Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease, and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here, we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, critically important aspects of tissue biology. Using novel OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies. †Authors to whom correspondence should be addressed: R.K.J (jain@steele.mgh.harvard.edu) or B.E.B (bouma@helix.mgh.harvard.edu). * Authors contributed equally to this work Author Contributions BJV developed OFDI technology, designed and performed most of the experiments, developed methodology, headed all data analysis and wrote the manuscript. RML designed and performed most of the experiments, developed methodology, headed all data analysis and wrote the manuscript. JAT contributed to vascular tracing of OFDI data. TPP performed lymphangiography experiments and contributed to data analysis and manuscript preparation. LAB performed VEGF-R2 blockade in vivo experiments. TS developed and performed fractal characterization and contributed to manuscript preparation. LLM contributed to vascular data analysis. GJT contributed to OFDI technology development. DF contributed to experimental design and manuscript preparation. RKJ and BEB contributed to the design of experiments, preparation of the manuscript, and supervised the project.

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