Notes on two-photon microscopy

Abstract

Two-photon microscopy allows for the imaging of both fixed and live tissues orders of magnitude deeper than what is possible with standard widefield microscopy techniques and does so at higher resolutions than those conventional techniques. There are numerous structures that were previously unable to be imaged in live specimens until the development of two-photon microscopy due to their depth in the tissue. While confocal microscopy can also image deeper into tissues than conventional widefield techniques, the penetration depth is still insufficient for many biological structures of interest. Before the development of two-photon microscopy, the primary methods for imaging such structures were tissue sectioning and scaling, i.e. making the tissues transparent via soaking in chemical solvents. Both of these methods are incompatible with living samples, and while they can also achieve two-photon-like resolutions, they cannot visualize any dynamics. Two-photon microscopy exploits the nonlinearity of its namesake effect, and by only exciting near the waist of the beam in the imaging plane, is able to achieve millimeter-depth imaging in tissues at higher resolutions.