How does a fluorophore work?

A fluorophore, also known as a fluorescent dye or fluorescent molecule, is a type of molecule that can re-emit light upon light excitation. Here’s a detailed explanation of how a fluorophore works:

Excitation

  • When a fluorophore absorbs photons (light energy) of a specific wavelength, it becomes excited. This means that its electrons are elevated from a ground state to a higher energy excited state.

What is the wavelength?

The distance between two consecutive peaks of a light wave. It determines the color of the light we see.

  • Shorter Wavelengths: Higher energy, often seen as blue or violet light.
  • Longer Wavelengths: Lower energy, often seen as red or orange light.

Excited State Lifetime

  • The excited state is not stable, so the fluorophore spends a very short time (typically nanoseconds) in this state. During this period, some of the absorbed energy is lost as heat or through other non-radiative processes.

Emission

  • The remaining energy is then released as a photon of light when the electron returns to its ground state. The emitted light has a longer wavelength (lower energy) than the absorbed light due to the energy lost during the excited state lifetime. This phenomenon is known as the Stokes shift.

Wavelength and Energy

longer wavelengths of light correspond to lower energy. This relationship is described by the equation E=hcλE=λhc​, where:

  • EE is the energy of the photon,
  • hh is Planck's constant (6.626×10−34 J⋅s6.626×10−34J⋅s),
  • cc is the speed of light (3×108 m/s3×108m/s),
  • λλ is the wavelength of the light.

From this equation, it is clear that energy EE is inversely proportional to the wavelength λλ. Therefore, as the wavelength increases, the energy decreases. Conversely, shorter wavelengths have higher energy.

This principle is fundamental in understanding fluorescence, as the emitted light from a fluorophore typically has a longer wavelength (and thus lower energy) than the absorbed light due to the energy loss that occurs during the excited state.

Fluorescence

  • The emitted light is what we observe as fluorescence. The specific wavelengths of the absorbed and emitted light are characteristic of the particular fluorophore, allowing for the identification and distinction of different fluorophores.


Key Properties of Fluorophores

  • Excitation and Emission Spectra: Each fluorophore has specific wavelengths at which it absorbs (excitation) and emits (emission) light.
  • Quantum Yield: This is the ratio of the number of photons emitted to the number of photons absorbed. A higher quantum yield means more efficient fluorescence.
  • Photostability: This refers to the resistance of a fluorophore to photobleaching (loss of fluorescence due to prolonged exposure to light).
  • Brightness: This is a measure of the intensity of the emitted fluorescence and is determined by the product of the extinction coefficient (how well the fluorophore absorbs light) and the quantum yield.

Applications

Fluorophores are widely used in various biological and medical applications, such as fluorescence microscopy, flow cytometry, and fluorescence resonance energy transfer (FRET), to study biological structures and processes at the molecular and cellular levels.

Key Takeaways

  • Fluorophores will absorb lower wavelength and emit longer wavelength
  • The reaction is super fast, usually you need to simultaneously excite fluorophore.
  • Fluorophores are usually named with the color it emits. (But better to specify the excite light it need)
  • fluorophore spectra is different from one and each other
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