Autofluorescents

Autofluorescents, or autofluorescence, is the phenomenon by which biological tissues and certain molecules emit light after excitation by light, without any external fluorescent dyes or additives. The emission arises from endogenous fluorophores such as reduced nicotinamide adenine dinucleotide (NADH) and its phosphate form NADPH, flavin adenine dinucleotide (FAD), lipofuscin, and structural proteins like collagen and elastin. Other contributors include porphyrins, heme metabolites, and, in pigmented tissues, melanin. The spectral profile depends on the tissue and the excitation wavelength; common emissions range from blue to green, with weaker signals in the red and infrared for some fluorophores.

Mechanism and properties: When illuminated, autofluorophores absorb photons and reach excited electronic states, then emit photons as they relax. The intensity and lifetime of autofluorescence depend on metabolic state, tissue age, and microenvironment. Emission lifetimes are typically in the nanosecond range; these properties can be exploited to distinguish autofluorescence from exogenous fluorophores in time-resolved imaging.

Applications: Autofluorescence imaging is used in biomedical research and clinical diagnostics. In ophthalmology, fundus autofluorescence highlights lipofuscin in the retinal pigment epithelium. Autofluorescence endoscopy and confocal imaging aid detection of neoplastic or inflammatory changes in the gastrointestinal tract, oral mucosa, and lungs. It is also used in histology to assess tissue composition and in dermatology for evaluating skin lesions. Autofluorescence can complicate fluorescence imaging when endogenous signals overlap with administered fluorophores.

Limitations and techniques: Overlap between autofluorescence and targeted fluorophores can reduce contrast. Strategies include selecting excitation wavelengths that minimize autofluorescence, applying spectral unmixing to separate signals, using time-resolved (lifetime) fluorescence to distinguish fluorophores, and employing near-infrared dyes that shift emission away from strong endogenous signals. In some cases, chemical bleaching or tissue processing reduces autofluorescence but can alter tissue properties.