MPM is also referred to as two-photon excitation microscopy, two-photon laser scanning microscopy, or nonlinear excitation microscopy and is regarded as an alternative to confocal microscopy.

Fluorescence microscopy is a technique that employs fluorescent tagging to illuminate specific parts of a biological sample rather than using an external light source. The fluorescent tags are then “activated” using electromagnetic radiation, illuminating thick specimens like organs and proteins. This allows image detection and magnification to occur, ultimately revealing structure and function.

However, while it is a powerful technique, fluorescent imaging has drawbacks. Background fluorescent light can negatively affect the image when imaging relatively thick samples. Suppose you only want to observe something on the surface of a thick specimen. In that case, the background fluorescent light from the thickness of the material often drowns out the fluorescent light from the material’s surface.

Confocal microscopy is notorious for causing photobleaching and photodamage throughout the specimen. It collects the fluorescence signal only from the focal plane, which causes the sample to absorb the remaining excitation light, creating significant problems in live samples.

Multiphoton microscopy solves this problem by using two photons as the excitation light, rather than just one photon, and by eliminating the contamination of the fluorescence signal by the excitation light. The photons used in MPM also have longer wavelengths, which have lower energy and penetrate further into the sample. This results in high-resolution, in vivo, 3D images of living cells and biological tissue, and reduces photobleaching and photo-toxicity.

MPM has applications in several fields, from biology, physiology, embryology, neurobiology, neuroscience, and cancer research to tissue engineering and dermatology.