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.