Multiphoton imaging of thick samples combining axicons and spherical aberration

Abstract

The presence of specimen-induced aberrations within a tissue limits the performance of multiphoton microscopy, especially at deeper layers. Optical sectioning capabilities and maximum penetration depth are noticeable affected, and both contrast and resolution of the recorded images are reduced. Adaptive optics has been used to correct or minimize these aberrations. However plane-by-plane measurements are challenging and wavefront sensors are usually avoided [1]. Although spherical aberration (SA) is the dominant term at deeper layers within a sample, each depth location presents a value. It has been recently reported that a “unique optimum” SA value could be used to increase the depth-of-focus across the sample in multiphoton imaging [2]. In addition, Bessel beams generated by means of axicons (AXs) have shown a similar behaviour [3]. Here, we propose a wavefront sensor-less approach based on combining AXs and SA to further improve the quality of multiphoton images obtained in thick samples.