Photophysics · BEGINNER LESSON
Fluorescence
Fluorescence is a two-step light process in which a molecule absorbs higher-energy light and then emits lower-energy light. It is measured by the light a sample gives off after excitation, often reported as emission intensity and emission wavelength.
What fluorescence is
Fluorescence is a physico-chemical process in which a fluorophore, meaning a molecule that can fluoresce, absorbs light and then releases part of that energy as light again. The absorbed light is the excitation light, and the outgoing light is the emission. The emission has lower energy and therefore usually a longer wavelength than the excitation light. [Source: 0, 1, 2]
[1] Fluorescence as a means of colour signal enhancement - PMC [2] Recommendations and Guidelines for Standardization of ... - NIST [3] Small-molecule fluorescent probes and their design - PMC
How it happens
A photon is absorbed and promotes an electron to an excited state, which is a higher-energy electronic state. Before emission, some energy can be lost by internal conversion, which is non-light energy loss, or by intersystem crossing, which changes electron spin. Fluorescence is the radiative return: the molecule emits a photon and falls back toward the ground state. Because some energy is lost first, the emitted light is red-shifted relative to the absorbed light; this difference is the Stokes shift. [Source: 1, 2]
[2] Recommendations and Guidelines for Standardization of ... - NIST [3] Small-molecule fluorescent probes and their design - PMC
Why it is useful
Fluorescence is useful because the excitation and emission wavelengths are different, so emitted light can be measured with less interference from the incoming light. This makes fluorescence methods highly sensitive, sometimes down to single-molecule detection, and often fast and non-destructive. Fluorescence spectroscopy is described as background-free because very little excitation light reaches the detector. [Source: 1, 2]
[2] Recommendations and Guidelines for Standardization of ... - NIST [3] Small-molecule fluorescent probes and their design - PMC
Limits and caveats
Fluorescence does not add net brightness in the physical sense; converting absorbed light always loses some energy. Apparent brightness can still increase relative to a dark background because emission occurs in many directions and at wavelengths different from the excitation light. Measurements can be distorted by high sample concentration, reabsorption, photobleaching, photodegradation, and temperature-dependent quenching. Fluorescence can also be confused with bioluminescence, phosphorescence, or simple reflection unless the mechanism is checked. [Source: 0, 1]
[1] Fluorescence as a means of colour signal enhancement - PMC [2] Recommendations and Guidelines for Standardization of ... - NIST
SEE THE IDEA
How fluorescence produces measurable emitted light
Shorter-wavelength light is absorbed by the fluorophore.
The molecule holds extra energy after absorption.
A photon is released at longer wavelength and lower energy.
Detectors record emitted light with reduced interference from excitation.
Excitation light is absorbed first, then the excited molecule relaxes and emits lower-energy light that can be measured.
A simple absorption-to-emission pathway with a wavelength shift.
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Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.