A. Cameron Varano

Real-time imaging of lead nanoparticles in solution – determination of the growth mechanism

In situ electron microscopy is a tool which offers great promise for studying the mechanisms responsible for nanoparticle growth. In aqueous solution, the reduction of Pb2+ by the electron beam results in the formation of lead nanoparticles. Here, we directly examined the fundamental processes that influence the growth of lead nanoparticles in solution using in situ transmission electron microscopy. Lead nanoparticle growth was directly monitored at the molecular level and followed the sequence of nucleation, Ostwald ripening, and aggregative growth. The aggregative growth phase resulted in macrostructures having micron-sized dimensions. Importantly, when combined with quantitative measurements, our direct imaging results suggested that the growth properties observed for lead nanoparticles were unique in comparison to other metallic entities.

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

Real-time Imaging of Protein Therapeutics Using Liquid Cell EM

Lynn M. DiMemmo1, A. Cameron Varano2, Jonathan Haulenbeek3, Madeline J. Dukes4, Steven P. Piccoli3 and Deborah F. Kelly2. . 1. Drug Product Science and Technology, Bristol-Myers Squibb Company, USA. 2. Virginia Tech Carilion Research Institute, Virginia Tech, Roanoke, VA 24016, USA. 3. Analytical and Bioanalytical Operations, Bristol-Myers Squibb Company, USA. 4. Application Science, Protochips, Inc., Raleigh, NC 27606, USA.

In Situ Liquid Cell Electron Microscopy: An Evolving Tool for Biomedical and Life Science Applications

During the last decade, Liquid Cell-Transmission Electron Microscopy (LC-TEM) has proved to be an increasingly valuable tool in the microscopists repertoire. LC-TEM techniques involve sandwiching a thin layer of liquid between two ultrathin electron transparent membranes, typically either silicon nitride or graphene. These membranes are contained within specially designed sample holders that hermetically seal the contained liquid from the vacuum of the TEM column. Thus, researchers have been able to observe dynamic processes and behaviors at nanoscale resolutions in their native liquid environment and take advantage of the powerful analytical tools available to the electron microscope [1]. Commercially available LC-TEM holders have an increasingly sophisticated suite of options available, including the capability to heat and maintain a given temperature or deliver electrical stimuli directly to the sample. During real-time imaging, processes such as particle growth, interactions, and fine movements can be observed at the nanoscale. Already widely embraced by the materials fields to observe nanomaterial properties and electrochemical mechanisms, LC-TEM also represents a growing opportunity to transform our understanding of many biological events.

Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy

Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

Quantitative Analysis of Viral Nanomachines in Liquid

Currently, there remains a critical need to develop innovative techniques for visualizing active biological processes at the nanoscale. In situ Transmission Electron Microscopy (TEM) provides the technical framework to address this critical need. Using a liquid cell platform, we developed a unique strategy to illuminate the dynamic movements of viral pathogens. We used rotavirus as a model system for these analyses due its established size, features, and chemically inducible enzymatic activity in vitro.