JEOL Resources

Atomic resolution structures of biological macromolecules using microED on JEOL TEMs

Wed, 09 Dec 2020 13:23:39 GMT

Also see: JEOL CRYO ARM™ Achieves Top Resolution August 2023.

Micro electron diffraction, or microED, is a technique aimed at solving structures of biological macromolecules by electron diffraction. Barn-storming work by the group from Prof. Gonen showed the impressive impact and promise of this technique¹. The technique borrows from X-ray crystallography in that precession techniques are used for data collection and that much of the well-established software for solving structures by X-ray crystallography can be used for microED. However, it differs in a fundamental way in that electrons are used, which, owing to the substantially larger scattering cross-section of electrons with biological matter, means much smaller crystals can be used. Thus, crystals the size of a few microns are perfectly adequate for microED. The current crop of cameras and levels of microscope automation allow for the rapid collection of a full data set in a matter of minutes. By combining microED with the principle steps of SPA as employed in SerialEM², a single overnight run can yield hundreds of microED data sets³. Note that because diffraction data is collected, the acquisition process is immune to mechanical vibrations and drift; only a shift caused by poor eucentricity that would move the crystal out of the selected aperture field of view would impact the data collection.

MicroED can be applied on every new electron microscope in the JEOL TEM line-up. Precise control of the goniometer’s tilting speed compatible with microED is standard and can be set through either SerialEM or Recorder, the latter being part of JEOL’s TEMography package (SIF). SerialEM was first introduced on a JEOL JEM-2200FS in 2006 and is now fully compatible with all of JEOL’s electron microscopes. A record-breaking 1.34Å structure of apo-ferritin was recently obtained from JEOL’s latest generation of cryo-TEMs, the CRYO ARM™, a microscope available at 200 and 300 kV with a cold field emission gun and in-column energy filter⁴.

This application note reports on microED results obtained on a JEOL JEM-F200 outfitted with a cold FEG and a Gatan Elsa holder to keep the sample at -177ºC for the sake of reducing radiation damage⁵. Figure 1 shows a gallery of principle checks whether crystals of L-histidine will deliver high-resolution diffraction patterns for microED. The micro-crystals are deposited on ultrathin carbon films overlaid on lacey carbon.

Fig. 1: Diffraction images of L-histidine in defocused diffraction mode without (left) and with (middle) a selected area aperture inserted showing the L-histidine crystals. The focused diffraction pattern is shown on the right showing strong Bragg reflections.

Figure 2 shows several frames from one of the microED data sets acquired under continuous rotation at 0.25º/sec from -30º to +30º. The beam stopper is used to prevent the central, undiffracted beam from hitting the camera’s sensor.

Fig. 2: Selected focused diffraction patterns from -30º to +30º microED series acquired at 0.25º/sec. Total dose was 2.4 el/Å2.

After unpacking the movies, the individual frames are processed using standard X-ray crystallographic packages – SIR2019 and SHELXL. Table 1 shows the experimental results whereas Figure 3 shows the structures calculated from the data using direct methods. The refined map after running SHELXL shows clearly the expected atoms in the structure.

Table 1: experimental results
Molecular weight (Da)	155.16
Measurement temperature (K)	96
Space group	P212121
a, b, c (Å)	5.27, 7.44, 18.99
# of reflections	417
R factor (%)	19.81

Fig 3: Structure of L-histidine after initial direct method calculation using SIR2019 (left) and after refinement using SHELXL (middle) with the structure formula shown right.

Conclusion: Micro electron diffraction data can be readily acquired using a JEOL cryo-electron microscope using relatively shallow tilt series. The resulting data can immediately be processed using available pipe lines for X-ray diffraction data enabling rapid structure determination of small molecules by electron diffraction.

Contact your local JEOL representative to learn more about JEOL TEMs and microED using SIF Recorder or SerialEM.

References:

B.L. Nannenga et al., Nature Methods 11 (2014) 927.
D.N. Mastronarde, J. Struct. Biol. 152 (2005) 36.
M.J. de la Cruz et al., Ultramicroscopy 201 (2019) 77.
M. Tegunov et al. (2020) https://doi.org/10.1101/2020.06.05.136341.
Guzmán-Afonso et al., Nature Comm 10 (2019) 3537.

Cryo-EM screening on JEOL JEM-1400Flash

Wed, 23 Aug 2023 10:19:49 GMT

Introduction

Cryo-EM has enjoyed an enormous ground swell in popularity ever since the advent of more stable and automated electron microscopes, suitable movie-type cameras, and improved acquisition software. Results obtained so far have been nothing short of spectacular as illustrated by several structures in EMDB and EMPIAR solved by cryo-EM to resolutions better than 1.5Å, such as EMD-31314, EMD-33707 and EMD-35984, the latter of which reaching true atomic resolution. This note describes screening of cryo-EM samples on a JEOL JEM-1400Flash with a Gatan ELSA holder before committing to an SPA run on higher-end electron cryo-microscopes, such as the JEOL CRYO ARM (Figs. 1-3).

Results

Figure 1 shows the JEM-1400Flash at UC Berkeley, which is a turbo-pumped 120 kV electron microscope capable of imaging from 10x to 1.2Mx and diffraction with camera lengths ranging from 15cm to 80m, as well as tomography (±80º) and microED. The microscope at UC Berkeley has a High Contrast pole piece (Cs 3.4mm, Cc 3.0mm), an LaB6 filament, a fully integrated cryo box, and is equipped with a Gatan/Ametek OneView camera controlled by DigitalMicrograph and SerialEM. Fig. 2 shows the Elsa cryo-holder, model STP, from Gatan/Ametek used in the experiment which is capable of tilting ±60º.

Fig. 1: The JEM‐1400Flash at the Donner laboratory of UC Berkeley is a 120 kV LaB6‐equipped TEM with a High Contrast pole piece and an integrated cryo box.

Fig. 2: The Elsa cryo‐holder from Gatan/Ametek used for the cryoEM screening.

Not necessarily required, but screening can include preparing negatively stained samples as this gives information on particle size and overall morphology of the molecular complex. Fig. 3 shows a typical micrograph acquired from a sub-100 kDa membrane protein-Fab complex under study, designated here as sample A, prepared on thin C-coated formvar at 60,000x scope magnification.

Fig. 3: Sample A imaged at 120 kV at 60,000x in the JEOL JEM‐1400Flash equipped with a OneView camera.

Figs. 4 shows images of the vitrified sample at various magnifications, which clearly reveal subtle variations in ice thickness on 1.2/1/3 UltrAuFoil grids, treated by glow-discharging with a Pelco easiGlow.

Fig. 4: Atlas of frozen‐hydrated sample A imaged at 80x in the JEOL JEM‐1400Flash. These 1.2/1.3 UltrAuFoil grids were flash‐frozen in liquid ethane using a Vitrobot (A). Same grid, but now imaged at 300x showing 4 grid squares that reveal the subtle variation in ice thickness across these UltrAuFoils (B). Single grid square of the same grid imaged at 800x. All the holes of the UltrAuFoil grid are filled with thin ice (C).

Fig. 7 shows two micrographs depicting vitrified sample A in ice that is presumably too thin (Fig. 7A), or in ice of the proper thickness (Fig. 7B).

Fig. 7: Images of frozen‐hydrated sample A at 50kx, 6 e‐/Å2 dose, and ‐2 μm defocus, in vitreous ice recorded under low dose conditions in SerialEM with ice presumably too thin (A), or of the proper thickness (B).

Conclusion

The JEOL JEM-1400Flash is an excellent choice for screening frozen-hydrated specimens and can easily be used in a mixed environment of high-end, autonomous cryo scopes using appropriate cryo-holders.

CryoNote

Sun, 13 Feb 2022 17:08:15 GMT

“Visualize the truth” is a hope of researchers who use various measuring equipment. Researchers who use electron microscopes as well have a desire to observe the real structure.

But actually, in experiments using electron microscopes, many problems arise: They include damage regions of the specimen when it is cut for the size suited to observation, artifacts due to the staining that is applied to enhance image contrast, deformation caused by substitution of water to resin for withstanding vacuum exposure, and thermal damage to the specimen with electron-beam irradiation. As a result, the visualization of the real structure in the microscope image becomes increasingly difficult.

One recommended solution is to cool the specimen, that is, “Cryo” techniques. This “Cryo Note” introduces some of the diversified cryo-techniques. We sincerely hope your challenge to observe the “real structure” will be solved by “Cryo” methods.

This CryoNote includes:

Cryo History
A wide range of Cryo-techniques
Freezing
Sectioning / Fracturing
Etching (Ice sublimation)
Cryo-TEM
Freeze Substitution
Freeze Replication
Cryo-SEM
Low-Vacuum SEM and Cooling with Peltier element
Cryo-FIB
Cooling CP (Cryo-CP)

EDM Synchrony - Electron Dose Modulation And So Much More

Sun, 20 Jun 2021 13:56:05 GMT

The Electrostatic Dose Modulator (EDM) is a fast beam blanking system with a pre-sample electrostatic deflector that includes electronics and software control. With EDM, the beam can switch on or off in less than 50 ns. This 100,000x improvement in blanking speed results in immediate enhancement in the clarity of data taken at fast exposure times. Moreover, EDM includes a desktop control knob that allows users to easily attenuate electron dose without affecting imaging conditions. The included software interface gives TEM and STEM users direct access to EDM’s pulse width modulation parameters providing exceptional control over the dose rate on their samples – invaluable feature for beam sensitive specimen imaging and analysis.

Synchrony works in conjunction with EDM to allow nanosecond timing control and synchronization of STEM for programable and precise localized dose control in both space and time. In Programmable Scan mode users can adjust the dose per pixel in a STEM scan using the “dose painting” software interface. Or they can switch to temporal mode to define arbitrary electron pulse timing on their specimens as well as trigger lasers or specimen holders to create “pump-probe” experiments. Key features include:

Lightning-fast speeds - EDM systems achieve switching times faster than 50ns.
Independent intensity adjustment - By rapidly turning the beam on and off with variable pulse widths, the EDM makes it easy to adjust the average beam intensity without changing the image conditions. A knob provides an intuitive interface to adjust the dose attenuation factor.
Dose structuring – Users can easily control their illumination by applying dose in pulses with variable durations as short as 100 ns and frequencies up to 500 kHz.
Synchrony - Program the dose per-pixel in STEM scans , laser timing control for pump-probe experiments.
Modern control software, open API, and integration.

About IDES
IDES is a JEOL company and the leader in the field of Ultrafast and Dynamic TEM, specializing in pulsed lasers and high-speed electrostatic beam blanking and deflection technologies. IDES products add time resolution to the TEM imaging capabilities enabling new applications and the exploration of the dynamics of specimens across a range of very fast time scales.

Lithium Ion Battery Note

Mon, 04 Mar 2024 10:42:25 GMT

The applications for lithium ion batteries (LIB) cover a wide range, from power sources for personal computers and mobile devices to automobiles, and there is always a demand for even better performance and safety. In order to ensure the performance and quality of LIB, analysis and evaluation using high-performance assessment systems is necessary. JEOL offers a full line-up of equipment to support the development of new LIB technologies and to improve product quality, including instruments for morphology observation and surface analysis, chemical analysis systems to perform structural analysis on a molecular level, as well as fabrication systems to create high-performance coatings and powders. This LIB note offers solutions for researchers and engineers who are looking for the best equipment for their application.

Luminary Micro – Precision Localized Laser Heating Without Special Holder

Sun, 20 Jun 2021 13:40:18 GMT

Luminary Micro is a Compact Specimen Photoexcitation System (CPXS) for JEOL TEMs. It is composed of a modulated laser, a compact optical delivery system, an inlet port, and a mirror. With this add-on, users can direct and focus the laser output onto the TEM sample in situ. Luminary Micro can induce a rich variety of reactions and dynamic processes in the specimen, thanks to its <40 μm FWHM focus size, adjustable peak power up to 3 W, and the modulated pulse widths ranging from a few microseconds to seconds. With Luminary Micro, users can study laser-induced phenomena in situ using fast cameras. Combined with IDES/JEOL EDM fast shutter and/or Relativity subframing systems, Luminary Micro allows users to perform time-resolved studies using pump-probe methods in the microsecond time scale. The extremely compact footprint of the system allows easy installation without affecting the TEM resolution. The user can heat specimens to thousands of degrees C while keeping the freedom to use the specimen holder of your choice.

The key features of Luminary Micro include:

Precise and localized laser heating without a need for a special holder
Specimen contamination removal
Up to 3W average output power
577nm wavelength (other wavelengths available)
Pulsed or continuous wave operation
Compatible with most specimen holders

Applicable models: ARM300F2, NEOARM (Cs corrector), 2100F, 2100Plus

Relativity – High Frame Rate Imaging With ANY Camera

Sun, 20 Jun 2021 13:49:49 GMT

The IDES Relativity Electrostatic Subframing System multiplies the frame rate of cameras on JEOL TEMs. Microscopes equipped with Relativity achieve exceptional time resolution, data throughput, and advanced automation capabilities. Addition of Relativity allows current JEOL TEM users to forego expensive camera upgrades to their existing systems, instead relying on installation of an electrostatic optics assembly in a wide-angle camera port. These optics rapidly deflect the image data to different regions (subframes) of the camera in a programmable sequence. Each camera readout contains a tiled array of crisp, blur-free subframes. Raw data is automatically analyzed to give a stack of open format images that are loaded back into the camera control software for viewing or further analysis.

The key features of Relativity include:

Simple field installation through accessory port
Time resolution – transition between subframe regions in less than 100ns
Continuous kHz-scale video with subframe rates up to 100kHz
Integration with in-situ holders, laser, EDM, other accessories
Optics pneumatically retracted when not in use
Automated data processing (Acuity Edge analysis server) – segmentation, denoising, drift correction – helps the user get the most of the collected data
Advanced control software – seamless transition between measurements with intuitive interface combined with ability to program and design unique experiments

Applicable models: ARM300F/300F2, ARM200F, NEOARM, F200, 2100Plus, 2100F