A simple introduction to multiphoton microscopy

Summary Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode‐locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two‐photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two‐photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two‐photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three‐photon excitation and second harmonic generation imaging.