Third Harmonic Generation microscopy distinguishes malignant cell grade in human breast tissue biopsies.

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.