OVERVIEW

There are a number of options and technologies available for digital imaging in transmission electron microscopy (TEM) applications today. Traditionally, high energy electrons could not be directly exposed to a sensor without excessively damaging the detector. As a consequence, conventional TEM cameras first expose the incoming electron beam to a scintillating film that converts the electrons into light (photons). These photons are then transferred to the sensor, either through a series of optical lenses or a coupled fiber optic plate. Finally, the light is collected by a sensor where the image is created pixel-by-pixel based on the amount of light detected at each position in the sensor.

CONVENTIONAL TEM IMAGE DETECTION ARCHITECTURE

There are four basic steps in TEM imaging to address incoming electrons:

1. Convert electrons to signal
2. Transfer signal
3. Detect signal with sensor
4. Electronically transfer signal and read-out to form image

WHAT IS DIFFERENT WITH DIRECT DETECTION?

There are only two steps in TEM imaging with direct detection:

1. Convert electrons to signal – not applicable
2. Transfer signal – not applicable
3. Detect signal with sensor
4. Electronically transfer signal and read-out to form image

One key difference between conventional and direct detection is a custom CMOS sensor that utilizes the only radiation hard architecture that can tolerate direct exposure to high energy particles. To add, extremely high speed electronics for data transfer and processing enable low-dose counting and super-resolution capabilities. Combined, this allows frame rates (4k x 4k) of 400 frames per second (fps) to be processed in real-time to achieve optimal results.

STEP 1) CONVERT ELECTRONS TO SIGNAL

Gatan uses proprietary phosphor scintillators to optimize signal conversion that enhances detector sensitivity (SENS) and resolution. When you select a scintillator, it is appropriate to know the performance trade-offs between SENS and resolution.

• Sensitivity (signal): Ideal for dose-sensitive use cases where you need to generate more photons per incoming electron (e.g., cryo-tomography, beam sensitive materials)
• Resolution (spatial detail): Favorable for applications where you require more information to resolve details, but you can increase the dose (signal) without harming the sample (e.g., semiconductors and other less-sensitive materials)

STEP 2) TRANSFER SIGNAL

Various coupling (lens- and fiber-coupled) mechanisms are available to optimize signal transfer and meet cost or performance targets for a given detector.

Lens-coupled: Lens optics transmit light to the sensor where it is converted into sensor electrons (signal)

• Pros: (Gatan) Uses real transmission scintillator; can be less expensive than higher performance fibers
• Cons: Light (information) loss with lensing, angular dependencies (<10% efficient); vignetting (light fall-off); image distortion for higher magnification

Fiber-coupled: Scintillator creates photons that subsequently create sensor electrons; fiber directly transmits light to sensor with high efficiency

• Pros: Most efficient transmission of light information to the sensor (1:1 coupling of scintillator:sensor (>50% efficient) with no image distortion); can trade-off SENS verses resolution (fiber configuration detail)
• Cons: Fiber optics are slightly higher cost; requires process optimization including cladding, sintering, bonding of fibers (Gatan proprietary)

Renal sample at 3000x magnification. Fiber-coupled (left) vs. lens-coupled (right).

STEP 3) DETECT SIGNAL WITH SENSOR

Sensor type (CCD vs. CMOS) offers significant trade-offs for TEM camera performance as there are fundamental differences in architecture.

• Charge-coupled device (CCD): Charge transfers between neighboring cells, and read-out (e.g., noise) is seen at final stage; binning minimizes the impact of read-out noise
• Complementary metal–oxide–semiconductor (CMOS): Charge immediately converts to voltage (read-out with digital output); supports high frame rates, low overall electronics noise

Both technologies possess inherent advantages, so the question arises about what unique performance characteristics arise from each choice. CCDs can have 100% fill factor that captures all incoming light, whereas part of the CMOS sensor is occupied by transistors and metal wiring associated with each pixel. Historically, CCDs provided higher quality images with low noise, at affordable prices. Recent design advancements and processing techniques now advance CMOS sensor performance so it is a viable choice for some applications. Note that CCDs still maintain an advantage for binning in terms of signal-to-noise. However, CMOS chips can scale the number of read-out ports and achieve very high frame rates.

Image courtesy of http://meroli.web.cern.ch/meroli/lecture_cmos_vs_ccd_pixel_sensor.html

STEP 4) TRANSFER SIGNAL AND READ-OUT IMAGE

When a charge converts to voltage, you typically generate noise

• CCD: Transfer data out of serial register
• CMOS: Converts to voltage per pixel

It is very important to optimize read-out noise (higher voltages) and speed (multi-port and fast read times) for CCDs.

• Optimize controller for low read-noise; leverage multi-port read-outs for faster speed
• Interline CCDs with binning have the fastest readouts (fps) – up to 30 fps due to 100% duty cycle

CMOS typically is seen as a fast sensor because you can run in rolling shutter mode verses the slow global shutter mode.

OVERVIEW

What is in-situ microscopy?

In-situ transmission electron microscopy combines the image formation capabilities of the transmission electron microscope (TEM) with application of some external stimulus to measure results real-time. Currently, a wide variety of systems and holders are available to apply different stimuli to evaluate kinetic reactions such as catalysis, phase transformations, other chemical reactions, electrical fields, mechanical strain or nano-indentation deformation, heating and cooling, or the effects of the electron beam irradiation damage in samples.

Historically, movies of various reactions and system kinetics could be recorded onto video tape. More recently screen capture programs allow low-resolution, qualitative capture for you to visualize what is happening at video frame rates, e.g., 30 frames per second (fps). This adds significant value to investigators who want to physically see and understand reactions in real-time.

With the advent of faster data transfer and processing capabilities, it is now possible to record and manage large datasets directly from the sensor output. You can store each pixel in each frame directly to disk, and treat them as an individual image. Alternatively you can apply various algorithms or scripts (e.g., summing, drift correction, binning, etc.) post-capture to quantify the stimulus applied to the system. This greatly enhances the flexibility of data management and gives you previously unseen resolution in time and space, with sub-ms time resolution to resolve reactions that previously were unknown.

OVERVIEW

TEM specimen beam interactions.Electron energy loss spectroscopy (EELS) is a family of techniques that measure the change in kinetic energy of electrons after they interact with a specimen. This technique is used to determine the atomic structure and chemical properties of a specimen, including: the type and quantity of atoms present, chemical state of atoms and the collective interactions of atoms with their neighbors. Some of these techniques include: spectroscopy, energy-filtered transmission electron microscopy (EFTEM), and DualEELS™.

Atom-scale view of electron energy loss.As electrons pass through a specimen, they interact with atoms of the solid. Many of the electrons pass through the thin sample without losing energy. A fraction will undergo inelastic scattering and lose energy as they interact with the specimen. This leaves the sample in an excited state. The material can de-excite by giving up energy typically in the form of visible photons, x-rays or Auger electrons.

As the incident electron interacts with the sample, it changes both its energy and momentum. You can detect this scattered incident electron in the spectrometer as it gives rise to the electron energy loss signal. The sample electron (or collective excitation) carries away this additional energy and momentum.

Core-loss excitations occur when tightly bound core electrons are promoted to a higher energy state by the incident electron. The core electron can only be promoted to an energy that is an empty state in the material. These empty sates can be bound states in the material above the Fermi level (so called anti-bonding orbitals in the molecular orbital picture). The states can also be free electron states above the vacuum level. It is the sudden turn-on of the scattering at the Fermi energy and the probing of empty states which makes the EELS signal sensitive to both the atom type and its electronic state.Correlation between EELS and specimen features.

You can visualize the initial spectral features in the core-loss excitations when you align the Fermi level with the zero-loss peak (ZLP) of the spectrum. The edges can now be seen as the point where the electrons lose enough energy to promote the core level atomic electrons to the Fermi level. This analogy fails to reproduce the scattering above the Fermi level, but is helpful to visualize the core level edge sudden increase in intensity.An EELS spectrum.

A typical energy loss spectrum includes several regions. The first peak, the most intense for a very thin specimen, occurs at 0 eV loss (equal to the primary beam energy) and is therefore called the zero-loss peak. It represents electrons that did not undergo inelastic scattering, but may have been scattered elastically or with an energy loss too small to measure. The width of the zero-loss peak mainly reflects the energy distribution of the electron source. It is typically 0.2 – 2.0 eV but may be as narrow as 10 meV or lower in a monochromated electron source.

For more information on the EELS family of techniques, please visit EELS.info, an educational site.

OVERVIEW

Energy-filtered transmission electron microscopy (EFTEM) is a family of imaging techniques that utilize properties of the energy loss spectrum to increase contrast, remove the effects of chromatic aberration and create unique contrast effects in the image. Key applications include:

• Contrast enhancement – Improves contrast in images and diffraction patterns when it removes inelastically scattered electrons that produce background fog
  • Including zero-loss filtering, most probable loss imaging, contrast tuning, and pre-carbon imaging
• Mapping – Creates elemental/chemical maps at nanometer resolution by forming images with inelastically scattered electrons
  • Including 2- and 3-window elemental mapping/jump-ratio imaging and chemical mapping – provides fine structure imaging
• Analytical – Records and quantifies electron energy loss spectra (and maps) to provide chemical analysis of TEM samples

The principle behind EFTEM is based on the illumination of a very thin specimen with a beam of high energy electrons. When the majority electrons pass unhindered through the specimen, some will interact with the specimen and result in elastic or inelastic scattering. Inelastic scattering results in both a loss of energy and a change in momentum, which in the case of inner shell ionization is characteristic of the element in the sample.

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For more information on the EELS family of techniques, please visit EELS.info, an educational site.

OVERVIEW

Cryo-EM is being led by the technology and innovation behind the K2 direct detection cameras.

“At present only the Gatan K2, when operated in counting mode, can produce a DQE(0) as high as 80% in conjunction with reasonably small exposure times. The K2 detector frame rate is about 10 times higher than that available with the two other detector brands.”– Subramaniam, S., Kuhlbrandt, W. & Henderson, R. (2016). IUCrJ 3, 3-7.

WHAT IS SINGLE-PARTICLE CRYO-EM?

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During cryo-EM, a transmission electron microscope (TEM) is used to record high-resolution images of thousands to hundreds of thousands identical, but randomly oriented, particles (molecules) from each specimen. These images are then grouped, aligned, and averaged with image classification algorithms to distinguish between multiple orientations of the 3D molecule. With a well-behaved sample, cryo-EM can solve molecular structures with a resolution below 2 Å; a resolution level that was inconceivable only a few years ago.

This rapid improvement of cryo-EM resolution has been called the “resolution revolution” and is the direct result of direct detection cameras. Conventional cameras for electron microscopy use a scintillator to convert the electron image to a light image, and a fiber optic face plate to transfer the image to a CCD or CMOS image sensor for analog recording. When the results are converted and recorded, there is a loss of high-resolution detail that has long held cryo-EM from achieving its true potential.

Direct detection cameras now directly measure the electron image to avoid the loss of detail from the conventional camera conversion steps. The Gatan K2 Summit camera is a unique direct detection camera that uses an electron counting with super-resolution technology to record the image. This technology recognizes and counts individual image electrons in real-time, while it rejects the analog read noise. As shown with the K2 Summit camera, the detective quantum efficiency (DQE) performance at half Nyquist is unparalleled at 52%; more than a six-fold improvement over conventional cameras.

EFFICIENT FRONTIER OF RESOLUTION

The K2 Summit camera, often in combination with the GIF Quantum LS imaging filter, continues to yield breakthrough results that define the efficient frontier of resolution. Cutting edge publications show these products along with improved data acquisition and image processing strategies remain at the forefront of higher resolution reconstructions for smaller molecules, such as the 2.2 Å structure of β-galactosidase.

The graph above is a comparison between the resolution and molecule size for published single particle cryo-EM structures. The K2 Summit or Quantum LS have been employed in all the structures which define the high-resolution frontier across a range of molecular sizes.

ADVANTAGES OF SINGLE-PARTICLE CRYO-EM

Now that single-particle cryo-EM provides structures with comparable resolution to x-ray crystallography, there are a number of unique advantages that are helping the technique gain traction amongst structural biologists:

OVERVIEW

What is cathodoluminescence?

Cathodoluminescence (CL) is the emission of light when a material is stimulated by an electron beam.

Cathodoluminescence in a scanning or scanning transmission electron microscope (SEM or STEM) is a unique tool to characterize the composition and optical and electronic properties of materials, then correlate them with morphology, microstructure, composition, and chemistry at the micro- and sub-nanoscale.

Cathodoluminescence microscopy is the analysis of luminescence (emitted light or photons) from a material when stimulated by the electron beam of an electron microscope; the luminescence ranges from ultraviolet to infrared wavelength ranges.

Cathodoluminescence occurs because the impingement of a high energy electron beam elevates the sample to an excited state, which can then induce it to emit a (cathodoluminescence) photon when the sample returns to the ground state. In a semiconductor, this excitation process will result in the promotion of electrons from the valence band into the conduction band, which leaves behind a hole. Therefore when the electron and hole recombine, a photon will emit from the semiconductor.

The photon energy (color) and the probability that a photon (not a phonon) will be emitted depends on the material, its purity, and the defects it contains. Therefore to measure cathodoluminescence, you can examine almost any non-metallic material. In terms of band structure, classical semiconductors, insulators, ceramics, gemstones, minerals, and glasses can be treated the same way. In metals, the excitation of the specimen may be by surface plasmons being launched, which when they decay can result in the emission of a cathodoluminescence photon.

The electron microscope holds distinct advantages over conventional light microscopy, as the spatial resolution of light microscopy is limited by fundamental physics to an approximate resolution of 200 – 300 nm (or half the wavelength of the illuminating source). However in an electron microscope, you can focus the electron beam to a very small spot that potentially gives sub-nanometer spatial resolution. You can also use the emitted signals from the specimen to reveal morphological information, such as size and shape, as well as composition, chemistry, crystallography, electronic properties, plus much more. Thus, the volume of information from a specimen combined with the ability to correlate it directly with information from luminescence (spectroscopy) is what makes cathodoluminescence such a powerful characterization technique.

ADVANTAGES

USES

The characterization of optical properties at extremely small length scales is critical for many areas of scientific research and technologies. Including:

ELECTRON MICROSCOPE SETUP

When the electron beam excites the specimen, this causes luminescence to be emitted from the near-surface region of the specimen. To collect the cathodoluminescence emitted in the upper hemisphere, a mirror is often inserted between the specimen and the pole piece. The mirror is specifically shaped to couple the light out of the microscope vacuum chamber to a spectrograph or photon detector(s). For thin samples, such as electron transparent TEM samples, mirrors above and below the specimen may be employed to collect light emitted in both hemispheres.

When you scan the microscope's focused electron beam in an X,Y pattern then measure the light emitted with the beam at each point, you can obtain an optical activity map of the specimen. The primary advantages of this electron microscope based technique is the ability to resolve features down to 1 nm and correlate the optical properties of an object with structural, compositional and chemical properties measured simultaneously or, at least within the same instrument.

Typical light levels emitted from a specimen can be extremely low and often require you to collect and detect as many photons as possible. Even in samples intended to efficiently emit light, it is important to optimize experimental conditions to collect and detect photons with the minimum of optical losses so you can attain the highest spatial resolution results.

WORKFLOW

To better understand the cathodoluminescence workflow, select the appropriate electron microscope to support you intend to use.

OVERVIEW

Spectrum imaging (SI) is a technique that generates a spatially resolved distribution of electron energy loss spectroscopy (EELS) data. A typical experiment involves the creation of a data cube where two of the cube axes correspond to spatial information, while the third dimension represents the energy loss spectrum. The resultant dataset is referred to as a spectrum image (or spectrum line scan for the 1D case), which you can acquire and visualize in a number of ways. To create this data cube, you can acquire a complete spectrum at each spatial pixel during scanning transmission electron microscope mode (STEM SI) or collect a complete 2D image over a narrow band of energies at a single energy slice of the data cube during energy-filtered TEM mode (EFTEM SI).

A key advantage of spectrum imaging is the ability to process decisions after acquisition. When a complete spectrum is available at each data point, this allows creation of quantitative images and profiles to identify and correct data artifacts, understand image contrast, as well as determine dataset limitations.

During a STEM experiment, the electron beam focuses into a small probe, then scans over the sample to acquire spatial information (X,Y) in a serial manner. In the STEM SI mode, you can acquire a complete spectrum at each pixel position to build the spectrum image up on a spectrum-by-spectrum basis.

Alternatively, EFTEM SI uses a broad parallel beam (e.g., TEM) to acquire spectral data image plane-by-image plane, while changing the energy of each plane. In this mode, you acquire the image in parallel while the spectrum is built in a serial manner.

Once acquisition is complete, you can visualize the spectrum image (either EFTEM SI or STEM SI) either spectrum-by-spectrum (X,Y) or plane-by-plane (E). The combination of spectral and spatial information in a single dataset opens up a wide range of data analysis possibilities. You can apply any single spectrum analysis method to the entire spectrum image dataset to perform a spatially resolved spectral analysis. This increase in information provides a powerful tool for material analysis and characterization.

OVERVIEW

WHAT IS SERIAL BLOCK-FACE IMAGING?

Serial block-face scanning electron microscopy (SBEM, SBSEM, and SBFSEM) is a way to reproducibly obtain high resolution 3D images from a sample. This method is particularly good at imaging large fields-of-view in X,Y,Z at nanometer resolution. SBEM typically relies on an in-situ ultramicrotome to be installed inside a scanning electron microscope (SEM). The SEM will collect an image of the block face, then the ultramicrotome will cut the sample to expose the next layer to be imaged, in steps as small as 15 nm.

ADVANTAGES OF SBEM

NEUROSCIENCE

A 3D reconstruction of a dendrite from a 15,625 μm³ (25 x 25 x 25 μm) volumetric data set containing 500 serial images of mouse cerebellum generated by the 3View® system. Dendrite structure (green), buttons (yellow), and vesicles (red). Inset images, clockwise from top left: Confocal image of a dendrite; wire frame traces rendered into a volumetric model; ultra-resolution dendritic spine model with synapses; and image showing wire frame traces.

Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented using Imaris to create a 3D model of a neuron of interest.

CELL BIOLOGY: COMPLEMENT CONFOCAL

Image of HeLa cells, stably expressing LC-GFP grown on gridded glass bottom coverslips dishes, starved for 2 h in serum-free medium and cells of interest identified by confocal microscopy. The cells were then processed in situ for electron microscopy and the coverslips dissolved from the epoxy resin with hydrofluoric acid. The cells were again identified in the resin block and in subsequent serial images generated by the 3View system.

Samples courtesy of David Dinsdale, MRC Toxicology Unit, University of Leicester, UK.

CELL BIOLOGY: DEVELOPMENTAL

Top left: High resolution, mouse kidney, 8192 x 8192 pixel image acquired with 1.5 nm pixels. Top middle: Large field-of-view, mouse kidney, 8192 x 8192 pixel image acquired with 80 nm pixels. Right: C. elegans prepared by high pressure freezing; 4096 x 4096 pixel image acquired with 25 nm pixels. Sample courtesy of Kent McDonald, University of California, Berkeley. Bottom left: Mouse sciatic nerve, 2048 x 2048 pixel image acquired with 5 nm pixels. Bottom middle: 3D visualization of mouse sciatic nerve axons.

Samples courtesy of Gabrial Corfas, Harvard University and Children’s Hospital Boston and Kent McDonald, University of California, Berkeley. Images generated by 3View system. 3D data set contains 1,000 2048 x 2048 serial images collected with 5 nm pixels and segmented using the program Imaris.

MATERIAL SCIENCE

Upper and lower left: Images of anodized coating on aluminum surface generated by 3View system. Middle: 3D visualization of aluminum alloy with manganese particles generated by 3View system. 3D dataset contains one thousand 1024 x 1024 serial images with a pixel size of 15 nm and a cut thickness of 15 nm. 3D model created in DigitalMicrograph® using the 3D Visualization plugin.

Sample and data provided courtesy of Teruo Hashimoto and George Thompson, The University of Manchester, United Kingdom.

WORKFLOW FOR SERIAL BLOCK-FACE IMAGING

3View system quick start guide

OVERVIEW

WHAT IS EBIC?

Electron-beam induced current (EBIC) technique enables the characterization of local electrical behavior of semiconductor materials and devices by measuring the electrical currents that flow when a sample or device is exposed to an electron beam.

When an electron beam strikes a semiconductor, electron-hole pairs are created. If the carriers, of said electron-hole pairs, diffuse into a region with a built-in electric field, the electrons and holes will separate and a current will flow. The EBIC technique measures that current when it flows into an external circuit. In a material with no recombination centers (locations where the free electrons and holes annihilate) the collected current would be uniform and of little interest. However, regions of the sample which cause electrons and holes to recombine, reduce the collected current to cause contrast in an EBIC map, thus reveal the flow of (minority) carriers in a semiconductor sample.

WHAT IS EBAC?

Electron-beam absorbed current (EBAC) is a related technique where the local resistance of a sample is revealed when you measure the absorbed current that flows between the electron beam and an electrical probe. The current measured in the external circuit is proportional to the resistance of the electrical path travelled. Thus, EBAC reveals breaks in copper lines on electronic devices or, shorts and breakdown channels in capacitors.

ADVANTAGES OF EBIC AND EBAC

OVERVIEW

WHAT IS EBSD?

Electron backscatter diffraction (EBSD) is emerging as a valuable analytical tool for materials science and earth sciences. The EBSD technique permits statistical data on grain size and grain texture, both of which are extremely important parameters in determining the strength of crystalline materials. A good general reference on all aspects of EBSD can be found at www.ebsd.com

The reflected signal from the sample surface used in EBSD is generated within the first 10’s nm of the sample surface. Thus it is critical to have a surface that is near perfect, with little or no damage. Broad argon beam tools have historically been used to produce damage-free transmission electron microscope (TEM) samples. The same techniques are now applied for scanning electron microscope (SEM) samples used to produce EBSD signals.

IPFZ map of stressed Zircaloy 2 alloy, FOV 1 mm x 0.7 mm. Results courtesy of Department of Materials, University of Oxford. Professor Angus Wilkinson and Dr. Hamidreza Abdolvand. Data acquired on a Zeiss Compact Merlin equipped with a Bruker Quantax EBSD System.

USES

3D EBSD WITH BROAD ARGON BEAM TOOLS

This can be done manually by returning the sample to the PECS II instrument and polish removing from 5 – 500 nm simply by varying the time. An example is shown below.

WCarbide sample 140 x 140 x 15 µm3, 25 slices 600 nm thick. Image and data courtesy of University of Manchester.

A recent development by Gatan is to automate this process by integrating a PECS II instrument onto a FIB/SEM. The system was installed in November, 2015 and data will be published shortly.

WORKFLOW

Step 1: Prepare the sample

Schematic outline of iPrep II system attached to a SEM.

Specimens must be prepared carefully for EBSD applications, as the technique requires a very smooth specimen to avoid shadows on the diffraction pattern. The best way to accomplish this is to utilize a fully automated argon ion polishing system, like the PECS II instrument. By using a PECS II instrument, you can produce damage free surfaces, cross sections, and deposit coatings to protect or eliminate charging. Typical conditions for planar polish in the PECS II are the guns set to 2 – 4° to the surface with a coarse polish at 4 – 6 keV for 1 – 2 h, followed by a lower polish voltage of 1 keV for 30 – 60 min.

Step 2: Transfer to SEM

Schematic of iPrep II system shows transfer of sample to home position on a SEM stage.

Step 3: Imaging

Schematic of iPrep II system shows the sample on a SEM stage with the transfer rod retracted. SEM and sample now in position for SEM analysis.

OVERVIEW

WHAT IS EDS?

Energy dispersive x-ray spectroscopy (EDS, EDX, XEDS, etc.) is an analytical technique used for analysis and characterization of a sample.

Elemental composition analysis is key to understanding foreign materials, coating composition, small component materials, rapid alloy identify, evaluating corrosion, plus phase identification and distribution. EDS tools from Gatan provide qualitative and quantitative insight, in addition to elemental maps for microstructure determination during these types of material studies.

Since the changing local chemical environment atomic bonding has little effect on EDS, it is not a preferred technique for specimen chemical analysis. However, it is excellent to determine the elemental distribution of a sample. Used in combination with electron energy loss spectroscopy (EELS), you can easily analyze both the chemical and compositional makeup of your specimen.

Unlike similar techniques, EDS requires minimal setup and is used in semi-thin to bulk specimens; thickness options are unlimited. EDS also offers a high signal-to-background ratio (SBR). Some limitations of EDS include: non-local fluorescence; low-Z limits; and limited thin film signal-to-noise ratio (SNR).

In the example below, the Sr L2,3-edges at 1940 eV were not within the 0 – 850 eV energy range (top) after an EELS analysis was run on a semiconductor sample. When spectrum imaging was used to obtain the EDS Sr elemental map in combination with the EELS Ti, Fe, and La maps (bottom), the collective color map shows the distribution of each element across an extended energy range.

Total exposure time: 118 s; EELS core-loss: 350 – 850 eV; EELS low-loss: 0 – 500 eV; EDS spectrum: 0 – 20 keV beam current: 200 pA.

Sr EDS: red; Ti EELS: green; La EELS: orange; Fe EELS: light blue.

For more information about the comparison between this technique and other analytical techniques, please visit EELS.info, an educational site.

OVERVIEW

What is in-situ microscopy?

In-situ transmission electron microscopy combines the image formation capabilities of the transmission electron microscope (TEM) with application of some external stimulus to measure results real-time. Currently, a wide variety of systems and holders are available to apply different stimuli to evaluate kinetic reactions such as catalysis, phase transformations, other chemical reactions, electrical fields, mechanical strain or nano-indentation deformation, heating and cooling, or the effects of the electron beam irradiation damage in samples.

Historically, movies of various reactions and system kinetics could be recorded onto video tape. More recently screen capture programs allow low-resolution, qualitative capture for you to visualize what is happening at video frame rates, e.g., 30 frames per second (fps). This adds significant value to investigators who want to physically see and understand reactions in real-time.

With the advent of faster data transfer and processing capabilities, it is now possible to record and manage large datasets directly from the sensor output. You can store each pixel in each frame directly to disk, and treat them as an individual image. Alternatively you can apply various algorithms or scripts (e.g., summing, drift correction, binning, etc.) post-capture to quantify the stimulus applied to the system. This greatly enhances the flexibility of data management and gives you previously unseen resolution in time and space, with sub-ms time resolution to resolve reactions that previously were unknown.