Third harmonic generation microscopy of cells and tissue organization

Abstract

The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra-and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in threedimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice.

Engineering and physical sciences in oncology: challenges and opportunities

“…SHG and THG provide minimal photodamage, enhanced optical penetration and quantitative information on collagen remodelling 165 . Combining SHG and THG with fluorescence microscopy provides insight into tumour tissue organization, cell–matrix interactions, blood flow dynamics, and the dissemination of microvesicles 65,166 . More specifically, the combination of all three imaging platforms enabled tracking of melanoma invasion into the dermis ~600 µm deep, along with reconstruction of the tumour cell invasion mode and tissue tracks to determine the invasion routes and outcome 65 .…”

Through-focus or volumetric type of optical imaging methods: a review

“…LSM is mainly used to study biological tissues. Because of the opacity of such tissues, threedimensional imaging is typically limited to a depth of 500 μm to 1000 μm (72, 73). However, in some studies LSM appears to outperform confocal microscopy (10).…”

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

“…1B) is a non-linear scattering process that originates from the polarization properties of the excited volume and from variations of the refraction index in that volume, such as in water-lipid and waterprotein interfaces found, for instance, in cellular membranes and extracellular matrix structures (Weigelin et al, 2016). THG is mostly used in tumor biology to visualize ECM structures (Alexander et al, 2013) but has also been used to image lipid bodies in Drosophila melanogaster embryos (Débarre et al, 2006).…”