Which wavelengths are associated with autofluorescence from flavin coenzymes and NADH, respectively?

• A. 500 nm and 600 nm; 350 nm and 420 nm
• B. 450 nm and 515 nm; 340 nm and 460 nm
• C. 600 nm and 700 nm; 500 nm and 600 nm
• D. 350 nm and 420 nm; 300 nm and 360 nm

Answer: (D)

Autofluorescence from these endogenous fluorophores follows distinct spectral regions. Flavin coenzymes (FAD/FMN) are best excited with blue light around 450 nm and emit in the green, roughly 515–520 nm. NADH, on the other hand, is excited most effectively in the near-UV around 340–360 nm and emits near 460 nm. So the correct pairing reflects flavin autofluorescence at about 450 nm excitation and ~515–520 nm emission, and NADH autofluorescence at about 340–360 nm excitation with ~460 nm emission. This helps distinguish true immunostaining from background autofluorescence during analysis.