A. Nardini

B. S. Kariman

C. Conci

G. Chirico

G. N. Cerullo

M. Bouzin

M. Collini

M. Marini

M. T. Raimondi

R. Martínez Vázquez

R. Osellame

English

Archivio istituzionale della ricerca - Politecnico di Milano

             28th Congress of the European Society of Biomechanics    9- 12 July 2023, Maastricht, The Netherlands      ESB2023 BOOK OF ABSTRACTS  26th Congress of the European Society of Biomechanics, July 11-14, 2021, Milan, Italy TWO PHOTON POLYMERIZATION OF AN IMPLANTABLE MICROSCOPE OBJECTIVE FOR INTRAVITAL MICROSCOPY  A. Nardini1, M. Marini4, R. Martínez Vázquez2, C. Conci1, M. Bouzin4, M. Collini4, R. Osellame2,  G.N. Cerullo3, B.S. Kariman3, G. Chirico4, M.T. Raimondi1  1. Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Milano, Italy 2. Istituto di Fotonica e Nanotecnologie (IFN)-CNR), Politecnico di Milano, Milano, Italy 3. Department of Physics, Politecnico di Milano, Milano, Italy 4. Department of Physics, Università di Milano-Bicocca, Milan, Italy  I. Introduction Regulations for biomaterial testing rely on histopathological analyses of animal biopsies leading to ethical issues(1). In this context, intravital microscopy techniques allow to quantify in-vivo the foreign body reaction to the implant reducing the number of animals required to statistically validate the biomaterial(2,3). However, traditional windows chambers are invasive, lead to strong inflammatory reactions and fail in the reliability of long-term evaluations. Therefore, we developed a system of microlenses, coupled to a microscaffold, both incorporated in a miniaturised imaging window.  II. Materials and Methods The device is microfabricated by two-photon polymerization (2PP) of a biocompatible photoresist called SZ2080(4). It is designed to act as an in-situ microscope objective with the aim to overcome the restrictions of in-vivo imaging related to tissue-induced spherical aberrations. The chip includes fluorescent beacons, allowing optical alignment for multiple observations. Firstly, we established the microlenses process of fabrication as 2PP of the outer shell followed by the UV bulk polymerization of SZ2080(5). We developed a dedicated protocol to fabricate both the microlenses and the 3D microscaffolds on the same chip. Then, we quantified the lenses dioptric power and magnification by coupling them to low numerical aperture objectives, to image stained cells cultured in-vitro both on flat substrates and inside the 3D microscaffolds.   III. Results We proved the reliability of the microfabrication process and independently validated the microlenses morphological and optical quality and obtained a prototype of the integrated smart microstructured imaging window (Fig1.A-C). Remarkably, the fabricated microlenses allow to efficiently excite the fluorescence of labelled cells (Fig1.D-F) and collect high resolution, magnified images of them on a conventional two-photon scanning microscope (Fig1.G-H). The chip enhanced the possibility to reduce optical aberrations related to intravital imaging.  IV. Discussion Thanks to these encouraging results, the chip will now be used for in-vivo observations of the host inflammatory response to the implant of biomaterials, in the aim (a) to reduce the number of animals sacrificed in biomaterial testing procedures and (b) strengthening the validation protocols in both a qualitative and quantitative manner.   Figure 1: Microlenses (A) and microscaffolds (C) fabricated on both sides of the device (B). Confocal images of stained human fibroblasts superimposed to the transmitted images of microlenses arrays (D), through a single microlens (E) and of cells at the glass coverslip focal plane (F). Fluorescence images of cells under two photon excitation through a single microlens (G) and at the glass coverslip focal plane (H).  References 1. Standardization IO 2018, (EU) 2017/745 normative: ISO 10993 series 2. Dondossola E. et al., Nat Biomed Eng, 2017;1(1):1–10 3. Diaspro A. et al., Q Rev Biophys, 2005;38(2):97–166 4. Conci C. et al., Adv Opt Mater, 2022;10(7) 5. Malinauskas M. et al., J Opt, 2010;12(3)   Acknowledgements This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 964481. Funded by the European Union (ERC, BEACONSANDEGG, G.A. 101053122). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. 

Two photon polymerization of an implantable microscope objective for intravital microscopy.

https://re.public.polimi.it/bitstream/11311/1286316/1/Nardini_ESB_2023.pdf

Two photon polymerization of an implantable microscope objective for intravital microscopy.

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