Microscopy Conference 2023

Session chairs

Technological advances steadily push the limits of cryo-electron microscopy (cryo-EM). In recent years, novel developments in cryo-EM instrumentation as well as newly established workflows enabled groundbreaking insights in both, life sciences and material sciences.
In this session we will discuss new developments in experimental cryogenic techniques, cryo-sample preparation as well as instrumental capabilities applied across different fields.
Contributions are welcome from, but not restricted to, the following areas: SEM, FIB, TEM, STEM and CLEM at cryogenic temperatures, including hardware improvements, cryo-stages, cryogenic sample preparation techniques used for EM, measurements of local temperature as well as image processing for cryo-EM.


Prof.

Cryo-STEM tomography – a powerful technique

IM 4: Development of cryo-EM instrumentation and techniques

LS 2: Cryo-tomography

Abstract text (incl. figure legends and references) For many years, life science cryo-EM has been practically synonymous with wide-field imaging in transmission. This modality has been famously successful in determination of macromolecular structure at near-atomic resolution, based on the combination of phase contrast generated by defocus with image averaging. The same wide-field TEM approach was further employed for pioneering works in 3D imaging of cells by tomography. An alternative modality is STEM, scanning transmission EM. STEM uses a focused probe and a variety of detectors to collect one or more signals pixel by pixel. Its popularity in the materials sciences has not yet been matched in cryo-EM for biological materials, due mainly to the impression that STEM is incompatible with beam-sensitive specimens. To the contrary, STEM may be more dose-efficient than TEM. A case in point is tomography of thick samples, where the energy filter required for conventional TEM can be avoided. 4D STEM and multi-segment detectors can collect every electron, but the means to combine the information they contribute are still under development. These include angle-resolved STEM, ptychography, and differential phase contrast in various configurations. The power of the scanning probe methods lies in their flexibility, and the fact that multiple image contrast modes may be generated in a single scan. The talk will describe progress and prospects for cryo-STEM in life science applications with an emphasis on cellular cryo-tomography.

Invited talk

Microsecond time-resolved cryo-electron microscopy

Abstract text (incl. figure legends and references) While cryo-electron microscopy (cryo-EM) is rapidly becoming the dominant method in structural biology, its time resolution is currently insufficient to directly observe proteins in action, leaving our understanding of these nanoscale machines fundamentally incomplete. Here, we demonstrate a novel approach to time-resolved cryo-EM that affords microsecond time resolution. Our method is illustrated in Fig. 1. It involves melting a cryo sample with a laser beam (a), which allows dynamics of the embedded particles to occur in liquid once a suitable stimulus is provided, for example by releasing a caged compound (b). While the dynamics occur, the heating laser is switched off at a well-defined point in time (c), causing the sample to rapidly recool, so that it vitrifies, and the particles are trapped in their transient configurations (d), in which they can subsequently be imaged (e). We demonstrate that our approach affords a time resolution of 5 µs or better. Moreover, near-atomic resolution reconstructions can be obtained from revitrified samples, showing that the revitrification process does not damage the protein structure. Finally, we present a microsecond time-resolved pH jump experiment, in which we observe the dynamics of the capsid of CCMV, an icosahedral plant virus. These results highlight the potential of our method to fundamentally advance our understanding of protein function through direct observation. Figure 1. Experimental concept for microsecond time-resolved cryo-EM.

Towards multimodal in situ cryo-FIB-SIMS imaging in biological systems

LS 5: Correlative and multimodal microscopy

Abstract text (incl. figure legends and references) Introduction: One of the struggles of correlative multimodal imaging in biological systems is the necessity for utilizing several analytical instruments to permit high-resolution and high-sensitivity morphological and chemical analyses. This induces time-consuming sample transfers and subsequent data processing and correlation. For cryo-investigations, not only is there a limited availability of cryo-compatible instruments, but there is the additional concern of contaminating or even damaging the frozen-hydrated sample during sample-transfer due to ambient temperature and humidity. Our group specializes in incorporating several complimentary techniques into one individual instrument to streamline such correlative analytical workflows, all the while focusing on the vital key characteristics, i.e. high spatial resolution, chemical sensitivity, dynamic range (for the detection and mapping of atoms and small clusters with concentrations varying over several orders of magnitude) and isotopic selectivity. Objectives: We present a novel instrument devised for multimodal in situ imaging and chemical analysis of biological samples in a close to native cryogenic state. Materials &amp; Methods: Previously, we described the in-house developed npSCOPE instrument [1], based on a Gas Field Ion Source using finely focused Helium and Neon ion beams and capable of performing: a) Secondary Electron (SE) imaging (providing morphological and topographical information; lateral resolution: &lt; 1 nm); b) Scanning Transmission Ion Microscopy (STIM, providing bright and dark field imaging; lateral resolution: &lt; 6 nm); and c) Secondary Ion Mass Spectrometry (SIMS, employing a compact high-performance double-focussing magnetic sector mass spectrometer equipped with a continuous focal plane detector, allowing a full mass spectrum to be recorded for each single pixel; lateral resolution: &lt; 15 nm). Here, we describe new developments that allow the investigation of frozen-hydrated biological samples at temperatures &lt; -140° C close to their native state on the npSCOPE under high vacuum conditions. To achieve this, we equipped the instrument with a custom-built cryogenic sample stage, and we developed a customized cryo-sample transfer system structured around a prototype cryogenic-glovebox, ensuring optimal environmental conditions during sample handling. Results: We present proof-of-principle results comparing data obtained on various chemically fixed room temperature (RT)-compatible samples which were investigated both at RT and at &lt; -140° C that show that all three imaging modes function artefact-free at cryogenic temperatures, as well as early results obtained from frozen-hydrated samples. Conclusion: The here presented cryo-workflow on the npSCOPE will allow the chemical analysis of biological samples from various fields of research under close to native conditions. Funding: This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 720964 and by the Luxembourg National Research Fund via the project INTER/DFG/19/13992454. References: [1] O. De Castro et al., "npSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles," Anal. Chem., vol. 93, no. 43, pp. 14417–14424, Nov. 2021, doi: 10.1021/acs.analchem.1c02337.

Abstract talk

Prof. Dr.

Mechanisms for radiation damage in biological specimens revealed by vibrational EELS

IM 2: Spectroscopy

Abstract text (incl. figure legends and references) INTRODUCTION Radiation damage fundamentally limits the attainable spatial resolution in the electron microscopy of biological specimens. To determine protein structures with the resolution needed to understand biological function it is necessary to average over multiple copies of a molecule of interest. The need for averaging limits studies of how macromolecules interact e.g. in an organelle. Traditionally, the effects of radiation damage have been assessed by measuring spot fading in crystals [1], or by monitoring mass loss from EELS spectra [2]. OBJECTIVES It is highly desirable to understand the mechanisms of radiation damage, which bonds are breaking, and when they can reform. Recent developments make it possible to record vibrational spectra in the electron microscope - equivalent to IR but at the nanometer scale. We acquired data on guanine and vitreous ice to quantitatively measure the structural changes through the vibrational signal from different chemical bonds as a function of electron exposure. MATERIALS &amp; METHODS We used a Nion HERMES microscope at 60kV with Dectris ELA direct detector attached to the IRIS spectrometer. We used guanine fish scales [3] and vitreous ice from plunge freezing. Spectrum images were recorded by repeatedly scanning a defocused probe over an area (typically 100x100 nm²). The spectra from each map were aligned and summed, yielding average spectra vs. fluence at below 8 meV energy resolution. The intensities of peaks corresponding to different bonds were extracted by fitting power laws over multiple windows. RESULTS Spectra of guanine and vitreous ice are given in Fig. 1 and show vibrational modes linked to different bonds. Fig. 2 shows different peak heights (normalized to zero loss) versus fluence. In the early stages, the C=O intensity from guanine at room temperature does not change while the XH intensity is drastically, cf. Fig. 2 (a). This indicates that the CH and NH bonds break first and is consistent with the observation of hydrogen bubbles [4]. At higher electron exposure up to 1000 e-/ Å2 the C=O bonds are broken as seen in Fig. 2 (b). Eventually, a steady state of broken bonds is reached but mass loss from sputtering continues (increased zero-loss intensity, not shown). Fig. 2 (c) shows that cooling to liquid N2 temperatures reaches the steady state earlier but does not change the general behavior. For vitreous ice, it appears from Fig. 2 (d) that single OH bonds break first releasing free H, as seen from the decrease in the OH stretch peak. CONCLUSION At a low electron fluence of up to 100 e-/ Å2 CH, NH and OH bonds are the first to break. They presumably release hydrogen that can accumulate as bubbles. As the electron exposure increases other bonds such as the C=O in guanine are broken. However, many of these bonds are also being reformed as damage proceeds. We are now studying hydrogen exchanges via deuteration. [1] H. Stark et al, Ultramicr. 63, 75 (1996). [2] P. Li and R.F. Egerton, Ultamicr. 101, 161 (2004). [3] P. Rez et al, Nat. Commun. 7, 10945 (2016). [4] R.D. Leapman and S. Sun, Ultramicr. 59, 71 (1995). Fig. 1 (a) Background subtracted spectrum of guanine and (b) spectrum of vitreous ice. Variation of peak intensities (normalized to zero-loss) versus fluence for (c) guanine at room temperature and low fluence, (d) increased fluence, (e) cooled and (f) for vitreous ice.

Cryo-STEM imaging for single particle structure determination

LS 1: High-resolution cryo-EM

Abstract text (incl. figure legends and references) In electron cryo-microscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission electron microcopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution 3D structure. Although scanning transmission electron microscopy (STEM) has been successfully applied to organic and inorganic materials and achieved exceptionally high resolution, its capability to resolve biological structures with high resolution is still to be determined. Here, we apply STEM using the integrated differential phase contrast mode also known as iDPC-STEM (1) to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) (2) and tobacco mosaic virus (TMV) (3). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The estimated B-factor from iDPC-STEM of 93 Å2 indicates that the accomplished micrograph quality exceeds CTEM recordings from 2015 using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules. Our proof-of-principle study introduces high-resolution STEM to biological cryo-EM (4). I. Lazić, E. G. T. Bosch, S. Lazar, Phase contrast STEM for thin samples: Integrated differential phase contrast. Ultramicroscopy. 160, 265–280 (2016). C. Gatsogiannis, J. Markl, Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units. J. Mol. Biol. 385, 963–983 (2009). S. A. Fromm, T. A. M. Bharat, A. J. Jakobi, W. J. H. Hagen, C. Sachse, Seeing tobacco mosaic virus through direct electron detectors. J. Struct. Biol. 189, 87–97 (2015). I. Lazić, M. Wirix, M. L. Leidl, F. de Haas, M. Beckers, E. V. Pechnikova, K. Müller-Caspary, R. Egoavil, E. G. T. Bosch, C. Sachse, &quot;Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution&quot; (preprint, Biophysics, 2021), , doi:10.1101/2021.10.12.464113. Accepted at Nature Methods Fig.1 Cryo-iDPC-STEM resolves biological test specimens up to near-atomic resolution. (a) Optical path of STEM, including the convergence semi-angle (α), the average electron path (red line) and the center of mass (COM) of the intensity at the detector. (b) Raster geometry and beam dimensions over the field of view. (c) iDPC-STEM micrograph of keyhole limpet hemocyanin (left) and corresponding 6.5-Å resolution map with superimposed atomic model (right). Scale bar is 100 nm. (d) iDPC-STEM micrograph of tobacco mosaic virus (left) and corresponding 3.5-Å resolution density (based on the 0.143 Fourier shell correlation (FSC) cutoff criterion) with superimposed atomic model. Scale bar is 100 nm. ADF, annular dark field; BF, bright field; E, electric field; f, focal length of the lens; rp, position of the probe in real space; V, electrostatic potential field.

New principle and robot for vitrification of cryo-EM samples with controllable sample thickness and real-time optical inspection

Abstract text (incl. figure legends and references) Keywords: Vitrification of biological samples, blotting-free ethane plunging, sample preparation robot, cryo-Electron Microscopy (cryo-EM), Single Particle Tomography (SPT), cryo-Electron Tomography (cryo-ET) Introduction Cryo-EM, cryo-tomography, Single Particle Tomography (SPT) and cryo-CLEM (Correlative Light and Electron Microscopy) have become standard tools for the investigation of protein 3D structures, cellular structures and more in the last decade. Despite the progress with cryo-EM instruments and automated sample loading and imaging the routine preparation of cryo-samples can be challenging. Ice layer thickness and uniformity are key parameters for obtaining high quality data. Cryo-plunging techniques most widely used for vitrification are based on paper-blotting to adjust the thickness of the vitrified layer. We have identified this as a key drawback in the process because the blotting paper soaks up moisture while inside the high-humidity chamber leading to characteristics that are challenging and not always repeatable [2][3]. Objectives In this work we aim to address the difficulties of cryo-sample preparation for cryo-EM and cryo-ET by means of automation and by introducing the principle of suction to adjust the film thickness on the grid before plunging. Materials &amp; methods: comparison with conventional blotting, robot design Fig 1 compares the traditional cryo sample preparation technique (top row, sample pipetted onto EM grid with support film followed by blotting in humidity chamber) with the method proposed here: sample application via dipping followed by film thickness adjustment via suction (bottom row) [1]. Filter paper used in the traditional technique soaks up moisture from the environment of the humidity chamber, changing properties. The suction used by our method can be adjusted (flow rate and duration) to control film thickness. It also gives access for direct optical real-time imaging that is used to assess sample conditions and to determine the best moment to trigger plunging [1]. The desktop plunging system is shown in fig. 2, the internal elements in fig. 3. Results We built a robotic cryo-plunger and demonstrated the use for protein suspensions, lipid vesicles, bacteria and human cells. See [1] for additional details. Conclusion We have identified the blotting process in the traditional plunging setups as a key drawback in the process and propose a novel method where fluid is removed for the sample thickness adjustment by means of a calibrated and programmed suction process. Key benefits are repeatability, straightforward automation and direct optical access to the sample for real-time monitoring and control of sample film thickness. The thin-film interference and the liquid surface characteristics observed in the optical microscope give control over the optimisation of film thickness (see [1]). Importantly, the optical image acquired immediately before plunging allows good prediction of sample ice quality – saving valuable EM beam time. The new setup improves repeatability, removes potential risks from manual sample handling and increases speed and throughput via automation. References [1] Nature Communications, (2022) 13:2985, https://doi.org/10.1038/s41467-022-30562-7 [2] Nature Methods, VOL 18, May 2021, 463–471, P. J. Peters, et al. https://doi.org/10.1038/s41592-021-01130-6 [3] J. of Microsc., Vol. 276, Issue 1 2019, pp. 39–45 doi: 10.1111/jmi.12834

IM 4
Development of cryo-EM instrumentation and techniques

Termin

Session chairs

Beschreibung

Programm

IM 4Development of cryo-EM instrumentation and techniques

Termin

Session chairs

Beschreibung

Programm

IM 4
Development of cryo-EM instrumentation and techniques