Maria Göppert-Mayer proposed in 1930 the use of two photons instead of one to excite fluorescent molecules, which occurred 60 years before the advent of two-photon microscopy. This system uses a tunable pulsed laser that utilizes half the energy of a continuous laser to excite fluorochromes, causing them to produce fluorescence with the same intensity as those emitted under the single-photon system, which uses a continuous-wave laser. The two-photon system does not require a pinhole because the interaction of the two photons with the fluorochromes only occurs in a very limited area of the sample; however, optical sectioning can still be performed for plane-by-plane reconstruction to generate three-dimensional images. Additionally, this system can be used for thick samples, those with low fluorescence emission, or samples containing highly unstable fluorochromes to extend their lifespan. The use of the two-photon microscope is not exclusive to samples containing fluorochromes; its use is also known in clinical pathology for diagnosis through a nonlinear light emission phenomenon known as second harmonic generation.