Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration

Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.