Two-photon microscopy for microrobotics: Visualization of micro-agents below fixed tissue.

Optical microscopy is frequently used to visualize microrobotic agents (i.e., micro-agents) and physical surroundings with a relatively high spatio-temporal resolution. However, the limited penetration depth of optical microscopy techniques used in microrobotics (in the order of 100 μm) reduces the capability of visualizing micro-agents below biological tissue. Two-photon microscopy is a technique that exploits the principle of two-photon absorption, permitting live tissue imaging with sub-micron resolution and optical penetration depths (over 500 μm). The two-photon absorption principle has been widely applied to fabricate sub-millimeter scale components via direct laser writing (DLW). Yet, its use as an imaging tool for microrobotics remains unexplored in the state-of-the-art. This study introduces and reports on two-photon microscopy as an alternative technique for visualizing micro-agents below biological tissue. In order to validate two-photon image acquisition for microrobotics, two-type micro-agents are fabricated and employed: (1) electrospun fibers stained with an exogenous fluorophore and (2) bio-inspired structure printed with autofluorescent resin via DLW. The experiments are devised and conducted to obtain three-dimensional reconstructions of both micro-agents, perform a qualitative study of laser-tissue interaction, and visualize micro-agents along with tissue using second-harmonic generation. We experimentally demonstrate two-photon microscopy of micro-agents below formalin-fixed tissue with a maximum penetration depth of 800 μm and continuous imaging of magnetic electrospun fibers with one frame per second acquisition rate (in a field of view of 135 × 135 μm2). Our results show that two-photon microscopy can be an alternative imaging technique for microrobotics by enabling visualization of micro-agents under in vitro and ex ovo conditions. Furthermore, bridging the gap between two-photon microscopy and the microrobotics field has the potential to facilitate in vivo visualization of micro-agents.

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Copyright: © 2023 Huaroto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Déclaration de conflit d'intérêts

The authors have declared that no competing interests exist.