Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry

A Geochronologic Study of a Granite Pluton from the Llano Uplift, Texas

Granite and related pegmatite and aplite from several localities within a pluton from the Llano uplift, Texas, are being studied geochronologically. Biotite, muscovite, hornblende, microcline, plagioclase, quartz, apatite, and fluorite have been analyzed by refined chemical and mass spectrometric methods in order to determine the consistency in ages between various minerals and between different localities within an individual pluton. Field and petrologic evidence suggests that this intrusive has had a simple history of emplacement and no later metamorphism. Quadruplicate analyses on a master biotite yield K-Ar and Rb-Sr ages reproducible to 1 per cent. In most cases K-Ar determinations on biotites, muscovites, and hornblendes and Rb-Sr determinations on biotites, muscovites, and microclines give ages which fall within a 5 per cent spread. A half-life of 1.307 X 10^9 years and a branching ratio of 0.124 are used to calculate the K-Ar ages, and Rb-Sr ages are calculated with a half-life of 50 X 10^9 years. The average age of the pluton thus determined is 1060 m.y. K-Ar determinations on microclines give ages which are 5 to 15 per cent lower. One plagioclase from the granite gives a K-Ar age of 920 m.y. Two suites of biotites, one from pegmatites and one from a border facies of the granite, give anomalously low Rb-Sr ages. The pegmatitic biotite also has a somewhat low K-Ar age; however, the biotite from the granite gives a normal age by this method, as do coexisting microclines from both these rocks by the Rb-Sr method. Geologic evidence suggests that meteoric or hydrothermal fluids may have been responsible for this age discrepancy. Ages determined on a gneiss, a pegmatite cutting the gneiss, and a granite porphyry all give results approximately equal to those of the granite. No evidence of a significantly older basement rock or a younger igneous or metamorphic event in the area has been obtained to date

Helium, argon, and carbon in some natural gases

Thirty-nine samples of natural gases representing varied chemical compositions and geological occurrences were analyzed for their helium, radiogenic argon, and atmospheric argon contents. The total range in the (He/A)_(rad) ratio was found to be 1.6 to 130 with most samples having values between 6 and 25. This range of values is essentially equal to the production ratio from the uranium, thorium, and potassium in average igneous rocks and a wide variety of sediments. This indicates that all of these natural gases have obtained their radiogenic gases from rather average rock types. This is true in spite of the fact that the gases range in helium content from 37 to 62,000 ppm. A theoretical discussion of the origin of helium and argon in natural gases is given. It can be shown from the ratio of nitrogen to atmospheric argon that most of the nitrogen in these gases cannot come from the entrapment of air. From a consideration of the concentration of atmospheric argon in natural gases it is possible to estimate the proportion of gaseous and aqueous phases assuming diffusive equilibrium. The isotopic composition of the carbon in the methane of these gases was found to be very light. It was shown that for coexisting CH_4-CO_2 pairs the carbon dioxide was always isotopically heavier

Insights on the systematics and morphology of Humiriaceae (Malpighiales): Androecial and extrafloral nectary variation, two new combinations, and a new Sacoglottis from Guyana

Humiriaceae have had little recent comparative morphological study except for their distinctive fruits. We surveyed the diversity of stamen structures in the family with consideration of dehiscence patterns and the evolutionary transitions between tetra- and disporangiate anthers. Novel interpretations of floral morphology support new combinations (Duckesia liesneri K.Wurdack & C.E.Zartman, comb. nov. and Vantanea spiritu-sancti K.Wurdack & C.E.Zartman, comb. nov.) for two species formerly in Humiriastrum. We investigated all eleven species of Sacoglottis for diagnostic features that may contribute to better species delimitations, and describe Sacoglottis perryi K.Wurdack & C.E.Zartman, sp. nov. as an endemic of the Pakaraima Mountains in western Guyana. Finally, our survey across Humiriaceae for extrafloral nectaries (EFNs) revealed their presence on leaves of all extant species as adaxial basilaminar and/or abaxial embedded glands, in addition to the frequent occurrence of marginal glandular setae. The significance of inter-generic variation in gland position and anther morphology within the family are discussed. © K.J. Wurdack, C.E. Zartman

Microfluidics on the fly: Inexpensive rapid fabrication of thermally laminated microfluidic devices for live imaging and multimodal perturbations of multicellular systems

Microfluidic devices provide a platform for analyzing both natural and synthetic multicellular systems. Currently, substantial capital investment and expertise are required for creating microfluidic devices using standard soft-lithography. These requirements present barriers to entry for many nontraditional users of microfluidics, including developmental biology laboratories. Therefore, fabrication methodologies that enable rapid device iteration and work “out-of-the-box” can accelerate the integration of microfluidics with developmental biology. Here, we have created and characterized low-cost hybrid polyethylene terephthalate laminate (PETL) microfluidic devices that are suitable for cell and micro-organ culture assays. These devices were validated with mammalian cell lines and the Drosophila wing imaginal disc as a model micro-organ. First, we developed and tested PETLs that are compatible with both long-term cultures and high-resolution imaging of cells and organs. Further, we achieved spatiotemporal control of chemical gradients across the wing discs with a multilayered microfluidic device. Finally, we created a multilayered device that enables controllable mechanical loading of micro-organs. This mechanical actuation assay was used to characterize the response of larval wing discs at different developmental stages. Interestingly, increased deformation of the older wing discs for the same mechanical loading suggests that the compliance of the organ is increased in preparation for subsequent morphogenesis. Together, these results demonstrate the applicability of hybrid PETL devices for biochemical and mechanobiology studies on micro-organs and provide new insights into the mechanics of organ development

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ's final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal–distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue

Robust cell tracking in epithelial tissues through identification of maximum common subgraphs

Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterizing cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a ‘maximum common subgraph’ to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as phototoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell–cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2-fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade