Third Harmonic Generation Microscopy (THG)

In Third Harmonic Generation Imaging microscopy, the fluorescent dye is omitted and the third harmonic signal is generated from the sample itself. In a homogenous sample, the THG signal from above the focus cancels the signal generated below the focus due to phase matching. Thus, a third harmonic signal is only generated when the focus is close to a gradient in the refractive index. As a result, THG microscopy is particularly well-suited for 3D label-free imaging of transparent specimens where the membrane or other interface is of interest.
Energy level diagram for the SGH mechanism
Figure 1. Energy level diagram for the THG imaging mechanism.

Third Harmonic Generation Bio-Imaging Examples

Zebrafish embryo (6 hours post fertilization) using label free THG at 1140 nm; imaged with InSight® DS+™
Figure 2. Zebrafish embryo (6 hours post fertilization) using label free THG at 1140 nm; imaged with InSight® DS+™ fs laser.
Courtesy of Dr. Nadine Peyrieras, CNRS, Gif sur Yvette, France

Mouse mammary gland, label free image of collagen (SHG) and adipocytes (THG)
Figure 3. Mouse mammary gland, label free image of collagen (SHG, magenta) and adipocytes (THG, yellow), imaged with InSight DS+™.
Courtesy of Dr. Marie Irondelle, Institut Curie/CNRS, Paris, France

3D images from a mouse brain cerebellum extending 1 mm deep into the tissue acquired via 3PF and THG
Figure 4. 3D images from a mouse brain cerebellum extending 1 mm deep into the tissue acquired via 3PF (left) and THG (right) microscopy at 1.3 μm using a Spirit laser with a Spirit-NOPA.
Courtesy of Chris Xu, with permission from SPIE Publications: Wang, et. al., “In vivo three-photon imaging of deep cerebellum,” Proc. SPIE: Multiphoton Microscopy in the Biomedical Sciences XVIII, vol. 10498, 2018.e