Journal Article

Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue

Visvader, Jane E., Lindeman, Geoffrey J., Rios, Anne C., Dawson, Caleb A., Mueller, Scott N.

2021

Overview

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. We present a protocol for long-term intravital imaging of mouse mammary tissue while maintaining tissue access using a skin flap and an imaging chamber. Strategies are provided for single-cell imaging, photomanipulation and multiplexed analysis.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/245/2225 / 631/1647/328/2057 / 631/1647/334/1874/345 / 631/80/2373 / Analytical Chemistry / Anesthesia / Animal tissues / Animals / Biological Techniques / Biomedical and Life Sciences / Cell research / Chambers / Composition / Computational Biology/Bioinformatics / Connective tissues / Dimensional analysis / Ducts / Epithelial Cells - cytology / Female / Fluorescence / Fluorescent Dyes - chemistry / Green Fluorescent Proteins - metabolism / Image processing / Image resolution / Imaging, Three-Dimensional / Intravital Microscopy - methods / Lasers / Life Sciences / Light scattering / Mammary gland / Mammary glands / Mammary Glands, Animal - cytology / Mammary Glands, Animal - surgery / Mathematical analysis / Methods / Mice / Mice, Inbred C57BL / Mice, Transgenic / Microarrays / Microenvironments / Microscope and microscopy / Microscopy / Multiplexing / Observations / Organic Chemistry / Protocol / Single-Cell Analysis / Skin / Stromal Cells - cytology / Surgery / Tissues / Video