Oral Presentation 26th ACMM “2020 Visions in Microscopy”

Correlative Light and 3-D SEM Techniques for Bio-Medical Research (#118)

Kevin Grant 1
  1. Carl Zeiss Pty Ltd, North Ryde, NSW, Australia

Scanning electron microscopy has emerged as a powerful tool in understanding biological ultrastructure. Modern SEMs offer improved opportunities to automate the generation of structural data at a wide range of resolutions. New technologies like focal charge compensation, allow high-resolution imaging even of challenging tissue samples without charging effects. Modern SEMs are crucial in many multi-modal imaging workflows, especially in the growing field of cryo-Electron Microscopy. With traditional methods, creating lamellae for cryo-TEM can be challenging since structures of interest are not visible during the sample preparation process. We present a new multi-modal workflow that allows researchers to identify and locate target structures based on confocal fluorescence imaging. Sample are then transferred to a focused-ion-beam SEM, where coordinates previously recorded are used to relocate the subsurface structures. Simultaneously imaging and milling then exposes the target region. This guided, simultaneous acquisition yields details of cellular contrast of the unstained frozen sample (ultimately providing a 3-D image of the surrounding ultrastructure) and, secondly it provides continual visual feedback while milling. This later aspect is critical to ensuring that the lamella truly contains the structure of interest and vastly improves the yield of quality lamella for subsequent structural examination in the cryo TEM.