Journal
ACS SENSORS
Volume 3, Issue 2, Pages 458-467Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.7b00887
Keywords
fluorescent probe; two photon excitation microscopy; zinc homeostasis; live cell imaging neuroscience
Funding
- National Science Foundation [CHE-1626172, CHE-1306943]
- National Institutes of Health [DK68096, GM067169, HD018655, EY024481]
- NINDS [NS079414]
- Baby Alex Foundation
- National Multiple Sclerosis Foundation
- William Randolph Hearst Foundation
- Genise Goldenson Research Award
- [NS070682]
- EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT [P30HD018655, U54HD090255] Funding Source: NIH RePORTER
- NATIONAL EYE INSTITUTE [R01EY024481] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK068096] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM067169] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R25NS070682] Funding Source: NIH RePORTER
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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.
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