Breaking the ice with new Samples: cryo sample preparation during field studies

Abstract text (incl. figure legends and references)

The set of tools combined in correlative light and electron microscopy have seen new developments in the last couple of years allowing to address new research questions or revisiting old questions in a different way. The ability to combine dynamic and targeted imaging in the light microscope to their ultrastructural composition in electron microscopy provides insights into functional traits, subcellular dynamics and ultrastructure on a single organelle level. At the same time new developments for software and hardware for high-end scanning and transmission electron microscopes have allowed the resolution of protein structures inside cells to be pushed to atomic level. Combining advanced sample preparation strategies and being able to target precisely subcellular regions with high-end transmission electron microscopy has allowed unprecedented insights into biological processes. Sample preparation in a standardized, well-controlled laboratory environment with humidity-controlled rooms and access to ideal set-ups has been becoming rather standard. As a new emerging field of research, planetary biology spanning multiple ecosystems has the aim to understand how microbes, plants and animals respond to each other and to their environment. To be able to achieve this goal, field studies with controlled laboratory research are combined to understand the influence of environmental parameters on the molecular mechanisms underlying biological processes.

To be able to use newest imaging technologies on field samples, we have been working on bringing different high-end sample preparation tools such as plunge freezer and high-pressure freezer to the field to prepare samples in the best possible way. Following pilot studies collecting Plankton from different ecological habitats in warmer and colder climates, during the TREC (Traversing European Coastline) project and beyond, EMBL mobile laboratories will bring this cutting-edge technology and methodology to the field.

Here I would like to discuss cryo workflows, different sample preparation strategies and challenges of sample preparation during field expeditions. I will show sample preparation for volume electron microscopy using the focused ion beam scanning electron microscope workflow under cryogenic conditions, using cryo light microscopy to select structures of interest in different species of Plankton, followed by focused ion beam scanning electron microscopy at cryogenic temperatures to acquire 3D data.

Figure 1. Overlay of a cryo confocal image and scanning electron microscopic image of Acantharia (marine skeleton-bearing planktonic protists) (left, scale bar 50 µm). Green: autofluorescence of host, red: chlorophyll of symbionts (microalgae). Overlay of cryo-volume imaging FIB-SEM and cryo-confocal imaging (right, scale bar 5 µm).