Measuring a Fluorescence Emission Spectrum

An excitation spectrum is measured by fixing a convenient wavelength at which to measure fluorescence and then following fluorescence intensity as the wavelength of illumination is varied. An excitation spectrum may be more easily measured than an absorption spectrum, as fluorescence measurements are inherently more sensitive than absorbance measurements, and so such measurements are useful if amounts of sample are small. If fluorescence is observed to decrease with time, various precautions can be instigated. With mild cases of photobleaching, it may be sufficient to decrease the time of illumination, by closing the excitation shutter when readings are not being made, and to stir the solution periodically, since only molecules in the light beam are bleached. The position of the peaks in a fluorescence emission spectrum can be determined as described for absorbance peaks — manually (by dropping a vertical on to the abscissa), on a visual display (using a cursor) or by computation (by determining the first derivative).