The development of intravital imaging with multiphoton microscopy has had a major impact on kidney research. It provides the unique opportunity to visualize dynamic behavior of cells and organelles in their native environment and to relate this to the complex 3-dimensional structure of the organ. Moreover, changes in cell/organelle function can be followed in real time in response to physiological interventions or disease-causing insults. However, realizing the enormous potential of this exciting approach has necessitated overcoming several substantial practical hurdles. In this article, we outline the nature of these challenges and how a variety of technical advances have provided effective solutions. In particular, improvements in laser/microscope technology, fluorescent probes, transgenic animals, and abdominal windows are collectively making previously opaque processes visible. Meanwhile, the rise of machine learning-based image analysis is facilitating the rapid generation of large amounts of quantitative data, amenable to deeper statistical interrogation. Taken together, the increased capabilities of multiphoton imaging are opening up huge new possibilities to study structure-function relationships in the kidney in unprecedented detail. In addition, they are yielding important new insights into cellular mechanisms of tissue damage, repair, and adaptive remodeling during disease states. Thus, intravital microscopy is truly entering an exciting new era in translational kidney research., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)