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

Nature Protocols volume 12, pages 1472–1497 (2017)Cite this article

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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 FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

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Figure 1: Fluorescence anisotropy principles and measurements.
Figure 2: One-photon versus two-photon excitation.
Figure 3: Results from HT1080 cells incubated in PARPi-FL.
Figure 4: Setup overview.
Figure 5: Polarization control unit overview.
Figure 6: Noise contribution to fluorescence anisotropy images.
Figure 7: Optical characterization of three calibration solutions, ruthenium tris(bipyridyl) [Ru(bipy)3] in PBS (Ru in PBS), fluorescein in PBS (FL in PBS), pyridine 1 in propylene glycol (Py1 in PG).
Figure 8: GUI interface of the AnisotropyCalculation program.
Figure 9: PMT intensity responses across their dynamic ranges at different voltage settings.
Figure 10: Measurements of fluorescein at varying viscosities.
Figure 11: Fluorescence anisotropy measurements for receptor/binding quantification.
Figure 12: Imaging phantom for fluorescence anisotropy imaging.
Figure 13: GUI interface of the OnLine program.
Figure 14: Fluorescence anisotropy time-lapse imaging.
Figure 15: Two-photon fluorescence anisotropy imaging of drug-target engagement.

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Acknowledgements

This project was funded in part by National Institutes of Health (NIH) grants RO1-EB010011, RO1-HL122208 and P50GM107618. Investigators at Memorial Sloan Kettering Cancer Center were funded in part by NIH grant P30 CA008748 and investigators in Europe were funded by the EC Seventh Framework Programme under grant agreement no. 622182.

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Authors and Affiliations

  1. Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Claudio Vinegoni, Paolo Fumene Feruglio, Sungon Lee, Antoinette E Nibbs, Shawn Stapleton, Ralph Mazitschek & Ralph Weissleder

  2. Department of Neurosciences, Biomedicine, and Movement Sciences, University of Verona, Verona, Italy

    Paolo Fumene Feruglio

  3. Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Christian Brand & Thomas Reiner

  4. School of Electrical Engineering, Hanyang University, Ansan, Republic of Korea

    Sungon Lee

  5. Institute for Molecular Medicine,

    Sunil Shah & Ignacy Gryczynski

  6. University of North Texas Health Science Center, Fort Worth, Texas, USA

    Sunil Shah & Ignacy Gryczynski

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  1. Claudio Vinegoni
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Contributions

All authors contributed to the writing of the paper. C.V., R.M., P.F.F. and R.W., along with others, primarily developed the technique. S. Shah and I.G. conducted one-photon experiments. C.V. and P.F.F. wrote the software. S.L. designed and machined the systems components. A.E.N., C.B., R.M. and T.R. performed the synthesis. S. Stapleton prepared cell cultures.

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Correspondence to Claudio Vinegoni.

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Integrated supplementary information

Supplementary Figure 1 PARPi-FL.

Chemical structure (a) and analytical HPLC chromatogram (b) of PARPi-FL.

Supplementary Figure 2 Image Acquisition Control window of the Olympus FluoView program.

The interface allows to control the PMT voltage settings, to initiate acquisitions and image averaging. Image of FLUOVIEW program adapted with permission from Olympus.

Supplementary Figure 3 Acquisition Setting window of the Olympus FluoView program.

The interface allows to set image acquisition dwell time, size, zoom factor, excitation wavelength selection and laser power. Image of FLUOVIEW program adapted with permission from Olympus.

Supplementary Figure 4 Acquired Image window of the Olympus FluoView program.

The two images (red boxes 1,2) show simultaneous acquisition of fluorescence emission at two orthogonal state of polarization as detected at PMT3 and PMT4, for a solution of fluorescein diluted in a mixture of water-glycerol (w/w) at 95%. Image of FLUOVIEW program adapted with permission from Olympus.

Supplementary Figure 5 Alignment polarization control unit.

Laser intensity as measured at the back-aperture of the objective, as a function of the half-waveplate angle. Light is measured after passing through a near infrared linear polarizer parallel to the microscope's Y axis (Fig. 4a).

Supplementary Figure 6 LightPath & Dyes window of the Olympus FluoView program.

The interface allows to select the dichroic, imaging path, and the imaging PMTs for simultaneous dual channel detection. Image of FLUOVIEW program adapted with permission from Olympus.

Supplementary Figure 7 GUI interface of the BackgroundEstimation.exe.

GUI interface of the BackgroundEstimation program, necessary to calculate the dark noise background correction.

Supplementary Figure 8 Closed bath imaging chamber.

(a-c) Different views of the closed bath imaging chamber for time measurements of drug target engagement. 1, inflow tubing connected to the perfusion manifold; 2, cell-seeded coverslip; 3, tubing connected to the vacuum outlet; 4, suction reservoir. The 2x dry objective, is used to find cell-seeded areas on the imaging coverslip.

Supplementary Figure 9 GUI interface of the OffLine.exe.

GUI interface of the OffLine program, for image processing, calculation and visualization of fluorescence anisotropy images for data previously acquired.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Figures 1–9. (PDF 1979 kb)

Supplementary Data 1

A .zip file containing the STL file for the assembly of the polarization control unit. (ZIP 8201 kb)

Supplementary Data 2

A .zip file containing the STL files for the 3D printing and drawing of the polarization filter cube. (ZIP 331 kb)

Supplementary Data 3

A .zip file containing a longitudinal data set acquired in a perfusion chamber during the drug loading and washing phase, calibration measurements for high and low values of anisotropy, and data for cells at different time points during the loading and washing time. (ZIP 13205 kb)

Supplementary Software

A .zip file containing the software for the OnLine and OffLine elaboration and visualization of the fluorescence anisotropy images, and for calculating the background noise subtraction and average anisotropy: OnLine.exe, OffLine.exe, BackgroundEstimation.exe, AnisotropyCalculation.exe. (ZIP 23538 kb)

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Vinegoni, C., Fumene Feruglio, P., Brand, C. et al. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging. Nat Protoc 12, 1472–1497 (2017). https://doi.org/10.1038/nprot.2017.043

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