Non-linear optical imaging techniques have been used to greatly enhance our understanding of issues with high biological significance and promise a strong impact on early and accurate detection of various diseases. In our current work, we employ Third Harmonic Generation (THG) and Second Harmonic Generation (SHG) imaging modalities for diagnosis of cancerous tissue limits and for obtaining additional quantitative information to supplement standard histopathology procedures. For this reason, unstained histological slides of breast tumor biopsies were irradiated. Cancerous and normal tissue areas could be distinguished based on cell morphology, size, and density. THG imaging reflects lipid bodies (LBs) in the intracellular compartments, cellular and nuclear membranes, while SHG shows collagen distribution in the tissue. By using THG microscopy, it is feasible to concentrate on specific cells in tissue and collect quantitative information. Our initial results showed that quantification of THG signaling can depict differences between healthy and cancerous tissue. This is a very promising observation, since the non-linear technique described here, allows fast, non-invasive, label-free imaging that does not require the use of fluorescent dyes or other preparations of tissues in order to detect specific structures and features. The significance of this work has a clinical potential since it can monitor quantitative changes in cellular behavior in healthy and pathological human tissues.
Third Harmonic Generation (THG) imaging was applied to mouse resting and activated T-cells.
Quantification of THG signal, which corresponded to lipid droplets, could distinguish activated Tcells,
allowing follow-up of immune response development.
Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field. Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the activation state of different cell types by using the THG imaging microscopy as a diagnostic tool.
BV-2 microglia cell line was used as a representative biological model enabling the study of resting and activated state of the cells linked to various pathological conditions. Third Harmonic Generation (THG) and Two Photon Excitation Fluorescence (TPEF) measurements were simultaneously collected from stained breast cancer cells, by employing a single homemade experimental apparatus and it was shown that high THG signals mostly arise from lipid bodies. Continuously, BV-2 microglia cells were examined with or without activation by lipopolysaccharide (LPS) in order to discriminate between control and activated cells based on the quantification of THG signals. Statistically quantification was accomplished in both mean area and mean intensity values of THG. The values for mean total area and mean THG intensity values have been increased in activated versus the non-activated cells. Similar studies of quantification are underway in breast cancer cells for the exact discrimination on different cell lines. Furthermore, laser polarization dependence of SHG and THG signal in unstained biological samples is investigated.