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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\u2009days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&amp;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 biomaterials in in vivo longitudinal studies.<\/jats:p>","DOI":"10.1063\/5.0165411","type":"journal-article","created":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T08:54:48Z","timestamp":1704790488000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":7,"title":["<i>In vivo<\/i>\n                    label-free tissue histology through a microstructured imaging window"],"prefix":"10.1063","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2335-9046","authenticated-orcid":false,"given":"Claudio","family":"Conci","sequence":"first","affiliation":[{"name":"Department of Chemistry, Materials and Chemical Engineering \u201cGiulio Natta,\u201d Politecnico di Milano 1 , Piazza L. da Vinci 32, 20133 Milan, 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Scienza 3, 20126 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0987-3345","authenticated-orcid":false,"given":"Donato","family":"Inverso","sequence":"additional","affiliation":[{"name":"Division of Immunology, Transplantation and Infectious Diseases IRCCS San Raffaele Scientific Institute, Vita-Salute San Raffaele University 3 , Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8728-5819","authenticated-orcid":false,"given":"Rebeca","family":"Mart\u00ednez V\u00e1zquez","sequence":"additional","affiliation":[{"name":"Institute for Photonics and Nanotechnologies (IFN), CNR and Department of Physics, Politecnico di Milano 4 , Piazza L. da Vinci 32, 20133 Milan, 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