Improved diffuse fluorescence flow cytometer prototype for high sensitivity detection of rare circulating cells in vivo

Abstract. Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×103 cells/mL. In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo

An authentic imaging probe to track cell fate from beginning to end

Accurate tracing of cell viability is critical for optimizing delivery methods, and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. Upon incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently, and the first color is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second color appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively

Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration

Summary Post-natal skeletal stem cells expressing PRX1 (pnPRX1+) have been identified in the calvaria and in the axial skeleton. Here we characterize the location and functional capacity of the calvarial pnPRX1+ cells. We found that pnPRX1+ reside exclusively in the calvarial suture niche and decrease in number with age. They are distinct from preosteoblasts and osteoblasts of the sutures, respond to WNT signaling in vitro and in vivo by differentiating into osteoblasts, and, upon heterotopic transplantation, are able to regenerate bone. Diphtheria toxin A (DTA)-mediated lineage ablation of pnPRX1+ cells and suturectomy perturb regeneration of calvarial bone defects and confirm that pnPRX1+ cells of the sutures are required for bone regeneration. Orthotopic transplantation of sutures with traceable pnPRX1+ cells into wild-type animals shows that pnPRX1+ cells of the suture contribute to calvarial bone defect regeneration. DTA-mediated lineage ablation of pnPRX1+ does not, however, interfere with calvarial development

Direct measurement of local oxygen concentration in the bone marrow of live animals

Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment

Significant quantum effects in hydrogen activation

Dissociation of molecular hydrogen is an important step in a wide variety of chemical, biological, and physical processes. Due to the light mass of hydrogen, it is recognized that quantum effects are often important to its reactivity. However, understanding how quantum effects impact the reactivity of hydrogen is still in its infancy. Here, we examine this issue using a well-defined Pd/Cu(111) alloy that allows the activation of hydrogen and deuterium molecules to be examined at individual Pd atom surface sites over a wide range of temperatures. Experiments comparing the uptake of hydrogen and deuterium as a function of temperature reveal completely different behavior of the two species. The rate of hydrogen activation increases at lower sample temperature, whereas deuterium activation slows as the temperature is lowered. Density functional theory simulations in which quantum nuclear effects are accounted for reveal that tunneling through the dissociation barrier is prevalent for H2 up to ∼190 K and for D2 up to ∼140 K. Kinetic Monte Carlo simulations indicate that the effective barrier to H2 dissociation is so low that hydrogen uptake on the surface is limited merely by thermodynamics, whereas the D2 dissociation process is controlled by kinetics. These data illustrate the complexity and inherent quantum nature of this ubiquitous and seemingly simple chemical process. Examining these effects in other systems with a similar range of approaches may uncover temperature regimes where quantum effects can be harnessed, yielding greater control of bond-breaking processes at surfaces and uncovering useful chemistries such as selective bond activation or isotope separation

Probing the composition of unexposed basement, South Portuguese Zone, southern Iberia: implications for the connections between the Appalachian and Variscan orogens

Geochemistry and Sm–Nd and U–Pb (magmatic zircon) isotope data from a postcollisional batholith that crosscuts the allochthonous South Portuguese Zone (SPZ) of southern Iberia suggest that the basement is compositionally more juvenile than the exposed upper crust. The SPZ is an allochthonous terrane of the late Paleozoic Variscan orogen. The oldest exposed units in the SPZ are Late Devonian continental clastics, and as a result, the origins of the SPZ are unknown. Multifaceted inherited zircon cores from a granitoid batholith (Sierra Norte Batholith, SNB) reveal Neoproterozoic (ca. 561–647 Ma) and Mesoproterozoic ages (ca. 1075 – ca. 1116 Ma). Granitoid samples are characterized by εNd values ranging from +1.4 to –9.6 and model ages ca. 0.76–1.8 Ga. Conversely, the exposed Late Devonian clastics of the SPZ are characterized by more negative εNd values (–7.5 to –10.4). Taken together, U–Pb and Sm–Nd data indicate the lower crust that melted to yield the SNB was (i) Neoproterozoic (ca. 560–650 Ma) to Mesoproterozoic (ca. 1.0–1.2 Ga) in age, (ii) was not compositionally similar to the overlying Devono-Carboniferous continental detritus but was instead more juvenile, with model ages between ca. 0.9–1.2 Ga. This unusual relationship is similar to the relationship between the relatively juvenile basement and ancient upper crust documented in the exposed portion of the Meguma terrane in the northern Appalachians, which paleogeographic reconstructions show was immediately outboard of southern Iberia in the Late Devonian.Department of Earth Sciences, Saint Francis Xavier University, CanadáInstituto Geológico y Minero de España, EspañaDepartment of Earth and Ocean Sciences, The University of British Columbia, Canad