label-free tissue histology through a microstructured imaging window.

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 , 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 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 i longitudinal studies.