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Detection of breast cancer in fresh tissue obtained from surgery is investigated using near-infrared autofluorescence imaging under laser excitation at 532 and 632.8 nm. The differences in intensity between the three main components of breast tissue (cancer, fibrous, and adipose) are estimated and compared to those obtained from cross-polarized light scattering images recorded under polarized illumination at 700 nm. The optical spectroscopic images for each tissue sample were subsequently compared with the histopathology slides. The experimental results indicate that the intensity of the near-infrared emission is considerably different in breast cancer compared to that of the adjacent nonneoplastic tissues (adipose and fibrous tissue). The experimental results suggest that 632.8-nm excitation offers key advantages compared to 532 nm excitation.