Live animal imaging in the far red
Methods to image whole, live animals are invaluable biomedicals tools for the study of various disease states, especially for investigating processes that underlie tumor growth and metastasis. While many fluorescent and luminescent methods have been devised for visualizing tumor development in live animals, many of these approaches are hindered by issues such as background signal from circulating probes or autofluorescence from surrounding tissue. Ming Zhang and colleagues at Carnegie Mellon University have improved upon previous reporter/probe systems and developed a far-red absorbing fluoromodule that circumvents imaging issues associated with tissue autofluorescence at lower wavelengths, such as are required for conventional approaches. Specifically, this system involves targeted reporter proteins, Mars1 and Mars1Cy, and an activatable fluorogenic synthetic dye probe called SC1. When SC1 encounters Mars1- or Mars1Cy-tagged proteins it becomes conformationally restricted in an excited state and increases in fluorescence by thousands of orders of magnitudes in the tissue of interest. Zhang et al. chemically modified the SC1 molecule to generate a membrane impermeant version (SCi1) suited for labeling cell surface markers. Furthermore, Mars1 has the ability to bind multiple types of probes that fluoresce at different wavelengths, which allowed for two-color fluorescence imaging in live cells. Moreover, the authors demonstrated the feasibility of using this fluoromodule system in live, nude mice, wherein HCT116 colorectal cancer cells expressing plasma membrane targeted Mars1Cy established both subcutaneous and intraperitoneal cavity tumors. SC1 or SCi1 was then injected into these mice where it illuminated the tumor tissue. The Mars1/Mars1Cy and SC1/SCi1 fluoromodule system establishes a platform on which to build more specific reporter/probe systems better suited for live animal imaging. The accompanying images show that the internal and external pools of a Mars1Cy-tagged AVPR2 receptor can be differentiated in HEK293 cells. Membrane-impermeant fluorogen (SCi1 (top row, pink) or MG-2p (bottom row, blue)) identifies the membrane-bound Mars1Cy-tagged AVPR2 receptor population and is followed by addition of a permeant fluorogen (MGE (top row, blue) or SC1 (bottom row, pink)), which labels the cytosolic pool (left hand column). Stimulation of these cells with desmopressin allows observation of Mars1Cy-tagged AVPR2 receptor endocytosis (right hand column, T=20 min).
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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
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)