Cross-Topic (Biological & Physical Sciences) Tutorial
X40 – Need for Speed: Imaging Biological Samples with the 64-Beams FAST-EM
Speakers: Jacob Hoogenboom Group, Delft University of Technology, The Netherlands
Large scale and volume electron microscopy (EM) has revolutionized the understanding of biological systems across different spatial scales, but the low throughput of EM limits further progress. In this tutorial, we will illustrate the working principles behind the FAST-EM, a novel multibeam scanning electron microscope with a footprint similar to regular single-beam SEMs. We will present results of faster imaging with FAST-EM and discuss sample preparation, imaging parameters, detection techniques, image post-processing and data analysis.
Physical Sciences Tutorial
X41 – Specimen Preparation for in-situ Transmission Electron Microscopy Experiments
Speaker: Sriram Vijayan, The Ohio State University
Micro electro mechanical system (MEMS)-based in situ heating devices have enabled the observation of thermally activated phenomena under high spatial resolution and low specimen drift inside the transmission electron microscope (TEM). These dynamic processes provide mechanistic insights on phase transformations, which are critical to understanding process-structure-property relationships in materials. These MEMS based devices are used to study materials systems ranging from nano-particulate samples dispersed across the sample support membrane to thin-foils extracted from site-specific locations in bulk materials via focused ion beam scanning electron microscope. The former is relatively straight forward, however, the latter has proven to be extremely challenging. In this tutorial, a review of different specimen preparation techniques for in situ TEM experiments will be discussed, in addition to, a ‘step by step’ guide for FIB based specimen preparation for MEMS-based in situ TEM heating experiments. The talk will also cover some important tips and tricks of specimen preparation for a wide array of materials systems and MEMS devices.
X42 - Biological Sciences Tutorial
CryoAPEX: Inception, Growth and Evolution of the Method
Speaker: Ranjan Sengupta, Angiex Inc.
The application of electron tomography in the functional dissection of membrane remodeling within the subcellular space requires localization of candidate proteins in 3D space. The precise localization of membrane proteins at nanometer resolution requires an electron microscopy based robust detection technology coupled with sample preparation that confer superior ultrastructural preservation. Here, I provide step-by step detail of our method, cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. The superior membrane preservation obtained by the cryoAPEX method makes it amenable to electron tomography, arming the versatile APEX tagging technology to answer questions in the realms of organelle biogenesis and in situ membrane remodeling. Question based evolution of the method in its application in virus cell biology will be discussed.
- Understanding APEX2 tagging in the context of the functional preservation of candidate protein
- APEX2 based localization of membrane protein and membrane interacting proteins
- Ultrastructural preservation of cells using hybrid chemical and cryofixation
- Marrying APEX2 tagging with electron tomography: a way to localize proteins in 3D space within the cellular ultrastructure