Three-dimensional microscopy of large biological samples (>0.5 cm³) is transforming biological research. Many existing techniques require trade-offs between image resolution, sample size and method complexity. A simple robust instrument with the potential to perform large volume 3D imaging currently exists in the form of the Optical HREM, however the development of the instrument to date is limited to single fluorescent wavelength imaging with non-specific eosin staining. This work presents developments to realize the potential of the HREM to become Multi-fluorescent High Resolution Episcopic Microscopy (MF-HREM). MF-HREM is a serial-sectioning and block-facing wide-field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image post-processing to enable multiple specific stains in large samples, and show how these enable segmentation and quantification of the data. The application of MF-HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm³ at resolutions of 2.75 μm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white matter track orientation in adult mouse brain.

中文翻译：

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多荧光高分辨率镜检显微镜（MF-HREM）用于成年鼠器官的三维成像

大型生物样品（> 0.5 cm ³的三维显微镜））正在改变生物学研究。许多现有技术需要在图像分辨率，样本大小和方法复杂性之间进行权衡。目前，以光学HREM的形式存在一种可能进行大体积3D成像的简单而坚固的仪器，但是迄今为止，该仪器的开发仅限于具有非特异性曙红染色的单荧光波长成像。这项工作提出了实现HREM成为多荧光高分辨率镜检显微镜（MF-HREM）的潜力的发展。MF-HREM是一种连续切片和面向块的广域荧光成像技术，不需要组织清除或光学切片。样品制备和图像后处理中详细介绍了多种进展，以使大样品中具有多种特定的污点，并展示它们如何实现数据的细分和量化。MF-HREM的应用在多种生物学环境中得到了证明：异种移植肿瘤中最大肿瘤血管网络的3D成像和肿瘤细胞侵袭性可达7.5 mm³的分辨率为2.75μm，对成年小鼠肾脏中的肾小球体积进行定量，对成年小鼠脑中的血管网络和白质径迹方向进行定量。