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

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 1

Properties of Mars1- and Mars1Cy-based fluoromodules.

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Properties of Mars1- and Mars1Cy-based fluoromodules. (A) Chemical struc...
(A) Chemical structures of fluorogens SC1 (membrane permeant) and SCi1 (membrane impermeant). (B) Absorbance spectra: SC1 in acidic (5% glacial acetic acid) methanol (mean, 4 samples) and Mars1:SC1 in PBS, pH 7.4 (mean, 2 samples); spectra are scaled to have peak absorbance of 1.0 units. Free and bound forms of SCi1 are similar but with a 3 nm bathochromic shift. (C) Excitation (dashes, mean of 5 samples) and emission (solid, mean of 4 samples) spectra of SCi1 in PBS and FBS with and without Mars1Cy; spectra of Mars1 and SC1 are virtually identical. Intensities are expressed as a fraction of the maximum signal. (D) Binding and fluorescence activation traces of Mars1:SC1, Mars1:SCi1, Mars1Cy:SC1, and Mars1Cy:SCi1 (1 acquisition per fluoromodule) recorded from solutions where [FAP] = KD and [fluorogen] = 10 KD. Respective on rates (3.3 × 106, 2.8 × 106, 3.7 × 105, and 1.5 × 106 M-1s-1) were calculated from activation observed at 4 different concentrations of fluorogen. (E) Fluorescence of Mars1 fluoromodules in buffers spanning pH 2.0 to 10.0 measured 1 and 30 hours after sample preparation. Points indicate mean sample fluorescence (n = 5: Mars1:SC1/1 hour, n = 4: others). Bars indicate SD. Values are scaled such that the highest mean signal is 1.0. Mars1Cy exhibits similar pH insensitivity. (F) Fluorescence from HEK293 cells that express cytoplasmic Mars1Cy-eGFP, and untransfected control cells maintained at 37°C prior to (0 minutes) and at intervals after addition of SC1 or SCi1. Points denote average of median population intensities (n = 3 separate samples prepared identically) analyzed by flow cytometry.