Microscope Image Processing

using appropriate narrow-band excitation filters such that the autofluorescence

excitation wavelength lies outside the excitation spectrum of the target fluoro-

phore. If this is not the case, an additional correction factor can be used. This factor

is computed by recording images of an unstained sample at both the autofluores-

cence and target fluorophore excitation wavelengths. The ratio R of the two images

at the desired emission wavelength of the target fluorophore is then used to

compute the specific fluorescence as [27]

SF ¼ TF  R  AFðÞ (12:16)

where SF is the specific fluorescence, TF is the total fluorescence, and AF is the

autofluorescence. An alternative method based on time-delayed fluorescence

imaging can also be used to eliminate autofluorescence