Fluorescence is a phenomenon where a substance absorbs light and then re-emits it as light of a different color. This process involves a fluorophore absorbing energy from incident light. The re-emitted light always possesses a specific wavelength, which provides information about the substance and its environment. Understanding what determines this emitted wavelength is important for many scientific and practical applications.
Light, Wavelength, and Color
Light is a form of electromagnetic radiation, which travels in waves. The distance between two consecutive peaks of a wave is called its wavelength.
Different wavelengths of light correspond to different colors. The visible light spectrum ranges approximately from 380 nanometers (nm) to 750 nm. Shorter wavelengths (380-450 nm) appear as violet or blue light, while longer wavelengths (620-750 nm) appear as red.
The Mechanism of Fluorescence
Fluorescence begins when a fluorophore absorbs a photon of light, causing an electron to jump from its low-energy “ground state” to a higher-energy “excited state”. The fluorophore then loses some excess energy through vibrations and interactions with its surroundings. Following this energy loss, the electron returns to its ground state, emitting a new photon of light. This emitted photon has less energy than the absorbed photon, meaning it has a longer wavelength.
This difference between the absorbed (excitation) wavelength and the emitted (emission) wavelength is known as the Stokes Shift. For example, a fluorophore might absorb blue light but emit green light, as green light has a longer wavelength. This shift to a longer wavelength is a characteristic of fluorescence.
What Influences Fluorescence Wavelength
The specific wavelength of light emitted by a fluorophore is influenced by several factors. The chemical structure of the fluorophore plays a primary role; molecules with more conjugated double bonds tend to absorb and emit light at longer wavelengths. Electron-donating groups like hydroxyl (-OH) or amino (-NH2) can increase fluorescence intensity and shift the emission wavelength, while electron-withdrawing groups like nitro (-NO2) can decrease or eliminate fluorescence.
The surrounding environment also significantly impacts the emission wavelength. Solvent polarity can affect the fluorophore’s dipole moment, leading to longer emission wavelengths in more polar solvents. Changes in pH can alter the fluorophore’s chemical form, causing shifts in its fluorescence spectrum. Temperature also plays a role, as higher temperatures can increase molecular collisions, leading to more energy loss as heat and shifts in the emission spectrum.
How Fluorescence Wavelength is Used
Understanding fluorescence wavelength is applied across many scientific and technological fields. In biomedical imaging, fluorescent dyes visualize specific cells, tissues, or molecules within biological samples. Fluorescent probes can stain bacteria or highlight structures in living cells for microscopy, providing real-time insights into biological processes.
Fluorescence wavelength is also employed in biosensing for detecting specific substances. Changes in fluorescence wavelength or intensity can signal the presence of pathogens, pollutants, or disease markers. These biosensors detect various analytes, including oxygen, pH levels, or metal ions, by coupling the target’s binding to a change in the fluorophore’s emission. Beyond biology, material science uses fluorescence to characterize new materials or identify defects, while environmental monitoring leverages it to detect contaminants in water or air.