Surgical Robotics Laboratory, Department of Biomechanical Engineering, University of Twente, Enschede, The Netherlands.
Surgical Robotics Laboratory, Department of Biomedical Engineering, University Medical Centre Groningen and University of Groningen, Groningen, The Netherlands.
PLoS One. 2023 Aug 10;18(8):e0289725. doi: 10.1371/journal.pone.0289725. eCollection 2023.
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.
光学显微镜常用于以相对较高的时空分辨率可视化微机器人（即微机器人）和物理环境。然而，微机器人中使用的光学显微镜技术的有限穿透深度（在 100μm 左右）降低了在生物组织下可视化微机器人的能力。双光子显微镜是一种利用双光子吸收原理的技术，允许以亚微米分辨率和光学穿透深度（超过 500μm）进行活组织成像。双光子吸收原理已广泛应用于通过直接激光写入（DLW）制造亚毫米级组件。然而，它作为微机器人的成像工具在最新技术中仍未得到探索。本研究介绍并报告了双光子显微镜作为在生物组织下可视化微机器人的替代技术。为了验证双光子成像在微机器人中的应用，制造并使用了两种类型的微机器人：（1）用外源性荧光染料染色的电纺纤维和（2）通过 DLW 打印的具有自发荧光树脂的仿生结构。设计并进行了实验以获得两种微机器人的三维重建，对激光与组织的相互作用进行定性研究，并使用二次谐波产生来可视化微机器人和组织。我们通过实验证明了在福尔马林固定组织下具有 800μm 最大穿透深度的双光子显微镜，以及以每秒一帧的采集速度连续成像磁性电纺纤维（视场为 135×135μm2）。我们的结果表明，双光子显微镜可以通过在体外和鸡胚条件下实现微机器人的可视化，成为微机器人的替代成像技术。此外，弥合双光子显微镜和微机器人领域之间的差距有潜力促进微机器人在体内的可视化。
