Cryo Subtitles English ((FULL))
This class covers the fundamental principles underlying cryo-electron microscopy (cryo-EM) starting with the basic anatomy of electron microscopes, an introduction to Fourier transforms, and the principles of image formation. Building upon that foundation, the class then covers the sample preparation issues, data collection strategies, and basic image processing workflows for all 3 basic modalities of modern cryo-EM: tomography, single particle analysis, and 2-D crystallography.
Cryo subtitles English
Atomic-resolution cryo-EM is finally here. Structural biologists used a Thermo Scientific Krios G4 Cryo-TEM equipped with a Thermo Scientific Selectris X Imaging Filter to achieve record-breaking resolution results, recently featured on the cover of Nature and other recent articles from Science and Nature . Researchers from the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, obtained a 1.2 ångström resolution structure of the iron-storing protein apoferritin. They also achieved a 1.7 ångström resolution of membrane protein (GABA receptor), with an even sharper resolution in key parts of the protein. This human membrane protein has target sites for general anesthetics, benzodiazepines, barbiturates and neuroactive steroids, making it very relevant to structure-based drug discovery. In addition to the resolution leap, the Selectris technology will also significantly impact productivity. By having a better filter and camera, scientists will be able to achieve a specific resolution with much less data.
Cryo-electron microscopy (cryo-EM) represents the next frontier in structural biology. This revolutionary technique can now resolve protein structures to true atomic resolution with the new Selectris and Selectris X Imaging Filters.
Discover what you can do with atomic-resolution cryo-EM through the Selectris Imaging Filters and latest Falcon 4 camera. See the record-breaking structure results for single particle and cellular cryo-tomography from MRC-LMB, MPI Biochemistry, IST Austria, and others. Learn the potential applications of the Selectris for small proteins (
For all users of the e-learning course we offer a preview of a video training on "WBC Treatment Safety". This video is the launch of a new training series by Antra Getzoff (Cryo Pros United) and will soon be available in its full version on cryoAcademy.
In this video, scientists from across the Harvard community describe how they are using the power of cryo-EM to study the inner workings of molecular machines involved in everything from cancer to inflammation to circadian clocks.
Based on cryogenically-cooled, RF coil design this cutting-edge technology delivers an increase in sensitivity of in-vivo proven factors of 2.5 to 5.3 over standard room temperature RF-coils in routine MRI applications.
On the Cryo tab, ensure Vitrified Grid is not selected, click on Queue Target, then on Plunge. Evaluate the upper and lower images to confirm the dispensers are functioning normally. Dilute two samples to the desired concentrations with an appropriate buffer, ideally using the same for both, and fill the cryogen bowl with liquid nitrogen.
Before embarking on a cryo-ET project and prior to collecting tilt-series, it is crucial to the success of the project to prepare the most suitable sample that is thoroughly vitrified and sufficiently thin for the electron beam to penetrate. Ideal samples will provide a 3D map revealing features of biological significance and/or for structural analysis. The unique strength of the diversity of biological samples accessible through cryo-ET comes with a challenge, since the sample preparation steps need to be tailored individually to each project.
In this chapter we focus on sample grid preparation and the major considerations and decisions one has to make to prepare a good starting sample. The modules are organized along the workflow to cover first the similarities to single particle workflows, such as for specimens involving viruses, virus-like particles (VLPs), or purified complexes. Additionally, we will cover common electron microscopy grid types used in cryo-ET, and other pre-freezing considerations such as fluorescent-tagging, micro-patterning, and inclusion of fiducial markers. The end of the chapter will summarize common freezing techniques and their applications.
Cryo-ET and cryo-EM share many commonalities, the most important of which is that frozen-hydrated specimens are imaged in their near-native states. Single-particle projects typically rely on having purified proteins or protein complexes as a starting point. In contrast, cryo-ET applications are used for a much broader range of specimens, from purified proteins to whole cells.
The type of workflow used is decided based on the goal of the project. In general, single-particle projects aim to achieve high-resolution reconstructions that are used for building atomic models. Cryo-ET projects can also achieve high-resolution reconstructions of purified materials, but it is more commonly used for imaging samples at the cellular or subcellular level. Specific details about single particle workflows are available on CryoEM 101, but more specific comparisons about the similarities and differences of the major steps in cryo-EM and cryo-ET are summarized below.
Sample and grid preparation: Samples for cryo-EM or cryo-ET need to be adsorbed onto a TEM grid. Cryo-EM and cryo-ET use identical grid preparation procedures for purified samples: a few microliters of sample are applied to the surface of a glow-discharged grid. After the sample is applied, the grid is blotted to remove excess liquid and then plunge frozen into a cryogen such as liquid ethane.
Exceptions to this procedure arise when preparing adherent cells for cryo-ET projects. Adherent cells are grown directly on gold grids (copper is not used due to cytotoxicity). Grids are then blotted only from the back side to prevent damaging the cells. For thick specimens, an extra step of focused ion beam-milling (FIB-milling) may be used to create thinned sections throughout the grid for imaging.
Data collection: Specimens are loaded onto the TEM stage and recorded using low-dose methods. For thicker specimens that are more commonly used in cryo-ET, image recording with an energy filter is often necessary to increase image contrast. Single-particle data are recorded by recording single exposures over each area, whereas cryo-ET tilt-series are recorded by recording multiple exposures over a single area at various tilt angles. Consequently, each cryo-ET tilt-series typically requires a higher total of electron beam exposure compared to single particle cryo-EM movies.
Data processing: Cryo-EM and cryo-ET data are typically recorded as movies. Movie frames are aligned to correct for beam-induced motion and then summed into a single image. From there, the data processing workflow diverges completely. In single-particle projects, particle images are extracted from micrographs and then used for downstream 2D classification and 3D reconstruction. In cryo-ET, summed images in a tilt-series are aligned to each other to generate a tomogram that is used for downstream processing.
Fiducial markers are used to aid tilt-series alignment, since they are of high contrast and embedded in the ice, and also for accurate correlation between fluorescent light, SEM and TEM images. Protein coated gold nanoparticles are most commonly used in cryo-ET applications and are added to the sample prior freezing to embed the beads into the ice. These beads can be used during tilt-series alignment to correct for x,y-shifts to produce high-resolution 3D reconstructions.
In cryo-ET, most projects are focused on a specific region or macromolecular complex inside a cell which is likely not abundant. Substantial screening of snapshots and data processing efforts would be needed to identify images and tomograms with relevant information. However, access to high-end microscopes is limited and costly. Similarly, data processing is computationally and time intensive. These issues can be mitigated on the sample level by labeling organelles or specific proteins fluorescently to identify relevant regions on the grid for tilt-series collection.
In addition to identifying regions of interest to image, the fluorescent label can also be used to correlate the final 3D reconstruction, the tomogram, to the area of the cell it was taken from providing cellular context. This workflow is referred to as Correlated Light and Electron Microscopy (CLEM). The fluorescent imaging can either take place prior vitrification (CLEM), after vitrification (cryo-CLEM) and sometimes both. Correlation between vitrified sample and tomogram is superior since the fluorescent images are taken directly from the vitrified samples and no changes can occur between imaging and freezing which one needs to take into account for RT CLEM. A dedicated upright fluorescent light microscope outfitted with a cryogenic stage and objectives that can be used at cryogenic temperature is crucial to image vitrified samples. Recently, Focused Ion beam (FIB) instruments which are used to produce thin lamella (Chapter 3) can be outfitted with fluorescent light microscope capabilities. The advantage is that ice contamination is minimized; however, the distance between the stage and objective is fixed allowing only for one magnification which may bring additional challenges if higher magnifications are needed. The developments in this field are rapid: some solutions offer confocal cryo-light microscopes amplifying signal strength, cameras with improved quantum efficiency with the ability to provide Z-stacks, and more magnifications (20x, 50x and 100x) to choose from for integrated fluorescent imaging within FIB instruments.
The grid forms the foundation of a successful cryo-ET experiment. When working with whole cells and large specimens for tomography applications, various grid properties including mesh sizes, grid types, types of support layer, and materials of the mesh need to be considered. 041b061a72