Multiphoton microscopy is an LSM utilizing a high-power, pulsed, near-infrared laser. The most frequently used application of this method – two-photon microscopy – relies on the simultaneous absorption of two photons by fluorescent dyes. The likelihood of this event taking place decreases rapidly with an increase in distance from the focal point. When applied in LSM, fluorescent dyes are exited in and light is emitted from a well-defined optical section. Moreover, imaging of biological tissues up to 1 mm is possible thanks to reduced scattering by the utilized infrared excitation light. Therefore, two-photon LSM is the method of choice when acquiring optical sections from turbid, scattering, thick samples and is often used to acquire thick tissue sections, organoids, spheroids or utilized during intravital imaging. However, depending on the sample, the energy of two excitation photons may be combined to create a photon with twice the energy in a process referred to as second-harmonic generation (SHG). Collagen may be visualized via SHG in a label-free fashion. A further case involves the interaction of three photons within the focal point, which is utilized in third harmonic generation (THG) microscopy. It may be used to visualize myelin label-free.