通过微结构成像窗口进行体内无标记组织组学研究

IF 6.6 3区 医学 Q1 ENGINEERING, BIOMEDICAL
C. Conci, L. Sironi, E. Jacchetti, D. Panzeri, D. Inverso, R. Martínez Vázquez, R. Osellame, M. Collini, G. Cerullo, Giuseppe Chirico, M. Raimondi
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引用次数: 0

摘要

组织病理学是对薄组织切片进行苏木精和伊红(H&E)染色,是评估生物材料植入后免疫反应的黄金标准。这种方法耗时长、成本高,无法进行纵向研究。在体内使用非线性激发显微镜,基本上不需要标记,有可能克服这些局限性。为此,我们开发并验证了一种植入式微结构装置,用于对植入生物材料的免疫反应进行非线性激发显微镜评估。这种微结构装置是通过双光子激光聚合技术获得的,其形状为规则的三维晶格矩阵。随后,它被植入胚胎鸡卵的绒毛膜(CAM)中 7 天,作为细胞计数和识别的内在三维参考框架。根据植入微结构周围组织切片的 H&E 图像进行组织学分析,并与非线性激发和共聚焦图像进行比较,以建立细胞图谱,将组织学观察结果与无标记图像关联起来。通过这种方法,我们可以量化在微结构中重组的组织中招募的细胞数量,并在微结构内外的无标记图像上识别粒细胞。此外,还可通过二次谐波发生和自发荧光成像技术识别胶原蛋白和微血管。分析表明,组织对植入微结构的反应类似于损伤后典型的 CAM 愈合,没有大规模的异物反应。这为使用与生物材料耦合的类似微结构,利用无标记非线性激发显微镜对组织与生物材料之间的再生界面进行活体成像开辟了道路。这有望成为一种替代传统组织病理学的变革性方法,用于生物工程和体内纵向研究中生物材料的验证。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
In vivo 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 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 biomaterials in in vivo longitudinal studies.
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来源期刊
APL Bioengineering
APL Bioengineering ENGINEERING, BIOMEDICAL-
CiteScore
9.30
自引率
6.70%
发文量
39
审稿时长
19 weeks
期刊介绍: APL Bioengineering is devoted to research at the intersection of biology, physics, and engineering. The journal publishes high-impact manuscripts specific to the understanding and advancement of physics and engineering of biological systems. APL Bioengineering is the new home for the bioengineering and biomedical research communities. APL Bioengineering publishes original research articles, reviews, and perspectives. Topical coverage includes: -Biofabrication and Bioprinting -Biomedical Materials, Sensors, and Imaging -Engineered Living Systems -Cell and Tissue Engineering -Regenerative Medicine -Molecular, Cell, and Tissue Biomechanics -Systems Biology and Computational Biology
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