Andreas Verch

Exceptionally Slow Movement of Gold Nanoparticles at a Solid/Liquid Interface Investigated by Scanning Transmission Electron Microscopy

Gold nanoparticles were observed to move at a liquid/solid interface 3 orders of magnitude slower than expected for the movement in a bulk liquid by Brownian motion. The nanoscale movement was studied with scanning transmission electron microscopy (STEM) using a liquid enclosure consisting of microchips with silicon nitride windows. The experiments involved a variation of the electron dose, the coating of the nanoparticles, the surface charge of the enclosing membrane, the viscosity, and the liquid thickness. The observed slow movement was not a result of hydrodynamic hindrance near a wall but instead explained by the presence of a layer of ordered liquid exhibiting a viscosity 5 orders of magnitude larger than a bulk liquid. The increased viscosity presumably led to a dramatic slowdown of the movement. The layer was formed as a result of the surface charge of the silicon nitride windows. The exceptionally slow motion is a crucial aspect of electron microscopy of specimens in liquid, enabling a direct observation of the movement and agglomeration of nanoscale objects in liquid.

Graphene Liquid Enclosure for Single-Molecule Analysis of Membrane Proteins in Whole Cells Using Electron Microscopy

Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.

Time lapse liquid phase scanning transmission electron microscopy of nanoparticles

Liquid-phase scanning transmission electron microscopy (STEM) is capable of dynamic imaging of processes taking place in liquid at the nanoscale [1]. Examples of such processes are the growth metallic nanoparticles, sintering of nanoparticles, dissolution of nanomaterials in liquid, structural changes of nanomaterials, nanoscale movements in liquid, and self-assembly processes of materials in liquid. The experimental setup typically consists of a liquid cell enclosed between thin and electron transparent windows placed in the vacuum of the electron microscopy. Both closed cells and cells connected to a microfluidic system capable of exchanging the liquid have been reported. Published liquid-phase STEM studies involved a rather wide range of sometimes unexpected phenomena occurring in the liquid. The most important factor is the interaction of the electron beam with the water causing the formation of radiolysis species such as eaq, H, OH, H2, H2O2, H, OH. These species locally change the chemistry of the liquid enclosed within the cell, triggering reactions such as reduction of metal ions. The growth of metallic nanoparticles from the solutions can thus be initiated [2], nanoparticles may instead dissolve [3]. The scanning electron beam of STEM can even be used to induce deposition of nanoscale structures in liquid [4] and change the shapes of nanomaterials in an anisotropic manner [5], so that arbitrary patterning of nanomaterials is possible in principle. When conducting experiments in liquid it is thus crucial to understand the electrochemical effects taking place. Experiments should be designed in such way that the parameters under observation can be extracted from the observed manifold of phenomena.

Graphene Enclosure Facilitates Single-Molecule Analysis of ErbB2 Receptors in Intact, Hydrated Eukaryotic Cells by Electron Microscopy

Although membrane receptors mediate important cellular functions, such as cell growth, division or cell death, techniques for the analysis of their distribution and assembly in the intact cell membrane of fully hydrated cells are limited. For the visual examination of molecular structures, electron microscopy (EM) represents the gold standard. Yet, conventional high resolution EM is based on the investigation of thin, solid sections of cells and typically does not reveal the membrane proteins in intact cells. Light microscopy, on the other hand, does not provide sufficient spatial resolution to resolve the subunits of protein complexes as needed to elucidate their functional state. A new approach is presented by liquidphase electron microscopy [1]. In this study, we introduced a graphene liquid-enclosure to study the assembly of the growth factor receptor ErbB2 in intact, hydrated breast cancer cells by scanning EM (SEM) and scanning transmission EM (STEM). ErbB2 is overexpressed in about 30% of all breast cancer cases and targeted by the antibody trastuzumab [2]. The high number of primary and acquired drug resistance (~ 70%) underlines the need for a reliable biomarker in the clinic, as well as a better understanding of underlying molecular mechanisms [3].

Aqueous ball milling of nacre constituents facilitates directional self-assembly of aragonite nanoparticles of the gastropod Haliotis glabra

A ball-milling approach was developed to investigate the constituents of isolated nacre tablets of the gastropod Haliotis glabra in aqueous suspension without additional chemical additives. The obtained particle mixtures were characterized using X-ray crystallography as well as scanning and transmission electron microscopy. Aragonite nanoparticles retained their crystal structure even after 14 h of ball milling. The long-term stability of the particle mixtures varied as a function of the ball-milling duration. An increased milling time led to rod-like stable assemblies of aragonite nanoparticles. Selected area electron diffraction investigations revealed that the longitudinal axes in about one-third of these nanoparticle rods were oriented along the crystallographic c-axis of aragonite, indicating oriented attachment of the aragonite nanoparticles. These in vitro observations support the idea that a two-stage process, separated into crystallization of nanoparticles and oriented assembly of nanocrystals, could also occur in vivo.

Depth Dependence of the Spatial Resolution in Scanning Transmission Electron Microscopy Experiments

Scanning transmission electron microscopy (STEM) is mostly used for very thin (few tens of nanometers or less) specimens. However, for a range of scientific applications, it is important to image through thicker samples. For example, STEM of specimens in liquid involves thicknesses of a few hundreds of nanometers to several micrometers. Beam broadening occurs when the thickness is larger than the mean free path length for elastic scattering in the materials under investigation. As a result, an electron beam not focused to the top layer (with respect to a downward traveling electron beam) becomes substantially broadened thus reducing the spatial resolution. But also the spatial resolution for objects in the top layer is influenced by the material beneath as the contrast and thus the signal-to-noiseratio decreases with increasing sample thicknesses. Both effects reduce the spatial resolution at a given electron dose in a STEM experiment. The resolution for imaging an object either at the top or at the bottom of thick specimen was determined experimentally, via simulations, and in various analytical models [1]. An important question is what happens for particles at a certain vertical position within a thick specimen. The effect of beam broadening on the resolution in this case was approximated by a preliminary analytical model [2] but not yet verified experimentally. Here, we show beam-broadening data obtained for nanoparticles at certain vertical distances within a thick sample.

The Stability of Gold Nanoparticles in Liquid Scanning Transmission Electron Microscopy Experiments Studied under Varied Conditions

Studying dynamical processes such as the self-assembly of nano-particles in liquid with nanometer resolution is possible using a recently established technology for transmission electron microscopy (TEM) in liquid [1]. The sample containing liquid and solid parts are placed in a microfluidic chamber between two electron transparent membranes. The principal setup is schematically shown in Fig. 1. However, interactions between the liquids and the electron beam dramatically increase the complexity of the system and complicate the analysis of the observed processes. High energy electrons excite electronic states in the specimen and the ample liquid resulting in the generation of highly reactive, transient species such as solvated electrons, hydrogen-, and hydroxide radicals. Their appearance initiates a cascade of reactions in which various other powerful oxidizing and reducing agents are formed. These species are often able to attack the solid specimen chemically, which might change its surface properties or actually dissolve it. In many cases this behavior is not desired and it might even render the experiment useless. In order to design experiments resembling the unperturbed specimen as closely as possible, it is thus essential to improve our understanding of the processes occurring in liquid electron microscopy experiments, and to learn how to reduce the impact of the electron beam on the specimen.