MULTIPHOTON MICROSCOPY

Nowadays, optical microscopy has evolved into three‐dimensional (3‐D) (x‐y‐z) and four‐dimensional (4‐D) (x‐y‐z‐t) analyses allowing researchers to probe even deeper into the intricate mechanisms of living systems. Within this scenario, two‐photon and multiphoton excitation (TPE, MPE) microscopy are probably the most relevant advancement in fluorescence optical microscopy since the introduction of confocal imaging in the 1980s. MPE microscopy has a 3‐D intrinsic ability coupled with almost five other interesting capabilities, namely: (1) MPE greatly reduces photo‐interactions and allows imaging of living specimens on long time periods; (2) MPE operates in a high‐sensitivity background‐free acquisition scheme; (3) MPE microscopy can image turbid and thick specimens down to a depth of a few hundred micrometers; (4) MPE allows simultaneous excitation of different fluorescent molecules reducing colocalization errors; and (5) MPE can be used to prime photochemical reactions within a subfemtoliter volume inside solutions, cells, and tissues. Besides, MPE fluorescence microscopy is not only revolutionary in its ability to provide the above‐mentioned features, together with other practical advantages, but also in its elegance and effectiveness of application of quantum physics. The MPE story, more specifically TPE, dates back to 1931, and its roots are in the theory originally developed by Maria Göppert‐Mayer that brought, passing through the scanning nonlinear microscope developed by Colin Sheppard in Oxford at the end of the 1970s, to the revolutionary and successful results on biological samples obtained at the Watt Webb laboratories by Winfried Denk and colleagues at the beginning of the 1990s. MPE can be considered a young microscopical technique. In general, it is more convenient to consider TPE, for the sake of simplicity.