A Miniaturized Imaging Window to Quantify Intravital Tissue Regeneration within a 3D Microscaffold in Longitudinal Studies

Abstract

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.

Microlenses fabricated by two-photon laser polymerization for cell imaging with non-linear excitation microscopy

“…The photoresist used for the 2PP of the microlenses was a negative hybrid organic-inorganic UV-curable resist known as SZ2080TM 36 , biocompatible and validated for biological applications 6,37 . To increase the efficiency of the polymerization 1% of Irgacure-369 (2-Benzyl-2-dimethylamino-1-(4-morpholinophenyl)-butanone-1) is added to the resist as photoinitiator.…”

“…We recently proposed a miniaturized (500 μm × 500 μm × 100 μm) imaging window, named Microatlas, which incorporates an array of micro-scaffolds, able to guide the regeneration of tissue at the interface with the implanted material 6 . Although the Microatlas has been validated up to now in embryonated chicken eggs 6 , it can also be implanted sub cute in mice, in which case non-linear excitation microscopy would be the best choice for direct imaging. Non-linear excitation microscopy with near infrared (NIR) laser sources can mitigate the penetration issues in tissues [7][8][9] by reducing the effect of scattering on the point spread function.…”

“…However, the majority of the existing optical windows influence and/or have a severe impact on the laboratory animal health and behavior, therefore limiting the medical relevance of the observations. We recently proposed a miniaturized (500 μm × 500 μm × 100 μm) imaging window, named Microatlas, which incorporates an array of micro-scaffolds, able to guide the regeneration of tissue at the interface with the implanted material [6] . Although the Microatlas has been validated up to now in embryonated chicken eggs [6] , it can also be implanted sub cute in mice, in which case non-linear excitation microscopy would be the best choice for direct imaging.…”

“…We further fully characterize the microlenses for widefield transmission and fluorescence imaging and for confocal and two-photon scanning microscopy. These microlenses can be included in implantable devices for the direct inspection of cellular dynamics in vivo [6] or in integrated in microfluidic setups for the in vitro monitoring of complex biosystems, such as organoids, directly in the organoid bioreactor.…”

Proteinaceous microstructure in a capillary: a study of non-linear bending dynamics

“…41 Since we are interested in soft microstructures capable of undergoing substantial (≥5%) bending motion under the action of fluid drag force, we opt for the use of hydrogels with Young's moduli in the MPa range, 41 much softer than silica based resists characterized by a GPa Young's modulus. 42 We rely on photoresists based on bovine serum albumin (BSA), which is one of the most employed proteins in the field of AOM 43,44 because of its well-studied functionalities, biocompatibility and low cost. 45 Aiming at the development of an integrated experimental platform enabling both the structure manufacturing and the characterization of their bending dynamics, we demonstrate the direct fabrication of proteinaceous micro-structures within borosilicate microfluidic channels.…”