Organizer: Elizabeth Wright, Emory University
Kent McDonald, Danielle Jorgens, Rick Webb, Helmut Gnaegi
In this course, we will review why cryo-techniques for biological specimen preparation are superior to conventional methods. We will discuss some low-cost cryo-methods, as well as some of the latest equipment and techniques for high-pressure freezing, freeze substitution, cryosectioning and correlative LM-EM. We will show how to recognize ice-damage artifacts and provide a library of reference materials that can be accessed online after the course. Persons taking this course should leave with a better understanding of these biological cryotechniques and their role in different applications such as correlating light and EM, EM tomography, EM immunolabeling, cryo-EM of vitrified sections, as well as their routine use for the best-available preservation of cellular fine structure.
Sjors Scheres, Tanmay Bharat
The course will begin with an explanation of the RELION algorithm. Next, the limitations of tomographic data will be discussed along with the missing wedge. Strategy for CTF estimation and CTF correction using the combined 3D CTF and missing wedge model in RELION will be explained.The practical course that will follow will show how to setup of files and directories for RELION sub-tomogram averaging, including generation of 3D CTF models. Tomographic data will then be classified in 2D and 3D. Finally, sub-tomogram averaging will be conducted using the auto-refinement program in RELION with an emphasis on how to assess refinement quality, Euler angle distributions and whether more data is needed.
Brendan J. Griffin
This short course aims to take the challenge out of imaging in variable-pressure SEM mode. We will sequentially address VPSEM column components and operation: electron (SE and BSE), and light (CL) imaging and x-ray analysis strategies and detectors for both biological and materials samples. Procedures for monitoring instrument performance and optimizing image quality will then be presented. Examples of the novel charge-related contrasts available in VPSEM will also be discussed. The appropriate use of hot, cool and cold stages is included. The course will conclude with invited manufacturer presentations on new developments and a final lecture comparing VPSEM with conventional SEM. Lecture pdfs will be available online.
The course first focuses on a wide range of practical topics in the field of image analysis, covered in an easy-to-understand format so that users with little or no experience can comprehend how image analysis can provide extensive quantitative measurements leading to better understanding of material performance. Treating image analysis as a problem-solving tool, along with discerning key metrics within a microstructure, will be discussed through several real-life examples. The course then will cover the use of the public-domain image-analysis package ImageJ/Fiji. This analysis tool will be explored in terms of its extensive capabilities and types of image-analysis projects, using several examples. Programming considerations will be discussed, with basic examples of batch-processing images for enhanced analysis of structural features. Finally, the strengths and weaknesses of image-analysis tools, and options for applying them in various types of imaging systems, will be described and discussed.
Lucille Giannuzzi, Joe Michael
The use of FIB instruments in materials and biological science laboratories is growing rapidly. The versatility and capabilities of these tools are also rapidly improving. This course will first cover ion/solid interactions, which are so important for a user of FIB instrumentation to understand. We will then discuss techniques of sample preparation for SEM and TEM, using conventional liquid-metal Ga+ ion FIBs. We will introduce liquid-metal alloy sources, the gas field-ionization source, the plasma-ion source, and the various ions that are now commercially available to benefit differing applications. The course will conclude with discussions of 3D applications and nanofabrication.
Lou Germinario, Phillip Russell, John Thornton
With the wide variety of analytical instrumentation available, the selection of the correct "tool" for analysis of nanomaterials is critical. By introducing various microscopy and microanalysis methods, this workshop should provide a solid foundation for nanomaterial characterization for the beginning to intermediate investigator. The importance of choosing the proper preparation technique, to minimize introduction of artifacts and to ensure that representative samples are identified for subsequent analysis, will also be discussed.