Michael M. Norton

Caenorhabditis-in-drop array for monitoring C. elegans quiescent behavior

STUDY OBJECTIVES To develop a method, called Caenorhabditis-in-Drop (CiD), encapsulating single worms in aqueous drops, for parallel analysis of behavioral quiescence in C. elegans nematodes. DESIGN We designed, constructed, and tested a device that houses an array of aqueous droplets laden with individual worms. The droplets are separated and covered by immiscible, biocompatible oil. We modeled gas exchange across the aqueous/oil interface and tested the viability of the encapsulated animals. We studied the behavior of wild-type animals; of animals with a loss of function mutation in the cGMP-dependent protein kinase gene egl-4; of animals with a loss of function mutation in the gene kin-2, which encodes a cAMP-dependent protein kinase A regulatory subunit; of animals with a gain-of-function mutation in the gene acy-1, which encodes an adenylate cyclase; and of animals that express high levels of the EGF protein encoded by lin-3. MEASUREMENTS AND RESULTS We used CiD to simultaneously monitor the behavior of 24 worms, a nearly 5-fold improvement over the prior best methodology. In support of our gas exchange models, we found that worms remain viable on the chip for 4 days, past the 12-h period needed for observation, but show reduced longevity to that measured on an agar surface. Measurements of duration of lethargus quiescence and total leth-argus quiescence showed reduced amounts as well as reduced variability relative to prior methods. There was reduced lethargus quiescence in animals that were mutant for kin-2 and for acy-1, supporting a wake-promoting effect of PKA in C. elegans, but no change in lethargus quiescence in egl-4 mutants. There was increased quiescence in animals that expressed kin-2 in the nervous system or over-expressed EGF. CONCLUSIONS CiD is useful for the analysis of behavioral quiescence during lethargus as well as during the adult stage C. elegans. The method is expandable to parallel simultaneous monitoring of hundreds of animals and for other studies of long-term behavior. Using this method, we were successful in measuring, for the first time, quiescence in kin-2(ce179) and in acy-2(ce2) mutants, which are hyperactive. Our observations also highlight the impact of environmental conditions on quiescent behavior and show that longevity is reduced in CiD in comparison to agar surfaces.

Controlling nanowire growth through electric field-induced deformation of the catalyst droplet

Semiconductor nanowires with precisely controlled structure, and hence well-defined electronic and optical properties, can be grown by self-assembly using the vapour–liquid–solid process. The structure and chemical composition of the growing nanowire is typically determined by global parameters such as source gas pressure, gas composition and growth temperature. Here we describe a more local approach to the control of nanowire structure. We apply an electric field during growth to control nanowire diameter and growth direction. Growth experiments carried out while imaging within an in situ transmission electron microscope show that the electric field modifies growth by changing the shape, position and contact angle of the catalytic droplet. This droplet engineering can be used to modify nanowires into three dimensional structures, relevant to a range of applications, and also to measure the droplet surface tension, important for quantitative development of strategies to control nanowire growth.

Automated analysis of evolving interfaces during in situ electron microscopy

In situ electron microscopy allows one to monitor dynamical processes at high spatial and temporal resolution. This produces large quantities of data, and hence automated image processing algorithms are needed to extract useful quantitative measures of the observed phenomena. In this work, we outline an image processing workflow for the analysis of evolving interfaces imaged during liquid cell electron microscopy. As examples, we show metal electrodeposition at electrode surfaces; beam-induced nanocrystal formation and dissolution; and beam-induced bubble nucleation, growth, and migration. These experiments are used to demonstrate a fully automated workflow for the extraction of, among other things, interface position, roughness, lateral wavelength, local normal velocity, and the projected area of the evolving phase as functions of time. The relevant algorithms have been implemented in Mathematica and are available online.

Radiolysis during Liquid Cell Electron Microscopy

Recent advances in liquid cell electron microscopy [1, 2] have enabled real time imaging of objects suspended in liquids and processes taking place in liquids with the nanometer resolution of the electron microscope. As ionizing radiation passes through the suspending medium, energy is transferred from the fast-moving electrons to the irradiated medium. This energy excites and dislodges orbital electrons, which results in the generation of radical and molecular species such as H2, O2, H2O2, and hydrated electrons [3-5]. The hydrated electrons, oxidizing agents, and gaseous species can cause, respectively, reduction and precipitation of cations from solution, dissolution of metals, and nucleation and growth of bubbles [6-10]. A quantitative understanding of electron beam-induced effects is critical to assess whether the electron beam significantly affects the imaged phenomenon, to correctly interpret experiments carried out with liquid cells, mitigate unwanted effects, and take advantage of beam effects.