Sardella Donato, Kristensen Anders M, Bordoni Luca, Kidmose Hanne, Shahrokhtash Ali, Sutherland Duncan S, Frische Sebastian, Schiessl Ina Maria
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.
Front Physiol. 2023 Apr 24;14:1176409. doi: 10.3389/fphys.2023.1176409. eCollection 2023.
Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.
对肾脏和其他腹部器官进行连续的活体双光子显微镜检查是一种强大的技术，可同时并随时间评估组织功能和结构。因此，连续活体显微镜检查可以捕捉健康和疾病过程中组织的动态变化，并具有以亚细胞分辨率表征（病理 - ）生理过程的巨大潜力。然而，成功的图像采集和分析需要大量专业知识，并带来多种潜在挑战。腹部器官会因呼吸运动而有节奏地移位，这会妨碍高分辨率成像。传统上，肾脏活体成像在倒置显微镜上进行，动物的重量会部分补偿呼吸运动。在此，我们展示了一种用于在正立显微镜上对肾脏和其他腹部器官进行活体成像的定制且易于实施的设置。此外，我们提供了图像处理协议以及用于免费图像分析软件FIJI的新插件，以处理多通道荧光显微镜数据。所提出的图像处理流程涵盖多种图像去噪算法、使用二维配准的样本漂移校正，以及使用基于地标的三维配准对数周内收集的连续成像数据进行对齐。所提供的工具旨在降低肾脏活体显微镜检查的入门门槛，生物医学从业者可轻松应用。
