Fluorophores

Fluorophores are molecules or molecular assemblies that absorb light at a specific excitation wavelength and re-emit light at a longer emission wavelength. They are widely used as reporters in biological research and imaging to visualize cellular structures, processes, and analytes.

Key properties include the excitation and emission spectra, the extinction coefficient, quantum yield, brightness (the product of extinction coefficient and quantum yield), and photostability. The Stokes shift describes the difference between peak excitation and emission wavelengths. Many fluorophores are sensitive to environmental factors such as pH, polarity, and binding state, which can alter brightness or spectrum.

Fluorophores come in several forms, including synthetic organic dyes (for example fluorescein and rhodamine), cyanine and Alexa dyes, fluorescent proteins such as GFP and its derivatives, and inorganic quantum dots. They are commonly attached to antibodies, nucleic acids, or other molecules to serve as labels.

Applications span fluorescence microscopy, confocal and super-resolution imaging, flow cytometry, fluorescence in situ hybridization, and fluorescence-based assays such as ELISAs and DNA microarrays. Fluorescence resonance energy transfer (FRET) enables probing of molecular interactions and distances.

Choosing a fluorophore involves balancing brightness, photostability, spectral separation from other labels, chemical compatibility with labeling chemistry, and potential toxicity. Spectral overlap can lead to bleed-through; proper filters and compensation are often required. Careful optimization is essential for reliable measurements.