Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.

多光子活体成像对于了解体内细胞行为和功能至关重要。由于光散射阻碍了高分辨率成像，乳腺富含脂肪的环境对体内显微镜提出了独特的挑战。在这里，我们提供了一个协议，可对整个小鼠乳腺和相关组织的区域进行高质量、六色 3D 活体成像，持续数小时，同时保持组织访问以进行显微切割和标记。在腹侧中线和沿着右后腿的切口形成一个皮瓣，然后将其固定在凸起的平台上，皮肤面朝下。这允许对结缔组织进行荧光引导显微切割，以提供乳腺导管的畅通无阻的成像。皮瓣上方的密封成像室创造了一个稳定的环境，同时保持对大组织区域的访问，以便用正置显微镜进行成像。我们提供了一种利用多色五彩纸屑谱系追踪和附加染料对单细胞和组织微环境进行成像的策略，使用定制设计的滤光片和双多光子激光器的顺序激发。此外，我们描述了使用奥林巴斯 SIM 扫描仪对单细胞同时成像和光操作的策略，并提供了单细胞行为的 3D 视频处理、可视化和高维分析的步骤。然后，我们提供了将活体成像与固定、免疫染色、组织透明化和 3D 共焦成像相结合的步骤，以将细胞行为与蛋白质表达相关联。皮瓣手术和腔室准备需要 1.5 小时，然后进行长达 12 小时的成像。应用范围从 1 天到 5 天的基本拍摄，用于多重分析和复杂分析。