Multiphoton imaging of thick samples combining axicons and spherical aberration

Summary form only given. 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.The experimental system is based on a custom-build research multiphoton microscope previously described [4]. A spatial light modulator (SLM) was introduced in the illumination path to manipulate the wavefront of the illumination beam before entering the microscope [5]. This was used to generate the corresponding SA patterns for different amount of aberration and to produce the AXs by means of 0-2π phase masks (that originate Besselshaped beams in the Fourier-domain). Individual or combined SA patterns and amounts of AX were systematically generated during image recording. Results show that specific combinations provide improved images, better that those obtained when using each profile separately. An example is shown in Fig. 1. The optimum aberration maps to be induced were investigated for different specimens, nonlinear signals and depth locations. The improvement was more noticeable at deeper locations within the samples.