Two-photon microscopy for microrobotics: Visualization of micro-agents below fixed tissue
Juan J Huaroto1, Luigi Capuano1, Mert Kaya1,2
1Surgical Robotics Laboratory, Department of Biomechanical Engineering, University of Twente, Enschede, The Netherlands.
Abstract:
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.
Insights
Two-photon microscopy enables deeper visualization of micro-agents within biological tissues, overcoming optical microscopy limitations. This advancement is crucial for microrobotics research and potential in vivo applications.
Area of Science:
- Biomedical Engineering
- Optical Imaging
- Robotics
Background:
- Optical microscopy offers high spatio-temporal resolution but is limited to ~100 μm penetration depth in biological tissues.
- Visualizing microrobotic agents (micro-agents) below tissue surfaces is a significant challenge in current microrobotics research.
- Two-photon microscopy (TPM) provides deeper tissue penetration (>500 μm) and sub-micron resolution, with applications in live tissue imaging and direct laser writing fabrication.
Purpose of the Study:
- To introduce and evaluate two-photon microscopy as a novel imaging technique for visualizing micro-agents beneath biological tissue.
- To demonstrate the capability of TPM for deep-tissue imaging of micro-agents in microrobotics applications.
- To explore the potential of TPM for advancing in vivo microrobotics visualization.
Main Methods:
- Fabrication of two types of micro-agents: exogenous fluorophore-stained electrospun fibers and autofluorescent resin-printed bio-inspired structures using direct laser writing (DLW).
- Experimental setup for three-dimensional reconstruction of micro-agents and qualitative study of laser-tissue interaction.
- Utilization of second-harmonic generation for simultaneous visualization of micro-agents and surrounding tissue.
Main Results:
- Demonstrated two-photon microscopy imaging of micro-agents below formalin-fixed tissue with a maximum penetration depth of 800 μm.
- Achieved continuous imaging of magnetic electrospun fibers at a rate of one frame per second within a 135 × 135 μm² field of view.
- Successfully visualized micro-agents alongside tissue structures, confirming TPM's utility.
Conclusions:
- Two-photon microscopy serves as a viable alternative imaging modality for microrobotics, enabling visualization of micro-agents beyond the limitations of conventional optical microscopy.
- The study validates TPM for micro-agent imaging under in vitro and ex ovo conditions, paving the way for deeper tissue exploration.
- Integrating TPM with microrobotics holds significant potential for future in vivo applications, enhancing the capabilities of micro-robotic systems in biological environments.
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