Fluorescence-based light microscopy has driven enormous progress in our understanding of biology. However, a majority of these studies have been carried out on cells in vitro or tissues ex vivo, which often do not reflect the 3D environment, complex signaling pathways, feedback networks, or natural dynamics of biological processes in vivo.

Intravital microscopy (IVM), the imaging of live animals at microscopic resolution, enables the study of these biological processes in vivo, and their native environs. Long considered a technically challenging art form, the past decade has seen a wide range of advances in technologies and tools for IVM, making it increasingly possible to conduct reproducible, mechanistic studies and derive unparalleled quantitative and dynamic insights into cell and tissue biology during development, homeostasis, and disease. These advances include imaging technologies that allow subcellular or functional IVM, specialized surgical protocols and hardware to enhance access to and stability of tissues for long-term image-acquisition, and the generation of new animal models and fluorescent probes to address an ever-increasing number of physiological questions.

Unfortunately, the nuances of many of these developments are not easily conveyed via written protocols alone and would benefit greatly from a visual depiction. To this end, we aim for this Methods Collection to focus on the latest IVM methods and technologies, from the subcellular to the tissue scale. Our goal is both to make these contributions accessible to adoption by the wider scientific community, as well as to highlight their impact on the fields of cell biology, immunology, cancer biology, nephrology, neuroscience, and developmental biology.