Invited Speakers' Abstracts

Structures of Zika virus captured during maturation and host recognition using cryo-EM

Geeta Buda1, Zhenguo Chen1, James E. Crowe2, Michael S. Diamond3, Estefania Fernandez3, Andrei Fokine1, S. Saif Hasan1, Thomas Klose1, Richard J. Kuhn1, Wen Jiang1, Feng Long1, Vidya Mangala Prasad1, Andrew S. Miller1, Theodore C. Pierson4, Jason C. Porta1, Michael G. Rossmann1, Gopal Sapparapu3, Devika Sirohi1, and LeiSun1

1Purdue University,2Vanderbilt University,3Washington University in St. Louis, 4NIH

The structures of the immature Zika virus, the mature Zika virus and the mature Zika virus complexed with a neutralizing antibody have been determined by cryo-electron microscopy to 3.8Å, 9Å and 6.2Å resolution, respectively. The biggest structural difference between Zika virus and dengue virus (another flavivirus) occurs at a glycosylated Asn residue that is probably a site where the virus can attach to a cellular receptor. The immature Zika virus structure shows vestiges of a viral capsid inside the viral membrane, not seen in other flaviviruses. A number of neutralizing antibodies are being studied when in complex with the virus. One of these neutralizes by cross-linking the surface glycoproteins. Because the three independent Fab attachment sites are closer together than the diameter of the Fab molecules, not all sites can be occupied simultaneously. This reduces the concentration of the antibody required for neutralization, thus increasing the antibody’s potency.

Overcoming resolution vs dose barriers in Electron Microscopy; A look at techniques that can be employed to maximize the efficiency and resolution of data collection

Jan Ringnalda, Thermo Fisher Scientific

Achieving High Resolution images and analytical resolution has been a goal for many Materials Scientists over the last few decades. Instrumentation has advanced to a level where any sample has a limited life time when examined in a modern corrected electron microscope system. So there has been much effort spent on optimizing the sample, optimizing the detectors both for imaging and elemental analysis in order to minimize the dose levels needed to collect images and data at the highest resolution. This talk will detail some of the methods used to achieve the best compromise.

Improving Our Vision of Nanobiology

Deborah F. Kelly, Ph.D.

Associate Professor Virginia Tech Carilion Research Institute Roanoke, VA 24016, USA

Understanding the properties of macromolecules is a common goal of physicians and engineers. Electron microscopes (EMs)are used to directly view the intricate details of molecular entities at the nanoscale. The revolution in EM phase-plates, direct electron detectors, and in-column energy filters offers premiere technology to record pristine images of weak-phase objects. Correspondingly, the next generation of EM substrates must also  be  developed  to  best  utilize  these  new  tools.As  new materials  are evolving worldwide, there  is a  prime  opportunity to  use alternative substrates  in  the  EM  field. Such materials include graphene films and silicon nitride microchips (cryo-SiN). One recent example is the use of cryo-SiN to trap active biological assemblies while preserving them for cryo-EM and liquid cell imaging. By decorating microchips or other  substrates with  specific  adaptor  molecules, we  create versatile tunable devices. We have recently used these devices to examine 3D structures of breast cancer assemblies isolated from human cancer cells (Fig. 1). With these new tools in hand, we are uniquely poised to peer into the active world of molecules and cells as never before.

Figure 1. Unlocking BRCA1 – Cracking the breast cancer code with state-of-the-art imaging.

Seeing is Believing:  Visualizing Active Nanoscale Processes in the TEM

Madeline J. Dukes

Protochips

In situ and operando microscopy has become an indispensable technique for researchers across many different disciplines. These techniques enable the transmission electron microscope (TEM) to function as a real-time nano-laboratory by providing the ability to observe dynamic processes such as growth or phase transformation in non-vacuum environments.  These experiments typically require dedicated sample holders and specimen supports which enable the user to control the environment of the sample and/or deliver stimuli directly to the specimen. Designed around semiconductor technology, Protochips' suite of in situ systems provide features which enable the end user to perform a variety of in situ and operando experiments spanning applications from basic materials characterization to batteries, catalysts and life sciences. Here, we highlight the features of, and applications from the Protochips' family of in situ systems:  Fusion (temperature, electrical and electro-thermal), Poseidon (liquid and electrochemistry) and Atmosphere (gas).  The robust features and straightforward design of the Protochips systems enables users to take advantage of the latest innovations of in situ research and explore the dynamic nanoscale world around them.

New sciences enabled by cryo-electron microscopyin the physical sciences

Lena F. Kourkoutis

School of Applied and Engineering Physics, Cornell University, Ithaca, NY, USA.

Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY, USA.

Recent advances in electron  microscopy  have  opened  a  new  era of  atomic  resolution  imaging and spectroscopy inside solids which can be studied at room temperature, in the vacuum of the electron microscope. Liquid/solid interfaces have yet to be imaged at high spatial resolution, but play a critical role in a  range  of  biological,  chemical  and  physical  processes  from  catalysis  to electrochemical energy storage to the formation of biominerals. Liquids and many soft materials including biological structures require them to be kept in a hydrated  state  to  ensure  structural preservation.  Vitrification by rapid freezing preserves  the  specimen  in  its  near-native,  hydrated environment  and  allows  thin  biological  specimens  to  be  studied  at near  atomic resolution  in  a cryo-electron microscope–a technique that has been awarded the Nobel Prize in Chemistry this year. In this talk, I will discuss applications in the physical sciences where cryo-EM could have similarly transformative impact. Operating close to liquid nitrogen temperature gives access to a range of emergent electronic states in solid materials, allows us to study processes at liquid/solid interfaces immobilized  by  rapid  freezing and  the  formation  pathways  of  nanostructures  in  solution. I will discuss our approach to study two processes at the anode-electrolyte interface in lithium metal batteries,  uneven  deposition  of  lithium  metal  leading  to  dendrite  growth  and  the  breakdown  of electrolyte to form a “solid-electrolyte interphase” (SEI) layer, processes which result in capacity fade  and  safety  hazards.  By  combining  cryo-scanning  transmission  electron  microscopy  and spectroscopy with cryo-FIB lift out, we provide nanoscale compositional information about intact SEI layers in cycled lithium metal batteries and track local bonding states at interfaces, leading to new insights into SEI and dendrite formation.

Recent instrumentation advances offer new opportunities in electron microscopy of polymers

Enrique Gomez

Penn State

Imaging of polymers by transmission electron microscopy (TEM) or scanning transmission electron microscopy (STEM) remains a challenge due to the low contrast between domains and sensitivity to the electron beam.  Recent advances in instrumentation for electron microscopy have aimed to push the resolution limit, leading to remarkable instruments capable of imaging at 0.5 Å.  But, when imaging soft materials, the resolution is often limited by the amount of dose the material can handle rather than the instrumental resolution.  Despite the strong constraints placed by radiation sensitivity, recent developments in electron microscopes have the potential to advance polymer electron microscopy.  For example, monochromatated sources enable spectroscopy and imaging based on the valence electronic structure, aberration correctors enable imaging of thick films, direct electron detectors minimize the required dose for imaging, and differential phase contrast imaging can map heterogeneities in electric fields within films.  Altogether, the field of polymer electron microscopy is poised to make significant advances in the near future.

Advantage of Direct Detection and Electron Counting for Electron Energy Loss Spectroscopy Data Acquisition and the Quest of Extremely High-Energy Edges Using EELS

Paolo Longo1, Jamie L. Hart2, Andrew C. Lang2, Ray D. Twesten1 and Mitra L. Taheri2

1Gatan Inc., 5794 W Las Positas Blvd, Pleasanton CA, 94588, USA

2Department of Materials Science and Engineering, Drexel University, Philadelphia PA, 19104, USA

Transmission electron microscopes primarily employ indirect cameras (IDC) for electron detection in imaging, diffraction and EELS modes. Such cameras convert incident electrons to photons which, through a fiber optic network or lens, are coupled to a light sensitive camera. This indirect detection method typically has a negative impact on the point spread function (PSF) and detective quantum efficiency (DQE) of the camera. Over the last decade, radiation tolerant CMOS active pixel sensors, which directly detect high-energy incident electrons and have the speed to count individual electrons events, have been developed. These detectors result in greatly improved PSF and DQE in comparison to conventional IDCs. Such direct detection cameras (DDCs) have revolutionized the cryo-TEM field as well as have strong advantages for in-situ TEM in both imaging and diffraction applications. EELS applications can benefit from the improved PSF and the ability to count electrons. The improved PSF allows spectra to be acquired over larger energy ranges while maintaining sharp features and greatly reduced spectral tails. The ability to count electrons nearly eliminates the noise associated with detector readout and greatly reduces the proportional noise associated with detector gain variations. This effectively leaves the shot noise as the limiting noise source present. The implication for EELS acquisition is that fine structure analysis becomes more straightforward for typical conditions and even possible for the case of low signal levels.

As example of the advantages due the reduced noise and PSF, Figures 1 show the EELS spectra extracted from the Ti L2,3-edges at 456eV and Sr L2,3-edges at 1940eV. Here both the DD and IDC detectors were set in such way that the resulting energy range is about 2000eV and this can be achieved specifically with a dispersion of 1eV/channel in the case of IDC and 0.5eV/channel for DD. The DD detector is made of 4k x 4k pixels, hence the use of dispersion 0.5eV/channel to generate a spectrum with an energy range of 2000eV. With such high energy range both the Ti L- and Sr L-edges can be collected in the same spectrum. In the case of DD, given the much reduced PSF, the energy resolution is such that the eg and t2g peaks in both L2 and L3 edges can be easily resolved and show that the Ti is in 4+ oxidation state. In the case of IDC, dispersion 1eV/channel does not allow enough energy resolution to resolve all the features in the Ti L and identify the oxidation state. Higher dispersion would be needed in order to cope with the high PSF and give enough energy resolution for chemical analysis. The much lower noise of the DD allows the collection of a spectrum where both the L2 and L3 edges of the Sr L can be easily resolved. In the case of the spectrum extracted using the IDC detector, the amount of noise is such that L2 and L3 edges cannot be resolved as nicely as in the DD case. All these data was acquired using exactly the same experimental conditions across similar areas in the same specimen.

Very high-energy edges have always been very hard or almost impossible to acquire using EELS due to the very limited amount of signal. With the introduction of DD detectors the amount of noise has been enormously reduced and as result low intensity signals can now be observed and detected. Figures 2 show EELS spectra of Cu K and N K-edges at about 9keV and 8.3keV. The spectra can be easily observed and the quality is such that high contrast elemental maps can be generated as shown in Figure 2c. Until now, such high energy edges have been collected using synchrotron based techniques such as XAS with very limited spatial resolution. Now, by acquiring EELS data in counting mode using DD detectors, high energy edges can be collected and their signal mapped out with high spatial resolution. A new world is about to open up.

In this presentation, we will review the current state of electrons counting detectors for electron microscopy with an emphasis on system for EELS measurements.

Figures 1a,b EELS spectra of Ti L- and Sr-edges at 456eV and 1940eV extracted from the same area in the specimen under the same experimental conditions using both the DD and IDC detectors. Both the Ti and Sr L signals are extracted from the same spectrum. The spectra extracted using the DD detector show higher energy resolution as well as much reduced noise that allows the clear observation all the main features in the near edge fine structure
Figures 2: a,b) EELS spectra of Cu K and Ni K at 9keV and 8.3keV acquired in STEM mode using the DD detector in counting mode. The signal-to-noise ratio is quite and allows the clear observation. C) EELS elemental maps of Ni K in red and Cu K in green. This proves that such high energy edges can be collected, observed and the signals extracted generating high-contrast elemental maps in STEM mode.