Abstract: The present invention provides methods and systems for evaluating biological materials for the diagnosis of disease, such as gland abnormalities, and the cancerous and precancerous conditions. Nonlinear optical microscopy techniques, such as MP microscopy and harmonic generation microscopy, are used to generate high resolution, three dimensional images of a test tissue, such as a biopsy tissue sample and tissue in whole organisms, that are analyzed, optionally in combination, to detect, identify and characterize tumor-associated collagen signatures. The presence, abundance and extent of histological features and structural motifs comprising tumor-associated collagen signatures may be directly and accurately correlated with the onset and progression of cancer, such as breast cancer. The present methods are capable of providing an accurate and selective diagnosis of cancer, and provide diagnostic information complementary to conventional diagnostic methods.