Second Harmonic (SHG) and Third Harmonic Generation (THG) Imaging

We can image SHG using the Zeiss 780 and Zeiss 510 multiphoton microscopy systems. The THG & SHG can be imaged using the Zeiss 780 multiphoton system.Third Harmonic Generation (THG) and second harmonic generation (SHG) microscopy is a non-fluorescent multi-photon technique that combines the advantages of label-free imaging.

SHG is based on a nonlinear optical effect known as second harmonics generation (SHG). SHG/THG has been established as a viable microscopy imaging contrast mechanism for visualization of cell and tissue structure and function. A SHG microscope obtains contrasts from variations in a specimen’s ability to generate SHG light (say 450 nm) from the incident light (say 900 nm) while a conventional optical microscope obtains its contrast by detecting variations in optical density and path length of the specimen. SHG requires intense laser light passing through a material with a noncentrosymmetric molecular structure. Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarized, and assembled into fairly ordered, large noncentrosymmetric structures. Biological materials such as collagen, microtubules, and muscle myosin can produce SHG signals. The SHG signal is propagated mostly in the forward direction but backward direction signals are also recorded for some of the specimens.

SHG is directional depending on the distribution and orientation of the nonlinear dipoles (b) whereas in general, fluorescence is isotropically emitted (c).

THG is a process in which the energy of three incoming photons (1200 nm) is combined to generate one out going photon (400 nm) with a wavelength of exactly one third of the excitation beam. All the photon's energy is converted. None is deposited in the specimen, as during a fluorescence excitation and emission cycle. THG signals were sufficiently strong for informative imaging of blood vessel walls and blood cells, muscle fiber sarcomeres, nerve fibers and nuclei of some cell types.

What is the difference between THG and SHG microscopy? In SHG, the energy of two photons is combined to create one with half the wavelength of the excitation beam. THG, on the other hand, is induced by several mechanisms. One is an interface in the specimen, such as a refractive index mismatch. As a result, structures are highlighted that also would come up in phase contrast or interference contrast microscopy. THG, however, is induced only at the focal point of the excitation laser, enabling 3D-imaging of tissues. Another THG mechanism is resonance enhancement. When a chromatic material absorbs the wavelength of the produced THG signal, contrary to intuition, this causes an increase in signal intensity by resonance processes.