Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons

Juan M Bueno,Martin Skorsetz,Pablo Artal,Geovanni Hernández

doi:10.3390/photonics8120573

Juan M Bueno, Martin Skorsetz + Show 2 more

Open Access

https://doi.org/10.3390/photonics8120573

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Journal: Photonics	Publication Date: Dec 13, 2021
Citations: 1	License type: CC BY 4.0

Affiliation: University of Murcia

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.

Highlights

Multiphoton (MP) microscopy techniques combine inherent confocality and minimized tissue damage [1,2]
The spatial light modulators (SLM) was programmed to generate controlled amounts of spherical aberration (SA) and variable axicons in order to increase the quality of images at different depth locations independently of the specimen-induced aberrations
Different authors have showed the benefit of using SA compensation in MP microscopy through both static and dynamic approaches [5,6,7,8,9,10]

Summary

Introduction

Multiphoton (MP) microscopy techniques (two-photon excitation fluorescence, TPEF, and second harmonic generation, SHG) combine inherent confocality and minimized tissue damage [1,2]. 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 [3,4,5,6]. The correction (or minimization) of this SA by using either objective correction collars [8,9] or adaptive optics [3,4,5,6,7,10] has been reported to improve the quality of MP images at deeper locations within the sample. Phase masks [11] and refractive axicons [12,13,14]

Methods

Results

Conclusion