Emission Ratiometric Imaging of Intracellular Zinc:  Design of a Benzoxazole Fluorescent Sensor and Its Application in Two-Photon Microscopy

Abstract

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.

Selective fluorescent probes for live-cell monitoring of sulphide

“…The development of innovative fluorescent imaging probes has revolutionized cell biology, allowing localization and dynamic monitoring of cellular metabolite and inorganic ion pools 15,16,[24][25][26][27][28][29] . A significant bottleneck in the emerging field of H 2 S/aqueous sulphide signalling is the absence of technology for effective in vivo detection and imaging, a problem that is exacerbated by the high intracellular thiol concentration.…”

Nutrient-gene interactions: a single nutrient and hundreds of target genes

“…To which extent an experimental zinc deficiency reduces the concentration of available zinc for coordination in Zn fingers is not known. Concentrations of free zinc in cells are difficult to determine but it has been suggested that they may vary between 10 y9 and 10 y4 M (Taki et al, 2004). Zinc dissociation constants reported for naturally occurring zinc sites in various transcription factors fall in the range of 10 y11 to 10 y9 M and nuclear concentrations were proposed not to drop below 10 y12 M (Payne et al, 2003).…”

Enhanced Two‐Photon Absorption of Organic Chromophores: Theoretical and Experimental Assessments

“…For example, it enables in vivo imaging of calcium dynamics [20,69,70] or intracellular zinc. [71,72] Carrying out two-photon instead of conventional one-photon excitation offers number of advantages. These include highly spatially confined excitation, three dimensional resolution, increased penetration depth in tissues, in particular thanks to reduced scattering losses, and reduced photodamage owing to excitation in the visible red-NIR region (typically 700-1200nm) as well as improved signal-to-noise ratio due to reduced background fluorescence.…”