The ability to visualize the presence, abundance, location and functional state of many proteins at once in cells and tissues with preserved morphology has long been a goal. This proposal seeks to optimize recently demonstrated mass-spec-based imaging methods that can, when fully developed, detect as many as 40 to 100+ molecular targets in cells and tissues using simultaneously applied mass-tag-labeled antibodies, with outstanding morphology. The technique is analogous to laser-scanning confocal microscopy, except that the scanning is performed using a tightly focused ion beam, rather than a similarly sized point of laser light. And instead of fluorophores, the labels detected are clustes of metal atoms attached to any given probe. Preliminary data using a highly focused, scanning oxygen ion beam and 10 antibodies applied simultaneously to clinical breast cancer tissues demonstrate histology-like images that reveal bound antibody location with subcellular resolution. Going forward, we would like to optimize sensitivity and reliability, with the goal of making this a standard, high-performance, imaging approach for basic and translational science discovery, drug development, and possible clinical deployment. The present project will address: reliable pre-analytical sample preparation methods and labeling techniques for protein analytes; ion-beam imaging method improvements based on current and projected instrumentation; powerful, easy-to-use software for equipment control, display and analysis; and validation in several important biological test cases.

Public Health Relevance

The biology and clinical course of cancers are often predicted by the presence and distribution of proteins within cells that are involved in cell division, invasion, metastasis and evasion of the host's immune system. We describe a new, non-optical microscopic technology that can visualize as many as 100 proteins simultaneously. We think this may prove to be a remarkable engine for research and clinical advances in the understanding and treatment of cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1)
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Knowlton, John R
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Stanford University
Schools of Medicine
United States
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