Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging

Claudio Vinegoni, Paolo Fumene Feruglio, Christian Brand, Sungon Lee, Antoinette E. Nibbs, Shawn Stapleton, Sunil Shah, Ignacy Gryczynski, Thomas Reiner, Ralph Mazitschek, Ralph Weissleder

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPEmicroscope, the protocol can be extended to other commercial or custom-built microscopes.

Original languageEnglish
Pages (from-to)1472-1497
Number of pages26
JournalNature Protocols
Volume12
Issue number7
DOIs
StatePublished - 1 Jul 2017

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    Vinegoni, C., Feruglio, P. F., Brand, C., Lee, S., Nibbs, A. E., Stapleton, S., Shah, S., Gryczynski, I., Reiner, T., Mazitschek, R., & Weissleder, R. (2017). Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging. Nature Protocols, 12(7), 1472-1497. https://doi.org/10.1038/nprot.2017.043