Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes

• Published in: ACS sensors Vol. 3; no. 2; pp. 458 - 467
• Main Authors: Bourassa, Daisy, Elitt, Christopher M, McCallum, Adam M, Sumalekshmy, S, McRae, Reagan L, Morgan, M. Thomas, Siegel, Nisan, Perry, Joseph W, Rosenberg, Paul A, Fahrni, Christoph J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.02.2018
• Subjects: Animals; Cell Differentiation; Cells, Cultured; Coordination Complexes - analysis; Coordination Complexes - chemistry; Cytoplasm - chemistry; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Mice; Microscopy, Fluorescence, Multiphoton; NIH 3T3 Cells; Oligodendroglia - chemistry; Pyridines - chemical synthesis; Pyridines - chemistry; Single-Cell Analysis; Spectrometry, Fluorescence; Zinc - analysis; Zinc - chemistry; zinc homeostasis; fluorescent probe; live cell imaging; neuroscience; two-photon excitation microscopy
• ISSN: 2379-3694; 2379-3694
• DOI: 10.1021/acssensors.7b00887

Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor–acceptor fluorophore architecture, we developed a membrane-permeant, Zn­(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn­(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a K d of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn­(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn­(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.