Related Experiment Video
Updated: Nov 16, 2025
Intravital Subcellular Microscopy of the Mammary Gland
Published on: June 24, 2022
Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue
Caleb A Dawson1,2, Scott N Mueller3,4, Geoffrey J Lindeman1,5,6
1Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Insights
This study presents a novel protocol for high-quality multiphoton intravital imaging in the challenging mouse mammary gland. The technique enables detailed, long-term visualization of cellular behavior and tissue microenvironments in vivo.
Area of Science:
- Biomedical Imaging
- Cell Biology
- Microscopy
Background:
- Multiphoton intravital imaging is crucial for in vivo cellular studies.
- Mammary gland's adipose tissue causes light scattering, hindering high-resolution microscopy.
- Existing methods lack comprehensive imaging of the mammary gland's cellular dynamics.
Purpose of the Study:
- To develop a protocol for high-quality, multi-color 3D intravital imaging of the mouse mammary gland.
- To overcome light scattering challenges in adipose-rich mammary tissue.
- To enable long-term cellular behavior analysis and multiplexing with other techniques.
Main Methods:
- A skin-flap surgical approach for unimpeded access to mammary ducts.
- Custom filters and sequential laser excitation for multicolor imaging.
- Integration with photomanipulation, 3D processing, and post-imaging analysis.
Main Results:
- Achieved high-quality, six-color 3D intravital imaging of the entire mouse mammary gland.
- Enabled several hours of stable imaging with continued tissue access.
- Successfully multiplexed imaging with fixation, immunostaining, and 3D confocal microscopy.
Conclusions:
- The developed protocol significantly enhances in vivo imaging capabilities in the mammary gland.
- This method facilitates detailed study of cellular behavior and tissue microenvironments.
- The protocol supports diverse applications from basic observation to complex, multiplexed analyses.
Abstract:
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.
