Wiley & ZEISS are proud to present: New Discoveries from the Ultrastructure of Life

Life is inherently three-dimensional (3-D). Thus, approaches that can acquire, visualize, and measure the spatial relationship of organelles, cells, tissues, and organisms allow for greatly improved interpretations and deeper insights for many important biological questions.

Until more recently, capturing cell/tissue volumes at the nanoscale level was a daunting, inefficient, and highly manual process. The development of volume electron microscopy (vEM), a broad collection of imaging strategies that capture hundreds or thousands of consecutive sections in an automated fashion, has extended the power and scope of 3-D cell ultrastructure far beyond traditional transmission electron microscopy (TEM) of individual ultrathin sections. One approach, array tomography (AT), collects a ribbon or “array” of serial ultrathin sections via an ultramicrotome onto a substrate (e.g., grid, glass slide/coverslip or silicon wafer). These sections can be probed with antibodies, stained with heavy metals for contrast, imaged by light, transmission or scanning electron microscopy and archived for future interrogation.

Alternatively, serial block-face scanning electron microscopy (SBF-SEM) employs a diamond knife with an ultramicrotome placed inside a scanning electron microscope which repeatedly removes a thin surface layer from a resin embedded specimen followed by imaging of its rigid block-face. In a related block-face approach, focused ion beam scanning electron microscopy (FIB-SEM) uses an ion beam to remove even thinner layers, enabling the acquisition of high-resolution isotropic 3-D volumes.

This presentation will i) provide an overview of key vEM workflows, how they work and when to use, ii) describe the fundamentals of sample preparation, iii) include examples of biological questions that can be answered, and iv) share community vEM resources to support vEM understanding and success. 

In this webinar, we aim to explore the impact of sample preparation on various aspects of volumeEM , from sample preparation, to data acquisition and processing. The complexity and breadth of the field preclude a one-size-fits-all approach, yet we aim to shed light on common challenges and necessary considerations. The session is structured into three segments:

First, Christel Genoud will delve into how factors such as fixation, contrast, and embedding type affect the resolution, acquisition speed, and overall quality of the resultant images. Next, Jean Daraspe will present practical examples illustrating the variability in sample mounting and trimming, contingent upon the specific goals of image acquisition and the desired outcomes. He will suggest strategies for targeting regions of interest on a stub or block, and discuss how mounting techniques can influence both charging and imaging conditions. Finally, Corrado Cali will focus on micrograph processing, demonstrating how sample preparation not only affects the interpretation of images but also the ease of reconstruction. He will specifically focus on how fine-tuning image parameters during pre-processing can aid in identifying different cell types, and improves image segmentation tasks.

Overall, this webinar aims to equip participants with tangible examples and insights to critically evaluate and enhance their sample preparation techniques, tailored to their unique research needs. 

Array tomography is a volume EM technique where serial ultra-thin sections are cut and collected onto a substrate, and then imaged in an SEM to build up a 3D representation of that sample. Due to the non-destructive nature of the technique, and the stability of the sections on substrate, arrays are amenable to repeated sessions of automated image acquisition, providing unlimited new regions of interest according to the user’s needs and data handling capabilities.

Firstly, Jemima Burden will introduce the Array Tomography workflow, demonstrate how these steps contribute to making it one of the more flexible volume EM techniques, and share how this offers real practical benefits to biomedical and clinical researchers, core facilities and community groups.

Ian White will explain how, with standard equipment that many EM labs will already have, array tomography is a volume EM technique that is genuinely accessible to all on some level and showcase some of the tips and tricks already shared in the growing array tomography community that could help you start accessing the benefits of array tomography for your research now!

Finally, Luke Noon will provide a researcher’s perspective and share how access to this remarkable technique has transformed his lab’s ability to sample liver tissue for the tracing and reconstruction of hepatic nerve endings.

Complete understanding of biological structures and functions needs three-dimensional imaging to better depict their original view. Serial Block Face Scanning Electron Microscopy (SBF-SEM) offers new opportunities to capture volume information in a slice-and-view automated fashion.

In this webinar, latest breakthroughs in SBF-SEM instrumentation will be shown, focusing on Volutome technology and its improvements in resolution, speed and automation. Next, three different en bloc staining procedures will be considered and compared to speed up tissue preparation while ensuring sample preservation and good resulting contrast. Strategies to maximize the final imaging outcome (i.e., mounting techniques, sample discharge with the focal charge compensation) will be suggested, as well as approaches for 3D reconstruction examined.

Advancements in microscopy have continually propelled the frontiers of biology, empowering scientists to see deeper into the intricate world of cellular structures. Among multiple microscopic techniques, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) stands out as a stellar technology that provides unprecedented insights into the three-dimensional morphology of cells with isotropic nanoscale resolution.

In this webinar, Dr. Ilya Belevich will discuss the principles and applications of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) within cell biology. The session begins with an introduction to the fundamental concepts of FIB-SEM followed by the criteria necessary for sample preparation. Subsequently, Ilya will present case studies highlighting the usage of FIB-SEM in cell biology, showcasing how it can be paired with other imaging techniques such as confocal light microscopy and serial block face SEM to broaden our perspective through correlative imaging approaches. Concluding the session, he will tackle the complexities of data processing, offering strategies to convert qualitative insights into quantitatively robust, visually compelling findings. 

The aim of this webinar is showing the benefits of 3-dimensional representation of images originally acquired as 2D stacks. Not only the electron microscopy sample preparation and imaging techniques like array tomography (AT), serial blockface (SBF) and focused ion-beam (Fib-)SEM have made huge steps over the past decade. 3D reconstruction methods, big data visualization capabilities and semi- to fully automated analysis tools using artificial intelligence have continuously raised the level of speed and sample-complexity that can be analyzed higher and higher.

Modern computing power paired with easy-to-use and A.I.-enabled software platforms, locally or cloud-based. build up a modern ecosystem in which the scientist can follow all the way from the precious sample, to image acquisition, segmentation of the objects of interest to the final the analysis proper visual representation of the results that ultimately might or might not support the initial hypothesis.