| Autor: |
Bourassa D; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Elitt CM; Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States., McCallum AM; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Sumalekshmy S; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., McRae RL; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Morgan MT; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Siegel N; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Perry JW; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Rosenberg PA; Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States., Fahrni CJ; School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States. |
| Abstrakt: |
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. |
