Search

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.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of

The possibility to fabricate microstructures to be used in the medical field is a reality. Further steps in this direction consist in the fabrication of active microstructures for implants and/or for cellular treatments. Two major areas of interest will be briefly treated here. The first is the one of stimulus-responsive optical polymers, especially hydrogels, that can be shaped by means of laser 3D printing and ablation. These can be used for thermal stimulation, energy transduction and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. A second field of interest is the microfabrication of rigid microstructures that can stand the tissue-induced stresses in implants. The increasing demand of microdevices for nanomedicine and personalized medicine has fostered the quest for an efficient combination of composite and hybrid photo-responsive materials and digital micro/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. The aim of this report is to provide a short overview of the basics of photo-polymerization induced by two-photon excitation and to discuss two case studies. In the first one, we discuss the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photo-thermal activity, chemical swelling and chemical sensing. In the second case, we outline the fabrication steps and the first tests of a novel chip which aims at enabling longitudinal studies of the reaction to the biomater

Intravital microscopy windows for in vivo observation of tissue are desirable in many biomedical fields, from immunology to the assessment of the foreign body reaction to biomaterials. Moreover, they allow one to reduce the number of animals required to statistically validate biomaterials if compared with traditional histopathological analyses of animal biopsies [1,2].

We report new methods of 2 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.

Non-linear excitation microscopy offers several advantages for in-vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, Fibroblast cell culture plano-convex microlenses to be used for non-linear imaging of biological tissue are developed and tested. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. Magnified fluorescence images can be retrieved through the microlenses coupled to commercial confocal and two-photon excitation scanning microscopes. The signal-to-noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro-optics for optical in-vivo inspection of biological processes.

We report new methods of 2 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 biocompatibility assessment of biomaterials or the dynamic response of implanted constructs entails inflammatory events primary reflected in cell behavior at the microcirculatory system. Current protocols are based on histopathology which are over 40 years old and require the sacrifice of a huge number of laboratory animal with an unsustainable ethical burden of animal research. Intravital microscopy techniques are actually used to study implantation outcomes in real time. However, no device providing a specific tracking geometry to reposition the field of view of the microscope, for repeated analyses, exists yet. The synthetic photoresist SZ2080 is characterized here, allowing the development and in vivo validation of a miniaturized imaging window, the Microatlas, that, fabricated via two-photon polymerization, is implanted in living chicken embryos and imaged by fluorescence microscopy 3 and 4 days after the implant. The characterization of their elastomechanical and fluorescence properties highlights planar raster spacing as the most important parameter in tuning the mechanical and spectroscopic features of the structures. The quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration and as beacon for 3D repositioning of the microscope field of view and correction of optical aberrations.

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 stem 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/pluripotency, compared to conventional flat culture. This result opens an avenue towards a safer use of these cells for stem cells therapies. Finally, we describe our new revolutionary concept of implanting the cell model in a living organism to regenerate a vascularized network anastomosed to the host, allowing for studies involving interactions with the host immune system.