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overview of electron beam lithography

An overview of electron beam lithography

An overview of electron beam lithography

Electron beam lithography uses a focused electron beam to pattern the surface of a material. As electron beams can be very tightly focused and small beam sizes achieved, electron beam lithography can be used to create very intricate structures for a wide variety of nanofabrication applications.1

What is the electron beam lithography process? 

The electron beam lithography process involves covering the surface of the substrate with a resist. Certain areas of the sample are then exposed to the incident beam of electrons that allow for the pattern's direct writing into the resist layer.
The sample is exposed by the electron beam in very fine steps to create the pattern. At each step, the resist must be exposed for sufficient time for the beam to activate the resist. Determining the optimal time for this in electron beam lithography is one of the steps in the process automation of the technique.
Once the resist layer has been exposed, the remaining resist is developed through chemical treatment of the sample, often with solvents such as acetone or alcohols that will dissolve the resist layers. Polymers such as PMMA (positive tone) and HSQ (negative tone) are popular choices for electron beam lithography resists and can be spun in very thin layers on top of the substrate of choice for the electron beam lithography process.

What is electron beam lithography used for?

Nanofabrication is one of the biggest applications for high-resolution direct-write methods like electron beam lithography.2 Many technologies, from new metalens structures toquantum computing, rely on techniques such as electron beam lithography to create the complex surface structures that these applications demand. One of the biggest challenges to be overcome with many new nanotechnology developments is reliable fabrication techniques for creating devices, and electron beam lithography is one potential solution for that.
Other applications of electron beam lithography include creating complex structures such as metal-organic frameworks (MOFs) that are now of interest for the miniaturization of solid-state devices.3 There are industrial applications using EBL for production of communication devices, due to the beam position’s nanometer-level accuracy and small linewidth capabilities.

What is the advantage of e-beam lithography over photolithography?

Photolithography involves the use of optical beams. As the diffraction limit of visible light is on the order of hundreds of nanometers, this limits the spatial resolution achievable with photolithography. While electron beam lithography is also diffraction limited, the diffraction limit of the high energy electrons that can be produced in various electron beam lithography experiments is on the order of nanometers or even sub-nanometer.
The improved spatial resolution of electron beam lithography means much more detailed, and complex structures can be created than with photolithography methods, though the latter has the advantage of higher throughput.

JEOL Solutions

JEOL are world leaders in electron beam technologies and has a number of products available to support electron beam lithography processes. Contact JEOL today to see how your direct-write processes could benefit from JEOL’s expertise in the development and optimization of electron beam lithography experiments.

References

  1. Nagashima, K., Zheng, J., Parmiter, D., & Patri, A. K. (2011). Biological Tissue and Cell Culture Specimen Preparation for TEM Nanoparticle Characterization. In S. E. McNeil (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (pp. 83–91). Humana Press. https://doi.org/10.1007/978-1-60327-198-1_8
  2. Chen, Y. (2015). Nanofabrication by electron beam lithography and its applications : A review. Microelectronic Engineering, 135, 57–72. https://doi.org/10.1016/j.mee.2015.02.042
  3. Tu, M., Xia, B., Kravchenko, D. E., Tietze, M. L., Cruz, A. J., Stassen, I., Hauffman, T., Teyssandier, J., Feyter, S. De, Wang, Z., Fischer, R. A., Marmiroli, B., Amenitsch, H., Torvisco, A., Velásquez-hernández, M. D. J., Falcaro, P., & Ameloot, R. (2021). Direct X-ray and electron-beam lithography of halogenated zeolitic imidazolate frameworks. Nature Materials, 20, https://doi.org/10.1038/s41563-020-00827-x
  4. Smith, D. J. (2008). Ultimate resolution in the electron microscope? Materials Today, 11, 30–38. https://doi.org/10.1016/S1369-7021(09)70005-7
  5. JEOL (2022) Scanning Electron Microscopes, https://www.jeolusa.com/PRODUCTS/Scanning-Electron-Microscopes-SEM, accessed April 2022
  6. Arenal, F. L., A., D., & R., M. (2015). Advanced transmission electron microscopy. Springer

Carrying Out Nanostructural Analysis with Focused Ion Beams

Carrying out Nanostructural Analysis with Focused Ion Beams

Carrying Out Nanostructural Analysis With Focused Ion Beams

Carrying Out Nanostructural Analysis with Focused Ion Beams
Focused ion beam technologies, considered to be an emerging field within electron microscopy, are used in a multitude of scientific disciplines for site-specific analysis, milling, micromachining, deposition, and imaging. This article will serve as an overview of focused ion beam technologies and how they are used in nanostructural analysis.

What are Focused Ion Beams?

Focused ion beam (sometimes referred to as FIB) is a method that is often used in materials science, semiconductor industry, and biological fields for the analysis, deposition, and ablation of materials. A focused ion beam setup is a scientific instrument that looks similar to a scanning electron microscope (SEM).
Whereas SEMs use a focused beam of electrons to image a given sample, FIBs use a beam of focused ions to image, mill, deposit, etc. Focused ion beams can be incorporated into a system in which both electron and ion beam columns are present, which means that the same feature can be investigated with either of the beams.
Dual-beam (SEM + FIB) platforms serve as multifunctional tools for direct lithography for nanostructural analysis. These platforms combine a high-resolution scanning electron microscope and a focused ion beam column which is also equipped with precursor-based gas injection systems, micromanipulators, and chemical analysis tools.

What is Nanostructural Analysis?

A nanostructure is a structure of a non-specific size between microscopic and molecular structures. Nanostructural detail refers to microstructure at the nanoscale, and when describing nanostructures it is important to differentiate between the number of dimensions in the volume of an object which are on the nanoscale. Nanotextured surfaces have one dimension which is on the nanoscale such as the thickness, whereas nanotubes have two dimensions that are on the nanoscale and spherical nanoparticles have three dimensions on the nanoscale.

Using Focused Ion Beams to Carry Out Nanostructural Analysis

Focused ion beam milling enables manipulation of the shape and size of nanostructures to create geometries that may be useful for thermoelectrics, optoelectronics, and quantum computing.
At JEOL, we are experts in metrology and analytical instruments. If you would like to find out more about how we can help with focused ion beam technology, contact us today for more information.

Achieving Pristine Cross Sections of Battery Samples for SEM

Achieving Pristine Cross Sections of Battery Samples for Scanning Electron Microscopy

Achieving Pristine Cross Sections of Battery Samples for Scanning Electron Microscopy

Lithium-ion batteries present unique challenges for sample preparation prior to imaging and analysis with the Scanning Electron Microscope (SEM). Batteries consist of layers of thin films that form multiple solid−solid interfaces and are sensitive to air when the inner structures are exposed. The ultrathin layers of cathode materials, separators, anode materials, metal current collectors, and electrolyte – all in the form of powders, sheets, and fluids – are each of different compositions and hardnesses. This layered composite of varying and sensitive materials requires special preparation techniques.
Broad beam ion milling is a robust method for obtaining pristine cross sections of such a complex system. Other, more traditional mechanical polishing tools introduce various artifacts, such as scratches and embedded polishing media, that obscure the original microstructure, crystallographic information and precise layer thickness measurements.
Figure 1. Scanning Electron Microscope image of Cross sectioned battery layers.
Figure 1. Scanning Electron Microscope image of Cross sectioned battery layers.
The JEOL Ion Beam Cross Section Polisher (CP) is widely used for preparing pristine samples prior to high resolution imaging and elemental analysis with the SEM. It produces pristine cross sections of samples – hard, soft, or composites – without smearing, crumbling, distorting, or contaminating them in any way.
The JEOL CP uses an Argon ion beam to mill cross sections or polish virtually any material that is affixed to the continuously rotating sample holder. During milling, the sample is rocked automatically to reduce creating beam striations on the cross-sectioned surface.
Figure 2. JEOL Ion Beam Cryo Cross Section Polisher
Figure 2. JEOL Ion Beam Cryo Cross Section Polisher
With the emergence of increased research on batteries, JEOL developed a special Cooling Cross Section Polisher (CCP) for preparation and polishing of materials that are sensitive to exposure to air or thermal damage. This new configuration is designed for cryo-preparation (down to LN2 temperature) and air-isolated transfer for atmosphere sensitive specimens The CCP allows long cooling periods while conserving liquid nitrogen.
The CCP is integrated into JEOL’s air-isolated workflow using a sample transfer vessel for air and heat-sensitive samples. The sample is initially prepared in an inert gas environment (such as in a glove box) then safely transferred to the Cryo Cross-section Polisher, and subsequently into the SEM (or Focused Ion Beam preparation and Transmission Electron Microscope) using the special air-isolated sample holder.
Figure 3. JEOL air-isolated workflow for battery sample preparation and imaging in the SEM.
Figure 3. JEOL air-isolated workflow for battery sample preparation and imaging in the SEM.
Using this technique, it is possible to examine morphological (surface) details and compositional characterization of battery materials without any air exposure. Every layer of detail can be examined clearly in the SEM. Ultra-low voltage imaging combined with signal filtering in the SEM allows direct imaging and analysis of battery constituents (anode and cathode) with nanometer resolution.
Figure 4. Scanning Electron Microscope image showing cross section of Lithium Ion Battery cathode. Larger image is magnified at 7500X for details of precise cross sectioning.
Figure 4. Scanning Electron Microscope image showing cross section of Lithium Ion Battery cathode. Larger image is magnified at 7500X for details of precise cross sectioning. 
Figure 5. Scanning Electron Microscope image showing cross section of Lithium Ion Battery separator.
Figure 5. Scanning Electron Microscope image showing cross section of Lithium Ion Battery separator.

Also See:

Further Reading:

cryo-electron microscopy images and microscope

What is Cryo-Electron Microscopy Used for?

What is cryo-electron microscopy used for?

What is cryo-electron microscopy used for?

Cryo-electron microscopy, or cryo-EM, is an imaging method that has revolutionized imaging of macromolecular complexes.1 Numerous atomic structures solved purely by cryo-EM and single particle analysis (SPA) have been deposited into data banks.

One of the older techniques in structural biology, protein x-ray crystallography, uses high energy photons that are diffracted from a high-quality crystal from different angles to build the 3D structure. However, not every protein can be crystallized and, although the drive to better beamlines allowed researchers to solve structures from ever smaller crystals, many samples are out of reach of this method
Cryo-EM by virtue of sub-millisecond vitrification does offer a way to obtain true or near-atomic resolution structures without requiring crystals. This approach has been highly successful on samples such as membrane proteins, reaction intermediaries, complexes that are fleeting, or those that exist in a mixture that can be heterogeneous in conformational and/or compositional in nature.

The field of structural biology was revolutionized with the advent of direct detectors cameras and highly stable and automated cryo-electron microscopes in cryo-EM. This resolution revolution propelled cryo-EM into becoming one of the most widely used tools in structural biology. In 2017 the Nobel prize in Chemistry was rightly awarded to Dr. Henderson, Dr. Dubochet and Dr. Frank for each of their contributions to the field of cryo-EM. Cryo-EM is currently applied to study variants of the SARS-CoV-2 virus, various membrane proteins and the immune system. 1 Understanding the structure of these species is key in understanding how some of these protein systems function, or malfunction in the case of disease, as well as in identifying potential drug-binding targets for therapies.

What is cryo-electron microscopy

Cryo-EM involves vitrifying the sample and imaging the vitrified, or frozen-hydrated specimen in the electron microscope under cryogenic conditions in transmission mode. The electrons that are scattered by the sample form an image modulated by phase contrast, which is magnified onto a camera. Although electrons have a much shorter wavelength than visible light photons, the final resolution in cryo-EM is governed by many factors, some of which are the aberrations in the entire system, both optically and environmentally; others are the radiation sensitivity of the sample2.

In SPA workflows, thousands of images of a macromolecular complex are recorded under low-dose conditions as movies on preferably a direct detector camera. Hundreds of thousands or even millions of particles are selected from motion-corrected movies and subjected to rounds of alignments, classification, and intermediate 3D model builds. After the final 3D reconstruction is scrutinized for correctness and resolution, the task is to try to understand the complex structure in terms of its functionality.
In tomography workflows, around 100 images are acquired under low-dose conditions from the same area of a vitrified sample at various tilts in the electron microscope. These images are recombined after corrections for tracking and focus changes into a 3D volume using a weighted-back projection scheme or a variant thereof. The resulting volume can be annotated and analyzed, but also subjected to sub-tomogram averaging techniques, whereby selected sub-volumes from the tomogram are processed using SPA techniques. Given that typically fewer sub-volumes are available the resulting 3D model of a complex is often obtained at a slightly lower resolution.

The biggest challenge in imaging vitrified specimens is radiation damage from the electron beam. This can show up in various forms, most easily recognizable as a loss of Bragg reflections of 2D crystalline samples, like catalase or purple membrane. However, other effects include charging as a result from secondary electron emission, bond breakage and even mass loss. Specimen charging is most often visible as beam-induced specimen motion and, in extreme cases, doming of the sample. These effects severely restrict any attempt to acquire a high resolution structure.4 Pioneering efforts at the MRC point to a new type of grid for SPA, hexafoils, to combat these effects with great efficacy5.

The success of cryo-EM applied to a macromolecular complex depends on many factors, some of which are biochemical in nature, others related to the vitrification process and yet others related to the automation on the microscope. Since many images are required to solve the structure of a complex, the best possible approach is to have as many images of the complex of interest as possible. Automation, gauged by the number of high resolution images per hour is important in any setting. A substantial fraction of the cycle time, i.e. the time to go from one area on the sample to the next, is taken up by either moving the stage and waiting for drift to settle, or simply the process of recording the movie. The current state-of-the-art puts this number around 20,000 movies/day. A clinical evaluation of all steps that factor into any throughput number is critical in the choice of equipment and imaging strategy. 

JEOL cryo-electron microscopes

Performing successful cryo-EM means having the right microscope set up. JEOL offers the CRYO ARM 300 II, a dedicated instrument for highthroughput, automated cryo-EM using either SPA or tomography. The CRYO ARM 300 II will achieve this throughput via high levels of instrument stability and automated data collection through SerialEM or JADAS.3  With an ever expanding arsenal of experimental techniques and the availability of commercial cryo-electron microscopy instruments like the CRYO ARM 300 II, cryo-EM is projected to become the leading technique in Life Sciences in the coming years1.

References

Choosing the right scanning electron microscope for your laboratory

Choosing the right scanning electron microscope for your laboratory

Choosing the right scanning electron microscope for your laboratory

Author: Noriyuki Inoue, JEOL USA

What is scanning electron microscopy?

Scanning electron microscopy (SEM) is an imaging technique that produces images of a sample by scanning the surface with a focused beam of electrons. SEM differs from optical microscopy, as it uses electrons instead of light to “see” into the material’s surface. When comparing SEM to optical microscopy, optical microscopy is limited by the wavelength of light, which is physically set in a defined range. SEM has the advantage of breaking this limit and allows for resolution that can reach the sub-nanometer level.
SEM has a large depth of field and higher magnifications than traditional optical microscopy. This, combined with its ability to conduct chemical analyses using spectroscopic methods, makes it a very powerful research tool. SEMs provide a high degree of analytical capability and reveal surface details at nanoscale resolution. A single image from SEM can often be enough to achieve critical objectives i.e., visualizing microstructures. There are many types of SEMs, ranging from the more common type, which use a tungsten filament as an electron source, to the more specialized type which, with a field emission (FE) electron gun mounted, attains higher resolution and magnification.

How does scanning electron microscopy work?

SEM uses deflector coils which alter the path of the electron beam, scanning it in a zig-zag type pattern (raster scanning). Typically, three detectors are positioned at angles in the sample chamber, these are an X-ray detector, a back-scattered electron detector, and a secondary electron detector. Sample thickness is not an issue as none of these elements is reliant on transmission.
When operating an SEM, a high-energy beam of electrons is scanned across the sample. Magnets focus the electron beam to a point several nanometers in diameter. As the electron beam interacts with the surface of the sample, signals are produced and compiled by various imaging and analytical detectors. Thus, high-resolution nanoscale images are achieved along with precise measurements. An SEM may detect backscattered electrons (to reveal morphology and topography and give insight as to composition), or secondary electrons (to reveal surface topography).
SEM is often considered a quick, versatile, and convenient option over other microscopy techniques. Recently, it has been shown to be useful in more and more applications and choosing the ideal SEM instrument is dependent on many factors. Below, we summarize some of the main considerations when selecting an SEM.

1. Microscope magnification

Since electron wavelengths are up to 100,000 times smaller than the wavelengths of visible light, SEMs resolve details hundreds of thousands of times smaller than optical microscopes.
The field of view (FOV) in a microscope defines the size of the feature to be imaged. This value can range between millimeters, microns and nanometers. To define the FOV required to image samples, first the end goal must be decided. If the number of particles in a sample is what is of interest, having multiple particles per image is not an issue, so an SEM that provides a FOV of 100 times greater is enough. However, if the structure of a particle is what is of interest, a closer FOV is needed to see the required detail. This is shown in Figures 1 and 2, which compare tabletop, tungsten, multipurpose FE and ultra-high resolution (UHR) FE SEM instruments.
To simplify choosing the right magnification for specific applications, a tabletop SEM can be very efficient. The relaxed vacuum requirements and small evacuated volume enable fast image production, without the extensive sample preparation. Additionally, tabletop SEM is normally carried out by the individual who requires the information, which thereby eliminates time required for a dedicated SEM operator to carry out analysis and produce a report. As well as obtaining answers quickly, it is also beneficial to be able to carry out analysis straight away and for the user to be able to manage it in real-time response to observations.
For instances where higher magnification is needed, but space is also a limiting factor, conventional tungsten SEMs are an option to simplify specimen navigation and advanced automation delivers crisp secondary and backscatter images in seconds. If a specimen is challenging to analyze, FE SEMs and UHR FE SEMs provide topographical and elemental information at magnifications of 10x up to 1,000,000x.
Figure 1 Materials SEM Comparisions
Figure 1: Materials SEM comparisons.
Figure 2 Biological SEM Comparisons
Figure 2: Biological SEM Comparisons.
Figure 3. SEM comparisons for imaging Titanium alloy cross section
Figure 3: SEM comparisons for imaging Titanium alloy cross section.

2. Microscope resolution

The word resolution indicates the smallest observable element in an image. For the human eye, that is about 0.2 mm. SEM resolution is usually between 0.5 and 4 nanometers, meaning it provides the opportunity for particle diameters and geometries to be studied in great detail. There are many contributing factors that can affect the maximum resolution obtained in an SEM, like the electron spot size and interaction volume of the electron beam with the sample.
SEM’s high resolution is attributed to the fact that the wavelength of the electrons becomes shorter because the accelerating voltage of the electrons used in the SEM is as high as several kV to several tens kV, and to the characteristic difference of the electromagnetic lenses used to converge the electron beams. By utilizing several images together with software, the size distribution of particles may be determined and a concentration versus particle diameter may also be calculated.
SEM images are stored in an image file (e.g., JPEG, TIFF) with a user-defined number of pixels. An SEM will scan small areas with an electron beam, which means the portions of the surface will become a pixel of the final image. More pixels result in a longer processing time; however, a long analysis process can have a detrimental effect on the sample.
Tabletop SEMs can generate an electron beam at the specimen surface with spot size of several nanometers, and a price range similar to that of a high-end optical device, thus they are slowly revolutionizing the industry, realigning production standards to a new level of miniaturization. Tungsten SEM is suitable for analysis of sub-micron structures (hundreds of nm), the lower kV allows for a smaller X-ray signal depth within the sample and thus higher X-ray spatial resolution. If ultra-high X-ray spatial resolution is needed to resolve ~50nm layers, then an FE SEM is the best option, since FE emitters maintain a very small spot size even at low kV. Table 1 shows a comparison of some relevant parameters between thermionic tungsten emitters and Schottky FE emitters.
Table 1: A comparison of parameters between thermionic tungsten and Schottky field emission emitters
Table 1: A comparison of parameters between thermionic tungsten and Schottky field emission emitters.

3. Microscope applications

SEMs are used in a wide range of industries including electronics, chemicals, machinery and pharmaceuticals and are used in research, quality control and product inspection.
SEM is very popular with scientists in the materials and life science research areas as its resolution and depth of field capabilities are a significant improvement over those of traditional optical microscopy. In materials science, investigations into nanotubes and nanofibers, high temperature superconductors, mesoporous architectures and alloy strength, all rely heavily on the use of SEMs for research and investigation. Many material science industries, from aerospace and chemistry to electronics and energy usage, have been made possible with the help of SEMs.
For pharmaceutical applications, SEM can play a pivotal role in the rapid and efficient characterization of new drug treatments, providing insights into their interactions with human cells and their applications in complex therapies.
A tabletop SEM can produce the necessary results at a lower cost as long as the application is routine and well defined. A key consideration is any future laboratory requirements, which may need resolutions unachievable by tabletop SEM. To combat this, outsourcing to a laboratory with a larger model is a potential option or tabletop SEM can be utilized to aid a future floor model system. For example, tabletop SEM can be used to screen capacity and to carry out routine analyses, leaving the floor model system available for more challenging uses. Due to the nearly unrestricted FOV of FE and UHR FE SEMs, high resolution imaging and high current analyses can be achieved without sacrificing performance. These SEMs are particularly suited for imaging and analyzing magnetic light element materials and nanostructures.

4. Microscope users

As electron microscopy has become more available, the user experience has been redesigned to suit any operator. SEM has historically been restricted to substantial laboratories with the budget and space to justify the installation of full-scale, floor standing SEMs. Traditional SEM instruments have offered unrivalled details of every material, from insects to crystals and bacteria, but can be complex to use, requiring expert understanding, and also necessitate a large, dedicated space. 
However, the arrival and advancement of compact and user-friendly tabletop SEMs is changing this picture. The invention of compact SEM instruments intended to fit on a tabletop and the onset of new technologies has made it feasible to incorporate further analysis equipment into SEMs, creating self-contained nano-laboratories through the addition of an assortment of electrical, mechanical, and chemical test equipment.
When considering what type of SEM to buy or use, it is worth thinking about the number of individuals who will be utilizing the system, how much training they have, and the amount of time it might take to train them. Whereas floor-based systems with their built-in automation are simple to use, training is recommended to optimize performance. Tabletop SEMs are uncomplicated, and, for the majority of applications, they require much less training.

Conclusion

SEM is an innovative tool with countless applications. However, it is important that the user has a defined idea of what type of analysis is required – and of how the different spot sizes, electron beams, and accelerating voltages will influence the SEM imaging quality. Selecting the best parameters for any given experiment is crucial in selecting the most appropriate SEM.
Overall, tabletop SEM meets the imaging requirements in most situations. The resolution limits in the several nanometer range is enough for 80-90% of all applications making them a smart choice for most laboratories.

Suiting Up with NanoSuit for Imaging in the SEM

Suiting Up with NanoSuit for Imaging in the SEM

Suiting Up with NanoSuit for Imaging in the SEM

Suiting Up with NanoSuit for Imaging in the SEM
FAU Owls Lab uses a unique biofilm for imaging microorganisms in the NeoScope Tabletop Scanning Electron Microscope
Applying the Nanosuit
They’ve just begun testing out the NanoSuit® using the NeoScope benchtop SEM in the school’s Owls Imaging Lab and are getting good imaging results. Their entomological specimens appear to be in a more natural state, and they are already on their way to helping students and researchers using the biofilm. 
Their first subject was a hairy spider that helped them learn what worked and what they needed to do.
“The spider was a pretty cool test,” says Knaub, who watched a video to learn how much of the solution to pipette onto the spider positioned on the sample holder. “Spiders don’t do well in a critical point dryer,” she says of the typical sample preparation tool for microorganisms. In the image one can see that the spider looks more realistic. “We don’t have a ton of experience yet. The sample just seems to absorb the solution. It has a very interesting viscosity; it seems to fold back onto itself. It just takes a thin layer on top of it. We’re trying to perfect that.” After fully coating the specimen, she took filter paper to draw off any excess. It was trial and error, but she learned that more of the Nanosuit solution could be applied as needed and reapplied later for additional imaging. Like their other specimens, they kept the spider in their repository to be used again.
Their success opened the door for research faculty member Dr. Jennifer Krill and her high school student mentee, Ms. Saachi Mody, to use the technique to observe tumor expression in fruit flies. The series of images, ranging from 100-200 µm made for a nice timeline showing changes over time.  The work is part of the high school student’s science research project. The school puts enormous emphasis on student research and begins using the tools of science, such as the SEM, in the lower grades. The microscope is used by researchers at the university as well. Many SEM images make their way into reports and posters.
Meredith and Knaub look forward to trying some plant materials, which tend to shrivel in the critical point dryer. They’ll experiment more during the summer and will be ready to give a new crop of students of all ages a special look into the biological materials they are studying.
Watch a demonstration video here:

How Cryo-EM Differs from TEM

How Cryo-EM Differs from TEM

How Cryo-EM Differs from TEM

Transmission Electron Microscopy or TEM is a generalized term for a suite of imaging techniques that uses electrons to acquire image data and/or analytical data from a specimen.  Cryo-electron microscopy or cryo-EM, a de facto sub-set of TEM, has as a goal acquiring information from biological samples whose native state has been preserved whilst in the vacuum of the electron microscope. Cryo-EM has yielded a surge of near- and real atomic structures solved for instance by single particle analysis (SPA) or sub-tomogram averaging, all deposited in the structure data banks around the world. 

As alluded to above, the key difference between cryo-EM and conventional TEM is the sample preparation method. Cryo-EM uses flash or slam or jet freezing of a liquid or suspension to create a specimen that can be observed in the microscope without the use of fixating or staining aids, whereas conventional TEM methods typically include approaches like chemical fixation or staining agents or even the use of polymers to immobilize the sample in place.1 This principal difference in sample preparation dictates what types of specimens can be studied by either technique. Conventional TEM has been widely used in cellular imaging for over fifty years and can reveal information on the structure of cell organelles.2 This approach is ideal for revealing ultrastructural elements e.g., cellular components, vesicles, ER etc, albeit with limited resolution. Cryo-EM, on the other hand, in avoiding the fixation and staining preserves the structure of macromolecular complexes to high resolution, which can be retrieved using SPA or sub-tomogram averaging applied to tomography data. Also, and more critically important, cryo-EM allows us to examine those complexes that are typically not amenable to other structural studies, such as NMR or x-ray crystallography, because either the complexes are simply too large or crystals are impossible to grow. Furthermore, cryo-EM can address those cases where the complex of interest is available in only minute quantities, such as reaction intermediaries, or the complex is present in a conformational mixture. The advent of direct detectors, highly automated electron microscopes and powerful image processing algorithms, responsible for the resolution revolution, have established cryo-EM as an increasingly popular tool in structural biology projected to eclipse x-ray crystallography as the technique to solve atomic structures in a few years (Kikkawa, 2022).

In-situ TEM, where biological samples are imaged in a liquid cell at room temperature, has the potential to yield information regarding fluctuations in the protein structure as a function of temperature.3 The level of detail, however, may be limited to domains owing to the vast increase in radiation sensitivity of samples studied by this technique, and the possible mobility of a complex in solution. On the other hand, recent computational approaches applied to cryo-EM data sets of ribosomes obtained by SPA have suggested various conformational states that were observed with substantially greater detail (cryoDRGN, 2022).

Instrumentation

The key components of the instrumentation for cryo-EM and TEM can be the same. Both approaches require an electron source – typically an electron gun capable of producing electron beams with different energies but whose characteristics depend critically on the type of source employed.
The electron beam must then be focused onto the sample using a series of lenses, which occurs in the condenser system. These lenses help to shape the beam and achieve the small spot sizes necessary for obtaining the best spatial resolution with the electron beam as well as achieve the proper dose rate for various studies.
The heart of the electron microscope is the objective lens. This optics piece, composed of an upper and lower pole piece, is responsible for the level of detail observed in an electron microscope. Worth noting is that owing to the presence of spherical aberration, high-resolution images can be acquired from unstained cryo-EM samples by simply defocusing the objective lens, typically by a fraction of a micron.
Inserted in the gap of the objective lens is the sample holder. For automated, high-throughput studies an autoloader is paramount, whereas a side-entry holder suffices for other cases. Imaging cryo-EM samples is principally done using a stationary beam. In Materials TEM, the preferred technique is to raster the electron beam across the sample.
Below the sample are magnifying lenses and finally, at the very bottom of the column, is typically a direct electron detector or DED. Largely responsible for the resolution revolution in cryo-EM, DEDs record movies of the sample rather than still images, thus providing for a way to eliminate beam-induced motion. Typical cameras in conventional TEM do not require this and are thus far cheaper and simpler.

JEOL Microscopes

Performing cryo-EM has never been so easy as with the JEOL CRYO ARM 300 II. With the capability of performing SPA and tomography for 3D reconstructions, the CRYO ARM 300 II makes it simple to harness the excellent spatial resolution and structure-solving capabilities of cryo-EM.
Contact JEOL today to find out if the CRYO ARM 300 II could be the instrument for you and how cryo-em and advanced TEM methods could benefit your application. JEOL offers a full suite of Transmission Electron Microscopes, with unique options such as the 120kV JEM-1400Flash, the LaB6 JEM-2100, and more.

References

  1. Nagashima, K., Zheng, J., Parmiter, D., & Patri, A. K. (2011). Biological Tissue and Cell Culture Specimen Preparation for TEM Nanoparticle Characterization. In S. E. McNeil (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (pp. 83–91). Humana Press. https://doi.org/10.1007/978-1-60327-198-1_8
  2. Winey, M., Meehl, J. B., Toole, E. T. O., Giddings, T. H., & Drubin, D. G. (2014). Conventional transmission electron microscopy. Molecular Biology of the Cell, 25, 319. https://doi.org/10.1091/mbc.E12-12-0863
  3. Nagashima, K., Zheng, J., Parmiter, D., & Patri, A. K. (2011). Biological Tissue and Cell Culture Specimen Preparation for TEM Nanoparticle Characterization. In S. E. McNeil (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (pp. 83–91). Humana Press. https://doi.org/10.1007/978-1-60327-198-1_8

Carbon Nanotubes imaged by TEM (L) and SEM (R)

Scanning Electron Microscopes Vs Transmission Electron Microscopes

Scanning Electron Microscopes Vs Transmission Electron Microscopes

Carbon Nanotubes imaged by TEM (L) and SEM (R)
Carbon Nanotubes imaged by TEM (L) and SEM (R)]

Electron microscopy has revolutionized the imaging of nanostructures. With its excellent sub-Angstrom spatial resolution, electron microscopies can be used to image objects as small as atoms on a surface.1 For high-precision manufacturing of nanoscale structures, such high spatial resolution is essential for quality control and checking for the presence of any defects in the structure that may impede performance.

The reason electron microscopy has such high spatial resolution in comparison to traditional optical techniques is due to the much shorter wavelength of an electron compared to that of a photon. In optical microscopes, this limits the apparatus to a spatial resolution of several hundred nanometers.
Two of the most popular electron microscopy methods make use of scanning electron microscopes (SEM) and transmission electron microscopes (TEM). Scanning electron microscopes and transmission electron microscopes require slightly different sample preparation and recover different imaging information but many of the basic operating principles are similar between the techniques.

Basics of Electron Microscopy

Most electron microscopes are composed of the electron source, typically a high voltage electron gun, a series of focusing optics, the sample holder, and a detector. High brightness electron guns produce the electron beam that is then focused onto the sample. The focusing optics in an electron microscope play an important role as changing these conditions affects the depth of field and illumination area of the electron beam. After interacting with the specimen, the scattered electrons are collected in various geometries to reconstruct an image.

What is the Difference Between SEM and TEM?

The positioning of the detector and the type of electrons detected is one of the key differences between scanning electron microscopes and transmission electron microscopes.2 In a transmission electron microscope, the electron beam passes straight through the sample where the change in the electron transmission is detected.

Transmission electron microscopy is ideal for thin layer samples (< 150 nm) and can be sensitive to not just the surface of the sample but the inner regions as well. It is commonly used for highly crystalline structures and nanotechnology applications but can be used for looking at tissues and bacteria as well.3

Scanning electron microscopes instead detect the backscattered and secondary electrons from the sample instead of just detecting the incident beam. When combined with raster stages to translate the sample, a scanning electron microscope can be used to reconstruct a full structural image.
Both scanning electron microscopes and transmission electron microscopes are ‘label-free’ microscopy techniques and image the sample as it is. However, for imaging with scanning electron microscopes, one of the big challenges is ensuring the sample can withstand the high vacuum conditions and is sufficiently clean to capture the incredibly detailed images the technique is capable of.

JEOL Microscopes

JEOL offer an extensive range of scanning electron microscopes and transmission electron microscopes.2 Contact JEOL today to find out what the right technique for your application is and how their instrumentation can benefit you.

References

  1. Smith, D. J. (2008). Ultimate resolution in the electron microscope? Materials Today, 11, 30–38. https://doi.org/10.1016/S1369-7021(09)70005-7
  2. JEOL (2022) Scanning Electron Microscopes, https://www.jeolusa.com/PRODUCTS/Scanning-Electron-Microscopes-SEM, accessed April 2022
  3. Arenal, F. L., A., D., & R., M. (2015). Advanced transmission electron microscopy. Springer

MALDI Imaging Solution

What is MALDI Imaging Mass Spectrometry?

What is MALDI Imaging Mass Spectrometry?

MALDI Imaging Solution
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a widely used soft ionization technique used to measure particularly fragile samples such as biomolecules. Compared to other mass spectrometry ionization methods, MALDI imaging mass spectrometry results in much less fragmentation of the molecular species. 
MALDI imaging mass spectrometry works by immobilizing the analyte of interest in an inert matrix. The MALDI imaging matrix should not induce any changes in the sample of interest, and simply act as a medium to absorb the laser irradiation used and subsequently ionize the analyte of interest. There are numerous methods to prepare samples in matrices, including spraying or sublimation.
The key improvement of MALDI imaging mass spectrometry, when compared to simple MALDI methods, is that as well as having the mass spectrum recorded for the analyte, the position in the sample is also measured. The sample is laser irradiated, ionized and a new spectrum recorded for each position, meaning that a full two-dimensional image can be built up of the sample with each data point containing the full mass spectrum recorded, thus giving the technique its spatial resolution.
MALDI imaging mass spectrometry has become a popular technique as, unlike many microscopy methods, it is a label-free way to study biological samples. MALDI imaging can achieve very high spatial resolutions and recent developments in the technique are making it possible to create three dimensional images to provide volume information on the sample in a similar way to magnetic resonance imaging (MRI).

Applications

Another advantage of MALDI imaging mass spectrometry is its suitability for various sample types. Tissue samples can be directly prepared on microscope slides for imaging, and the reduced fragmentation from the soft ionization process makes it ideal for detecting complex profiles of small molecules to profile drug distributions in biological samples. The spatial element of the information recovered in MALDI imaging makes it possible to identify which regions of the tissue certain drugs may be concentrated in and build an understanding of drug transport and uptake in the body.

JEOL’s MALDI Imaging Solutions

JEOL’s MALDI imaging instruments offer excellent mass-resolution, one of the key parameters for the unambiguous identification of molecular species and discrimination between compounds in complex mixtures.1 The JMS-S3000 SpiralTOF Plus is an all-in-one MALDI imaging system that can be paired with JEOL’s SCiLS Lab MVS to both capture and analyze all your data. The volume of data generated by MALDI imaging datasets can be challenging to handle, but SCiLS Lab MVS comes with many built-in options for visualization and processing to streamline this process.
The JMS-S3000 MALDI imaging instrument can manage the complexity of even spectrally congested brain tissue samples with large numbers of lipids. Combining high mass resolution with high spatial resolution makes it possible to identify even minor lipid families in the sample. This is aided using electric sectors in the analyzer to improve chemical noise, particularly in low m/z regions, by removing any post-source ions.
Are you looking for a robust technique to provide a wealth of information on complex biological samples? Contact JEOL today to discover how their MALDI imaging solutions could support your workflows.

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