Unlocking the Potential of Chemiluminescence Imaging

The availability of bright fluorescent dyes haspositioned fluorescence microscopy to dominatelive cell molecular imaging for more than a hundred

years. Now, a team lead by Doron Shabat offers a potentialalternative in the form of elegantly designed small moleculescaffolds that open the gates for live cell chemiluminescencemicroscopy.1

Fluorescence microscopy offers a high throughputtechnique that can be used to image cellular structures,enzyme activities, and chemical species. Core fluorescentscaffolds like fluorescein, rhodamine, and boron-dipyrrome-thene (BODIPY) have been widely adopted to expandthe scope of microscopy with new imaging techniquesconstantly emerging.2 Despite wide success, problems suchas photobleaching, light scattering, and autofluorescencehave plagued microscopy and stymie translation of the tech-nology to thicker specimens and animal models. Chem-iluminescence, the direct generation of light from a chemicalreaction, overcomes these problems by eliminating the needfor an excitation light source. Indeed, organisms engineeredto express luciferase and emit bioluminescence have becomea critical tool with new innovative applications in continualdevelopment.3 The need for genetic modification, however,can be a drawback in many experimental systems. For thisreason efforts have been made to develop nonenzymaticchemiluminescent imaging agents, but up until now lowlight emission in water has necessitated stabilization withspecialized supramolecular systems.4,5

Sterically stabilized 1,2-dioxetanes, initially disclosed inthe 1980s by Paul Schaap, are versatile scaffolds thatremain inert until triggered in a chemically initiated electronexchange luminescence mechanism to emit light.6−8 Thisdesign has been used for chemiluminescent agents to detect

β-galactosidase, phosphatase, and other analytes in vitro.More recently, chemiluminescent probes based on thisscaffold have been engineered for whole animal imagingof parameters like tissue oxygenation in wild-type tumormodels.9 Still, low light emission levels require the use ofpolymeric “enhancer” solutions that can prevent cell uptakeand have cellular toxicity that makes them incompatible withlongitudinal studies. In order to overcome the complicationsof these polymeric systems, previous work from DoronShabat’s laboratory demonstrated that tethered fluorophorescould enhance chemiluminescence efficiency for wholeanimal imaging, but chemiluminescent microscopy wasonly achieved in fixed cell samples.10

In their latest work published in ACS Central Science,1 theauthors have systematically investigated modifications anduncovered a key structural motif that unlocks high chemi-luminescent emission, even in water (Figure 1). Hypothesiz-ing that increasing the fluorescent quantum yield would

Published: March 24, 2017

Figure 1. An acrylonitrile substituted phenol endows a spiroadman-tane 1,2-dioxetane with high chemiluminescent emission in water,unlocking chemiluminescence microscopy in living cells.

Chemiluminescence, the directgeneration of light from a chem-ical reaction, overcomes these

problems by eliminating the needfor an excitation light source.

Alexander R. Lippert

Department of Chemistry, Center for Drug Discovery, Design, and Delivery (CD4), and Center for Global Health Impact(CGHI), Southern Methodist University, Dallas, Texas 75275-0314, United States

Improved chemiluminescence probes mayhelp transition the technology into a widelyemployed tool for whole animal imaging andmicroscopy.

© 2017 American Chemical Society 269 DOI: 10.1021/acscentsci.7b00107ACS Cent. Sci. 2017, 3, 269−271

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This is an open access article published under an ACS AuthorChoice License, which permitscopying and redistribution of the article or any adaptations for non-commercial purposes.

ultimately translate into increased chemiluminescent emission,the authors discovered that appending an acrylonitrile groupat the appropriate position provides a dramatic increase influorescence quantum yield and a remarkable 3 order ofmagnitude increase in chemiluminescence emission. Theversatility of this bright chemilumiphore allowed rapidpreparation of responsive chemiluminescent imaging agentsfor hydrogen peroxide, glutathione, and β-galactosidase.Importantly, this enhanced brightness was instrumental inallowingthe team to perform live cell chemiluminescentimaging of HEK293-LacZ cells.This key result clears the path for small molecule chemi-

luminescence microscopy (Figure 2). Only recently has the

appropriate instrumentation become commercially available,and thus far it has been limited to bioluminescence micro-scopy of luciferase expressing cell lines. The reportedchemiluminescent structure may play a similar role forchemiluminescence microscopy as fluorescein has played forfluorescence microscopy: a highly emissive compound thatcan be tailored to different types of imaging experiments.The tractable synthetic approach and simplicity of thesystem will make it straightforward for other researchers toadopt these techniques. Although it remains to be seen whatadvantages chemiluminescent cellular imaging will haveover more traditional fluorescence microscopy, the low

background and increased imaging depth may be particularlyuseful for imaging tissue slices without a reliance ontwo-photon absorption techniques. At present, imaging inliving cells requires overexpression of a marker enzyme,β-galactosidase, but the gates have been opened and a newday of chemiluminescent microscopy probes can be seen onthe horizon.

The increased tissue imaging depth granted by chem-iluminescence will also have distinct advantages for imagingat the whole animal level, where problems with tissueautofluorescence are particularly limiting. The moleculardesigns lend themselves to further red-shifting of emissionwavelengths, and eliminating the need for polymeric encap-sulation dramatically simplifies experiments and helps toalleviate toxicity concerns. Much of the extensive progress inresponsive fluorescent molecules can be readily translated tochemiluminescent designs. While the focus of this workhas been on biological imaging, the ability to make bright,tunable, and responsive chemiluminescent molecules couldfind applicability in a range of disciplines from optical displaysto photonic logic gates, portending the empowering centralityof this important chemical advance.

■ Author InformationE-mail: alippert@mail.smu.edu.

■ REFERENCES(1) Green, O.; Eilon, T.; Hananya, N.; Gutkin, S.; Bauer, C. R.;Shabat, D. Opening a Gateway for Chemiluminescence Cell Imaging:Distinctive Methodology for Design of Bright ChemiluminescentDioxetane Probes. ACS Cent. Sci. 2017, DOI: 10.1021/acscents-ci.7b00058.(2) Lavis, L. D.; Raines, R. T. Bright Building Blocks for ChemicalBiology. ACS Chem. Biol. 2014, 9, 855−866.(3) Jones, K. A.; Porterfield, W. B.; Rathbun, C. M.; McCutcheon, D.C.; Paley, M. A.; Prescher, J. A. Orthogonal Luciferase−Luciferin Pairsfor Bioluminescence Imaging. J. Am. Chem. Soc. 2017, 139, 2351−2358.(4) Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.;Petros, J.; Taylor, W. R.; Murthy, N. In vivo imaging of hydrogenperoxide with chemiluminescent nanoparticles. Nat. Mater. 2007, 6,765−769.(5) Baumes, J. M.; Gassensmith, J. J.; Giblin, J.; Lee, J. J.; White, A. G.;Culligan, W. J.; Leevy, W. M.; Kuno, M.; Smith, B. D. Storable, thermallyactivated, near-infrared chemiluminescent dyes and dye-stained micro-particles for optical imaging. Nat. Chem. 2010, 2, 1025−1030.

Figure 2. Chemiluminescent probes (top left) are 3000 times brighterthan luminol (top right), allowing for the first time nonenzymaticchemiluminescent microcopy (bottom right, bright field image bottomleft). Reproduced with permission from ref 1. Copyright 2017American Chemical Society.

The reported chemiluminescentstructure may play a similar rolefor chemiluminescence micros-copy as fluorescein has played forfluorescence microscopy: a highlyemissive compound that can betailored to different types of

imaging experiments

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REFERENCES

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(6) Schaap, A. P.; Chen, T.-S.; Handley, R. S.; DeSilva, R.; Giri, B. P.Chemical and enzymatic triggering of 1,2-dioxetanes. 2: Fluoride-induced chemiluminescence from tert-butyldimethylsilyloxy-substi-tuted dioxetanes. Tetrahedron Lett. 1987, 28, 1155−1158.(7) Matsumoto, M.; Watanabe, N. Structural Aspects of 1,2-Dioxetanes Active toward Intramolecular Charge-Transfer-InducedChemiluminescent Decomposition. Bull. Chem. Soc. Jpn. 2005, 78,1899−1920.(8) Augusto, F. A.; de Souza, G.; de Souza Junior, S. P.; Khalid, M.;Baader, W. J. Efficiency of Electron Transfer Initiated Chemilumi-nescence. Photochem. Photobiol. 2013, 89, 1299−1317.(9) Cao, J.; Campbell, J.; Liu, L.; Mason, R. P.; Lippert, A. R. In VivoChemiluminescent Imaging Agents for Nitroreductase and TissueOxygenation. Anal. Chem. 2016, 88, 4995−5002.(10) Hananya, N.; Eldar Boock, A.; Bauer, C. R.; Satchi-Fainaro, R.;Shabat, D. Remarkable Enhancement of Chemiluminescent Signal byDioxetane−Fluorophore Conjugates: Turn-ON ChemiluminescenceProbes with Color Modulation for Sensing and Imaging. J. Am. Chem.Soc. 2016, 138, 13438−13446.

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