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Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging
Ming Zhang, … , Marcel P. Bruchez, Alan S. Waggoner
Ming Zhang, … , Marcel P. Bruchez, Alan S. Waggoner
Published September 8, 2015
Citation Information: J Clin Invest. 2015;125(10):3915-3927. https://doi.org/10.1172/JCI81086.
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Technical Advance Oncology

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

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Abstract

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Authors

Ming Zhang, Subhasish K. Chakraborty, Padma Sampath, Juan J. Rojas, Weizhou Hou, Saumya Saurabh, Steve H. Thorne, Marcel P. Bruchez, Alan S. Waggoner

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Figure 3

Mars1Cy-tagged AVPR2 is responsive to desmopressin-stimulated (DDAVP) endocytosis and demonstrates the feasibility of 2-channel separation of external and internal protein pools using impermeant and membrane-permeant fluorogens that emit in the far-red.

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Mars1Cy-tagged AVPR2 is responsive to desmopressin-stimulated (DDAVP) en...
(A) HEK293 cells expressing Mars1Cy-AVPR2 were incubated at 37°C with the indicated membrane-impermeant fluorogens for 5 minutes prior to stimulation with DDAVP. Images were acquired immediately upon agonist application and at 20 minutes after addition. (B) Sequential addition of impermeant fluorogen (either SCi1 or MG-2p, 10 minutes) followed by permeant fluorogen MG-ester (MGE) or SC1, respectively, for 30 minutes. Each panel is representative of 3 replicates. Scale bars: 16 μm. Magnification is constant across all time points.

Copyright © 2021 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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