Final answer:
The emission line of a laser does not need to exactly match the absorption maximum of a fluorophore but must be within the absorption spectrum for efficient excitation. Synchronous spectroscopy helps detect specific fluorophores like tyrosine and tryptophan, which have different energy shifts between their excitation and emission wavelengths.
Step-by-step explanation:
The emission line of a laser does not have to match exactly the absorption maximum of a fluorophore, but it often falls within the range of the fluorophore's absorbance spectrum in order for efficient excitation to occur. When measuring fluorescence, one sets the wavelength of light irradiating the sample (excitation wavelength) and the wavelength of the light emitted (emission wavelength). The emission wavelength is typically at a lower energy (longer wavelength) than the excitation due to the phenomenon of Stokes shift, where part of the excitation energy is lost as heat before the fluorophore re-emits light. Furthermore, synchronous spectroscopy is sensitive to the energy difference between excitation and emission wavelengths. For-instance, setting a Δλ = 15 nm is used for detecting tyrosine, which fluoresces after a small shift in wavelength, while a Δλ = 60 nm is used for tryptophan, which exhibits a larger shift.
Specifically, tryptophan's emission peak is around 350 nm and has a broader energy change, indicating a higher energy loss before light emission, compared to tyrosine, which has a narrower emission peak around 305 nm and less energy change from absorption to emission. When using a helium-neon laser, the pure wavelength emission shown by the sharp spike in the emission spectra is key for targeted fluorescence excitation.