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.

Magnetic Soft Helical Manipulators with Local Dipole Interactions for Flexibility and Forces.

Magnetic continuum manipulators (MCMs) are a class of continuum robots that can be actuated without direct contact by an external magnetic field. MCMs operating in confined workspaces, such as those targeting medical applications, require flexible magnetic structures that contain combinations of magnetic components and polymers to navigate long and tortuous paths. In cylindrical MCM designs, a significant trade-off exists between magnetic moment and bending flexibility as the ratio between length and diameter decreases. In this study, we propose a new MCM design framework that enables increasing diameter without compromising on flexibility and magnetic moment. Magnetic soft composite helices constitute bending regions of the MCM and are separated by permanent ring magnets. Local dipole interactions between the permanent magnets can reduce bending stiffness, depending on their size and spacing. For the particular segment geometry presented herein, the local dipole interactions result in a 31% increase in angular deflection of composite helices inside an external magnetic field, compared to helices without local interactions. In addition, we demonstrate fabrication, maneuverability, and example applications of a multisegment MCM in a phantom of the abdominal aorta, such as passing contrast dye and guidewires.

Embedded 3D printing of dilute particle suspensions into dense complex tissue fibers using shear thinning xanthan baths.

In order to fabricate functional organoids and microtissues, a high cell density is generally required. As such, the placement of cell suspensions in molds or microwells to allow for cell concentration by sedimentation is the current standard for the production of organoids and microtissues. Even though molds offer some level of control over the shape of the resulting microtissue, this control is limited as microtissues tend to compact towards a sphere after sedimentation of the cells. 3D bioprinting on the other hand offers complete control over the shape of the resulting structure. Even though the printing of dense cell suspensions in the ink has been reported, extruding dense cellular suspensions is challenging and generally results in high shear stresses on the cells and a poor shape fidelity of the print. As such, additional materials such as hydrogels are added in the bioink to limit shear stresses, and to improve shape fidelity and resolution. The maximum cell concentration that can be incorporated in a hydrogel-based ink before the ink's rheological properties are compromised, is significantly lower than the concentration in a tissue equivalent. Additionally, the hydrogel components often interfere with cellular self-assembly processes. To circumvent these limitations, we report a simple and inexpensive xanthan bath based embedded printing method to 3D print dense functional linear tissues using dilute particle suspensions consisting of cells, spheroids, hydrogel beads, or combinations thereof. Using this method, we demonstrated the self-organization of functional cardiac tissue fibers with a layer of epicardial cells surrounding a body of cardiomyocytes.

Visualization of micro-agents and surroundings by real-time multicolor fluorescence microscopy.

Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 [Formula: see text] 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.

SonoTweezer: An Acoustically Powered End-Effector for Underwater Micromanipulation.

Recent advances in contactless micromanipulation strategies have revolutionized prospects of robotic manipulators as next-generation tools for minimally invasive surgeries. In particular, acoustically powered phased arrays offer dexterous means of manipulation both in air and water. Inspired by these phased arrays, we present SonoTweezer: a compact, low-power, and lightweight array of immersible ultrasonic transducers capable of trapping and manipulation of sub-mm sized agents underwater. Based on a parametric investigation with numerical pressure field simulations, we design and create a six-transducer configuration, which is small compared to other reported multi-transducer arrays (16-256 elements). Despite the small size of array, SonoTweezer can reach pressure magnitudes of 300 kPa at a low supply voltage of 25 V to the transducers, which is in the same order of absolute pressure as multi-transducer arrays. Subsequently, we exploit the compactness of our array as an end-effector tool for a robotic manipulator to demonstrate long-range actuation of sub-millimeter agents over a hundred times the agent's body length. Furthermore, a phase-modulation over its individual transducers allows our array to locally maneuver its target agents at sub-mm steps. The ability to manipulate agents underwater makes SonoTweezer suitable for clinical applications considering water's similarity to biological media, e.g., vitreous humor and blood plasma. Finally, we show trapping and manipulation of micro-agents under medical ultrasound (US) imaging modality. This application of our actuation strategy combines the usage of US waves for both imaging and micromanipulation.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy.

Multicolor fluorescence microscopy is a powerful technique to fully visualize many biological phenomena by acquiring images from different spectrum channels. This study expands the scope of multicolor fluorescence microscopy by serial imaging of polystyrene micro-beads as surrogates for drug carriers, cancer spheroids formed using HeLa cells, and microfluidic channels. Three fluorophores with different spectral characteristics are utilized to perform multicolor microscopy. According to the spectrum analysis of the fluorophores, a multicolor widefield fluorescence microscope is developed. Spectral crosstalk is corrected by exciting the fluorophores in a round-robin manner and synchronous emitted light collection. To report the performance of the multicolor microscopy, a simplified 3D tumor model is created by placing beads and spheroids inside a channel filled with the cell culture medium is imaged at varying exposure times. As a representative case and a method for bio-hybrid drug carrier fabrication, a spheroid surface is coated with beads in a channel utilizing electrostatic forces under the guidance of multicolor microscopy. Our experiments show that multicolor fluorescence microscopy enables crosstalk-free and spectrally-different individual image acquisition of beads, spheroids, and channels with the minimum exposure time of 5.5 ms. The imaging technique has the potential to monitor drug carrier transportation to cancer cells in real-time.