Third Harmonic Generation microscopy as a diagnostic tool for the investigation of microglia BV-2 and breast cancer cells activation

Abstract

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) andTwo 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.

Detection of the T cell activation state using nonlinear optical microscopy

“…THG advantage is that the main signal is generated within intact live cells and tissues, when there is a change in the refractive index or change of the third order susceptibility values, without the need of any staining . Previous studies have shown that lipid bodies are the main intracellular sources for the high THG signals , while others have indicated that polarized THG microscopy can discriminate triacylglycerols from cholesteryl esters in intact mammalian cells .…”

Third‐harmonic generation imaging of breast tissue biopsies

“…This paper introduces backward THG imaging microscopy to image unstained healthy and malignant breast biopsy tissue. Previously THG microscopy has been used to study the resting and activated states of microglial cells present in breast tissue (Gavgiotaki et al ., ). However, to the best of our knowledge, the work in this paper will be the first application of THG imaging on unstained breast tissue biopsies.…”

Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue

“…Using the spectral information cancerous breast tissue can be distinguished from normal and benign tissue, both ex‐vivo and in‐vivo . Studies using SHG/THG microscopy to image breast tissue have also shown very promising results, but were applied on stained and fixated breast tissue , unstained but fixated breast biopsies or breast cell lines . Tu et al applied label‐free multimodal multi‐photon microscopy (including SHG/THG microscopy) to unprocessed breast tissue to visualize the micro‐environment of mammary tumors induced in rats, and to visualize extra‐cellular vesicles in the stroma tissue of a human tumor specimen.…”