Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Synchrotron X-ray fluorescence (SXRF) microtomography has emerged as a powerful technique for the 3D visualization of the elemental distribution in biological samples. The mechanical stability, both of the instrument and the specimen, is paramount when acquiring tomographic projection series. By combining the progressive lowering of temperature method (PLT) with femtosecond laser sectioning, we were able to embed, excise, and preserve a zebrafish embryo at 24 hours post fertilization in an X-ray compatible, transparent resin for tomographic elemental imaging. Based on a data set comprised of 60 projections, acquired with a step size of 2 μm during 100 hours of beam time, we reconstructed the 3D distribution of zinc, iron, and copper using the iterative maximum likelihood expectation maximization (MLEM) reconstruction algorithm. The volumetric elemental maps, which entail over 124 million individual voxels for each transition metal, revealed distinct elemental distributions that could be correlated with characteristic anatomical features at this stage of embryonic development.

Full elucidation of the functions and homeostatic pathways of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as Cu . Buffered at attomolar concentrations, intracellular Cu is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, we reveal a chelator design strategy in which phosphine sulfides aid in Cu coordination while simultaneously stabilizing aliphatic phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissociation constant and 10 -fold selectivity for Cu over Zn , Fe , and Mn . As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring Cu in a broad range of biological systems.

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs. diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound fluorescent proteins.

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 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.