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 October 1, 2015; First 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 2

Fluoromodules appear brighter than IFP1.4 and are functional when expressed within mammalian cells.

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Fluoromodules appear brighter than IFP1.4 and are functional when expres...
Channels are labeled DIC or formatted excitation:emission/bandwidth. (A) HEK293 cells expressing Mars1Cy-eGFP or IFP1.4-eGFP fusion proteins, from otherwise identical vectors, were imaged in the presence of 400 nM SC1 and 50 μM biliverdin, respectively, to compare observed brightness under identical conditions and display settings. Images are representative samples of data analyzed in B. (B) Box plot of fluoromodule/eGFP signal intensity ratios measured from protein coexpressed with eGFP in HEK293 cells as fusion proteins or separate polypeptides (P2A). Whiskers indicate maximum and minimum values; for boxes, top, line, and bottom indicate first, second, and third quartiles, respectively. Differences between mean ratios were analyzed by 1-way ANOVA: F(3,614) = 5332.14, P < 0.001. A Games-Howell post-hoc test shows differences between paired group means as denoted by lowercase letters: a (IFP1.4-eGFP fusion versus separate IFP1.4/eGFP peptides, P = 0.650); other pairs (ab, ac, bc) describe different means with high confidence, P < 0.001. Number of measurements (n) per group (IFP1.4/eGFP: 159, IFP1.4-eGFP: 180, Mars1Cy/eGFP: 102, Mars1Cy-eGFP: 177) indicate the number of unique cells measured. (C) Mars1Cy anchored to the plasma membrane by a CD80 transmembrane domain (TMD) can be labeled with membrane-impermeant SCi1. (D) Signal peptide–prefixed (COXIV) Mars1Cy-eGFP is directed to the mitochondria and becomes labeled upon addition of the membrane-permeant fluorogen SC1. (E–H) Mars1- and Mars1Cy-based fluoromodules can be used to tag proteins such as β-actin, vimentin, histone H1.1, and IGF1R in living cells. Images in C–H represent at least 10 fields of view per transgene-hosting cell population. Scale bars: 16 μm.