A team from the Walter and Eliza Hall Institute of Medical Research in Australia has shown the ability to image tissue cells in the breast, without harming them, to observe their behavior at microscopic levels. The team used multiphoton microscopy, a technique using infrared lasers to illuminate fluorescently labeled cells. The protocol enabled the researchers to observe how developing and formed cells in the breast rearrange, interact, and sense their environment over the development of the breast itself. It also allowed them to capture high-resolution images of the affected cells’ movement in hard-to-reach regions of breast tissue, improving understanding of cell function. The ability to image different stages of breast development, immune cells, lymph nodes, and hair follicles, observing how each individually labeled cell functions, establishes an avenue for a range of targeted research opportunities, said Caleb Dawson, who is credited with developing the protocol. Currently, the filming component of the microscopy system helped the developing team increase its ability to study the terminal end buds (TEBs) in breast tissue. The club-like structures are located at the tip of mammary ducts and grow to produce the structure of breast tissue. “The unique cells inside the TEBs have never been filmed like this before, so it was fascinating to watch this process for the first time,” Dawson said. “We have watched a cell behavior inside the TEB that was hypothesized in the 1980s but was never proven, and which has implications for breast stem cell function.” The study of TEBs previously involved the dissociation of individual cells, filming them outside the breast or capturing still images. Those approaches restrict the ability to see the cells’ actual behavior and interactions inside living tissue. “By filming the moving cells inside intact breast tissue in laboratory models, we are able to grasp a better understanding of how the cells behave and cooperate to help the breast to form and function properly,” Dawson said. The approach supported imaging of up to six fluorescent colors at the same time, meaning that in deployment it allowed users to study a greater number of cell and cell-type interactions than with other methods. The clear and detailed images revealed information about the cells’ physical appearances, functions, and interactions, as well as their evolution over time. The team additionally consisted of Jane Visvader and Anne Rios, and worked in collaboration with Scott Mueller from the Peter Doherty Institute for Infection and Immunity. Geoffrey Lindeman is a co-author of the paper introducing the protocol. This work was made possible by the WEHI Centre for Dynamic Imaging. The National Health and Medical Research Council, the Australian government, the National Breast Cancer Foundation, the Australian Cancer Research Foundation, and the government of Victoria funded the work. The research was published in Nature Protocols (www.doi.org/10.1038/s41596-020-00473-2).