation
Practical benefit
Separate excitation from emission
FRET consideration
Affects spectral overlap

Origin of the Shift

The Stokes shift arises from rapid vibrational relaxation:

  1. Photon absorbed, electron excited to S₁ with excess vibrational energy
  2. Picoseconds: vibrational relaxation to S₁ minimum (energy lost as heat)
  3. Nanoseconds: fluorescence emission from S₁ to S₀ vibrational levels
  4. Picoseconds: vibrational relaxation in S₀ (more energy lost)

Both relaxation steps (in S₁ and S₀) contribute to the total energy loss between absorption and emission.

Simplified

Why It Happens: After absorbing light, the molecule vibrates intensely. This vibration quickly settles down, releasing some energy as heat. By the time light is emitted, less energy remains—so the light is a different color (longer wavelength).

Practical Implications

Stokes shift affects experimental design:

Large Shift (>50 nm)
  • Easy filter separation
  • Low background
  • May require different optics
Small Shift (<30 nm)
  • Harder to separate
  • Potential excitation contamination
  • Common in red fluorophores
Simplified

Bigger shift = easier to separate excitation light from emission.

Small shift makes filtering harder but some useful fluorophores have small shifts.

Practical Considerations

  • Filter selection: Shift determines dichroic and emission filter requirements
  • Background reduction: Larger shifts enable better excitation rejection
  • FRET pairs: Donor emission position affects acceptor spectral overlap
  • Multiplex imaging: Stokes shift affects channel separation in multi-color experiments

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