Turn ideas into Turn ideas into discoveries3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman Imaging Imaging

Let your discoveries lead the scientifi c future. Like no other system, WITec’s confocal 3D Raman microscopes allow for cutting-edge chemical imaging and correlative microscopy with AFM, SNOM, SEM or Profi lometry. Discuss your ideas with us at info@witec.de.

3D Raman image of a pharmaceutical ointment.

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www.witec.deRaman • AFM • SNOM • RISEMADE IN GERMANY

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HELLO FRIENDS, I hope you’re all keeping well and safe during the current global situation and its impact on all our lives. Clearly things are far from normal, but we’re doing our best to bring you quality content to educate, engage and inspire you.

Science is a unifier and its always been our mission to bring science to everyone, which seems more relevant than ever at this time. Alongside the changes in our world, we’ve been implementing some changes in the way we connect with you.

Some of you will undoubtedly noticed that online we have incorporated Microscopy and Analysis and its content into the Wiley Analytical Science website. This means that readers can more easily engage with the full portfolio of editorial articles, peer reviewed journals and news articles to explore the areas of interest to them. Let me assure you that we’re still publishing and distributing M&A (clearly, you’re reading this) and all this content is online too.

In the background also I’ve been working on revising the editorial board, I wanted to ensure a gender balance and breadth of areas of microscopy expertise covered its members. I’m pleased to unveil that board in this issue – see page 11 for more information. The board will be assisting me in keeping the content representative of what’s going on in microscopy in all its various applications. Please read and see who these individuals are. My thanks go the outgoing board members for their service to the publication over the years.

The content in this issue covers the news from around the world and our profile is with outgoing M&A board member and veteran of electron spectroscopy Christian Colliex, as he recounts his four-decade career at the leading edge of electron spectroscopy. In the LabFocus Rebecca Pool takes us to the Animation Lab,

in Utah, US, to meet the team of scientific animators who help to generate stunning animations that support scientific articles and movies.

Not forgetting our own articles, new board member, Annalena Wolff shares her insight on the various Ion Microscopes that are currently available. Eva Olsson and Andrew Yankovich show their work on determining site specific strain in nanoparticles using scanning transmission electron microscopy.

In the supplement, Grigore Moldovan serves up an article on the emerging area of electrical analysis using microscopy. This text should serve as a primer into this area of microscopy and has particular relevance to the semiconductor and energy markets. This ties in nicely with the supplement topic which is: Energy. Don’t forget the product focus is your ‘go to’ place for products and services relating to this field from global suppliers.

Following on, we have the What’s New section, here you can catch up on what our friends in the microscopy companies are doing. Within What’s New, we have a special editorial piece from Rebecca Pool on the microscopy sector’s response to the global shutdown during the Coronavirus pandemic.

As a final thought, I’m interested to hear from all of you on what the Coronavirus has meant to you and what you’ve been doing in our new existence. Have you become a carer to a family member? Has this given you the opportunity to do something you couldn’t have before? Have you refocused your work onto virus related work? Please let me know.

I wish you all a safe spring and early summer until next issue.

Chris

3 Editor’s letter4 News8 ProfileChristian Colliex11 Meet the board12 High-precision stem for imaging and quantifying local strain Andrew B. Yankovich, Torben Nilsson Pingel, Mikkel Jørgensen, Henrik Grönbeck, Eva Olsson15 Focused Ion Beams: an overview of the technology and its capabilities Annalena Wolff20 Lab focus24 Coronavirus spotlight26 What’s new

ENERGY SUPPLEMENTS3 The emergence of electrical analysis in electron microscopy Grigore MoldovanS6 Product focus

Editor Dr Chris Parmenter commed.ma@gmail.comNews editor Dr Rebecca Pool rebeccapool@microscopy-analysis.comSales EMEA Dr. Stefanie Krauth + 49 (0) 6201 606 728 mkrauth@wiley.comSales North America A-K Bob Zander 312-925-7648 bzander@wiley.comSales North America L-Z Joe Tomaszewski 908-514-0776 jtomaszews@wiley.comSales APAC Yosuke Sato +81-3-3830-1234 ysato@wiley.comAdvertising production Kerstin Kunkel kkunkel@wiley.comReader Services wiley@vuservice.dePublisher Dr Heiko BaumgartnerDirector of publishing Roy OpieAssistant editor Simon EvansAssistant web editor Felix David

MICROSCOPY AND ANALYSIS ISSN 2049-4424 © 2020 John Wiley & Sons, Ltd. Issued in: January, March, May, July, September, NovemberPublished by: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, UK Tel: +44 (0)1243 770443Fax: +44 (0)1243 770432Email: microscopy-analysis@wiley.co.uk Web: www.microscopy-analysis.comWhile every effort is made to ensure accuracy, John Wiley & Sons, Ltd and its agents cannot accept responsibility for claims made by contributors, manufacturers or advertisers.

INSIDEMAY-JUNE 2020 EMEA 48

INTROEDITOR’S LETTERDR CHRIS PARMENTER

Business as usual and business as unusual

A STILL image from an animation of the HIV life cycle, revealing the structure and composition of a single virion. Science of HIV

REGISTRATION The journal is free of charge, worldwide, to users who pur chase, specify or approve microscopical, analytical or imaging equipment at their place of work, and marketing exe cutives who make advertising decisions. To register, go to http://www.microscopy-analysis.com/user/register. To amend your address details, go to http://www.microscopy-analysis.com/user/login. The registered address entered must be an organisational address.Subscription charges for non-qualifying readers: $110 per annum (UK) or $195 (Europe); all other countries $280 by airmail.NON-USA returns should be sent to Readerservice Wiley & Sons Ltd, 65341 Eltville, Germany Tel. Germany: 06123/9238-290 Tel. International: 0800/0961137 Fax. Germany: 06123/9238-244 Fax International: +49/6123/9238-244Microscopy and Analysis (ISSN No: 2043-0639 USPS NUMBER 010-289) is published Bi-monthly by Wiley, and distributed in the USA by Asendia USA, 701 Ashland Ave, Folcroft PA. Periodicals postage paid at Philadelphia, PA and additional mailing offices. POSTMASTER: send address changes to Microscopy and Analysis, 701 Ashland Ave, Folcroft PA 19032

EDITORIAL BOARDPeter Hawkes CNRS, Toulouse, FrancePaul Verkade University of Bristol, UK Debbie Stokes Nanoviz, Netherlands Keith Duncan Danforth Inst, St Louis, USA Annalena Wolff Queensland University of Technology, Australia Nestor Zaluzec Argonne National Lab, IL, USA Dalia Yablon SurfaceChar LLC, Boston, USA Philip Moriarty University of Nottingham, UK Louise Hughes Oxford Instruments, UKKerry Thompson National University of Ireland Galway, IrelandGail McConnell University of Strathclyde, Scotland Erin Tranfield Instituto Gulbenkian de Ciência, Portugal

Turn ideas into Turn ideas into discoveries3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman3D Raman Imaging Imaging

Let your discoveries lead the scientifi c future. Like no other system, WITec’s confocal 3D Raman microscopes allow for cutting-edge chemical imaging and correlative microscopy with AFM, SNOM, SEM or Profi lometry. Discuss your ideas with us at info@witec.de.

3D Raman image of a pharmaceutical ointment.

speedsensitivity

resolutiioon

www.witec.deRaman • AFM • SNOM • RISEMADE IN GERMANY

Contents

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In a modern twist to the well-known ‘coffee ring effect’, US-based researchers have used time-lapse microscopy to see what happens when American whiskeys evaporate from glass.

On evaporating droplets of diluted American whiskey onto a clean glass coverslip, Professor Stuart Williams from the University of Louisville Speed School of Engineering and colleagues discovered that hierarchical layers emerge, forming a web-like pattern that they call a ‘whiskey web’.

The latest investigations on more than 80 different whiskeys reveal that different American whiskeys show unique web patterns that can be correctly matched to unknown samples more than 90% of the time.

Past studies from Williams and colleagues had shown that whiskey webs form in diluted American whiskeys, but not Scotch or Canadian whiskeys.

As Williams writes in ACS Nano: “Thousands of chemicals are found in whiskeys, and many of them may contribute toward the molecular assembly of the collapsed monolayers [we observed].”

“The unique concentration and combination of congeners that comprise each American whiskey’s flavor profile ultimately guide their correspondingly distinctive whiskey web patterns,” he adds.

TANGLED WEBSWhen a drop of liquid evaporates, solids are left behind in a pattern that depends on what the liquid is, what solids are in it and the environmental conditions.

A classic example of this is the ‘coffee ring effect’, which takes place when water evaporates from coffee, and has fascinated scientists for decades.

However, Williams and colleagues have now analysed an equally fascinating phenomenon involving American whiskey.

As Williams points out, more than 80 whiskey samples were either bought, generously donated by colleagues or provided by distilleries.

These included well-known brands such as Jack Daniel’s, Wild Turkey Rare Breed and Jim Beam Single Barrel as well as more niche tipples including Whiskey Row, Kentucky Artisan, and Pappy Van Winkle, Buffalo Trace.

Initial microscopic images were captured using a digital camera – Canon EOS Rebel T7i – mounted to either an inverted Nikon Ti-U microscope or an upright Zeiss Axio Imager. The researchers also added fluorescent microparticles to diluted whiskey samples for microparticle image velocimetry.

Image pairs were acquired every 0.24 s with a high-speed Fastec Imaging camera mounted to the inverted microscope, while particles were illuminated using an LED ring light.

Analyses revealed that during liquid evaporation, non-volatile organic compounds, such as phenols, aromatics and esters, cluster together and are driven to the surface of the droplet, where they form monolayers.

As the surface area of the droplet decreases, the monolayers collapse, creating strands of the web.

The researchers showed that different American whiskeys develop unique web patterns that could be correctly matched to unknown samples more than 90% of the time.

According to the researchers, the distinctive webs arise from the unique combination of solutes in each whiskey.

The researchers reckon that their latest results suggest that these distinctive ‘whiskey webs’ could someday be used to identify counterfeit spirits.

Research is published in ACS Nano.

A team of international researchers has used two-photon fluorescence microscopy to record the millisecond electrical signals in the neurons of an alert mouse.

Professor Kevin Tsia from the University of Hong Kong and Professor Ji Na, from the University of California, Berkeley were able to pinpoint individual neurons and trace their firing paths, millisecond by millisecond.

“This is really an exciting result as we now can peek into the neuronal activities, that were once obscured but can provide the fundamental clues to understanding brain functions and more importantly brain diseases,” says Tsia.

To understand information processing in the brain, neuronal activity needs to be monitored at high spatio-temporal resolution.

With this in mind, Tsia, Na and colleagues developed a method that they call FACED – free-space angular-chirp-enhanced delay imaging.

This uses an ultrafast two-photon fluorescence microscope with all-optical laser scanning to image neuronal activity in vivo at up to 3,000 frames per second and with submicrometer spatial resolution.

As part of this, a pair of parallel mirrors generate a shower of laser pulses to create the super-fast sweeping laser beam, which they say is at least 1,000 times faster than existing laser-scanning methods.

In the latest experiment, the microscope swept the laser over the mouse’s brain and captured 1000 to 3000 full 2D scans of a single mouse brain layer at the neocortex every second.

To probe the genuine electrical signals that pulsed between the neurons, the researchers also inserted protein molecule biosensors – developed by Dr Michael Lin, Stanford University – into the neurons of the mouse brain.

“These engineered proteins fluoresce whenever a voltage signal passes through the neurons. The emitted light is then detected by the microscope and formed into a 2D image that visualises the locations of these voltage changes,” explains Tsia.

“This is so far a one-of-its-kind technology that can detect millisecond-changing activities of individual neurons in the living brain,” highlights Tsia. “We are working to further combine other advanced microscopy techniques to achieve imaging at a higher resolution with also a wider view and deeper into the neocortex.”

Research is published in Nature Methods.

EXAMPLES of the surprisingly diverse “whiskey web” patterns. The patterns, approximately 2 mm in diameter, were formed by drying droplets from various off-the-shelf whiskey products diluted to 20–25% alcohol by volume. Scale bar is 0.5 mm. Stuart J. Williams et al Phys. Rev. Fluids 4, 100511

What happens when whiskey evaporates?

High-speed microscopy captures brain neuroactivities

PROFESSOR KEVIN TSIA from the University of Hong Kong has pioneered two-photon fluorescence microscopy to record neuron signalling. HKU

TIME-LAPSE of a 0.75 μL drop of diluted (25% ABV) bourbon whiskey evaporating on the surface of an ITO-coated glass slide under ambient conditions. Adam D. Carrithers et al, ACS Nano 2020, March 25, 2020

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Using super-resolution microscopy, Germany- and UK-based researchers have discovered that glutamate receptors usually appear in small groups at the synapses and are in contact with other proteins.

These receptors are crucial to neuron communication and have been linked to neurodegenerative disease.

“For the first time we now have insights into the molecular organisation of the complex protein machines that control the signal transmission at the synapses of our brain,” says Professor Davide Calebiro from the University of Birmingham, UK. “Only with this knowledge will we be able to understand how the brain functions and how it processes information on different time scales.”

Glutamate is a key messenger substance in the nervous system of humans, essential to learning processes and memory.

It acts as a signal transmitter at the synapses, binding to several specific

receptors, including the metabotropic glutamate receptor of type 4 (mGluR4), which plays a decisive role in this system.

However, little has been known about the distribution of the mGluR4 receptor in the active zones of synapses, until now.

Using two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM), Calebiro and his colleague, Professor Markus Sauer from the Biocenter of Julius-Maximilians-Universität (JMU) Würzburg, Germany, studied the nanoscale organization of mGluR4 within very small and densely packed active zones of synapses in the mouse cerebellum.

The researchers acquired images using an inverted fluorescence wide-field setup custom-built around an Olympus IX-71 microscope. This was equipped with an oil-immersion objective, a nose-piece stage to reduce stage vibration and drift, and 514 nm and 639 nm solid-state lasers.

For each color, 40,000 frames were acquired at 60 Hz, with this single-molecule localization data being analyzed using the open source software, rapidSTORM.

Analyses revealed that the majority of mGluR4 receptors were located in groups of one to two units, on average, in the presynaptic membrane.

Here, the receptors were seen to often be in direct contact with calcium channels and the protein, Munc-18-1, which is important for the release of

Using transmission electron microscopy, Brazil-based researchers have captured the exact moment that the coronavirus infects a cell.

A series of three TEM images taken by Debora Barreto-Vieira, from Latin America’s largest medical research center, the Oswaldo Cruz Foundation, and colleagues, shows how the coronavirus first attempts to invade a cell followed by its successful invasion.

Dark points within the image are actually the viral particles of the pathogen.

According to the researchers, the cells shown here were not human but instead derived from the African green monkey, routinely used in cell cultures in laboratories.

At the time of writing, global cases of COVID-19 is more than 3m while the death count was over 210,000.

The moment Covid-19 infects a cell

New insight into synapses

THE DISTRIBUTION of the glutamate receptor mGluR4 and other proteins in the presynaptic membrane Left a high-resolution dSTORM image. Right: the result obtained with conventional fluorescence microscopy; molecular details are not visible here Lehrstuhl Markus Sauer/Universität Würzburg

messengers.“Our data indicate that the direct

contact of mGluR4 receptors with other key proteins plays a major role in the regulation of synapse activity,” says Sauer.

The research teams will now use dSTORM to find out how all the proteins are distributed in the active synaptic zone – past research indicates that more than 100 proteins are involved in signal transmission in the active zones.

Research is published in Science.

CORONAVIRUS PARTICLES (black) attempting to enter the cytoplasm of the cell, top.

AN ARTISTIC CROSS-SECTION through forming crust approximately 3-4bn years ago

ONCE IT HAS entered the cell, the virus approaches the outer edge of the cell nucleus to start the infection, centre.

VIRAL PARTICLES within the cell, bottom. Images: Debora F Barreto-Vieira/IOC/Fiocruz

Using a quantum diamond microscope to study magnetism in ancient rock, Harvard researchers have uncovered proof that plate tectonics may have have started at least 3.2bn years ago.

Analyses of the magnetic field strengths and directions within core samples from the Pilbra Craton in Western Australia – one of the oldest pieces of the Earth’s crust – revealed latitudinal drift of about 2.5 centimeters a year, and dated the motion to 3.2bn years ago.

“We’re trying to understand the geophysical principles that drive the Earth,” highlights Professor Roger Fu. “Plate tectonics cycles elements that are necessary for life into the Earth and out of it.”

When Earth’s first tectonic plate shifts started has long been an issue of debate in geology. Some researchers theorize it happened around four billion years ago while others think it was closer to one billion. To find out more, Fu and colleagues turned to their newly-developed quantum diamond microscope (QDM), developed with colleagues at MIT.

The instrument uses a transparent diamond chip, containing nitrogen vacancy defects sensitive to miniscule magnetic fields, to map local magnetic fields within rock samples. These magnetic fields – detected within grains within the rock – are shaped by the Earth’s magnetic field at the time the mineral formed.

Using QDM alongside superconducting quantum interference device microscopy, the researchers scrutinised the magnetic fields within a series of core samples from the 3.2bn-year-old Honeyeater Basalt at the Pilbara Craton. Their core samples had formed over a period of around 180m years.

The researchers’ analyses revealed changes in the direction of magnetic fields in the rock, over time, that indicated the rocks were shifting by around 2.5 cm a year, over the 180m year period.

Fu and Brenner now plan to keep analyzing data from the Pilbara Craton and other samples from around the world in future experiments.

Research is published in Science Advances.

Diamond microscope revealswhen plate tectonics started to shift

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Using electron microscopy and X-ray analysis, France-based researchers have worked out how French Physicist, Edmond Becquerel, obtained colors in the world’s first color photograph.

Captured in 1848, Becquerel’s ‘photochromatic image’, created at the Muséum d’Histoire Naturelle in Paris, seems to show purple gradients on a silver-ionized sheet.

Much controversy has ensued over how the physicist achieved his colors, but using X-ray analysis, SEM, STEM, UV-visible spectroscopy and low-loss EELS, researchers from the Centre de Recherche sur la Conservation, the SOLEIL synchrotron and the Laboratoire de Physique des Solides, CNRS, have finally pinned down his process.

According to Professor Bertrand Lavédrine and colleagues, Becquerel’s colors result from the presence of metallic silver nanoparticles in a matrix of silver chloride grains that form the photograph’s light-sensitized layer.

Several researchers had thought that the colours were due to pigments formed via a light-reaction but this hypothesis was rejected after the spectroscopy methods showed no deviations in chemical composition from one color to another.

Other researchers had put the colours down to interference but electron microscopy revealed a distinct lack of a periodic pattern within the layer’s microstructure, disproving this hypothesis.

Instead, Lavédrine and colleagues noted that photosensitive silver nanoparticles within its colored layers were organized differently with specific localizations and sizes distributions for each color.

In a plasmonic hypothesis, they propose that the silver nanoparticles had reorganised within the light-sensitised layer, according to color – and energy – of the light.

This new nanoparticle configuration gives the material the ability to absorb all colors of light, apart from the colour that caused the reorganisation – and that is the color that we see.

As Lavédrine writes in Angewandte Chemie International Edition: “A charge transfer mechanism at the interface between silver nanoparticles and silver chloride would lead to the transfer of silver from the nanoparticles, which absorb the incident radiation, to existing nanoparticles or new ones by coalescence.”

Research is published in Angewandte Chemie International Edition.

Secrets behind first color photograph exposed after 172 years

EDMOND BECQUEREL, Solar spectra, 1848, photochromatic images, Musée Nicéphore Niépce, Chalon-sur-Saône.

Australia-based researchers have achieved unprecedented resolution in single-molecule microscopy, detecting interactions between individual molecules within intact cells.

The single-molecule localization microscope – Feedback SMLM – corrects for drift in real-time and can directly measure the distance between two molecules, within intact cells, on a scale of 1 to 20 nm.

“It’s a really simple and elegant solution to a major imaging problem,” says Professor Katharina Gaus, from University of New South Wales. “We just built a microscope within a microscope, and all it does is align the main microscope.”

“That the solution we found is simple and practical is a real strength as it would allow easy cloning of the system, and rapid uptake of the new technology,” she adds.

While individual molecules can be observed and tracked with super-resolution microscopy, interactions between these molecules cannot be resolved using existing single-molecule microscopes.

“The reason why the localisation precision of [current] single-molecule microscopes is [limited to] around 20-30 nanometres is because the microscope actually moves while we’re detecting that signal, which leads to an uncertainty,” explains Gaus.

To circumvent this problem, the team built autonomous feedback loops inside a single-molecule localization microscope (SMLM) so it could detect and re-align the optical path and stage.

A first feedback loop was implemented between the sample and stage position, with an optical feedback loop was also used in the microscope’s emission path.

The researchers went on to accurately measure distances between molecular species, recording high quality raw data with ultra-high localization precision and increased accuracy.

They were able to show a 4 to 7 nm difference in spatial separation between signalling T cell receptors and phosphatase molecules in active and resting T cells.

“We could determine separation distances directly without resorting to post-acquisition processing including drift correction, grouping, filtering, averaging and summation,” points out Gaus.

“It doesn’t matter what you do to this microscope, it basically finds its way back with precision under a nanometre,” she adds. “It’s a smart microscope. It does all the things that an operator or a service engineer needs to do, and it does that 12 times per second.”

Research is published in Science Advances.

A T CELL with precise localisation of T cell receptors (pink) and CD45 phosphatase (green) Single Molecule Science

Single-molecule localization microscope smashes super-resolution microscopy limits

Surprise biological packages from bacteria

Using high-speed atomic force microscopy, Japan-based researchers have discovered that the physical properties of bacterial extracellular membrane vesicles are unexpectedly diverse.

Azuma Taoka from the Nano Life Science Institute, Kanazawa University, Nobuhiko Nomura from Tsukuba University, and colleagues, reckon their AFM phase imaging methodology opens the door to single-vesicle analysis of many more membrane vesicles.

Bacteria release nanometer-scale extracellular membrane vesicles – biological packages wrapped in a lipid-bilayer membrane – for a variety of biological processes.

These membrane vesicles are being more widely used in nanobiotechnological applications, including drug delivery, so researchers are keen to better understand the structures’ physical properties.

Taoka, Nomura and colleagues used AFM phase imaging to interrogate the membrane vesicles produced by E. coli, P. aeruginosa, P. denitrificans and B. subtilis.

They recorded phase images of many membrane vesicles, and went on to color-code the structures according to the levels of adhesion and friction. On analyzing their color-coded maps, the researchers discovered a high diversity of

physical properties across the membrane vesicles, which they believe reflects the chemical composition of the vesicles.

According to the researchers, the latest results not only present important insights into the properties of membrane vesicles produced by different bacteria, but also show the power of phase shift AFM as a tool for biological vesicles.

Research is published in Nanoscale.

Mapping of the physical properties of bacteria extracellular membrane vesicles using phase imaging atomic force. The vesicles are color-coded on a scale ranging from “non-adherent/hard” (reddish-coloured spheres) to “adherent/soft” (greenish-coloured spheres).

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MACHINE-LEARNING enabled characterization of 3D microstructure showing grains of different sizes and their boundaries. [Argonne National Laboratory]

3D IMAGES of platinum particles between 2-3 nm in diameter shown rotating in liquid under an electron microscope. Each nanoparticle has approximately 600 atoms. White spheres indicate the position of each atom in a nanoparticle. [IBS]

In a breakthrough for fuel cell catalyst research, an international team of researchers has developed atomic-resolution 3D liquid-cell transmission electron microscopy to capture images of nanoparticles tumbling in liquid between sheets of graphene.

So-called 3D SINGLE – Structure Identification of Nanoparticles by Graphene Liquid cell Electron microscopy – was used to image and analyse the 3D atomic arrangements of individual platinum nanocrystals in solution.

“This is an exciting result,” says Peter Ercius, staff scientist at Berkeley Lab’s Molecular Foundry, US. “We can now measure atomic positions in three dimensions down to a precision six times smaller than hydrogen.”

When fabricating nanoparticles, even particles with a uniform size will still exhibit differences in atomic arrangements that can affect physical and chemical properties.

To better understand these differences, Ercius and colleagues devised a way to image individual nanocrystals in solution, and reconstruct the structure.

They first sandwiched a nanocrystal solution between a two graphene sheets, known as a graphene window, which allowed the nanoparticles to freely rotate within the liquid.

Then using a FEI Titan 80/300 TEM equipped with a post-specimen geometric- and chromatic aberration corrector and Gatan K2 IS direct electron detector, they captured thousands of images of the nanoparticles.

The researchers were able to obtain movies comprising 400 images per second of each nanoparticle freely rotating in liquid.

They then adapted computer algorithms originally designed for biological studies to combine many 2D images into atomic-resolution 3D images.

Ercius and colleagues observed that even though each of the particles was synthesized in the same batch, they displayed important differences in their atomic structures which would affect the physical and chemical properties.

“We have developed a groundbreaking methodology for determining the structures that

govern the physical and chemical properties of nanoparticles at the atomic level in their native environment, which will provide important clues in the synthesis of nanomaterials,” says Ercius’ colleague, Hyeon Taeghwan from the IBS Center for Nanoparticle Research, South Korea.

Research is published in Science.

US-based researchers have developed a machine-learning algorithm to characterize materials with microstructural features as small as nanometers in 3D and in real-time.

Professor Subramanian Sankaranarayanan from the Center for Nanoscale Materials at Argonne National Laboratory, and colleagues, reckon they can use the tool to analyze most structural materials of interest to industry.

“What makes our algorithm unique is that if you start with a material for which you know essentially nothing about the microstructure, it will, within seconds, tell the user the exact microstructure in all three dimensions,” says Professor Sankaranarayanan, also at the University of Illinois at Chicago.

“For example, with data analyzed by our 3D tool, users can detect faults and cracks and potentially predict

Machine learning analyzes 3D microstructures in real-time

TEM detects critical differences in nanoparticles

the lifetimes under different stresses and strains for all kinds of structural materials,” adds Dr Henry Chan, Center for Nanoscale Materials.

Most structural materials are polycrystalline, containing critical microstructural features that affect physical, mechanical, optical, chemical and thermal properties.

These microstructural features are typically analysed by capturing and processing 2D slices that are then pasted together to form a 3D picture.

However, the process can be time-consuming and inefficient, so Sankaranarayanan and colleagues set out to create a different 3D analysis method.

They developed an unsupervised machine learning-based technique to characterize 3D samples, which combines topology classification, image processing, and clustering algorithms.

As the researchers write in Nature Computational Materials: “Our

technique... can handle a wide range of microstructure types including grains in polycrystalline materials, voids in porous systems, and structures from self/directed assembly in soft-matter complex solutions.”

The algorithm doesn’t requires a prior description of the microstructure of its target sample and is insensitive to disorder, such as extended polycrystal defects arising from line and plane defects.

“Our technique provides unbiased microstructural information such as precise quantification of grains and their size distributions in 3D polycrystalline samples, characterizes features such as voids and porosity in 3D polymeric samples and micellar size distribution in 3D complex fluids,” says Chan.

“For researchers using our tool, the main advantage is not just the impressive 3D image generated but, more importantly, the detailed characterization data,” adds Sankaranarayanan. “They can even quantitatively and visually track the evolution of a microstructure as it changes in real-time.”

Importantly, the machine-learning algorithm is not restricted to solids and can be used to characterize the distribution of molecular clusters in fluids.

Research is published in Nature Computational Materials.

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When Christian Colliex first set foot in the wonderful world of electron microscopy, it was a very different place to today. Working on his PhD with French physicist and founding father of electron microprobe analysis, Professor Raimond Castaing, at the University of Paris, Orsay, he was tasked with identifying vortices in superconductors.

It was the late 1960s, Colliex was using a state-of-the-art Hitachi HU-11B electron microscope, and despite fabricating a liquid helium-cooled stage to hold his superconducting materials, he just couldn’t find these triangular lattices of magnetic flux lines. However, several other researchers had already floated the idea of combining electron microscopy with Electron Energy Loss Spectroscopy – by adding an energy analyser to the electron microscope column – and Castaing thought it might help.

“At the time, the contrast was too weak so Castaing suggested using an energy loss filter,” recalls Colliex. “While this didn’t bring any success either, it was the starting point of my quest in electron energy loss spectroscopy.”

Those elusive superconductor vortices would eventually be observed by Dr Akira Tonomura from Hitachi’s Advanced Research Laboratory in Japan more than twenty years later. But in the meantime, Colliex had just made his own important discovery.

While looking at lithium with his

energy loss filter and analyser, the young researcher had discovered unexpected features in the spectra. And from here on in, he was fascinated with EELS.

“I wanted to answer many questions – what is that, how can I understand it? So I started to analyse other materials,” he says. “At the time Castaing said ‘Oh I’m not so interested in spectroscopy’ but I continued to investigate this myself with the daily advice of my close colleague Bernard Jouffrey .”

Colliex completed his PhD two years later, remaining in Orsay to take on the role of head of electron microscopy in 1972, replacing Jouffrey, who was leaving to become the director of the Laboratoire d’Optique Electronique, (LOE now CEMES), in Toulouse. The concept of analytical electron microscopy – rather than purely qualitative electron microscopy – was emerging and the researcher started to grow what would become one of the world’s foremost electron microscopy groups.

In these early days, Colliex and his small team spent a lot of time experimenting with EELS as well as carrying out spectroscopy on numerous materials, publishing the entire set of spectra on rare-earth materials. Using his liquid helium-cooling stage, Colliex had also found a way to deposit noble gases on an amorphous layer and by combining this with spectrometry, could measure energy losses in

had just developed the very first field emission STEM – the highest resolution microscope to date – and had taken images of individual atoms, with the first motion pictures of atoms soon to follow.

Drawn to this field and keen to learn more about STEM Colliex spent six months at the Cavendish Laboratory, University of Cambridge, in 1976. Here he worked closely with Professor Archie Howie – now known for his pioneering work on using TEM to image crystals – and Dr Alan Craven, originally Howie’s PhD student. Together, the researchers studied many crystalline materials, observing, for example, the fringes in plasmon-loss images .

“They had one of the very first commercial STEM Vacuum Generators and under the guidance of Alan Craven I learnt all about STEM,” says Colliex. “Archie and Alan became my greatest friends in the microscopy world.”

“When I got back to Orsay I said, ‘I really want a STEM’ as it was the best instrument for combining imaging and spectroscopy,” he adds. “It took me three years with the VG HB501 being delivered and installed in 1980.”

RAPID PROGRESSFor Colliex, the 1980s were a period of rapid progress. With his STEM up and running, he and colleagues were quick to operate and understand the physics behind the instrument, and of course, perform EELS.

Adventures with atoms and molecules

In 2000, Professor Christian Colliex demonstrated single-atom electron energy loss spectroscopy for the first time. Rebecca Pool finds out how he pioneered EELS in transmission electron microscopy, and more.

EELS SPECTRUM of 100 kV through a thin layer of solidified argon gas on a thin carbon layer, shown in Colliex Ph.D. dissertation (1970) or published in J.Physique (1971). Note that in the early days of recording EELS spectra, the energy loss axis was directed to the left, contrary to the present common practice

CHRISTIAN COLLIEX in front of the VG STEM microscope, which was installed in 1980 but is still in daily operation in 2020

solidified neon, argon and krypton.Still, at this time, EELS remained

relatively unknown, with the great and the good in electron microscopy largely focusing on scanning transmission electron microscopy. British-born American physicist, Albert Crewe,

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Come the mid-1980s, Colliex had published his reference paper ‘Electron energy loss spectroscopy in the electron microscope’ in Advances in Optical and Electron Microscopy, recently digitised by Elsevier. And at the same time he was publishing EELS analyses of dislocations in germanium, precipitates in superconducting V3Si, small metallic clusters and much more.

Colliex had also joined forces with molecular biologists, Professor Eduard Kellenberger and Dr Eric Carlemalm, then at University of Basel, Switzerland, to analyse unstained biological specimens. As he highlights: “We applied Albert Crewe’s contrast method to unstained biological section and I remember these beautiful images of phages around the cell in annular dark-field, without staining.”

“This was a successful three or four years – and maybe we were a little early here as everyone is doing this now,” he adds.

At this time, Colliex was also collaborating with Dr Ondrej Krivanek, who had been building EELS spectrometers at Arizona State University, US. As Colliex puts it: “Krivanek had said, ‘you have the VG microscope so maybe it could be a good thing to test my new spectrometer on your machine’, so we decided to do something together.”

Colliex and Krivanek initially worked together at Orsay, testing a serial EELS spectrometer, then, in 1986, Krivanek left academia to head up research and development at Gatan. Here he developed the first EELS spectrometer with parallel detection which increased detection efficiency by several orders of magnitude.

“Krivanek brought his first parallel EELS detector to our VG, to replace our first generation serial EELS detector,” says Colliex. “He spent, in parts, more than a year at Orsay – this was the starting point of a big adventure with Krivanek and myself, using parallel detection more efficiently.”

Indeed, by 1991, Colliex, Krivanek and colleagues were approaching single-atom detection levels, publishing how to detect EELS signals from only a few atoms on thin substrates.

With parallel detection developed and intent on raising EELS resolution ever-further, Colliex had also started formulating a new concept in EELS digital acquisition and processing with his student Christian Jeanguillaume and the group engineer Marcel Tencé.

Their idea – STEM spectrum imaging – was to generate a spatially-resolved distribution of EELS data, or a spectrum image, that would provide more sample information than every before. On scanning the electron beam across a sample, a complete spectrum would be acquired at each pixel position, which could then be used to build up the spectrum image. The technique had huge implications for nano-sized structures.

“This was an important step as we could now collect thousands of spectra during a single STEM [session] instead

of just one spectrum, and then we could analyse the entire data-set,” says Colliex. “We introduced the idea in 1989 and we were ready to practically run the system by the mid-1990s.”

With these technology breakthroughs as well as combined efforts from many colleagues, Colliex would eventually extend the sensitivity and spatial resolution of EELS to the single-atom limit in 2000. This time, he and team members were working alongside Japanese physicist and ‘inventor’ of carbon nanotubes, Professor Sumio Iijima from Meijo University, Nagoya, and NEC Corporation, Japan.

Together they generated a chemical map of gadolinium that clearly showed single and multiple atoms inside carbon C82 fullerene molecules

aligned along the core of a single walled carbon nanotube. Results were published in Science. “These results were only possible with spectrum imaging as using this we could make a detailed quantitative analysis of all the pixels separated by two ångströms,” says Colliex.

“Reaching single-atom detection took 25 years and I like this story,” he adds. “It began in 1975 with our prediction with Vernon Ellis Cosslett, then there were all the developments with Krivanek in the 1980s, and finally, with Iijima and my post-doc, Kazu Suenaga, we reached success... Science and technology takes time.”

NANOTUBE SUCCESSAlong the road to single-atom detection a new material was

emerging; the carbon nanotube. Iijima had discovered carbon nanotubes in 1991, and Colliex had, as he puts it, ‘the machine to study them’.

In the ensuing years, Colliex and his team used STEM and EELS to characterise mixed boron nitride and carbon nanotubes, filled nanotubes, looking, publishing paper after paper in Science and Nature, and other journals. In one instance, Colliex together with Pulickel Ajayan, now Professor in Engineering at Rice University, also stumbled across a method to align arrays of carbon nanotubes while trying to slice the molecules within a polymer resin-nanotube composite.

“We had this failed lab experiment but Ajayan realised this was also a new way to align the nanotubes instead of cutting them,” says Colliex. “We submitted a paper on this to Science and it was accepted – so one of the most cited papers in my career is a description of a failed experiment!”

At the time, carbon nanotubes were also suspected to exhibit plasmons with a range of unusual properties. Prior to the discovery of the material, Colliex and his team had been using EELS to study these optical excitations in the bulk and at the surface of small spheres in their STEM. While the research was important in its own right – Iijima had provided specimens of silicon spheres – it laid the groundwork for studying the all-important plasmons in carbon nanotubes and then in metallic nanoparticles.

As Colliex says: “We were beginning to calculate the contrast of surface plasmons [in silica], and this really was the beginning of our work in this area.”

By this time, the team included as PhD students Odile Stéphan, now leading the STEM group at the Orsay Solid State Physics Laboratory, and Mathieu Kociak, now research director in the group, Alexandre Gloter and more. “We had this new generation of researchers that was really active so it was a very productive time,” says Colliex. “The machine was running well and we wrote a succession of papers.”

For example, the team used EELS to look at plasmons in curved, ‘onion-like’ nanotubes, as well as layered nanospheres and nanotubes, nanobundles and nanocylinders. And then came the breakthrough that Colliex believes brought plasmonics into the mainstream and opened the door to nanophotonics.

To drive optoelectronics and nanophotonics forward, researchers were keen to investigate how light interacted with matter at the nanoscale, and more specifically, understand how surface-bound plasmons varied across the surface of metallic nanoparticles, as a function of size, shape and environment. However, so far, no experimental method had managed to capture these localised optical excitations at sufficient resolution.

With this in mind, Colliex, Kociak, Stéphan and other Orsay colleagues had been working closely with Javier Garcia de Abajo, from the

TimelineBorn in 1944, Christian Colliex graduated from the École Nationale Supérieure des Mines de Paris in 1965 and received his PhD in Physics from the Faculté des Sciences d’Orsay in 1970 at the Laboratoire de Physique des Solides (LPS), Orsay, France. As a CNRS staff member, from Attaché de Recherches in 1965 to Directeur de Recherches at his retirement, he has spent his entire career, apart from short periods, at LPS. He was Head of the Electron Microscopy group until 2009, and from 2007 to 2010 served as President of the International Federation of the Societies for Microscopy (IFSM). He is currently CNRS Emeritus Research Director.

VINTAGE Colliex stands with Archie Howie, right next to the early VG STEM on which the images, left, were recorded in 1976. It is on display in the Cavendish Museum Christian Colliex and Archie Howie

ENERGY FILTERED images of graphite lattice fringes (elastic 0 eV, graphite plasmon loss at 25 eV) Craven & Colliex, 1977

VG STEM Z contrast images of unstained biological sections showing phages with filled (dark) or empty (bright) heads around a cell wall. Collaboration with E. Carlemalm and E. Kellenberger (1985)

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Institute of Optics at the Spanish National Research Council, and Luis Liz-Marzan, from the Department of Physical Chemistry at the University of Vigo, Spain. Garcia de Abajo had the necessary theoretical modelling expertise while Liz-Marzan could synthesise the nanoparticles.

So, using EELS on their VG-HB501 STEM, fitted with a Gatan spectrometer, they recorded spectra in spectrum-imaging mode and were able to map the surface plasmons on a single silver nanotriangle with unprecedented resolution. Their final results were published in Nature Physics and the researchers had without a doubt, demonstrated the power of EELS in the development of nano-optics.

“We had the modelling, the materials and the instrument and together we were successful,” highlights Colliex. “From here, a new field really grew.”

It was 2007, Colliex’s latest publication was recognised as the paper that pushed plasmonics into the spotlight, but for the researcher, it was time to step away from experimental work. Electron microscopy was relying more and more on computing, and as Colliex puts it: “I was keeping an eye on the results but was no longer running the machine as younger people were now more expert at this.”

Still, thanks to Colliex, the Orsay research group received one of the first aberration-corrected Nion STEMs, as developed by Krivanek, equipped with a 200 kV cold-field emission gun in 2011. On a daily basis it has provided atomically-resolved spectrum-images of the fine structures on core-loss

edges, giving access to maps of site-symmetry or of electron bonds on atomic columns,

Unsurprisingly, Colliex has since co-authored many EELS-related research papers, and written myriad review papers and comments on breakthrough research in the likes of Science. Group research has continued with a focus on plasmonics and also cathodoluminescence, with a multi-signal strategy and nanolaboratory coupling in the STEM also being developed along the way, as advocated by Colliex for many years. Thanks to the group’s recent acquisition of a Nion Chromatem instrument equipped with an electron energy monochromator and designed for in situ experiments, researchers are on the cusp of putting these developments into practise (see schematic).

Looking back, Colliex believes that a key highlight of his long career in electron microscopy has been the many people he was worked alongside. As he points out, the Orsay group now comprises some 20 researchers and is world renowned for its cutting-edge electron microscopy-related research, which of course, is a huge legacy.

“During my career, people migrated from all over the world to work with me and have since kindly expressed their thanks for the influence I had on them,” he says. “I have friends in Japan, Germany, Brazil, everywhere in the world and many are now top leaders in this field.”

“I see their research as a continuation of my own work and have a lot of hope for a bright future,” he adds.

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Exploiting newphysical events andsignal combinations

EELSspectroscopy

HAADF imaging

CLspectroscopy

EDXspectroscopy

HAADFimaging

EELSspectroscopy

HAADFdetector

HAADF image

EDX map (Sr L line)

HAADF image

EELS map (Sr M line)

CL map (plasmon)

Field emission source,gun and monochromator

EELS map (plasmon)

EELSspectrometer

EELS detector

Specimen

Probe forminglensScanning coils

Aberration corrector

CL mirror

CL spectrometerand detector

Multi-signal strategy in amodern STEM instrument

EELS COUNTING of isolated Gd atoms in Gd@C82@SWCNT. The signal to noise ratio on the Gd N45 edge is sufficiently high to identify single Gd atoms, see on (c) superposed maps of the Gd N45 (in red) and C K (in blue) signals extracted from a 32x128 pixels spectrum-image (K. Suenaga et al. (adapted from Science 2000)

EELS ANALYSIS of a BxCyNz nanotube with sub-nm scale resolution, reveals a strong phase separation between BN and C layers along the radial direction. Adapted from K. Suenaga et al. (Science 1997)

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Meet the boardKEITH DUNCANDanforth Plant Science Center

Keith spent over 25 years in a microscopy and imaging facility in Wilmington, Delaware,US, studying plant biology with light, fluorescence, laser, and electron microscopy. In 2016 he was hired as manager of the X-ray imaging facility at the Danforth Plant Science Center in St. Louis and is a research scientist in Dr. Christopher Topp’s lab. He coordinates X-ray computed tomography and X-ray microscopy studies with colleagues both within and outside the Danforth Center, with imaging projects covering a wide range of agricultural biology samples.

PHILIP MORIARTYUniversity of Nottingham

Philip’s research focuses on pushing, pulling, prodding and poking single atoms and molecules using scanning probe microscopes operating under ultrahigh vacuum and low temperature conditions. He has a particularly keen current interest in integrating machine learning with atomic resolution imaging, although he also complements his microscopy work with synchrotron-based spectroscopy and analysis. Unlike his infamous namesake, he has never been particularly enamoured of the binomial theorem. Philip blogs at muircheartblog.wordpress.com.

PAUL VERKADE University of Bristol

Paul works on the development and application of microscopy techniques mainly for the study of sorting mechanisms in intracellular transport pathways and in the area of Synthetic Biology. The main tools in his lab are Electron microscopy (EM) and Correlative Light Electron Microscopy (CLEM). He has published more than 70 papers in these fields and edited four books on CLEM.

PETER HAWKESCavendish Laboratory, University of Cambridge and CNRS Toulouse retired

Peter has spent his life in electron optics, in aberration studies especially, and digital image processing for electron microscopy. He has been extensively involved in disseminating information to the electron microscope community, as editor of Advances in Imaging & Electron Physics and frequent contributor to Ultramicroscopy as well as co-author of Principles of Electron Optics. He attempts to ensure that the history of the electron microscope is not forgotten.

DEBBIE STOKESNANOVIZZ

Debbie has a background in ESEM and FIB-SEM, mainly applied to soft and/or insulating materials, and the development of methods for imaging or milling of challenging samples. Currently, in her capacity as an independent scientific consultant, she is interested in a wide range of visualisation and measurement techniques for correlating structure-property functions of various materials and systems.

ANNALENA WOLFFQueensland University of Technology

Lena has a background in FIB-SEM and HIM and enjoys the broad range of applications from nanofabrication to sample analysis. She works across a broad range of materials which are commonly studied using Focused Ion Beams but specialises in working with soft materials such as polymers and biological specimens. Her research focuses on FIB/SEM and HIM technique development and to understand ion beam induced artefacts.

LOUISE HUGHESOxford Instruments

Louise has a background in biological electron microscopy, specialising in the influence of sample preparation on ultrastructure and in 3D biological electron microscopy (SBFSEM, FIBSEM, array tomography and electron tomography on cryo- and room temperature samples). She is currently working with energy dispersive x-ray spectrometry to understand elemental distributions in biological systems.

KERRY THOMPSONNational University of Ireland, Galway

Kerry is a lecturer in Anatomy, having spent a number of years working in and running a multidisciplinary multimodal core facility. Her current research is focused on the development of correlative light and advanced electron microscopy techniques and technologies. She is keenly involved in the development of adequate training and career progression pathways for Imaging Scientists and Core Facility Staff. She is the current Honorary Secretary for Education and Outreach of the RMS. 

DALIA YABLONSurfaceChar LLC

Dalia has worked in SPM for over 20 years, starting in STM and switching to AFM in 2002. Her research focuses on advanced AFM imaging techniques for soft matter, specifically polymers, and development of accurate and robust nanomechanical characterization methods.  In 2013 she founded SurfaceChar, an AFM and nanoindentation measurement and consulting company in Greater Boston, US.

GAIL MCCONNELUniversity of Strathclyde

Gail McConnell has a background in laser physics and nonlinear optics. Her research involves the design, development and application of linear and nonlinear optical instrumentation for biomedical imaging, from the nanoscale to the whole organism. 

ERIN TRANFIELDInstituto Gulbenkian de Ciência

Since 1999 Erin has applied an ever growing electron microscopy technique portfolio to study cardiovascular and pulmonary pathologies, and fundamental cell biology questions. She has been running the IGC EM Facility (Oeiras, PT) since 2013 which supports a broad spectrum of Portuguese biological and biomaterials research. Erin’s main areas of expertise are electron tomography, and correlative light and electron microscopy. Erin is the 2020/21 president of the Portuguese Microscopy Society.

NESTOR ZALUZECArgonne National Laboratory

Nestor has been developing and using electron optical beam lines for hyperspectral imaging of hard and soft matter for over four decades.  His interests include:  metals, ceramics, semiconductors, polymers, catalysts, geological and bio-systems. He is an adjunct professor at several universities,  a member of numerous  international societies and serves on editorial boards of multiple microscopy journals and publications.

The new Microscopy & Analysis editorial board has been finalised and we are pleased to introduce the new members

Board news

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INTRODUCTIONSince the advent of aberration correction, scanning transmission electron microscopy (STEM) has been providing atomic-scale views into the structure and composition of materials that were previously unattainable. Once atoms are clearly resolved, the question becomes how precisely their location can be measured.

Currently, for many STEM applications, the information that can be extracted from the data is no longer limited by the resolution of the instrument, but by image precision that is degraded by environmental and experimental factors.

These factors include instabilities in the microscope, sample stage, and environment that create distortions within STEM images and reduce the quality and quantifiability of the data. Emerging data science techniques offer the possibility to overcome some of these limitations, enabling higher quality data that is richer in materials information.

Supported metallic nanoparticles (NPs) are critical materials for catalytic applications[1,2]. Presently, NP size, composition and support material are parameters routinely used to tune the catalytic efficiency, but the effect of strain is not completely understood, partly due to the difficulty of measuring local NP strain.

Atomic resolution S/TEM can reveal local strain in NPs[3-5], but high precision is also required for a more detailed view of atomic site-specific strain variations.

HIGH-PRECISION STEM IMAGINGHere, the strain within a platinum (Pt) NP is measured using a combination of high-precision STEM imaging and strain analysis techniques. The alumina-supported Pt NPs were synthesized by incipient-wetness impregnation[6] and dispersed on ultra-thin carbon films. The NPs were imaged in side view to resolve the

support interface. STEM imaging was performed using a probe-corrected FEI Titan 80-300 instrument at 300 kV. The high angle annular dark field (HAADF) detector was used because the atomic number contrast is well suited for imaging small metallic NPs.

To enhance the precision in locating the Pt NP atom columns in STEM images, we used a post-acquisition non-rigid registration (NRR) and averaging technique that has been shown to enable sub-pm precision[7,8].

As shown in Fig. 1a, this technique involves acquiring a series of tens to hundreds of consecutive HAADF images of the same NP using short pixel dwell times (2-3 µs) to sample fast instabilities. The beam current was reduced to ~3 pA to mitigate beam-induced sample damage.

The pixelwise NRR algorithm corrects the image distortions within each frame that are created by instrumental and environmental instabilities. The distortion-free image series was averaged to enhance the image precision.

Fig. 1b shows the resulting high-precision side-view image of a Pt NP on an alumina support. This technique has been demonstrated to improve the image precision for single crystals and nanocatalysts, the quality of 3D atomic structure information, and atomic-scale composition information[8-11].

In order to assess image precision

and measure strain, the atom column positions were determined by fitting a two-dimensional Gaussian function[8] to each atom column. In each NP grain, a precision area was defined that was at least four atomic layers away from any grain boundary, interface or surface. These precision areas were used as reference areas and assumed to be free of strain. The image precision, defined as the standard deviation of the atomic column separations within the precision area, was measured to be 1-2 pm for the data in Fig. 1b.

EXTRACTING ATOMIC SITE-SPECIFIC STRAIN INFORMATION

In order to visualize the lattice deformations, we used two methods [12]:1. Projected displacement maps (Fig. 2a,d) show the displacement of atom columns from their unstrained position[8]. They are calculated by first generating an ideal periodic lattice for each grain that represents the unstrained atom positions. The ideal lattice is created from the average lattice parameters in the precision area, registered to the precision area fit positions, and extended over the whole grain.

For shared sites at twin boundaries, multiple displacements are calculated from each grain’s ideal lattice. The displacement of each atomic column fit position from the corresponding

High-precision STEM for imaging and quantifying local strainANDREW B. YANKOVICH1, TORBEN NILSSON PINGEL1,2, MIKKEL JØRGENSEN1,2, HENRIK GRÖNBECK1,2, EVA OLSSON1,2

1. Department of Physics, Chalmers University of Technology, 41296 Gothenburg, Sweden2. Competence Centre for Catalysis, Chalmers University Of Technology, 41296 Gothenburg, Sweden

BIOGRAPHYEva Olsson is Professor of Experimental Physics at Chalm-ers University of Technology, Sweden. She obtained her Ph.D. at Chalmers and was a post doc at IBM Thomas J. Watson research Center, Yorktown Heights, New York, USA. She was appointed full Professor at Uppsala University, The Ångström Laboratory, and then at Chalmers. She is a mem-ber of the Royal Swedish Acad-emy of Sciences, Physics Class, and she is the General Secretary of the International Federation of Societies for Microscopy. Her group performs advanced micros-copy of nanostructures to enable new sustainable solutions for energy harvesting and storage, catalysis, quantum technology and health.

Dr. Andrew Yankovich is a research scientist and faculty member in the department of physics at Chalmers University of Technology in Sweden. His academic background includes earning a Ph.D in materials science from the University of Wisconsin-Madison and Bachelor’s degrees in physics, mathematics, and materials science & engineering from Saint John’s University (Minnesota) and the University of Minnesota. His research focuses on developing and using quantitative STEM and EELS techniques to elucidate structure-property relationships in a variety of technologically relevant materials. In 2020 Andrew received the Albert Crewe Award from the Microscopy Society of America in recognition of his contributions.

ABSTRACTThis article presents a method for quantitatively measuring strain in crystalline materials with atomic-scale resolution directly from high-precision scanning transmission electron microscopy images. The method is applied to platinum nanocatalysts to reveal atomic site-specific strain patterns that are predicted to influence catalytic activity. This technique opens up possibilities for imaging and in-depth studies of strain-related phenomena in a wide range of crystalline material systems.

FIGURE 1 (a) Selected images from a HAADF STEM image series of an alumina-supported Pt NP viewed along [110]. (b) The high-precision image produced from the series shown in (a). The support interface is at the bottom of the NP. The blue and red boxes mark the {111} and {311} twin boundaries respectively. [adapted from[12]]

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Microscopy and Analysis 34(3): 12-14 (EU), June 2020

Determining atomic site-specific strain

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ideal lattice position is measured and indicated by a colored arrow in the displacement direction.

Projected displacement maps are an intuitive way of visualizing the global NP lattice deformation behavior, but they tend to not reveal the small-scale local lattice deformations because the displacements accumulate away from the precision area.2. Projected strain maps (Fig. 2b,c,e,f) are a more accurate and quantitative way to assess local lattice deformations between nearest neighbors. They are calculated by comparing each individual nearest-neighbor distance to reference values in different crystallographic directions.

Here, strain is defined as the deviation from the average atomic column spacing within the precision area divided by the average spacing. On this data, the technique enables a strain precision of <0.7%. The local strain is indicated as a colored region in the strain maps between each of the two neighboring atomic columns, and separate strain maps are created for each crystallographic direction.

NANOPARTICLE STRAIN BEHAVIORThese strain analysis tools reveal pm-scale crystallographic deformations (Fig. 2). The displacement maps (Fig. 2a,d) reveal large global lattice deformations at a {311} twin boundary and at specific surface and interface sites.

The strain maps (Fig. 2b,c,e,f) reveal moderate expansive and compressive strain of atoms at the free surfaces. These strains vary in magnitude

depending on whether the site is at an edge, corner, or facet.

The {111} twin boundaries show ~1% lattice expansion perpendicular but not parallel to the boundaries, while the {311} twin boundary induces much larger strains. The interface shows strong and localized strain that could originate from lattice mismatch and interface roughness. See reference[12] for visualization of strain in other Pt NPs.

Finally, we used a theoretical density functional theory-based scaling relation kinetic Monte Carlo method[13] to predict how the experimentally observed strain patterns affect the

attainable catalytic activity[12].SUMMARY AND CONCLUSIONSWe developed a high-precision STEM imaging method and strain analysis technique that allow for quantitative atomic site-specific strain measurements with <0.7% strain precision.

We have applied this to reveal the complex intrinsic and extrinsic strain behavior in supported Pt nanocatalysts that are present at twin boundaries, surface sites, and the support interface.

Combining precise and site-specific strain measurements with kinetic simulations opens up new possibilities for understanding and controlling

the relationships between strain and catalytic properties.

Article and references available online at: analyticalscience.wiley.com/publication/microscopy-and-analysis ©John Wiley & Sons Ltd, 2020

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FIGURE 2 (a) Atomic column displacement map showing the direction and magnitude of lattice deformations in the NP shown in Fig. 1. (b-c) Projected strain maps for the labeled crystallographic planes that are marked by arrows. Red and blue signify compressive and expansive strain, respectively. Bright yellow and light blue signify large values off the color scale. (d-f) Magnified views of the black rectangles in Fig. a-c. [adapted from[12]]

ACKNOWLEDGEMENTSThe authors acknowledge funding from the Chalmers Competence Centre for Catalysis, the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Chalmers Excellence Initiative Nano, and the European Network for Electron Microscopy (ESTEEM2). This work was performed in part at the Chalmers Material Analysis Laboratory.

CORRESPONDING AUTHORDr. Andrew B. YankovichDepartment of PhysicsChalmers University of TechnologyGothenburg, Swedenandrew.yankovich@chalmers.se

Prof. Eva OlssonDepartment of PhysicsChalmers University of TechnologyGothenburg, Swedeneva.olsson@chalmers.se

Determining atomic site-specific strain

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Contents

S3 The emergence of electrical analysis in electron microscopy Grigore MoldovanS6 Product focus

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MICROSCOPY AND ANALYSIS ISSN 2049-4424© 2020 John Wiley & Sons, Ltd. Issued in: January, March, May, July, September, NovemberPublished by: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, UK Tel: +44 (0)1243 770443Fax: +44 (0)1243 770432Email: microscopy-analysis@wiley.co.uk Web: www.microscopy-analysis.comWhile every effort is made to ensure accuracy, John Wiley & Sons, Ltd and its agents cannot accept responsibility for claims made by contributors, manufacturers or advertisers.

INSIDEMAY-JUNE 2020 EMEA ENERGY SUPPLEMENT

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The cover shows an image of the internal structures from a commercial 18650 battery. The spiral “jelly roll” structure of the electrode layers inside the battery can be seen in the

background. The colored foreground image shows a zoomed in volume imaged at much higher resolution (1.5 µm/voxel) where individual inclusion particles and electrode layer defects are clearly resolved. Images were acquired by laboratory-based 3D X-ray microscopy using ZEISS Xradia 620 Versa.

The ZEISS Xradia 620 Versa X-ray microscope used to generate the cover image uses a two-stage magnification system to enable sub-micron resolution 3D imaging at large working distances, so-called Resolution at a Distance (RaaD). This unique architecture where the X-ray image is projected onto a scintillator before being optically magnified onto a CCD camera means that high resolution imaging (down to 500 nm spatial resolution) is not restricted to extremely small samples, unlike conventional microCT. A seamless user experience allows you to create efficient workflows. Easily scout a region of interest with the Scout-and-Scan control system and acquire high quality data quickly and easily, especially when you are in a central lab where users may have a variety of experience levels.

With LabDCT (laboratory based diffraction contrast tomography) as an add-on you will non-destructively map 3D grain orientations in polycrystalline materials.

Using the Xradia Versa X-ray microscope enables materials researchers to develop the next generation of batteries that charge faster, hold more charge, last longer, and operate safer.

REGISTRATION The journal is free of charge, worldwide, to users who pur chase, specify or approve microscopical, analytical or imaging equipment at their place of work, and marketing exe cutives who make advertising decisions. To register, go to http://www.microscopy-analysis.com/user/register. To amend your address details, go to http://www.microscopy-analysis.com/user/login. The registered address entered must be an organisational address.Subscription charges for non-qualifying readers: $110 per annum (UK) or $195 (Europe); all other countries $280 by airmail.NON-USA returns should be sent to Readerservice Wiley & Sons Ltd, 65341 Eltville, Germany Tel. Germany: 06123/9238-290 Tel. International: 0800/0961137 Fax. Germany: 06123/9238-244 Fax International: +49/6123/9238-244Microscopy and Analysis (ISSN No: 2043-0639 USPS NUMBER 010-289) is published Bi-monthly by Wiley, and distributed in the USA by Asendia USA, 701 Ashland Ave, Folcroft PA. Periodicals postage paid at Philadelphia, PA and additional mailing offices. POSTMASTER: send address changes to Microscopy and Analysis, 701 Ashland Ave, Folcroft PA 19032

Contact DetailsCarl Zeiss MicroscopyEmail sabine.lenz@zeiss.comWeb zeiss.com/microscopy

EDITORIAL BOARDPeter Hawkes CNRS, Toulouse, FrancePaul Verkade University of Bristol, UK Debbie Stokes Nanoviz, Netherlands Keith Duncan Danforth Inst, St Louis, USA Annalena Wolff Queensland University of Technology, Australia Nestor Zaluzec Argonne National Lab, IL, USA Dalia Yablon SurfaceChar LLC, Boston, USA Philip Moriarty University of Nottingham, UK Louise Hughes Oxford Instruments, UKKerry Thompson National University of Ireland Galway, IrelandGail McConnell University of Strathclyde, Scotland Erin Tranfield Instituto Gulbenkian de Ciência, Portugal

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INTRODUCTIONElectrical analysis in electron microscopy is a field emerging alongside the traditional structure and composition characterisation in SEM, FIB-SEM and STEM. There are several fundamental topics within electrical analysis, reflecting a large cross-section of applications that ranges from electrical failure analysis to fundamental device research.

Development of some electrical analysis techniques goes back in time to the origins of electron microscopy, but development has intensified in recent years due to transition from optical to electron microscopy techniques, driven in part by the Moore’s Law.

Perhaps the most important topic in electrical analysis is electrical resistance or conductivity. The ability to map resistance in devices with high resolution was a major discovery in electron microscopy and failure analysis – a breakthrough that now enables a new generation of electrical failure analysis techniques for nanometre-sized device technology[1]. It provides means for identifying locations of defects in complex 3D interconnects, thus continuing the heritage of optical techniques to bridge between device characterisation and physical analysis.

A second topic for electrical analysis is direct imaging of internal fields in semiconductor devices, whether for technology development, failure analysis of reverse engineering [2]. This spans across from solar cells, optoelectronics to high-power HEMT and CMOS. Third, but not least, is research into electrical activity of defects, as part of material science and electronics engineering, in particular for high-volume low-cost devices such as photovoltaics [2].

KEY CONCEPTSIn order to bring into view the many original strands that make up today’s electrical analysis, here is an overview

of key concepts required to track its development and understand its breath of reach.• Specimen Current: arguably the

precursor of electrical analysis, it describes the current measured from the specimen. It may be used for very general imaging, similar to the conventional signals and detectors; so much so that not all specimen current signals are useful for electrical analysis, and therefore the term has been superseded.

• Charge Collection: was a term used to describe the condition where specimen current is charge absorbed, induced or otherwise originating from the application of the electron beam. It may be thought of as an operation mode of the microscope where the specimen itself acts as a detector for charge, i.e. the charge collector.

• Absorbed Current: somewhat a misnomer, originally intended to describe a specimen current produced by simple absorption of electron from the primary beam into the specimen. Perhaps less obvious at the time was that an electron beam can also remove electrons from the specimen, but the term has stuck and now describes all such basic charge exchange.

• Resistive Contrast: describes a condition where contrast in specimen current reveals resistance changes across the specimen. It’s one of the founding concepts for electrical analysis in electron microscopy, first to reveal that resistance can be mapped with very high spatial resolution.

• Resistance Change: a term that has crossed over from optical probing techniques, which describes a condition where the total resistance measured across a sample is temporarily changed due to the action of the electron beam. The origin of this reversible change may be local heating, charging or others.

• Induced Current: describes a

specimen current collected from the electron-hole pairs generated by energy loss of the primary electrons into the specimen. This only applies to materials with a band gap, and thus also able to support an internal electric field, and thus it’s generally applied to semiconductor devices.

• Electron Hologram: describes a holographic image of the specimen that includes information on electric fields, as well as magnetic fields and thickness. Extraction of a thin lamella from the device is necessary, as this is a transmission technique.As is always the case, more advanced

concepts follow, such as biased analysis with lock-in amplification, or transient currents with beam blanking, but these are beyond the purpose of this introduction.

TECHNIQUESVarious approaches and workflows have developed over the years, sometimes to fit the needs of a particular device or fundamental research, but a new microscopist or analysis approaching this field today must manage a collection of complementary techniques. Only the basic techniques are presented here.

RESISTIVE CONTRAST IMAGINGWith regards to electrical resistance, the most important technique is Resistive Contrast Imaging (RCI), see Figure 1. It makes use of the absorbed current and two electrical connections to the specimen to set up a current divider circuit, where the fraction of charge collected at each terminal is proportional its resistance to the position of the electron beam [1].

Resistance between the beam position and the electrical connection is thus revealed, at the same spatial resolution as the resolution of the microscope. For CMOS and MEMS devices, RCI is used in a lateral configuration (Figure 1) to image opens and shorts in tracks and vias across the device; and it’s used in a

The emergence of electrical analysis in electron microscopy

Dr. Grigore Moldovanpoint electronic GmbH, Erich-Neuss-Weg 15, D-06120 Halle (Saale), Germany

BIOGRAPHYGrigore (Greg) Moldovan is the Chief Technology Officer of point electronic GmbH – a supplier of electronics and software for electron microscopy. He manages technology, development and marketing. Greg holds a PhD from University of Nottingham, and has previously worked for Oxford Instruments, University of Oxford and University of Cambridge. He is a scientist and a microscopist.

ABSTRACTA new kind of analysis is emerging in electron microscopy, breaking away from the traditional boundaries of structure and composition, and establishing itself firmly within the area of imaging electrical properties. As a field in its own right, it comes packed with a coherent collection of dedicated techniques, electronics and software; and it’s applied across scales from bulk materials to nanodevices, from fundamental research to routine failure analysis. What is this field, where it comes from, and what is driving this development?

CORRESPONDING AUTHORpoint electronic GmbH Erich-Neuss-Weg 15, D-06120 Halle (Saale), Germany.email: grigore.moldovan@pointelectronic.de

Microscopy and Analysis 34(3): S3-S5 (EU), June 2020

Electrical Analysis in Electron Microscopy

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Electrical Analysis in Electron Microscopy

normal configuration (see Figure 2) to reveal leakages in capacitive structures and transistors.

ELECTRON BEAM INDUCED RESISTANCE CHANGESecond with regards to electrical resistance, is the technique of Electron Beam Induced Resistance Change

(EBIRCh), see Figure 3. Similar to Optical Beam Induced Resistance Change (OBIRCh), it forces a current through the device using a voltage source (i.e. bias) and detects locations where the electron beam is able to change the resulting current [4].

This is not a charge collection technique, as the absorbed current

plays no role in the resulting contrast, but it may still be considered a specimen current technique as it measures the current from the specimen.

In contrast with RCI, the physical origin of EBIRCh is open to interpretation, local heating, fields or charge density, but the technique does

provide easy localization of defective material in CMOS transistors, and it’s therefore important.

VOLTAGE CONTRASTFor completeness, Voltage Contrast (VC) is an older technique also able to provide information on local resistance or conductivity, see Figure 4. VC works by manipulating the yield of secondary electrons, backscatter electrons, or indeed absorbed current, due to local surface fields at the specimen, which originate either from an external bias or due to self-charging under the beam.

However, VC is difficult to obtain, especially for smaller technology, and lacks spatial resolution, therefore has been largely superseded by RCI and EBIRCh.

ELECTRON BEAM INDUCED CURRENTA further key technique for electrical analysis is Electron Beam Induced Current (EBIC), see Figure 5. This is somewhat more complex, as a sum of currents is present in the resulting images, including absorbed current from the electron beam, induced current from electron energy loss into the semiconductor, a biasing current from an applied voltage source, and also potentially a light induced current from in-situ illumination.

Briefly, the induced current is only collected at, and in the vicinity of, the probed junctions, and therefore EBIC maps the presence and shape of internal fields due to implantation or contacts [2]. Further, collection efficiency is reduced in the presence of electrically active defects, thus revealing location and strengths of grain boundaries, staking faults, dislocations and other crystalline defects [3].

SCANNING TRANSMISSION ELECTRON BEAM INDUCED CURRENTFollowing the ever-shrinking technology nodes into the nanometre range, there is a renewed interest in TEM-based techniques. The new generation of biasing holders, in combination with FIB-based lamella preparation, are now placing this into easy reach.

Perhaps given the past developments, the first technique to make this transition is EBIC, which gives rise to the aptly named Scanning Transmission Electron Beam Induced Current (STEBIC), see Figure 6. It’s conceptually similar with EBIC in SEM, with the notable difference that the charge carriers are confined within the thickness of the lamella, which results in a much-desired increase in spatial resolution, at the expense of increasing complexity in sample preparation and modelling [5].

ELECTRON HOLOGRAPHYWithin TEM-based techniques, most explored is however Electron Holography. Electron Holography is able to reveal electric, and

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The RCI technique uses two electrical connections and the electron beam to set up a current divider circuit, where absorbed current reveals relative resistance to beam position.

In lateral current divider configuration, it’s used to map connected networks in CMOS and MEMS devices.

Schematic of RCI setup, showing device with two electrical connections in a lateral current divider configuration

Current measured depends on the resistance between electrical connection and electron beam position

Typical line scans across resistors, showing that areas of constant sheet resistance give constant gradients in this lateral configuration

An open (high resistance area) is identified as a sudden drop in intensity

The Resistance Contrast Imaging (RCI) technique - lateral geometry

Colour mix of black-white SE and black-red RCI signals showing a CMOS network ending in an open failure point(plan view, 282.1 µm image width)

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RCI in normal geometry is an advanced case of RCI used when the resistance mapped is constructed between overlapping layers in the device, such as capacitors defined between metal layers or poly.

It requires careful use of acceleration voltage, such that charge from the electron beam is deposited into the lower layer.

Schematic of RCI setup, showing device with two electrical connections in a normal current divider configuration

Current is now divided in a direction normal to the beam, i.e. depth of the device

Typical line scans across resistors, showing that areas of constant sheet resistance give constant signals in this normal configuration

A leakage (low resistance area) is identified as a sudden peak in intensity

The Resistance Contrast Imaging (RCI) technique - normal geometry

Colour mix of black-white SE and black-red RCI signals showing a CMOS capacitor with a leakage failure point(plan view, 38.5 µm image width)

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EBIRCh reveals device locations that are sensitive to the electron beam. Such locations present variations in the current forced through the specimen by an external voltage.

Physical origin of change may be varied, including local heating and charging. High beam currents tend to be used, but are not always necessary.

Schematic of EBIRCh setup, showing device with two electrical connections, one used for a voltage source and the other for current measurement

Current measured is given by total resistance, and thus sensitive to resistance change due to the beam

Typical line scan profile, where constant current indicates no change, and current variations indicate beam sensitive material

For failure analysis, such local changes produce bright sports and thus localise failure sites

The Electron Beam Induced Resistance Change (EBIRCh) technique

Colour mix of black-white SE and black-red EBIRCh signals showing a leakage point in a high power transistor (plan view, 5.1 µm image width)

FIGURE 1 The Resistance Contrast Imaging (RCI) technique – lateral geometry

FIGURE 2 The Resistance Contrast Imaging (RCI) technique – normal geometry

FIGURE 3 The Electron Beam Induced Resistance Change (EBIRCh) technique

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Electrical Analysis in Electron Microscopy

magnetic fields with a high spatial resolution that approaches atomic detail[6]. In contrast with the other techniques presented here, numerical reconstruction is used in order to retrieve maps of electric fields from experimental holograms. Electron Holography also provides the ability to characterise space charge layers, such as those formed at grain boundaries.

EQUIPMENTIn terms of provision of equipment, all these techniques have undergone a transition from the hands of pioneering microscopist or analysts, into commercial supply and support. Electrical performance of current equipment far surpasses that of the early days – imaging speed and beam currents are no longer major limiting factors. Recent development focus has been moving towards ease of use, automation and service, with an ever-increasing role played by software.

Necessary equipment primarily consists of dedicated calibrated electronics and quantitative software, with the variations required by each method. For current techniques, this means one or more dedicated preamplifiers, a second-stage amplifier for digitisation, embedded voltage source for biasing, current source for compensation, an imaging unit for scanning and image acquisition. All these are available as turn-key solution for most SEM, FIB-SEM and (S)TEMs.

For SEMs, hardware typically includes a dedicated nanoprober, which may be removable to allow use of the microscope for other techniques, and may further include beam blanker, plasma cleaner, heating/cooling stages and other such dedicated equipment. For TEMs, a biasing holder is used to provide electrical connections to the specimen, and Electron Holography further requires a biprism.

Software for electrical analysis follows a well-established tradition for quantitative work, which exceeds the requirements for general imaging, where pixel values do not present a physical value or indeed have a physical unit. This is required not only for an adequate comparison between devices or processing parameters, but also for modelling.

Modelling for electrical analysis may include a wide range of parameters, including the interaction between the electron beam and the specimen, electrical fields at junctions, recombination activity of defects. This enables a very revealing comparison between calculated and measured parameters, which has been amply explored to extract diffusion lengths of minority carriers, edges of depletion regions, recombination strength, amongst others.

The modelling approach has been extended to account for temperature and charge density, and thus provide further information on nature and density of physical defects and corresponding energy levels.

SUMMARY AND CONCLUSIONSAs concluding remarks, development of all these concepts, techniques and equipment in electron microscopy is driven in part by the need for increased resolution, which continues to bring in electrical analysis traditionally done with probing stations and optical technology.

Arguably a more important trend is the increasing role and application of electronic devices, where electrical analysis is just as important as physical characterisation and microanalysis.

As always, the ability of microscopy to bring images to previously invisible concepts and theories is finding

successful audience, now within the electronic and electrical industry.

Article and references available online at: analyticalscience.wiley.com/publication/microscopy-and-analysis ©John Wiley & Sons Ltd, 2020

www.pointelectronic.depoint electronic GmbH © 2019

VC takes advantage of changes in the yield of secondary electrons, backscatter electrons or absorbed electrons in the presence of surface electric fields.

Surface fields spread across entire conductive networks or areas, and therefore VC has a degree of resistance contrast, but lacks spatial resolution

Typical setup for VC for CMOS devices, including electrical connection for voltage source (biasing)

Surface electric fields may arise because of charging from the beam, or an applied voltage

Electrically isolated regions tend to charge under the beam, giving rise to surface electric fields and therefore voltage contrast

A degree of resistance quantification may be obtained by measuring the absorbed current, however charging must be controlled

The Voltage Contrast (VC) technique

Two false-colour SE images of a chip operating in-situ, showing Voltage Contrast on tracks due to biasing (plan view, 650 µm image width)

Electron beam

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Conductive layers

Electrical connection

Isolating layers

Electrical connection

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region

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current

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EBIC relies on collection of charge carriers induced into the device due to energy loss from the primary beam. Induced electrons, or holes, are collected only in the presence of an electric field, typically a pn junction.

It also reveals electrical activity of defects inside the field, such as grain boundaries or dislocations.

Schematic of plan-view setup showing device with two electrical connections, induced charge and collection at the internal electric field

Electron-hole pairs are induced in the entire bulk, but only collected at the probed junction

Typical line scan profile across an electric field, showing current collected at junction

Unsharp edges are given by diffusion of charge outside the field

The Electron Beam Induced Current (EBIC) technique

Colour mix of black-white SE and black-red EBIC signals showing electric field in a textured solar cell (cross-sectional view, 45 µm image width)

www.pointelectronic.depoint electronic GmbH © 2019

STEBIC is an advanced variant of EBIC, where a thin lamella is extracted from the device in order to limit the motion of charge carriers and increase spatial resolution.

It provides direct correlation with other high-resolution TEM techniques, such as HAADF or EELS.

Schematic of STEBIC setup, showing biasing holder with charrier chip for electrical connections, and lamella specimen placed above a window.

Lamella is prepared using FIB-SEM with in-situ lift out

Electrical connections from the carrier chip to the lamella are made using GIS deposited interconnects

Final thinning is made with the lamella mounted on the carrier chip

The Scanning Transmission Electron Beam Induced Current (STEBIC) technique

Colour mix of black-white HAADF and black-red STEBIC of induced current in a GaAs pn junction, and absorbed current in interconnects (cross-sectional view, 30 µm image width)

Electric field contrast

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FIGURE 4 The Voltage Contrast (VC) technique

FIGURE 5 The Electron Beam Induced Current (EBIC) technique

FIGURE 6 The Scanning Transmission Electron Beam Induced Current (STEBIC) technique

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INTRODUCTIONIn 1959, Richard Feynman proposed that one day there will be technology that can be used as our eyes and hands in the microscopic world. In his speech “There is plenty of room at the bottom’[1], which is often considered as the origin of nanotechnology, he predicted the use of focused ion beams to help see and manipulate matter at the tiniest scales.

It would take another 40 years until the first Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) would be available commercially[2]. Since then, Gallium FIB/SEMs, which combine a scanning electron microscope with a focused ion beam in a single device have become one of the key instruments in many facilities and labs.

The rise of this technology can be attributed to their incredible versatility. This includes the unrivalled ability to cut a wide variety of materials site specifically with nanometre precision. Since FIB/SEMs can reveal sub-surface features with such high precision and are able to analyse the sample and prepare cross-sections in situ, they are often equipped with various detectors, including energy dispersive x-ray spectroscopy- (EDS) and electron back scatter diffraction (EBSD) detectors.

The majority of systems are additionally equipped with a manipulator (to pick up and move nano/microsized objects and move them with nanometre precision) as well as gas injections systems. The later attachments are used for additive manufacturing [3] (e.g. deposition of a platinum rich solid on the sample surface) or enhanced etching and many different gas chemistries are available today [4].

A typical setup of a FIB/SEM is illustrated in Figure 1. Today, FIB/SEMs are the go-to tool for TEM lamella preparation as they allow to prepare site specific sub 100nm

thin foils of bulk specimen that can subsequentially be analysed using Transmission Electron Microscopes (TEM). In addition, FIB/SEMs excel when it comes to site specific micron sized cross-sectioning and 3D reconstruction as well as nanofabrication[5].

Throughout the past 20 years, Ga FIB/SEMs have dominated the market. This is predominantly due to the long life time as well as robustness of the gallium liquid metal ion source[6].

Gallium, with its low meting point of 29.8°C, low volatility, low vapor pressure, low surface free energy, good

emission characteristics and vacuum properties has been the material of choice for ion sources for many years[7] Furthermore, gallium is sufficiently heavy and efficiently sputters a wide variety of materials. A focused beam of gallium ions, however, can cause significant issues when processing some materials. This becomes apparent when considering the underlying ion-solid interactions.

As the ion beam hits the sample, the incident ions interact with the sample atoms in various ways. The ions, no matter what ion species, lose their energy in collisions with the sample

Focused Ion Beams: an overview of the technology and its capabilities

Dr Annalena WolffCentral Analytical Research Facility, Institute for future environments, Queensland University of Technology

BIOGRAPHYAnnalena Wolff is a Research In-frastructure Specialist for Focused Ion Beams at the Queensland University of Technology (QUT), Australia. She manages the univer-sity’s new Xe plasma FIB/SEM as well as the Helium Ion Microscope and supports the instrument user groups. Her research interests are the physics behind the systems and the development of novel FIB approaches and techniques. Lena received her PhD in Physics at Bielefeld University and then worked as an electron microscopist at the Monash Centre for Electron Microscopy before establishing herself as the go to person for focused ion beams at QUT.

ABSTRACTFocused Ion Beams (FIBs) are considered a key technology. Today, different FIBs, including stand-alone FIB systems, gallium Focused Ion Beam Scanning Electron Microscopes (FIB/SEMs), plasma FIB/SEMs as well as the Helium Ion Microscope (HIM) help answer research questions and drive nanofabrication like no other technology can. This article looks at the physics of the FIB and the underpinning ion solid interactions to help understand the difference between the ion species (Ga, Xe, He, Ne) and to see what makes these different FIB systems special.

AKNOWLEDGEMENTSThe author acknowledges the facilities (Tescan S8000X plasma FIB/SEM, Zeiss Orion Nanofab HIM) of the Australia Central Analytical Research Facility operated by the Institute for Future Environments at the Queensland University of Technology. The author acknowledges the facilities (FEI Helios Nanolab 600) at Bielefeld University. The Monte Carlo simulations were run using the programs SRIM and CASINO and the author would like to thank the programmers for making the software available. The author would like to thank Prof. Raynald Gauvin for illuminating discussions about electron solid interactions.

CORRESPONDING AUTHORDr Annalena Wolff, Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technologyemail: Annalena.wolff@qut.edu.au

FIGURE 1 Commonly found FIB/SEM setup. FIB/SEMs combine a SEM and a FIB in a single device and are often equipped with multiple detectors incl ETD, BSE, EDS, EBSD and in lens detectors. Gas injection systems as well as manipulators are commonly found on FIB/SEMs.

Microscopy and Analysis 34(3): 15-18 (EU), June 2020

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atoms (Figure2).Throughout this statistical process,

energy is transferred (and/or lost) in every interaction. The energy loss 𝑑𝐸/𝑑𝑥 mostly occurs via the nuclear (elastic) energy losses [𝑑𝐸/𝑑𝑥]nucl and the electronic (inelastic) energy losses [𝑑𝐸/𝑑𝑥]elec:𝑑𝐸/𝑑𝑥= [𝑑𝐸/𝑑𝑥]nucl + [𝑑𝐸/𝑑𝑥]elec

The ion species, ion energy, the energy transfer as well as the sample itself influence what collision type occurs (Figure 2) in each of these interactions. The nuclear collisions can lead to sputtering of the sample surface atoms, secondary ion emission as well as backscattered ions, sample atom displacements such as vacancies, a collision cascade, replacement collisions and phonons. Secondary electron emission as well as polymerization are caused by the electronic ion-solid interactions. Multiple interactions occur until the incident ion has lost all its energy and comes to rest in the sample at a certain depth, leading to ion implantation [7-9].

Significant problems can arise when processing samples with a Ga FIB/SEM as a result of ion implantation [10]. It is well known that Ga changes the physical properties of semiconductors (doping). It is less recognized though that Ga FIB/SEM processing can alter the physical properties of many other materials.

For example, Ga accumulates along grain boundaries in aluminium. This leads to a completely different deformation and fracture behaviour of the material [11] after the sample was processed using Ga FIB/SEM. Furthermore gallium ions can cause phase transformations and alloying (such as Ga in Cu -> Cu3Ga) [12].

To avoid the drawbacks which are associated with Ga FIB/SEMs, different ion species are becoming increasingly popular these days. There is a trend towards multiple focused ion beams within facilities and labs which can be attributed to the fact that each ion species and FIB system excels at different tasks.

Today, systems with a conventional gallium liquid metal ion source (LMIS), liquid metal alloy ion sources (multi-species ion sources, LMAIS) as well as systems with plasma sources and gas field ion sources (GFIS) are the most commonly found systems [13,14].

Advances in low temperature ion sources (LOTIS) and magneto optical trap ion sources (MOTIS) have recently sparked interest and information on these emerging technologies can be found in the following report [6].

With so many different options to choose from, how do you approach the vastly changing landscape of focused ion beams?

Which is the tool that you might be most likely to use? It all comes down to the applications. This is illustrated in Figure 3 which shows how the different FIB applications come out of the ion solid interactions and how the system setup (including available beam currents and focused probe sizes) determine the feasible structuring/

milling size range.This article compares the Ga FIB/

SEM, Xe plasma FIB/SEM and the Helium Ion Microscope (HIM) and describes how these instruments excel at specific applications based on their ion solid interactions and setup. References to the other systems as well as emerging technology are provided throughout the article though to allow a more complete overview of focused ion beams beyond microscopy and analysis applications.

MATERIALS AND METHODSThe ion solid interactions for the different ion species were simulated (Monte Carlo simulations) using the program SRIM. 10.000 ions for each He, Ne, Ga, Xe with 30keV ion energy were simulated impacting on a silicon sample using the monolayer collision cascade TRIM calculation type. The plotting window was set to ensure that all ion trajectories were completely followed.

To allow comparison between the interactions of helium ions and electron with matter, electron solid interactions were simulated (Monte Carlo simulations) using the program CASINO. The ion solid interactions were simulated using SRIM. 30keV He ions

and 30keV electrons as well as 1keV electrons impacting on a Fe sample were simulated.

RESULTS / DISCUSSIONPLASMA FIB/SEM TECHNOLOGY (XE)The plasma FIB/SEM technology (available since 2012) is becoming increasingly popular with Xe as the most frequently used ion species today [13].

One attraction to this technology is that Xe is inert, avoiding doping of semiconductors or alloying. Furthermore, the combination of a higher sputtering yield for a Xe PFIB compared to Ga (~factor 1.5) and the possibility of using µA range currents (rather than nA range currents for the Ga FIB) makes patterning more than 30 times faster in comparison to a Ga FIB/SEM [16] (the actual increase in speed can be significantly higher and depends on the system).

This makes large area cross-sectioning or 3D volume reconstructions, with dimensions around 500 µm (in each direction) feasible while maintaining nm precision of the cross-section placement (see Figure 3).

This capability opens up new

opportunities, especially in materials engineering, life sciences as well as geology where very site specific larger area removal is often required and which was not feasible with Ga FIB/SEMs previously. In addition, Xe plasma FIB/SEMs make it possible to prepare TEM lamella with reduced amorphization layer thickness, improving the TEM lamella quality significantly [17]. The plasma FIB/SEM technology is rapidly developing and Ar, N, O have recently become available as ion species for plasma FIB/SEMs and hold promise to improve results when processing different materials [20].

The plasma FIB/SEMs have larger final probe sizes at lower beam currents compared to Ga FIB/SEMs [17], making sub 200 nanometre structuring problematic. There are other FIB systems which are better suited for structuring such small features, like Ga FIB/SEMs (if Ga implantation is not a problem) or the HIM. Processes (such as TEM lamella thinning or polishing of cross-sections) which require beam currents below 10nA are of course possible and good results are achieved, however, the processing times tend to be slightly longer here in comparison to a Ga FIB/SEM.

FIGURE 2 Illustration of the ion solid interactions that occur when a beam of focused ions hits a sample. The electronic interactions lead to secondary electron emission (1) as well as polymerization (2). The nuclear interactions lead to sample surface sputtering as well as secondary ion emission (3), dislocations and vacancy formation (4), interstitials (5), phonons (6), backscattered ions (7) as well as ion implantation (8). Rproj is the projected range where the majority of ions come to rest in the sample. The amount of backscattered ions, secondary electrons, sputtered atoms as well as vacancies for different ion species when simulating 10.000 ions impacting a silicon sample are given in the illustration. The simulated values demonstrate difference between the difference ion species during the ion solid interactions.

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Overall this technology excels when it comes to large area removal where larger beam currents are required for processing. A key feature is of course that samples can be processed without Ga poisoning. Ga induced sample changes [11,12] are usually the main reason to use this technology for processing samples where smaller beam currents are required despite slightly increased processing times.

HELIUM ION MICROSCOPE (HE, NE, GA)The ion solid interactions that occur when lighter ion species like He and Ne interact with sample atoms unravel what makes the HIM special when using He. A lighter ion species like He predominantly interacts with the sample atom electrons (inelastic collisions) in contrast to heavier ion species like Ga or Xe. The predominantly electronic interactions of He directly underneath the sample surface lead to the production of many ‘secondary electrons’ (roughly 10 times more signal than is created in comparison to an electron beam in an SEM) [18].

These secondary electrons can be detected using an Everhart-Thornley detector (ETD). Less sub-surface

Nuclear interactions for He ions still occur statistically and become significant leading to sputtering when using higher ion doses. This combined with the small spotsize and sharp beam profile gives rise to the sub-10 nm fabrication capabilities of the HIM and make it the ideal tool for nanofabrication (see Figure 3). It is not well recognized within the science community, that nuclear interactions become the dominant interaction type for He below the sample surface once the He ion’s energy has fallen below ~1 keV as a result of the previous ion solid interactions (towards the end of the range). Since atoms cannot be removed from deep within the material, dislocations are created (see Figure 5) and this becomes statistically significant when using elevated ion doses. These dislocations can, however, be used for defect engineering, creating novel material properties [19].

Figure 3 (bottom right) shows a defect engineered aluminium oxide layer on silicon which was turned from a brittle material into a superplastic material by He ion irradiation. Operating the HIM with He is therefore about carefully controlling the ion dose with different ion dose regimes for imaging (< 1014 ions/cm2), defect engineering (1014 ions/cm2 – 1017 ions/cm2) and nanofabrication (>1018 ions/cm2).

Ne, which is available for the Orion Nanofab series, is often used for nanofabrication due to its slightly higher sputtering yield than He (it interacts equally via electronic and nuclear interactions). This makes Ne a suitable ion species to create sub 20nm to micrometre sized structures using an inert ion species within a feasible time range. Ne, however, struggles to fabricate sub 10nm features. This places Ne in between He and Ga/Xe when it comes to feasible structuring sizes. A Ga FIB column is found on many systems to further

extend the structuring range of the tool.

A significant drawback of this technology used to be the missing analytical capabilities (incl. EDS and EBSD) which an electron column brings to the FIB/SEM. This issue has since been addressed with the addition of a secondary ion mass spectrometer (SIMS) attachment [20]. SIMS, which is drawing increasing attention within the FIB community is discussed in a separate section below, since secondary ion mass spectrometers are now available for the different FIB systems.

GA FIB/SEM (GA)If a plasma FIB/SEM and the HIM can cover inert ion species nanofabrication from sub 10nm to larger than 500µm why is there still a need for a Ga FIB/SEM?

The larger final probe sizes for plasma FIB/SEMs, when using sub 30nA beam currents [17], make sub 200nm nanofabrication, as well as small volume removal, more difficult and indeed less efficient than the Ga version of the system. Small volume removal efficiency is an important factor for small area 3D reconstructions as well as TEM lamella preparation, especially when high throughput is required. If the use of Ga does not pose a problem for the material, then the higher efficiency in these tasks may well be the determining factor for choosing a Ga FIB/SEM.

When it comes to nanofabrication, the HIM creates higher quality nanostructures [21] when using He and Ne. It is, however, limited to roughly 100pA beam current making it a slow option for pattern sizes larger than several microns. Furthermore, the lighter ion species penetrate deeper into the sample and cause sub sample surface damage and bubble formation in Si [22] which can be problematic and a reason to switch to Ga.

FIGURE 3 The underlying ion solid interactions that occur when a beam of focused ions hits a sample (central illustration) create a multitude different interactions. The various FIB applications come out of the different interactions: The sputtering (top) can be used for patterning the sample on the micro- to nanometre scale. The sputtering yield of the different ion species as well as the available beam current for different systems dictate the feasible patterning size range for each instrument as well as the feasible range where different systems and ion species (He, Ne, Ga, Xe) overlap. Helium ions predominantly interact via electronic interactions in contrast to Ne, Ga and Xe which is highlighted by using a different colour scheme. The emission of secondary electrons (electronic interactions) gives rise to the imaging capabilities of focused ion beams. The imaging capabilities of He ions are shown in a colorized HIM image of a wasp wing (bottom left). The created defects can be used to engineer novel material properties. This is often referred to as defect engineering (bottom right). The HIM image of He irradiated alumina on silicon, which was transformed from a brittle material to a superplastic material is reprinted from [19]. Secondary ion emission gives rise analytical capabilities (SIMS) for FIBs. The common applications requiring attachments such as manipulator or GIS are shown on the right hand side of the figure.

beam spread occurs for the HIM in comparison to a SEM, as evident in the Monte Carlo simulations in Figure 4. This in combination with a very low backscatter rate for He (the created secondary electrons are almost entirely SE1) provide surface specific and local information which can be collected with the ETD, rather than having to resort to in-lens detectors in the SEM.

The HIM’s atomically sharp and cryogenically cooled high brightness source means that the beam can be focused to a smaller final probe size (the ion beam probe size is not limited by diffraction due to the de Broglie wavelength λ=0.08 pm for a He ion) and gives the HIM its superior depth of field and surface imaging capabilities (Figure 4) [18].

A key feature of this technology is the ability to image uncoated non-conductive samples. The He ion neutralizes within the first few nm of the sample during the ion solid. This, in combination with the SE emission leaves a positive net charge on the sample surface for non-conductive samples that can easily be neutralized using a low energy electron floodgun.

An example of floodgun imaging a non-conductive sample is given in Figure 3 (bottom left).

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SECONDARY ION MASS SPECTROMETRY (SIMS)SIMS has sparked great interest within recent years and spectrometers are now available as attachments for a wide range of FIB systems, incl. the HIM, Ga FIB/SEMs and (Xe) plasma FIB/SEMs [20,23]. Secondary ions are created during the ion solid interactions (Figure 2) and these ejected sample particles can be detected using a secondary ion mass spectrometer.

SIMS offers several advantages over EDX. Elemental as well as isotope mapping is possible for bulk specimen with high spatial resolution, and resolved feature sizes as small as 15nm have been reported [24]. Furthermore, light elements such as Li and H can be detected and mapped. While the mass range and mass resolution of these attachments cannot compare with dedicated SIMS systems, the lateral resolution of the SIMS systems on FIBs and the HIM as well as the ability to correlatively image samples with nanometre resolution (in the SEM or HIM) makes this technology attractive for many application fields such as battery materials research, failure analysis, biology and geology.

SUMMARY AND CONCLUSIONSUsing different ion species and focused ion beams (FIBs) presents new opportunities to study as well as create materials of tomorrow. From site-specific cross-sectioning and 3D reconstructions, to TEM lamellae preparation and nanofabrication or defect engineering, there is a FIB tool which is ideally suited for each application space. The underlying physics of the ion solid interactions as well as the available attachments on the system (such as gas injection systems, manipulators and detectors) helps choose the appropriate ion species/system for the job. Focused ion beams are the eyes and hands in the microscopic world and will help create and understands tomorrow’s materials. But Richard Feynman already knew this in 1959.

Article and references available online at: analyticalscience.wiley.com/publication/microscopy-and-analysis ©John Wiley & Sons Ltd, 2020

FIGURE 4 Monte Carlo simulations of 1keV electrons (top) and 30keV electrons (bottom right) using Casino as well as 30keV He ions using SRIM (bottom left) impacting on Fe. 1keV electrons and 30keV He ions are often compared on the basis of surface sensitive imaging settings [18]. The ion beam (middle) stays collimated throughout the SE escape depth and only creates SE1. This gives rise to the fantastic surface sensitive imaging capabilities of the HIM. A comparison of 30keV He ions and 30keV electrons shows that the electron beam stays collimated for 30keV electrons throughout the SE escape depth, however, the backscatter electrons which are created during the electron solid interactions (displayed in red) create BSE and SE2 several nm (1keV electrons) to several hundreds of nm (30keV electrons) away from the incident point. They form a non-localized signal when recording images with an ETD in the SEM. In lens detectors in SEMs are often used to minimize collection of these signals to address this problem and should therefore be used for a better overall comparison of HIM and SEM imaging capabilities.

FIGURE 5: SRIM simulations of the energy loss rate for He ions impacting an aluminium oxide sample (left). The He ions predominantly lose their energy via electronic interactions which give rise to the secondary electron emission and imaging capabilities. He ions, with energies below 1keV (indicated by the black arrow), however, interact predominantly via nuclear interactions and lead to vacancies, interstitials, dislocations underneath the sample surface around the projected range, as shown in the STEM image (right). The sample alterations (black arrow) caused by the nuclear interactions of He ions with the sample are clearly visible in the FIB prepared cross-section of a He irradiated sample.

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Animation Lab: A new view of science

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Dr Janet Iwasa animates complex scientific concepts to tell the stories of our molecular world. Rebecca Pool finds out more.

From HIV breaking into a T cell to the intricacies of cell division, molecular animator, Dr Janet Iwasa, is making a real difference to how the world understands cell biology. At the Animation Lab, based in the Department of Biochemistry at the University of Utah, US, she and colleagues create animations of molecules and processes, so small and complex, that until recently, true portrayals have not existed outside a researcher’s imagination.

“I believe animation is so important in all of this as it can provide a very accessible view of what I call the movie playing inside a researcher’s head,” she says. “We’re seeing growing interest in researchers wanting to create these sorts of visualisations, and it’s mostly at the molecular scale.”

For Iwasa, it all started with actin. Studying for her PhD in Dyche Mullin’s lab, at the University of California San Francisco, she was immersed in the intricacies of the actin networks in motile cells while also learning about motor proteins from neighbouring cell biologist, Ron Vale. But somehow she felt something was missing.

“We’d have these joint group meetings every week and most of the researchers in the Vale Lab were studying kinesin at the time,” she says. “This complex motor protein would get visualised with just two circles, a stringy stalk and a line for the microtubule... and it just didn’t seem great that we were all summarising our findings with stick figures.”

But then Vale hired medical illustrator, Graham Johnson, to animate kinesin walking. His video was shown at a later group meeting and Iwasa’s perception of cell biology changed.

“I suddenly realised that before seeing this molecular animation, I hadn’t really understood how kinesin worked,” she says. “We were missing so much by using our highly simplified depictions to show something that is three dimensional and dynamic.”

“So I got to thinking, why aren’t more of us doing this, why aren’t we trying to make something more similar to what we are were picturing inside our heads?” she adds. “I felt that if we have complex information, especially in three dimensions, shouldn’t our representations be in 3D too? So I really got into animation at that point.”

Iwasa was quick to enroll in an animation course one day a week - her supervisor, Dyche, was incredibly supportive - and was soon making animations of actin and other projects from her lab. By the end of her PhD she’d amassed an entire portfolio of animations, Dyche was routinely using these in his talks, and she knew that her future lay in molecular animation.

Iwasa went on to study animation at the Gnomon School for Visual Effects in Hollywood, California, and was soon working on visualizing molecular processes in the Department of Cell Biology at Harvard Medical School.

While at Harvard, she also worked on adapting crowd simulation software

for video game animation, Massive, for complex biological systems. The software had wowed audiences in the spectacular battle scenes of The Lord of the Rings trilogy, but Iwasa had realised that it might also work for proteins.

And at the same time, she designed and animated the multimedia exhibit ‘Exploring the Origins of Life, for the Museum of Science, in Boston, US. Here, she created a series of beautifully accurate animations to portray a timeline of life’s origins, dating back to 4.5 billion years ago, as well as describing folding RNA, ribozymes and the protocell life-cycle. Importantly, her vivid imagery helped the public understand research on the origins of life conducted in the labs at Harvard University and Massachusetts General Hospital.

ANIMATIONS FOR EVERYONEAround seven years ago, Iwasa moved from Harvard Medical School to the Department of Biochemistry at the University of Utah, the first step on the

road towards the Animation Lab. Taking up the position of Research Assistant Professor, she was also working on the ‘Molecular Flipbook’, a two year project to develop a free, open source and easy-to-use 3D animation tool-kit, funded by the US National Science Foundation.

Here, she brought together a team of biologists, animators and programmers to create open source software – the Molecular Flipbook – for biologists to create molecular animations.

“Most 3D animation packages have this incredibly steep learning curve where it takes upwards of a year to even start to make some kind of reasonable animation,” she says. “I really believed, and still do, that everyone should be animating what they are thinking and thought that we needed software that would allow people to create models of their molecular complexes, moving in three-dimensional space.”

Iwasa reckons that with the Molecular Flipbook, even a biologist that had never used the software before could create his or her first molecular animation to match their hypothesis in only fifteen minutes. “I had seen this need in the community, so set out to create something that was intuitive, easy to learn, and easy to use to create a video,” she adds.

But simplicity wasn’t enough. Iwasa also envisaged that users would be

able to share their all-important scene files, which generate the animation, so their creations could be altered and evolve over time. With this in mind, she included an online database in the original Molecular Flipbook prototype, where anyone could view, download and then contribute their own animations.

“I could create an animation of kinesin that shows one type of walking,” she explains. “Then if someone else wanted to show how a mutant kinesin walks differently or even had a different kinesin hypothesis, they could download the file [from the database], add their protein and then load this back up.”

“With this I wanted to create something that would allow these different visual hypotheses to become a personal reflection of how people think, and these could also change over time, as we get new data,” she adds.

Fast forward several years, and Molecular Flipbook is providing inspiration for many of today’s cell biology projects (see ‘The Animated Cell’). And in the interim, Iwasa has set up the Animation Lab to bring the wonderful world of molecular animation to everyone.

The aim of the Animation Lab is to create scientifically accurate and visually stunning depictions of molecular and cellular processes. As well as Iwasa, the University of Utah

JANET IWASA, second left, with colleagues from the Animation Lab

A 3D VIEW of a model protocell (a primitive cell) approximately 100 nanometers in diameter, taken from the Origins of Life project. Janet Iwasa/Origins of Life

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group includes scientific animator, Grace Hsu, and two post-doctoral researchers, Shraddha Nayak and Ann Liu, each with backgrounds in biosciences as well as animation and illustration. Together, the team works with biologists and other researchers to explore and communicate ideas and hypotheses through illustrations and animations.

According to Iwasa, the majority of collaborators right now are structural biologists or researchers associated with structural biology. And as such, to create an illustration or animation, she, and her colleagues, are typically given biological structures that haven’t yet been published.

“We’ll receive [data] as a program database [PDB] file or, say, a Chimera scene file and then our collaborator will describe everything that is happening around it,” she says. “Once we’ve got the context, we model all of this information into our animation.”

Iwasa and colleagues tend to use 3D animation package, Autodesk Maya, to create the elements of an animation. They rely on UCSF Chimera to create a molecular model from electron microscopy data, which is then imported into Maya. They then use Adobe After Effects to layer, or composite, many elements into a single on-screen image.

According to Iwasa, the team generally work more with electron microscopy images and data, rather than light microscopy content. As she puts it: “I would argue that if you can see something pretty well using light microscopy then I’m not sure you would need an animation for it.”

“But there are some scales where we simply can’t see molecules or structures moving around,” she adds. “We often have to imagine what is

happening here and so this is where I like to apply animation.”

Case in point is cryo-electron microscopy and tomography. Iwasa and her team have worked closely with biochemist, Professor Peter Shen from the University of Utah, who uses cryo-EM to understand molecules in action, particularly protein homeostasis. Together, the researchers created Cryo-EM 101 a training curriculum for cryo-EM users which includes a

host of animations as well as videos covering the main steps in the cryo-EM workflow.

Meanwhile, Iwasa is also part of a large research group, called CHEETAH, that is focused on understanding the structural biology of HIV. As part of an outreach project, called the Science of HIV, she has worked with numerous HIV researchers, including Professor Grant Jensen from CalTech and colleagues from the University of Utah

A STILL IMAGE from an animation of the HIV life cycle, revealing the structure and composition of a single virion. Janet Iwasa/Science of HIV FROM THE HIV life cycle: HIV cell proteins cause the membrane of the T cell to break at which point the viral bud can enter the bloodstream. Janet Iwasa/Science of HIV

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and other US institutions.The Jensen Lab uses cryo-ET to study

the molecular architecture of microbial cells and HIV in their native state, and drawing from this wealth of data as well as many other collaborators, Iwasa and colleagues created stunning videos of many intricate HIV virus processes. Importantly, many of their stunning animations are a work-in-progress, and will be further updated, refined and augmented over the

coming years.“Our big animation was the HIV

lifecycle which included current research on the virus as well as data from thousands of researchers collected from the past several decades,” says Iwasa. “This included data on what virus looks like, how it is able to infect cells in our body and how therapeutics are helping to combat infections.”

“Many researchers are using parts of

this huge animation to communicate ideas to other researchers while other animations are also getting into publications,” she adds.

Right now, the Animation Lab team is also working on visualising cancer and tumor metabolism, plant cytokinesis as well as transcription regulation in human development and disease. And past projects include animating how the Type IV Pilus Machine in bacteria moves, chromatin remodelling and the structural transitions of centromeric nucleosome complexes during mitosis.

“I’m mainly interested in how animation can help the scientific process, so for example, how can we help people wrap their heads around these complex processes that have multiple moving parts,” highlights Iwasa. “The really fun projects are those where we are in it from the beginning and where we are helping researchers visualise these processes even before they’ve published a paper.”

“In many of these projects, the collaborator will have this idea that visualization is a key part of the research, which makes a big difference,” she says.

So what now for Iwasa and her team at the Animation Lab? Iwasa will be expanding her team with a new post-doctoral researcher joining later

this year, and is continually looking out for new projects and collaborations. Looking forward, she is certain that animations will play an increasingly important role in scientific research and communication.

“Most of our collaborators have studied their processes for years, even decades, and have these visual hypotheses of how it all works,” she adds. “And while nobody else can see this movie, we can animate it.”

The Animated CellFor the past several years, Janet Iwasa has been working with Graham Johnson, now head of the ‘Animated Cell’ team at the Allen Institute for Cell Science (AICS).

Johnson and his team have helped to produce a visual animation guide, designed for cell biologists and students, that looks at cell function and the relationships between function and structure. For example, while looking at a cell, users can select individual cellular structures, such as mitochondria, and the guide will show what the organelle cell looks like in the different stages of cell division or mitosis.

AICS researchers have gene-edited human induced pluripotent stem cells to express fluorescent-tagged proteins that can localize at specific cell structures. Spinning disk and other fluorescence microscopies have been used to image the cells, taking horizontal slices from the bottom to the top, with the resulting z-stacks used to reconstruct 3D structures.

To date, thousands of images of fluorescently-labelled cells have being compiled to create myriad renderings of a vast range of cellular structures. With these and other resources,

Johnson, Iwasa and colleagues are exploring how to develop visualizations of a multiscale cell, where users will be able to “zoom in” on a 3D cell model and, for example, view motor proteins walking along microtubules. They are also broadly interested in creating tools that will be freely

available to the community in order to help build an animated cell.

“We’re really trying to figure out how we can build a one size fits all software that works for different structures and proteins, so researchers can build models without having to always start from scratch,” says Iwasa.

LEADING EDGE ACTIN NETWORKS: this was the cover for Janet Iwasa’s PhD thesis

MOLECULAR FLIPBOOK, below, a screenshot of the open source and and easy-to-use 3D animation tool-kit. Janet Iwasa

JANET IWASA produced this image to accompany breakthrough research from Professor Peter Shen, Utah, and colleagues, that showed amino acids could be assembled by another protein, and without genetic instructions. The Rqc2 protein (yellow) is binding tRNAs (dark blue, teal) which add amino acids (bright spot in middle) to a partially made protein (green). The complex binds the ribosome (white). Janet Iwasa/University of Utah

Lab focus

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Since the outbreak of coronavirus, structural biologist, Professor David Goodsell, from US-based Scripps Research, has released stunning watercolour images of the virus, depicting its inner structures in glorious color and accurate detail.

One image, Coronavirus, captures the moment the virus enters the lungs, revealing a particle cross-section engulfed by respiratory tract mucus, antibodies and other immune system molecules. Then a second image, Coronavirus Life Cycle 2020, details what happens to a cell as it succumbs to the virus, with ribosomes, viral replicase, spike proteins, membrane vesicles and RNA strands all laid bare.

Considered by many to be the father of scientific and molecular illustration, Goodsell has been painting cells and viruses since the 1990s, with Coronavirus following Ebola, Measles, Zika and more. And while his stunning illustrations have made the scientific headlines, he isn’t alone in his endeavours to throw a spotlight on coronavirus.

Electron microscopy images from China’s National Resources Bank for Pathogenic Microorganisms were amongst the first to emerge.

These images show the first-ever SARS-CoV-2 specimen obtained by medics from a patient in Wuhan on January 6.

Then, in February, the US National Institutes of Health revealed a series of electron microscopy image of the virus, isolated from patients in the US.

At around the same time, researchers from The McLellan Lab, University of Texas at Austin, released the first 3D atomic map of the part of the SARS-CoV-2 that attaches to and infects human cells. Only days later, The Veesler Lab at the University of Washington School of Medicine unveiled their image of the spike-protein, with both structures swiftly funnelled into vaccine development efforts.

A host of TEM images have followed from India, Brazil (see The moment coronavirus infects a cell on page 5 ) and elsewhere, capturing the virus in action. But perhaps by now, the most familiar images come from US-based Fusion Medical Animation, and the US Center for Disease Control and Prevention. Each image shows the ultrastructural morphology of SARS-CoV-2 particle with its unmistakable spikes dotted across the surface.

Coronavirus up closeSARS-CoV-2 has spawned a host of coronavirus imagery from microscopists, scientific illustrators and many others. Rebecca Pool takes a closer look.

CORONAVIRUS LIFE CYCLE 2020 shows a time point when the virus is actively replicating, and new viruses are being created David S. Goodsell, RCSB Protein Data Bank

IMAGE of the first-ever 2019-nCoV specimen obtained by medics in Wuhan, China. National Pathogen Library

What’s new

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CORONAVIRUS David Goodsall’s painting is based on information about the SARS virus. The virus is enclosed by a membrane that includes the spike protein, membrane protein and envelope protein. David S. Goodsell, RCSB Protein Data Bank

SEM IMAGE from the NIH shows SARS-CoV-2 (yellow) emerging from the surface of cells (blue/pink), right, and a second NIH image, far right, shows SARS-CoV-2 (round gold objects) emerging from the surface of cells cultured in the lab. NIAID-RML

3D MODEL, for this overall representation, the surface protein density has been reduced to help show spike, envelope, and membrane proteins. Fusion Medical Animation

CDDC RENDERING of the surface of the coronavirus: the spikes that adorn the outer surface of the virus impart the look of a corona surrounding the virion when viewed electron microscopically. CDC/Alissa Eckert, MS; Dan Higgins, MAMS

MOLECULAR structure of the SARS-CoV-2 spike protein. McLellan Lab

What’s new

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What’s new

• High sensitivity: Newly optimized scintillator and 1:1 fiber optic coupling Gpixel CMOS sensor with 100% duty cycle and ultra-low readout noise • High resolution: 9 µm pixel, optimal for 30 – 200 kV operation • Large field of view: Up to 16 megapixels (4k x 4k) • High speed: Up to 20 4k x 4k resolution frames per second and 160 1k x 1k frames per second

Rio – The clear new performance benchmark for scintillator cameras up to 200 kV operation.

Cilia sample courtesy of Hitachi.

With Rio, you no longer have to choose between field of view, resolution, speed, and sensitivity for electron microscopy.

With Rio, you get it all.

With the Rio, You Get it All

As the coronavirus pandemic continues, shuttered organisations around the world have been delivering online advice and services to keep the microscopy and analysis community up and running. Leica Microsystems has released an article How to sanitize a microscope which includes information on a range of infectious agents, including enveloped viruses such as coronavirus, as well a range of disinfection methods.

Leica is also one of several companies offering licences to data analysis software so microscopists can better cope with lab closures – its LAS X Software Suite is currently available with a free 90-day licence.

At the same time, Zeiss is offering a free 90-day version of ZEN (blue edition) or ZEN Core so users can process and analyze microscopy data at home. Huygens is also offering free trials of its deconvolution and analysis software while Oxford Instruments is extending access to Imaris Satellite Licences.

Meanwhile, GerBI-GMB – the Society for Microscopy and Image Analysis – in Germany, and supported by the Carl Zeiss Foundation, has delivered recommendations for operating Core Facilities in a research environment during the SARS-CoV-2 pandemic.

The guidelines aim to help imaging core managers to protect themselves and their users if running their facility is

mandatory and also looks at how to resume operations in a post-peak situation as safely as possible.

The organisation’s website is also home to a wealth of information on online courses, teaching materials and web-based company services.

In the UK, the Royal Microscopical Society has launched a dedicated website for its members to share information on events, training, webinars and other items. It has also provided information on online talks, including Python for BioImage Analysis, Teaching data sicence, biostats and bioinformatics and Introduction to Machine Learning.

And still in the UK, optical imaging equipment manufacturer, Aurox, has already hosted its first virtual on-line Aurox Conference on Microscopy. The conference followed the cancellation of FOM2020, and all sessions are available online.

Wiley is also providing free access to Covid-19: Novel Coronavirus content. As Dr Charles Young, Editor-in-chief of International Journal of Clinical Practice, says: “It’s in these times of crisis where communities come together even more... we’ve made the relevant research articles, book chapters and entries in our major references freely available in support of the global efforts in diagnosis, treatment, prevention and further research in this disease and similar viral respiratory infections.”

The latest range of SuperK FIANIUM supercontinuum white light lasers promises to deliver high brightness diffraction-limited light in the entire 390-2400 nm region.

By adding one of NKT Photonics’ filters, the SuperK can be converted into an ultra-tunable laser.

The lasers are maintenance-free and the full fiber monolithic architecture promises to ensure excellent reliability and a lifetime of thousands of hours, as well as maintenance-free and alignment-free operation.

The lasers have upgraded electronics and new fiber technology to provide improved performance and reliability. A modular design makes it easy to upgrade features and performance.

The SuperK series is based on NKT Photonics’ Crystal Fibre technology that has delivered supercontinuum to all fields for more than 15 years.

According to NKT Photonics, operating the SuperK FIANIUM is easy and intuitive for users from any discipline and no laser expertise is needed.

An emission button lets you switch the light on and off without the need to reduce the optical power or wait for the system to warm up or cool down.

The jog wheel system is said to makes it easy to operate the laser. In standby mode, the laser remembers the latest power or current setting and returns to the same level when the emission is re-activated.

Users can get access to all laser functions from the SuperK CONTROL graphical user interface on a PC.

Community action on coronavirus

NKT Photonics delivers SuperK FIANIUM super-continuum lasers

Olympus showcases image analysis with easier segmentationOlympus is using deep learning in cellSens imaging for microscopy to offer improved segmentation analysis, such as label-free nucleus detection and cell counting, for more accurate data and efficient experiments.

Image analysis is a critical part of many life science applications and analyses that rely on segmentation to extract targets, such as cells and organelles, from the rest of the image are commonplace.

However, conventional thresholding methods that depend on brightness and color can miss critical information or may not be able to detect the targets at all.

CellSens software’s deep-learning technology enables users to quickly train the system to automatically capture this information, improving the speed and accuracy of label-free

object detection, quantitative analysis of fluorescent-labeled cells and segmentation based on morphological features.

CellSens software can identify and segment nuclei from simple transmission images so that fluorescent labeling is not required.

The software’s deep-learning technology allows users to get accurate analysis data from low signal-to-noise ratio images.

According to Olympus, the technology produces outstanding accuracy while significantly reducing the amount of excitation light the

cells are exposed to.This enables high-resolution

segmentation while helping to keep the cells healthy.

According to the company, deep-learning technology also saves time by identifying and counting mitotic cells automatically.

CELLSENS imaging offers improved segmentation analysis for more accurate data and efficient experiments.

Relevant coronavirus links

leica-microsystems.com/science-lab/how-to-sanitize-a-microscope

leica-microsystems.com/las-x-home-office-license/

zeiss.com/microscopy/int/products/microscope-software/zen/free-90-day-version-of-zen.html

svi.nl/SVI-About

oxinst.com/news/expanding-access-to-our-satellite-licenses /?sbms=bitplane

gerbi-gmb.de/

rms.org.uk/study-read/news-listing-page/online-microscopy-talks-list.html

aurox.co.uk/aurox-confocal-microscope-conference.php#

novel-coronavirus.onlinelibrary.wiley.com/

ZEN CORE software from Zeiss

• High sensitivity: Newly optimized scintillator and 1:1 fiber optic coupling Gpixel CMOS sensor with 100% duty cycle and ultra-low readout noise • High resolution: 9 µm pixel, optimal for 30 – 200 kV operation • Large field of view: Up to 16 megapixels (4k x 4k) • High speed: Up to 20 4k x 4k resolution frames per second and 160 1k x 1k frames per second

Rio – The clear new performance benchmark for scintillator cameras up to 200 kV operation.

Cilia sample courtesy of Hitachi.

With Rio, you no longer have to choose between field of view, resolution, speed, and sensitivity for electron microscopy.

With Rio, you get it all.

With the Rio, You Get it All

TESCAN AMBER X Ĭ High throughput, large area FIB milling up to 1 mm

Ĭ Ga-free microsample preparation

Ĭ Ultra-high resolution, field-free FE-SEM imaging and analysis

Ĭ In-column SE and BSE detection

Ĭ Spot optimization for high-throughput, multi-modal FIB-SEM tomography

Ĭ Superior field of view for easy navigation

Ĭ Essence™ easy-to-use, modular graphical user interface

Take gallium-free

microsample preparation

further

Array of micro-compression test pillars in UFG aluminium alloy

50 µm

TESCAN AMBER X is based on the S8000 platform.