KEYWORDS: In vivo imaging, Tissues, Windows, Animal model studies, Nonlinear optics, Microlens, Optical microscopy, Histopathology, Tissue optics, Signal generators
Tissue histopathology, reliant on costly and time-consuming hematoxylin and eosin (H&E) staining of thin tissue slices, faces limitations. Label-free non-linear optical microscopy in vivo presents a solution, allowing work on fresh samples. Implantable microstructures prove effective for systematic longitudinal in vivo studies of immunological responses to biomaterials using label-free non-linear optical microscopy. Employing two-photon laser polymerization, we implanted a matrix of 3D lattices in the chorioallantoic membrane of chicken embryos, establishing a 3D reference frame for cell counting. H&E analysis is compared to label-free in vivo non-linear excitation imaging for cell quantification and identifying granulocytes, collagen, and microvessels. Preliminary results in higher animal models demonstrate the transformative potential of this approach, offering an alternative to conventional histopathology for validating biomaterials in in vivo longitudinal studies.
Deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. In this framework we propose a miniaturized in-vivo imaging window composed of high dioptric power microlenses coupled to micro-scaffold, which were fabricated by two-photon polymerization (2PP) in the biocompatible photoresist SZ2080. We adopt a single-irradiation strategy for the fabrication of the whole structure: We first irradiate the micro-scaffold on the bottom side of the chip and afterwards the microlenses on the top. For the microlenses we adopted a hybrid approach by combining the 2PP of the micro lenses surface with a subsequent UV crosslinking of the inner volume. We explored different lenses profiles (plano-convex and parabolic) with variable parameters like diameter and focal lengths, to optimize the imaging characteristics. We envisage that these imaging windows will open the way to direct and continuous optical inspection of biological processes in vivo.
In the context of biomaterials, small-molecules and drugs testing, intravital microscopy allows to quantify in-vivo the immune reaction, reducing the number of laboratory animals required to statistically validate the product. However, fluorescence microscopy is affected by limited tissue penetration due to light scattering and by optical aberrations, induced on focused beams, by the animal tissue surrounding the implant. In this framework, we developed a system of microlenses coupled to microscaffolds, both incorporated in a miniaturized imaging window. The chip is designed to act as an in-situ microscope objective with the aim to overcome the restrictions of in-vivo imaging (i.e. spherical aberrations) and to allow multiple biological observations in the same animal (by including fluorescent beacons). The device is fabricated by two-photon polymerizing a biocompatible photoresist called SZ2080. The microlenses are manufactured by the concentric polar scanning of the laser beam to realize their outer surface, followed by the UV bulk polymerization of their inner SZ2080. We preliminarily characterized the imaging capabilities of our implantable system on live cells cultured on flat substrates and 3D microscaffolds by coupling it to low magnification objectives. The microlenses optical quality is sufficient to induce non-linear excitation and collect two-photon excitation images with the same level of laser intensity and signal-to-noise ratio. Remarkably, they allow to efficiently excite the fluorescence of labelled human fibroblasts collecting high resolution magnified images. These results will open the way to the application of implanted micro-optics for the real-time and continuous in-vivo observation of complex biological processes.
We report new methods of two-photon polymerization of microlenses with high numerical aperture, large diameter and good optical quality. We characterize the aberrations of these lenses that, coupled to raster scanning optical microscopes, allow two-photon excitation imaging of cells. In-vivo non-linear imaging will be also discussed, opening the possibility to use these micro-lenses in implants for the continuous inspection of biological dynamics in vivo.
The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration.
We describe several bioengineered cell models developed by our group. We develop tools for cell culture allowing fluorescence diagnostics on the cellularised constructs cultured within, both in 3D and prolonged culture times extending to several weeks. These cell models proved able to recapitulate in vitro several slowly developing biological processes such as the regeneration of a cartilaginous tissue by cartilage cells, the formation of a bone metastasis by breast cancer cells, the instruction of adaptive immune cells as occurs in a lymphnode, and the neuroprotective effect on pathological neurons of mesenchymal stem cell secretome. We also scaled down these tools in the aim to better control stem cell function in our models, by applying two-photon laser polymerization to fabricate micro scaffolds for cell expansion. We were able to condition mesenchymal stem cells, neural precursor cells and embryonic stem cells towards maintenance of a greater stemness and multipotency or pluripotency, compared to conventional flat culture. This result opens up an avenue towards a safer use of these cells for stem cells therapies. Finally, we describe a revolutionary concept to implant the cell model in a living organism, to be revascularized by the host allowing for studies involving interactions with the host immune system.
Recent studies have shown that mechanical factors can direct stem cell fate in vitro, even in the absence of biochemical
factors. Two-photon laser polymerization was applied here to fabricate ultra-precise 3D micro-scaffolds with different
architectures and pore sizes able to structurally interact with cells at the single-cell scale. Our experiments have shown
that randomly seeded mesenchymal stem cells systematically colonize the internal volumes of 3D scaffolds and
proliferate, while showing a roundish morphology. Even if stem cell mechanobiology is a very complex field, this study
shows how mechanical interactions studied in a 3D micro-architecture at a single cell scale may influence stem cells
response.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.