photonics hv Article Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons Juan M. Bueno * , Geovanni Hernández, Martin Skorsetz and Pablo Artal   Citation: Bueno, J.M.; Hernández, G.; Skorsetz, M.; Artal, P. Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons. Photonics 2021, 8, 573. https:// doi.org/10.3390/photonics8120573 Received: 11 November 2021 Accepted: 9 December 2021 Published: 13 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Laboratorio de Óptica, Instituto Universitario de Investigación en Óptica y Nanofísica, Universidad de Murcia, Campus de Espinardo (Ed. 34), 30100 Murcia, Spain; geov.hernandez@ugto.mx (G.H.); martin.skorsetz@gmx.de (M.S.); pablo@um.es (P.A.) * Correspondence: bueno@um.es Abstract: Multiphoton (MP) microscopy is a well-established method for the non-invasive imaging of biological tissues. However, its optical sectioning capabilities are reduced due to specimen-induced aberrations. Both the manipulation of spherical aberration (SA) and the use of axicons have been reported to be useful techniques to bypass this limitation. We propose the combination of SA patterns and variable axicons to further improve the quality of MP microscopy images. This approach provides enhanced images at different depth locations whose quality is better than those corresponding to the use of SA or axicons separately. Thus, the procedure proposed herein facilitates the visualization of details and increases the depth observable at high resolution. Keywords: spherical aberration; axicon; multiphoton microscopy 1. Introduction Multiphoton (MP) microscopy techniques (two-photon excitation fluorescence, TPEF, and second harmonic generation, SHG) combine inherent confocality and minimized tissue damage [1,2]. However, the penetration depth is limited in thick samples mainly due to specimens’ aberrations [3,4]. To overcome this loss of MP effectiveness, different adaptive optics configurations have been used [3–6]. Among all the aberration terms appearing in thick samples, spherical aberration (SA) is the dominant one [3,5–7]. The correction (or minimization) of this SA by using either objective correction collars [8,9] or adaptive optics [3–7,10] has been reported to improve the quality of MP images at deeper locations within the sample. In addition, the manipulation of the SA pattern of the incident beam while performing fast tomographic MP imaging is able to extend the imaging depth [7]. Phase masks [11] and refractive axicons [12–14] have also been reported to increase the depth-of-field in MP imaging microscopy without compromising lateral resolution. A refractive axicon is a conical lens able to transform a Gaussian beam into a non- diffracting Bessel beam [15], which is characterized by both the refractive index and the apex angle of the axicon. These axicons produce an axially elongated focus with a fixed axial length [12,13]. However, when using real-life biological samples, it is often desirable to have Bessel foci of different axial lengths to investigate thick volumes. Different approaches have been reported to adjust this length [14,16]; however, they all require additional optical elements and/or moving parts, which might enlarge the size of the experimental system and complicate its design. To improve the versatility of Bessel beams, computer-generated holograms and vari- able diffractive optics elements have been proposed [17–19]. In particular, spatial light modulators (SLM) provide a flexible and dynamic way of creating variable non-diffracting beams that can be changed in time [19–23]. This has been implemented into MP micro- scopes to obtain higher axial resolution and improved penetration depth [24–26]. Photonics 2021, 8, 573. https://doi.org/10.3390/photonics8120573 https://www.mdpi.com/journal/photonics