Justus Hermannsdörfer

The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.

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.

Anisotropic Shape Changes of Silica Nanoparticles Induced in Liquid with Scanning Transmission Electron Microscopy

Liquid-phase transmission electron microscopy (TEM) is used for in-situ imaging of nanoscale processes taking place in liquid, such as the evolution of nanoparticles during synthesis or structural changes of nanomaterials in liquid environment. Here, it is shown that the focused electron beam of scanning TEM (STEM) brings about the dissolution of silica nanoparticles in water by a gradual reduction of their sizes, and that silica redeposites at the sides of the nanoparticles in the scanning direction of the electron beam, such that elongated nanoparticles are formed. Nanoparticles with an elongation in a different direction are obtained simply by changing the scan direction. Material is expelled from the center of the nanoparticles at higher electron dose, leading to the formation of doughnut-shaped objects. Nanoparticles assembled in an aggregate gradually fuse, and the electron beam exposed section of the aggregate reduces in size and is elongated. Under TEM conditions with a stationary electron beam, the nanoparticles dissolve but do not elongate. The observed phenomena are important to consider when conducting liquid-phase STEM experiments on silica-based materials and may find future application for controlled anisotropic manipulation of the size and the shape of nanoparticles in liquid.

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.

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

Visualizing the Distribution and Stoichiometry of Growth Factor Receptors in Intact Cells in Liquid Phase with Correlative Fluorescence and Scanning Transmission Electron Microscopy

The epidermal growth factor receptor (EGFR) family consists of four similar members that play important roles in various cellular processes such as cell proliferation and survival. Over-expression of these proteins, especially EGFR and HER2, is involved in many types of cancer. After activation by its ligand EGF, activated EGFR proteins can form homodimers, thus activating specific signaling pathways for cell growth. The HER2 is considered as an orphan receptor because it needs no ligand to form homodimers, thereby contributing significantly to uncontrolled intracellular signaling. Heterodimers are also formed in some cases.

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.

Radiation Damage of Biological Specimen in Environmental Electron Microscopy

Proteins labeled with nanoparticles can be studied in their native context of the plasma membrane in intact cells in liquid using scanning transmission electron microscopy (STEM) [1,2]. One approach is the usage of a microfluidic chamber enclosing the cell entirely in liquid and imaging at 200 keV beam energy [1]. An alternative approach is to study the cell maintained under a thin liquid layer using environmental scanning electron microscopy (ESEM) at 30 keV [2]. An important question is to which extent the effect of electron beam induced radiation damage changes the structure of cells in liquid.