Microscopy Conference 2023

Session chairs

Prof. Dr. phil. nat.

Sample preparation is a crucial and critical step for all following analyses by electron microscopy. Although a whole variety of sample preparation protocols is available, the development and improvement of electron microscopy techniques necessitates adjustment of existing protocols and design ideas. This session will focus on advances in sample preparation for the different life sciences electron microscopy applications.

Prof.

Combining cryo-microscopy and expansion microscopy to reveal the molecular architecture of the cell

Abstract text (incl. figure legends and references) The centriole (also called basal body) is an evolutionary conserved organelle that coordinates fundamental biological processes including cell division, cellular signaling, and cell motility. This organelle, 500 nm long and 250 nm in diameter, is composed of about 100 different proteins, some of which have been associated with human diseases. How these proteins are organized at the level of the centriole architecture and how associated mutations could be involved in pathologies is still poorly understood. I will present the latest work from my laboratory, which tackle these fundamental structural cell biology questions using cryo-tomography, cell biology, and ultrastructure expansion microscopy (U-ExM) methods.

LS 7: Advances in sample preparation

Invited talk

New ways to tackle challenges in sample preparation in single particle cryoEM for improved sample quality and time-resolved studies

Abstract text (incl. figure legends and references) Recent developments in the electron microscopy (EM) field have led to a step change in our ability to solve the &quot;high resolution&quot; structure of previously intractable systems. This is exemplified by the sudden expansion in membrane protein structures solved revealing new insights into structure, mechanism and regulation. Advances in the size of data sets that can be collected and processed by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. This talk will describe the ongoing developments of a cryo-electron microscopy grid preparation device that permits rapid mixing and vitrification of samples within ~10 ms allowing us to pull out non-equilibrium states on a range of systems from membrane proteins to viruses. Moreover, rapid grid preparation has also provided new insights into factors that can be detrimental to sample preparation and the talk will discuss ways to improve grid making for single particle cryoEM. A significant focus of the talk will be in our new findings in factors that are detrimental to sample preparation, especially those caused by interactions with the air-water-interface. The talk will conclude with our current overview of the challenges in sample preparation for both conventional cryoEM and time-resolved studies and look towards future developments in the field.

LS 1: High-resolution cryo-EM

Scanning electron microscopy (SEM) preparation and imaging techniques to visualize cell-material interaction on hydrogels and thermoplastic printed scaffold

Abstract text (incl. figure legends and references) Introduction Among several imaging methods to study cell-material interaction, Scanning Electron Microscopy (SEM) is capable of resolving both materials and cellular structures down to the nanometer scale. However, Scanning Electron Microscope typically works under high vacuum conditions, which requires special sample preparation. Cells and water containing substrates need to be dehydrated and dried before investigation at room temperature. Dehydration using a row of water/ethanol mixtures with increasing ethanol concentration and drying using hexamethyldisilazane (HMDS) is an established preparation route to visualize cells on vacuum stable substrates by SEM. For water-containing substrates like hydrogels, cryo-SEM or ESEM can be used to study cells material interactions without morphological changes of the substrate due to drying (1). Objectives Here we show different preparation and imaging protocols to visualize with minimalized artifacts resulting from sample preparation, cells&quot; performance (i.e. distribution and morphology) on two types of bio-fabricated substrates commonly used for tissue engineering – hydrogels and thermoplastic (polycaprolactone, PCL) printed scaffolds. Materials &amp; methods 3-dimensional cell culture scaffolds were obtained using 3D printing: hydrogels samples were obtained via extrusion process of alginate-based ink, PCL constructs were prepared using melt electrowriting approach. Mouse fibroblast (NIH3T3) cells were cultured on the scaffolds and visualized using SEM. Cell-seeded PCL scaffolds were investigated by SEM after dehydration and drying using a row of water/ethanol mixtures and HMDS. Hydrogel samples were imaged using cryo-SEM and ESEM following optimized protocols. Results SEM of PCL scaffolds allowed detailed observation of cell-material interactions. Focal adhesion points after 3 days of growth were frequently observed with high resolution. Cells grown inside the PCL scaffold can be imaged by embedding the dehydrated and dried sample in frozen ethanol and cutting thin slices by a blade under a stereomicroscope. Cryo-SEM of hydrogel samples, following the proposed preparation procedure, allowed to study cell attachments without morphological change of substrate and cells. For the investigation of cells and substrates in fully wet/swollen conditions, ESEM was performed at 3°C and 750 Pa water vapor pressure. Due to the limited field of view (diameter of the pressure limiting aperture is 500 µm) stitching of individual images is necessary to visualize the complete scaffold. Using ESEM starting at 3°C and 750 Pa water vapor pressure an in-situ freeze-drying experiment shows the morphological change of gelatin from a dense hydrogel to a porous polymer structure during temperature and pressure decrease according to the pressure-temperature phase diagram of gelatin (see Fig. 1). Conclusion SEM is a powerful tool to study cell-material interactions. For water-containing substrates like hydrogels cryo-SEM or ESEM can be used to prevent morphological changes of the substrate due to drying inside vacuum/under atmospheric conditions at room temperature. SEM of freeze-dried samples should be avoided when characterizing hydrogel-based materials in detail. Koch, M. and Wlodarczyk-Biegun, M. K., Bioprinting 20, e00098 (2020) Fig. 1: Morphological change of aqueous 1.8 wt.% gelatin during in-situ freeze-drying using ESEM.

IM 4: Development of cryo-EM instrumentation and techniques

Abstract talk

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).

LS 5: Correlative and multimodal microscopy

Tokuyasu sample preparation for TEM, STEM and SEM

Abstract text (incl. figure legends and references) Cell biologist are frequently looking for tools to localize proteins of interest in biological samples. Specific antibodies are used to solve the questions on a fluorescent light microscopy level. But one would prefer to resolve the intracellular structure together with the immunolabeling pattern at higher resolution (e.g. electron microscopy). For IEM, samples are chemically fixed and embedded in a support layer. Ultrathin sections of 70 nm are cut and immunolabelled with the antibody of choice followed by a binding step with an electron dense Gold particle (visible in EM). The most efficient immunolabelling procedure is the Tokuyasu cryosectioning technique. Hereby a biological sample is fixed with mild chemicals like glutaraldehyde and paraformaldehyde (either directly or by High Pressure Freezing and Automatic Freeze Substitution). After fixation the sample is embedded in a gelatin support, infused in sucrose and frozen in LN2. Thin sections are cut in a ultracryotome at -120˚C using a diamond knife. Sections are retrieved, thawed, placed on EM-grids and immunolabelled followed by EM analysis. Unfortunately, high antigenicity only goes together with mild fixation and therefor good morphology is sacrificed. The protocols are challenging and skill demanding. The Tokuyasu method can be applied to a large range of samples including small organisms, tissue samples, and cells. Sections can be used for immunofluorescent- and high-resolution Gold labeling. In this presentation the focus will be on the pitfalls and difficulties of Tokuyasu protocols.

High temporal resolution electron microscopy (flash-and-freeze) reveals vesicle compound fusion at ribbon synapses of zebrafish retina

Abstract text (incl. figure legends and references) Our eyes offer us a window into the world. This is made possible first and foremost by the photoreceptor neurons in our retinas. These neurons perceive light and translate it into electrical nerve signals that our brain can understand and process. This translation from light to nerve signals is achieved at so-called ribbon synapses. There, neurotransmitter is released from vesicles that are lined up at ribbon structures. The amount of transmitter release over time is inversely correlated with light intensity: more light, less transmitter. In the dark, the rate of release is at maximum. In particular, right at light-dark transition, a huge amount of transmitter is released within milliseconds. It is unknown how such bursts of release can be achieved in such a short amount of time. The field has been arguing about possible models for decades. The process is too fast, and vesicles are too small to visualize by live-cell fluorescence microscopy. However, with a new technique called flash-and-freeze, this question can finally be answered. The flash-and-freeze technique is uniquely able to visualize cellular processes with extremely high spatial and temporal resolution. Retinas of adult zebrafish were subjected to light-dark transitions to elicit the transmitter bursts. During the bursts, the retinas were frozen under high pressure within milliseconds. This halts the cellular processes that produce the bursts, thus enabling us to examine these extremely fast events. We then use electron microscopy to visualize the ribbon synapses with nanometer-resolution. Our data show unusually large vesicles at synaptic ribbons several milliseconds after light-off. Fusion events where sometimes captured in action. At later time points, these large vesicles are not observed anymore. In light-on controls, all vesicles remain their usual size. Our data strongly suggest that vesicles undergo so-called compound fusion: They are lined up at the ribbon to fuse with each other to form super-vesicles. These giant vesicles then release a huge amount of transmitter at once, thus explaining the bursts. We are now investigating the molecular mechanisms of compound fusion. The flash-and-freeze technique can be adapted to investigate other fast cellular processes at extremely high spatial and temporal resolutions.

LS 7
Advances in sample preparation

Appointment

Session chairs

Description

Programme

LS 7Advances in sample preparation

Appointment

Session chairs

Description

Programme

LS 7
Advances in sample preparation