A microscope-based HSI system (for more details, see Hsieh et al.13) was used to produce the HSI data of thin, frozen sections cut from excised oral cancer specimens. Briefly, a regular inverted fluorescence microscope was connected to a custom-designed and manufactured embedded relay-lens system capable of decomposing the two-dimensional (2-D) image of a thin-sliced object, one line at a time, into continuous spectra. By scanning through the entire 2-D object space, the relay-lens system produced a data cube with two dimensions in the spatial domain and one in the spectral domain (thus, x by y by ).8,13 The embedded relay-lens system consisted of a moving relay-lens set with a fixed slit of 30 μm width and a hyperspectrometer, spanning a spectral range of 400 to 1000 nm, to convert the slit image into continuous spectra. The slit image was relayed from the sliced object placed on the object platform of the microscope, which was lit by a Xenon light source filtered into two wavelength ranges, 330 to 385 nm and 470 to 490 nm, for excitation. Inside the embedded relay-lens system, a relay-lens was mounted on a stepping motor-controlled platform that could move the relay-lens in 10-μm steps to cover the entire field of view of the slice sample. On the other side of the embedded relay-lens system, an electron multiplying CCD (EMCCD) was mounted to take a snapshot of the HSI data projected from the image slit. The image resolution produced by the EMCCD was with 10-μm pixel size. As a result, the devised microscope-based HSI system generated a three-dimensional data cube with an in-plane resolution of in the spatial domain and 2.8 nm in the spectral domain. Finally, since a objective was used in the fluorescence microscope, this resulted in a spatial resolution of for this study.