Considering its non-linear nature, two-photon excitation may generate very different spectral response in samples when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of samples, so that the two-photon fluorescence microscopic images can be properly interpreted. Fluorescence spectra obtained from bulk samples may not provide useful information for microscopy. For instance, due to the relatively small contribution to the total fluorescence intensity, a small number of fluorescent particles in a generally fluorescing specimen may escape detection when the spectrum of the specimen as a whole is obtained. Under two-photon excitation, the background noise can be greatly reduced due to the naturally limited excitation volume of focused laser beam. In addition, signals resulted from second harmonic generation (SHG) may be mixed with low level broad-band background autofluorescence which is commonly found in biological specimen. Therefore, measuring fluorescence spectrum from a micro-focused volume is essential for the proper interpretation of multi-photon fluorescence images.