ISSUE 146 JUNE 2020

Smashing Super-res Limits

COVID-19 and Microscopy 3616

AMMS JUNE 2020 1

The Australian Microscopy and Microanalysis Newsletter is published by the Australian Microscopy and Microanalysis Society Incorporated and affiliated special interest groups (LMA, AMAS, CryOz) © 2019.All the materials published in this publication are protected under copyright and remain the property of the authors. It is therefore an offence to copy this material without the express permission of the author/s.Subscription to this newsletter is by membership of the Australian Microscopy and Microanalysis Society Inc (AMMS) or by private subscription.Visit www.microscopy.org.auEnquiries (including advertising rates):Dr Jeremy Shawjeremy.shaw@uwa.edu.auPhone: (08) 6488 8059

Fax: (08) 6488 1087Postal Address:CMCA, The University of WACrawley, PerthWestern Australia 6009The newsletter is normally published in the first week of March, June, September and December. The deadline for submission of all material is the middle of the preceding month.The views expressed in this newsletter are those of the authors and not necessarily those of the AMMS Inc for which this newsletter is the official publication. No responsibility can or will be taken for the accuracy of articles published in this newsletter but all reasonable attempts have been made to ensure the information is factual. Any technical articles are reviewed by our editor and specialist reviewers.

Designed by Studio Mood

www.studiomood.net

Cover: Compilation of transmission electron micrographs of the COVID-19 virus. Images taken by Dr Jason Roberts (Victorian Infectious Diseases Reference Laboratory – Doherty Institute) and Dr Andrew Leis (Bio21). From the ABC article “Fighting the Invisible Enemy”, by Stephen Hutcheon and Alex Palmer.

ISSN 1446-6090

CONTENTSEditorial 2

President’s Report 3

Vale: Andy Johnson 5

LMA President’s Report 6

AMAS President’s Report 9

Society Office Bearers 11

AMMS Corporate Members 13

New Special Interest Group 14

Unveiling the potential of Cryo-EM for life scientists in 2020 15

Fighting the invisible enemy 16

Multiscale 3D imaging solutions for li-ion batteries 26

Transmission electron microscopy in the physical sciences 29

Multi-line lasers simplify biomedical imaging instrumentation 31

Next generation Phenom XL G2 desktop SEM brings advanced automation to the forefront 35

Self-aligning microscope smashes limits of super-resolution microscopy 36

TESCAN has Installed their 3000th microscope to help scientists develop fuel cells and special prostheses 39

Hirox release new digital microscope with increased resolution 40

FIB stub 3.0 from DENSsolutions simplifies TEM lamella on MEMS nano-chips 41

New possibilities in lab-based time-resolved micro-CT imaging – webinar report 42

Preparing for the hydrogen economy 43

Grid preparation for detergent- solubilized GPCR samples 44

A ZEISS initiative for an open digital microscopy platform 49

ZEISS lightsheet 7 allows multiview imaging of both living and cleared specimens 51

AMMS JUNE 2020AMMS JUNE 20202 3

GREETINGS EVERYONE. WELL. IT WOULD BE fair to say it’s been an interesting time since the last issue. I would use the obvious simile and say it was like a rollercoaster, gripping the rails in anticipation while on the upwards “curve” at the start of the ride, followed by the peak and rush down the other side. However, the COVID-19 ride has not been a fun one and, as I write this, I acknowledge that much of the world is still experiencing very difficult times. I do not need to tell any of you of the impact this has had on universities and research in Australia, but I do hope this newsletter finds you, your families and colleagues happy and well.

All branches of science, including microscopy, have played a pivotal role during the pandemic. Epidemiologists have given us the understanding needed to model and mitigate the spread of the virus (we are all sick of graphs now), labs around the world are feverishly attempting to develop a vaccine and doctors and nurses are applying their science derived skills at the front line. Even those in the humanities and social sciences have been and will be important for navigating our path back to “normal”. My hope would be that COVID-19 has gone some way to building the public reputation of scientists. Other than the front

cover, this issue has a number of COVID-19 elements throughout, as I thought it would be fitting to at least mark such a historical event. Specifically, we have reproduced an article published by the ABC on the role some of our microscopy facilities have had on characterising the virus.

Before moving to my fishy thought for this issue I did want to acknowledge another sad event, the passing of Professor Andrew Johnson. I will remember Andy for his keen and genuine interest in what everyone (including me) was doing, but mostly for his kind and friendly nature. We hope to prepare a more detailed tribute to Andy in the September issue. Many thanks to Professor Philip Nakashima for agreeing to gather information on Andy for this.

Intrastate travel restrictions in Western Australia meant that it was difficult for me to visit my usual fishing haunts for a couple of months. However, these were eased from the 18th of May, which allowed me to hit the road on a “boys” trip north to Sandy Cape. One of my companions is a rather good photographer and caught some great shots during the trip. In fact, our group was rather multicultural, comprising an Australian (me) an Englishman, a German and a Frenchman. There is a joke there somewhere…

WHAT AN INTERESTING TIME! I AM inclined to leave my little quarterly column to this but it would probably be taken as laziness.

The last few weeks have definitely been a roller coaster. COVID-19 has certainly rocked our world in more ways than one. My heart goes out to those of you directly impacted by the disease, by either contracting it or losing their job in the wake of the pandemic. For one among us, post-ACMM was supposed to be the holiday of a lifetime with a very exciting trip overseas. It turned out to be exactly that and more, but probably not for the right reasons. Hopefully she will write a little report (I am sure she recognises herself if I write: “Antarctic” and “boat”).

For those of us who were “spectators”, which means, not losing our job or not being sick, it was interesting, likely stressful and at a minimum challenging. Juggling with work, home school, family and a very different, if not inexistent social life has likely been an eye opener.

Most of the microscopy facilities around the country have either been shut down or running at reduced capacity for the last couple of months. I hope that Jeremy’s call to send stories about what happened in your facility will have been answered. In our case we were told to shut down and only take COVID-19 work. Some of this work was featured in a story run by the ABC with one of our team at its core and reproduced in this issue of the newsletter.

For me, working from home has also been a blessing, to some extent. Time was saved by not riding my bike in the traffic (which is probably riskier than catching the new virus), but

lost in the process of having to deal with home schooling. This made me realise why I am not a school teacher and gave me even more respect for those who have embraced this calling. It also allowed for some holidays were I couldn’t go anywhere, probably to the damn of my parents, and finally caught up on renovations that were planned 10 years ago.

As far as timing of the pandemic goes, it was probably not so bad, for a scientist. It is grant writing season and we are likely finding more time than other years for that. I know I have been in my basement for the last two months, writing and assessing grants in a more relaxed way since I had very little disturbance from the day to day life of a core facility.

The turmoil and the impact created by shutting down Australia’s economy has already been felt by most universities and the financial losses are incommensurable and ongoing for the next couple of years at a minimum. It will be inevitable that jobs will be lost or at least redefined as a consequence of COVID-19. I think this is when our community should remain very close and help whenever possible. I am guessing job advertisements will be low for the foreseeable future but if something arises please do not hesitate to reach out to the community and share your job vacancy. Or indeed if you are seeking a position.

As always I would like to finish on a positive note congratulating all of you who have caught up on all the environmental health and safety paperwork due 5 years ago. I wish you a happy new post pandemic standard operating procedures writing for access to labs and facilities.

My hope would be that COVID-19 has gone some way to building the

public reputation of scientists. Other than the front cover, this

issue has a number of COVID-19 elements

throughout, as I thought it would be

fitting to at least mark such a historical event.

My heart goes out to those of you

directly impacted by the disease, by either contracting

it or losing their job in the wake of the

pandemic.

“ “

EDITORIAL PRESIDENT’S REPORT

Dr Jeremy ShawNewsletter Editor

AMMS

Assoc. Prof. Eric HanssenPresident

AMMS

Sandy Cape night sky and fishing drone taking off. Courtesy Carsten Weckend.

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THIS IS THE LAST WRAP UP from me after a great ACMM conference in February. This was drafted just after the conference but somehow missed the boat for the last newsletter. It has been a pleasure to see all the preparations for the ACMM week coming together. As Canberra braved the smoke, the hailstorm and the bushfires, I cannot deny that all these events added a certain level of anxiety. It was a relief that the smoke was gone and the fires extinguished, and the weather throughout the week was most pleasant. Some people were reminiscing about the last ACMM in Canberra, ACMM2000.

I can remember that during the Sydney 2000 Olympics, we had the conference dinner at the Australian Institute of Sport arena where we were trying wheelchair basketball and other sports. I remember having to go to the physio the next day after the volleyball. I think I wasn’t the only one.

I want to thank the organising committee and program committee for all their hard work and efforts to create a fantastic scientific and social program. I

would also like to thank the pre-conference workshop convenors and lecturers for their hard work putting together great workshops during the week-end. This was much appreciated by many attendees at the workshops.

I would myself have liked to be in many of them to learn something. I would also like to thank the sponsors and vendors for their support and the fantastic trade show they put together for the conference.

On behalf of the organising committee, I would like to thank all the plenaries and invited speakers for travelling from distant corners of the world, the contributed speakers (international and interstate) and make the scientific program, a great discussion forum. I tried to attend as many sessions as possible during the week.

Perhaps the most memorable moments for me were the high attendance at all sessions including the last day, the vibrant discussions within the trade show during coffee times and lunches following up from the sessions, a nice evening at the National Gallery dinner where everyone managed to arrive at the NGA to enjoy drinks

outside and a happy atmosphere throughout the conference. So thank you to all members and the Australian microscopy community for making this meeting a success with your participation and contributions. Finally, I thank Liz Micallef and the ASN Events team (our Professional Conference Organiser) for doing all the behind the scenes administration to make sure that all the attendees to the pre-workshops and conference have a pleasant time.

So, it is with great pleasure that I handed Brian over to Martin Saunders. Brian instigated a lot of interest from PhD students visiting my office as he was looking so dapper with his loop and lab coat. I wish Martin and his team the best in their preparation for the next two years and hope to see you at ACMM27 in Perth 2022.

We have been extremely lucky to have held a successful ACMM in 2020 (a year that will now be remembered for Covid19) so I would like to wish everyone the very best, to keep safe, sane, fit and healthy throughout this crisis.

Jenny Wong-LeungACMM26 Conference Convenor

ANDY JOHNSONVale Professor Andrew W.S. Johnson (1936 – 2020)

Director of the Centre for Microscopy, Characterisation and AnalysisThe University of Western Australia (1983 – 2000)

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THIS IS A DIFFICULT REPORT TO write because we are all facing hard times since the last newsletter. Covid-19 has changed the lives of so many around the world. I do hope that you and your families are all managing to stay safe and well with all of the social distancing and strict hygiene rules in place. I know for many of you the challenges of working from home and/or home-schooling have not been easy but personally I have developed a stronger connection with my teenage son as a result and have had to change my mindset slightly!

Microscopy, that is, how we do microscopy, has also been impacted. Many institutes and Universities including core imaging facilities, have closed their doors to both students and researchers in Australia and around the world. Some remaining open for essential activities and research only. Conferences and meetings, nationally and internationally have been cancelled. Borders have closed and companies have been extremely hard hit.

How are we managing to keep microscopy going, to offer teaching and training at the microscopes, to conduct research

at the microscope and to continue to connect with our employers, staff, researchers, students and collaborators with Covid-19 restrictions in place?

I am extremely encouraged by how the microscopy community in Australia and across the world have come together through Zoom, Skype, Teams and other means, to share knowledge and resources (Check out MyScope Microscopy Australia; https://myscope.training/). And if you are not on Twitter, now is the time to join!

At the MIF (Microbial Imaging Facility at the ithree institute, UTS) we have replaced training sessions with remote training material by adding audio to PowerPoint Masterclasses, recording training sessions at the microscope and developed remote training Zoom sessions.

I am particularly grateful to Paul McMillan for allowing MIF members to attend the Biological Optical Microscopy Platform (BoMP), University of Melbourne, online FIJI training workshops earlier this month. I believe half of the attendees were from UTS! I am also grateful to Cytiva (formerly GE Healthcare Australia) for assisting

LMA PRESIDENT’S REPORT

Assoc. Prof. Louise ColePresident

LMA

I know for many of you the challenges

of working from home and/or home-

schooling have not been easy but personally I have

developed a stronger connection with

my teenage son as a result and have had to change my mindset slightly!

“

Zoom meetings are the norm. Microscopy Australia Linked Labs discussing Covid-19 coping strategies in core facilities.

us with training on deconvolution microscopy, 2D and 3D SIM, and INCell high content analysis training workshops at UTS.

I am sure you all have people to thank including microscope and software companies for offering free temporary software licenses for image management, visualisation, analysis and processing (!).

I would like to take this moment to acknowledge everyone for adapting and supporting the microscopy community across Australia. We can certainly all learn from each other about how to provide remote training and how we cope with the slow return to one-on-one training at the microscope.

Strategies, such as using a remote mouse and laser pointer

to ensure social distancing during training at the microscope, investing in a camera to allow live recording during training, effective cleaning of the microscope before and after use and the use of clingfilm on keyboards, eyepieces and touch pad, are some of the ways facilities and staff are coping.

What hasn’t changed is that for Light Microscopy Australia we are still committed to growing our community and are planning for a stellar National Meeting next year in 2020.

The next National Meeting is currently planned to take place at UNSW in Sydney in early March, organised by Dr Renee Whan (Biomedical Imaging Facility) and her team.

The local organising committee and LMA executive members are

now discussing options of how this meeting will go ahead. Planning for a virtual (online) meeting has been considered and we are all taking notes from how current national and international virtual meetings are being delivered.

I am excited about the new LMA Executive team (effective October 1st 2020) and the new roles that have been created (Image competition convenor, Workshop co-ordinator, Outreach person, Student representatives) in order to provide new ways of how we can better communicate with you all as well as report and deliver all our activities. Please do not hesitate to reach out if you want to get involved in any way (by Zoom of course). In the meantime, keep calm and carry on with the microscopy!

Creating remote training resources by recording training sessions at the microscope.

As international conferences and meetings are cancelled around the world, the imaging community stay connected by developing online training events and meetings.

Adapting to one-one training at the microscope with social distancing and safe hygiene rules in place.

AMMS JUNE 2020AMMS JUNE 20208 9

MUCH HAS HAPPENED SINCE THE FEBRUARY ACMM Conference in Canberra and all of our lives both at work and home have changed. I have been in correspondence with our colleagues at EMAS (Europe) and MAS (USA) to give them our support and let them know we are thinking of them.

One of the most significant changes we are all facing is the challenges associated with the training of our users, which is not easy to do with social distancing. Clever and innovative are our members however and hence there is now a growing list of online resources that many of our laboratories are both accessing and helping to develop that provide remote training and upskilling.

Many of our vendors are also providing online seminars and programs. Some of the online courses include the following:

Microscopy Australia Myscope training modules, which can be found: https://myscope.training

Microscopy Australia Youtube Channel: https://www.youtube.com/channel/UCSigbQY3GCS2f62p7gHeYCg

Plus many online training courses in microanalysis in 15-18 June: https://micro.org.au/news-events/events/

For the kids who are stuck at home there is the MyScope Explore for outreach with primary school children: http://myscope-

explore.orgThe AMAS Executive met last

month and will meet again at the end of June to decide on the form of the AMAS Symposium planned for Curtin University in February 2021. The executive are looking at a range of options for the meeting. Meanwhile our colleagues in Europe regrettably had to cancel their Regional EMAS workshop planned for Bruno in May.

Our friends in the USA have moved the Microscopy & Microanalysis meeting from Milwaukee WIS to an online conference, which I am sure will make it more accessible and less expensive for many to attend.

The new online registration link can be found at: https://www.microscopy.org/MandM/2020/registration/

Finally, I wish to thank a long serving member of the AMAS Executive the honourable Ron Rasch who stands down from the AMAS Executive as the Queensland representative after more than 20 years.

Ron has co-convenved AMAS meetings and multiple workshops at many AMAS symposia. Ron’s role at the University of Queensland is Material Science Microscopy & Novel Imaging Laboratory Manager, whilst his role on the AMAS Exec has been to keep us honest and entertained over many years. Thanks Ron for all of your work and wisdom.

AMAS PRESIDENT’S REPORT

Angus NettingPresident

AMAS

One of the most significant changes

we are all facing is the challenges

associated with the training of our users,

which is not easy to do with social

distancing.

“

AMMS JUNE 2020 11

AUSTRALIAN MICROSCOPY & MICROANALYSIS SOCIETY

Executive CommitteePresident

Eric Hanssenpresident.amms@microscopy.org.auSecretary

Sarah Ellissecretary.amms@microscopy.org.auTreasurer

Dr Jamie Richesjamie.riches@qut.edu.auPublications Officer

Colin Veitchpublications@microscopy.org.auNewsletter Editor

Jeremy Shaweditor@microscopy.org.auACMM26 Convenor

Jenny Wong Leungjenny.wongleung@anu.edu.auAMAS Representative

Angus Nettingangus.netting@adelaide.edu.auLMA Representative

Louise Colepresident.lma@microscopy.org.auCryOz

Georg Rammgeorg.ramm@monash.edu

AMMS State Representatives ACT – Felipe Kremerfelipe.kremer@anu.edu.auNSW – Renee Whanr.whan@unsw.edu.auQLD – Sandrine Roysandrine13.roy@gmail.comSA – Ashley Slatteryashley.slattery@adelaide.edu.auTAS – Oliver Bibarioliver.bibari@utas.edu.auVIC – Paul McMillanpaul.mcmillan@unimelb.edu.auWA – Zakaria Quadirzakaria.quadir@curtin.edu.au

Register of Members: the Register of Members is held by the Public Officer and can be viewed by arrangement with them.

NON-EXECUTIVE OFFICERSPublic Officer – John Fitz Geraldjohnfitz9_m@bigpond.comWeb Editor – Flame Burgmannweb@microscopy.org.au

Liaison Officer – Ray Withers

withers@rsc.anu.edu.au

AUSTRALIAN MICROBEAM ANALYSIS SOCIETY

EXECUTIVE COMMITTEEPresident

Angus Nettingpresident.amas@microscopy.org.auSecretary

Karsten Goemannsecretary.amas@microscopy.org.auTreasurer

Matthew Glenntreasurer.amas@microscopy.org.au

AMAS State Representatives ACT – Jeff Chenjiang.chen@anu.edu.auNSW

Timothy Murphytimothy.d.murphy@mq.edu.au

Richard Wuhrerrichard.wuhrer@westernsydney.edu.auQLD – Ron Rasch

r.rasch@mailbox.uq.edu.auSA – Benjamin Wade

benjamin.wade@adelaide.edu.auTAS – Karsten Goemann

karsten.goemann@utas.edu.auVIC – Flame Burgmann

web@microscopy.org.auWA – Malcolm Roberts

malc.roberts@uwa.edu.au

CRYOZ

Executive CommitteePresidentGeorg Rammgeorg.ramm@monash.eduSecretaryMelanie Rugmelanie.rug@anu.edu.au

CRYOZ State RepresentativesNSW – Nick Ariotti n.ariotti@unsw.edu.auGökhan Tolun gokhan@uow.edu.auQLD

Matthias Floetenmeyer m.floetenmeyer@uq.edu.au

Michael Landsbergm.landsberg@uq.edu.auSA – Ashley Slattery ashley.slattery@adelaide.edu.auVIC – Isabelle Rouiller

isabelle.rouiller@unimelb.edu.auWA – Peta Clode peta.clode@uwa.edu.au

LIGHT MICROSCOPY AUSTRALIAPresident

Louise Colepresident.lma@microscopy.org.auVice President

Trevor Hinwoodvicepresident.lma@microscopy.org.auSecretary

Paul McMillansecretary.lma@microscopy.org.auTreasurer

Ian Harpertreasurer.lma@microscopy.org.au

Council membersACT – Luke Wensingluke.wensing@optusnet.com.auNSW – Philip Ramsdenelizandphil@gmail.comVIC – Paul McMillanmpj@unimelb.edu.au

LMA State RepresentativesACT

Daryl Webbdaryl.webb@anu.edu.auCathy Gillespiecathy.gillespie@anu.edu.auQLD

Nigel Waterhousenigel.waterhouse@qimrberghofer.edu.auSanjleena Singhs4.singh@qut.edu.auSA

Agatha Labrinidisagatha.labrinidis@adelaide.edu.auNSW

Sue Linsday sue.lindsay@mq.edu.auKalev Maevalikalev.maevali@gmail.comVIC

Sarah Ellissarah.ellis@petermac.orgBetty Kouskousisbetty.kouskousis@burnet.edu.auWA – Tamara Abeltamara.abel@telethonkids.org.auTAS – Oliver Bibarioliver.bibari@utas.edu.au

SOCIETY OFFICE BEARERS

AMMS JUNE 2020 13

The Australian Microscopy and Microanalysis Society Inc.

Secretary: Sarah Ellis secretary.amms@microscopy.org.au

The Society is a non-profit organisation dedicated to the promotion and advancement of the knowledge of the science and practice of all microscopical imaging, analysis and diffraction techniques useful for elucidating the ultrastructure and function of materials in diverse areas of biological, materials, medical and physical sciences. The Society’s major activities are the convening of multidisciplinary conferences and workshops, the publishing of a quarterly newsletter, and the co-ordination of activities with similar groups within Australia and overseas. AMMS Inc. is a member of the Federation of Australian Scientific and Technological Societies (FASTS), a lobby group that seeks to influence government decision-making about science and technology. Special Interest Groups are represented in FASTS by AMMS Inc.

The Australian Microbeam Analysis Society (AMAS)

Secretary: Angus Netting secretary.amas@microscopy.org.au

AMAS concentrates on the important area of chemical analysis using microbeams and runs a symposium and workshop programme. AMAS operates its own office bearers and committee. AMAS closely co-operates with its US counterpart - the Microbeam Analysis Society - for example with exchange visits by society presidents and by students.

Light Microscopy Australia (LMA)

Secretary: Paul McMillan secretary.lma@microscopy.org.au

Light Microscopy Australia is a national special interest group holding an annual conference along with local meetings, workshops, seminars or keynote lectures independently or in conjunction with AMMS. Members are notified of meetings, activities and microscopy information. Our objective is the advancement of the science and techniques of light microscopy including; specimen preparation, optics and Image formation, image analysis and visualisation,

instrumentation and, training and career development. Applications include Biology, Biotechnology, Pathology, Physics, Chemistry, Electronics, Metallurgy, Minerology, Materials, Nanosciences, Teaching or other sciences. Membership of the LMA is free after joining AMMS and ticking the LMA membership box on the AMMS application form.

CryOz - Cryo-EM down under

The aim of the new CryOz group is to sustain and grow the Australian cryo-EM community, share expertise and resources, and provide a forum for early career and established researchers in the Cryo-EM field. CryOz runs the CryOz conferences (CRYOZ.org) and dedicated Cryo-EM hands-on workshops.

The Australasian Electron Microscopy Email Newsgroup (AUSTEM)

AUSTEM provides a rapid means of communication between Australasian microscopists for meeting and course announcements, problem solving, equipment for sale, donation or wanted, and other issues of regional concern (as compared to the world wide Microscopy Listserver).

For more information visit www.microscopy.org.au

Affiliated Special Interest Groups

Copies of the respective Constitutions /Articles of Association are available from the relevant Secretary. Membership in each of the Special Interest Groups is administered via membership applications to AMMS Inc. Membership of AMMS Inc is a necessary requirement for membership of any of the Special Interest Groups.

WANT TO BECOME AN AMMS MEMBER?Email Jamie Riches, AMMS Treasurer: jamie.riches@qut.edu.au

Professional membership: 1 year AU$50 // 2 years AU$90

Student | Junior Technical | Retired: 1 year AU$25 // 2 years AU$45

AMMS CORPORATE MEMBERS

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FOR OVER 10 YEARS VARIOUS TECHNICAL advances have taken microscopy and microanalysis into the 3D world. There now exists a broad range of imaging modalities that are generating volumetric data in addition to more well established techniques such as X-ray computed tomography and magnetic resonance imaging.

Recognising the diversity of imaging platforms and the challenges associated with the collection and analysis of 3D data, the Australian Microscopy and Microanalysis Society would like to support the formation of a volume imaging special interest group. The SIG would cover all techniques and applications where the aim is to generate, combine and/or process

3D data, including, but not limited to, the following areas:• Tomography (X-ray, electron,

optical)• Serial electron microscopy

(e.g. focused ion beam/

plasma/block face SEM)• Light sheet microscopy• Confocal microscopy• Data Reconstruction• Data visualisation and

analysis.

NEW SPECIAL INTEREST GROUPExpressions of Interest are being sought for a new SIG focusing on Volume Imaging

Any researchers wishing to register their interest in the Volume Imaging SIG can contact jeremy.shaw@uwa.edu.au

UNVEILING THE POTENTIAL OF CRYO-EM FOR LIFE SCIENTISTS IN 2020By Dr William Close – Australian Centre for Microscopy & Microanalysis, The University of Sydney

IV NSW CRYOEM USER GROUP MEETING for 2020 was held at University Sydney with a resounding success whereby academics, facility project managers also arrived from the University of NSW and Wollongong along with the Victor Chang Institute and additional fellow enthusiasts could attend to see how high-resolution structures of molecular proteins can answer big scientific questions. These include key presentations from Claudia Kielkopf from the University of Wollongong, who used Cryo-EM techniques to study the structure and dynamics of the 25kDa apolipoprotein-D, and Ichia Chen from the University of Sydney, revealing a new conformational structure of Glutamate transporters by Cryo-EM.

The keynote speaker was Dr. Tristan Croll from the Cambridge Institute for Medical Research, UK where he provided a hands-on workshop on the use of ISOLDE a program that assists in the building of high-quality macromolecular models into experimental maps. One recent result from this program was the development of a model that depicts the spike protein of the new coronavirus strain (COVID-19) shown below.

The Nobel Prize awarded in 2017 for the development of cryo-electron microscopy highlights the significance of the research technique. The uptake in the use of state-of-the-art electron microscopes to determine atomic resolution protein structures in their native form, as opposed to forming protein crystals for X-ray diffraction, has been overwhelming. CryoEM is now skyrocketing, with an ever increasing number of protein structures being uploaded to structural databanks at higher and

higher resolution and addressing new biological questions.

The sponsorship provided by Thermo Fisher enabled us to gather key players with expertise in these fields and discuss how future ground-breaking scientific breakthroughs can be achieved.

Figure 1 (top): Group photo of everyone who attended the meeting bringing together USyd, UNSW, UoW, Victor Chang Institute and many other enthusiasts.Figure 2: (A) Structure of 2019-nCoV S in the prefusion conformation (Adapted from D. Wrapp et al., Science 10.1126/science.abb2507 (2020)). (B) Dr. Tristan Kroll gave a detailed workshop on the use of ISOLDE which has been instrumental in the modelling of high-quality macromolecular structures. Such work includes the new spike protein of the coronavirus.

A

B

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FIGHTING THE INVISIBLE ENEMYBy Stephen Hutcheon and Alex Palmer, Digital Story Innovation Team

FOR MORE THAN TWO DECADES, DR Jason Roberts has been routinely peering into microscopic netherworlds and observing the behaviours of some of nature’s most deadly nano-sized insurgents.

A senior scientist at the Victorian Infectious Diseases Reference Laboratory in Melbourne, Roberts is also a consultant virologist with the Australian Government’s polio eradication program.

In early February, he was among the first Australians to observe the virus, which, a few days later, would be given the official name of SARS-Cov-2.

And as an accomplished scientific illustrator, he is also skilled in the use of powerful electron microscopes (EM) to capture images of viruses and then use those to recreate intricate three-dimensional models.

Although he didn’t know it at the time, he was snapshotting a virus that would transform into the mother of all disruptive pathogens, a biological wrecking ball that has become an existential threat to our way of life.

“You’re looking at the enemy,” says Roberts, recalling those first encounters. “It’s invisible to everyone else in the world.

“But to us in virology, especially in EM, it’s not invisible. We see it. We know what it looks like.”

To eyeball the virus is to know its shape and structure. Armed with that knowledge, scientists can begin to decipher its genomic

mission, a vital first step in understanding how to neutralise this invisible enemy.

FRAGMENT OF GENETIC CODE

As with all viruses, SARS-CoV-2 exists in a limbo somewhere between the animate and the inanimate.

Like a seed, a virus will only spring into action if it finds the right kind of conditions. Otherwise it will perish.

A virus in this stage is known as a virion. As they cannot replicate on their own, virions must find a suitable host in order to survive.

“It is really just a short fragment of genetic code,” explains Roberts. “[But] it’s amazing that such a little bit of code can wreak such havoc.”

Roughly spherical with a fringe of projections called spike proteins or peplomers, a SARS- CoV-2 virus particle typically measures about 100 nanometres in diameter. That’s about 10,000 times smaller than a grain of salt.

Each virus comes equipped with an outer layer, or viral envelope, that is designed to penetrate a cell and deliver its cargo of genetic material into the host.

JASON ROBERTS

Roberts describes that cargo as being “spring-loaded, like a jack-in-the-box”. When it finds the right target, “it just goes ping and delivers it”.

“Once that code gets into a cell, it hijacks the [cell’s] replication machinery. It’s now able to reproduce; it’s able to evolve; it’s able to metabolise energy.”

And there’s no escaping them. Our biosphere is teeming with viruses.

A 2011 paper in the research journal Nature Reviews Microbiology calculated there were approximately ten nonillion viruses on earth — that’s 1 x 1031 or a number with 1 followed by 31 zeros.

Despite the bad rap they get, not all viruses are bad for us.

Roberts says the human race couldn’t survive without some of them, like those found in our guts which help to keep our digestive systems in balance.

“Viruses,” he says, “are the oil that lubricates the gears of evolution.”

‘REALLY QUITE TERRIFYING’

Roberts’s lab is part of the Peter Doherty Institute for Infection and Immunity. That’s the same organisation which in late January became the first lab outside China to successfully grow a sample of the SARS-CoV-2 virus.

They used a specimen taken from one of the first people in Australia to test positive to COVID-19 in Australia, on Friday, January 24.

A few days later, Roberts and Dr Andrew Leis, an electron microscopist and viral expert at Melbourne University’s Bio21

Molecular Science & Biotechnology Institute, were putting samples under the lens of a transmission electron microscope (TEM).

These were the first, blurry images they produced. They were not published or distributed because of the image quality.

Roberts vividly recalls that first impression and the mood in the lab that day.

“Initially, there was excitement … and then it was just like this dread:

This thing’s really quite terrifying.”The sample showed the presence

of many virus particles, much more than they expected to see.

“We had this moment of clarity and sort of urgency that you need to get on to what’s got to be done, get results out there, collaborate…”

Around January 31, the Doherty Institute had also delivered a small vial containing a sample of the virus it had cultivated to the CSIRO’s Australian Centre for

Disease Preparedness (ACDP) in Geelong. The sample was taken by members of ACDP’s dangerous pathogens team into the most secure part of the facility where they grew the virus from the original culture.

ACDP is one of the few facilities that includes a certified Physical Containment Level 4 (PC4) lab area, the place where dangerous pathogens go to be cultivated, examined and stored.

January 31, 2020 February 3, 2020 February 7, 2020

Jason Roberts, VIDRL – Doherty Institute in collaboration with Bio21 EM laboratory (technical assistance, Dr Andrew Leis)

The intense worldwide research activity centred on the development of a vaccine for COVID-19 has certainly included contributions by number of our fellow society members. The following article was produced by the ABC and focuses on how microscopy is playing an important role in characterising the virus. Dr Jason Roberts of the Victorian Infectious Diseases Reference Laboratory (VIDRL), which now forms part of the Doherty Institute, has been a lead researcher in this space. Dr Andrew Leis and Associate Processor Eric Hanssen of the Bio21 Advanced Microscopy Facility and Monash University have also played key roles. This is a great example of why science and scientists play such a critical role in the global community.

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The facility has been constructed on a box-within-a-box principle.

Sections are air-locked, ensuring physical containment is duplicated or triplicated, in case one fails.

The secure area is surrounded by a 30cm-thick concrete wall and held at a lower air pressure than the outside world, to keep any airborne infectious agents inside.

ACDP is also where Sandy Crameri has worked for 30 years as electron microscopist specialising in viruses. She’s also one of the most experienced.

SANDY CRAMERI

Over the years, she has seen and imaged the worst of the worst: pathogens such as SARS, MERS, HIV, Hendra, Ebola and H1N1, the virus that triggered the 2009 swine flu pandemic.

Around February 10, Crameri recalls receiving her first sample to examine.

“When I first looked at the peplomers, which are the crown-like things, I thought, ‘wow, they’re really obvious’,” she says.

“Then I invited my colleagues … to come and have a look because everyone was interested to clap eyes on it when we first got it here.”

Crameri quickly turned around her first image, a monochromatic version of this image below, which after colourisation — which took a day or so more — was published by the CSIRO.

Her image of a SARS-CoV-2 virion was taken with a transmission EM, which fires electrons through the specimen to reveal the structure, producing a two-dimensional image.

It was prepared using what is called the negative staining method, which enhances the contrast of the specimen. It’s among the oldest and most widely used EM imaging techniques and produces relatively quick results.

Instead of using visible light, electron microscopes fire an accelerated beam of electrons at the specimen. As electrons have a much shorter wavelength, they can deliver higher magnification and better resolution, producing much more detailed imagery.

All electron microscope images

come out in greyscale with colouring added later.

While colour is used in research to highlight certain features, Crameri says it’s also added with an eye to making something that will catch the public’s attention.

“I didn’t want to pick red, for example, because red is a sort of alarmist colour,” she says, explaining her choices. “So I asked my colleague what colour she liked and she said burnt orange.”

So burnt orange it became and Crameri chose the complementary colour — which was a blue — to colour the background, allowing the virion to pop out more.

The image is similar to one in a series of images produced by Roberts, with assistance from Andrew Leis on February 7.

It shows a single virion, with its distinctive spike proteins clearly visible. This too, is a negative stain image captured on a transmission EM.

“If you look at the first images that came out, they were pretty crappy, to be honest,” he says. “It’s through fine-tuning the virus growth and tweaking that we managed to get some really amazing particles.”

Roberts chose to colour the yellow-orange hues with a dark background to reflect the danger posed. The background is what he calls the peacock effect — a purple, blue and green gradient that reflects the bird’s plumage.

LIMITED DIAGNOSTIC VALUE

Another type of virus imaging is one produced using a scanning electron microscope (SEM), which is not a piece of equipment used by either Crameri or Roberts.

As the name suggests, it scans the surface of an object rather than looking through it. And it gives a much more contoured, and — after colours are applied — textured, effect.

Scanning EMs were used to take images like the one below produced by the US National Institute of Allergy and Infectious

Source: Jason Roberts, VIDRL – Doherty Institute.

Source: Jason Roberts, VIDRL – Doherty Institute.

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Diseases (NIAID) by its Integrated Research Facility.

Each of the yellow dots on the image below is a coronavirus particle and corresponds to the images of the single virus particles shown above that were captured by Sandy Crameri and Jason Roberts.

This image shows a cell taken from a patient sample (in blue) heavily infected with SARS-CoV-2 virus particles.

Roberts says the scanning EM has limited diagnostic value when researching a new virus.

“Whilst the images from NIAID are interesting visually, they are not high enough resolution to give any real structural information regarding the virus,” he says.

Scanning EM micrographs of SARS-Cov-2 virions budding from a cell.

NIAID, which is headed by Dr Anthony Fauci, one of US President Donald Trump’s chief advisers during the current coronavirus crisis, has produced a series of stunning scanning EM of the SARS-CoV-2 virus.

A NEW FRONTIER IN MICROSCOPY

Roberts is also using a process known as cryogenic electron microscopy — or cryo-EM for short — an advanced molecular imaging technique.

The 2017 Nobel Prize for Chemistry was awarded to three scientists for their work in developing cryo-EM, which, according to the citation, had “moved biochemistry into a new era”.

Although the technique was developed in the years between

the 1970s and 1990s, it’s only in more recent times that the technology has caught up, allowing the images to be transformed into sophisticated 3D models.

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The process involves snap-freezing the virus in a thin layer of ice about 1,000th of a human hair thick, essentially capturing the specimen in suspended animation.

Roberts produced this image, which shows a single virion suspended in that ice layer, which appears as the darker background area.

With cryo-EM imaging, it is also possible to combine thousands of these pictures together — all taken at slightly different angles — to form a three-dimensional image, or tomogram.

Tomography is the T in CT imaging, sometimes known as CAT scanning, which produces a cross-sectional image of anatomy and is a form of diagnostic imaging used by doctors.

The only difference is that EM uses electrons instead of x-rays.

Roberts says tomography using cryo-EM images is a recent technique and is the process his team is currently using to build their models.

“It will give you a very good idea of how the virus is laid out and what’s its generalised structure.”

This image (bottom of page 19) is an example of a tomogram taken by Roberts on April 17.

It shows virus particle cores,

coloured red, encased in their yellow-coloured viral envelopes.

The parts coloured green and purple are part of the host cell, which is from an animal kidney.

One of the virus particles can be seen exiting the cell in a process known as budding, which is a form of replication.

This is a short 3D-effect video of the tomogram which shows that same slice of infected cell as the image (top of page 19).

It was produced by stitching together 126 individual two-dimensional EM images taken at different angles.

The thousands of EM images collected become parts of a jigsaw puzzle, enabling scientists to piece together the virus’s atomic structure.

That will then help to explain its tactics — its strategy for entering and exiting the cell, how it reproduces and what its weaknesses are.

Researchers can then either use existing drugs or develop new drugs to try and disrupt those viral processes.

The key lies in those atoms, Roberts says.

“Once you understand the atomic structure of a virus, you have a pretty good idea of how to knock it over.”

CREDITS

[1] Reporting: Stephen Hutcheon[2] Design and production:

Alex Palmer[3] Images courtesy of Jason

Roberts / VIDRL – Doherty Institute (with technical assistance from Andrew Leis / Bio21 Institute), Sandy Crameri / CSIRO and NIAID-RML.

DEAR AUSTRALIAN MICROSCOPY COMMUNITY, I HOPE that you are all well, and that the challenges we are all facing in the current environment are only temporary.

In our current climate of focus on our global struggle against the SARS CoV 2 virus, I wish to reinforce our individual support for those carrying out essential research, and our organisational commitment to maintaining the highest level of services to our customer base as possible.

Our service teams remain active, and are dealing with issues as efficiently as possible.

Travel restrictions make this a difficult scenario in all cases, but we are able to make exceptions for critical circumstances, if and when they might occur.

Although we hear about

the critical work that is being undertaken on the most advanced instruments that are available, Krios and Glacios, it should also be noted that all EM can be used to provide information on the action of viruses and that valuable information can be collected by all EM instruments.

The following is a summary of the different methods, and the type of data which can be found, from existing installations, to

provide a ready reference to user communities within our universities and research institutes.

I understand that many people have probably seen the individual papers in the past, but a collection to show to potential users, asking what they can achieve in the context of COVID research seemed like a useful exercise, and one that will facilitate user uptake of the appropriate techniques as rapidly as possible.

Method 1 (Na Zhu et al, 2020, New England Journal of Medicine)

VIRUS ISOLATION � NEGATIVE STAINING � TEM IMAGING

Supernatant from human airway epithelial cell cultures that showed cytopathic effects was collected, inactivated with 2% paraformaldehyde for at least 2 hours, and ultracentrifuged to sediment virus particles. After that they are stained with phosphotungstic acid and transferred to the transmission electron microscope (Thermo Scientific Tecnai G2 Spirit) for the imaging and data collection.

Please feel free to reach out to myself, or anyone at ThermoFisher Scientific for any further discussion, support or any other issue, at ryan.shaw1@thermofisher.com

Dr Ryan ShawSales Account Manager – Analytical InstrumentsMaterials & Structural Analysis – Thermo Fisher Scientific5 Caribbean Way, Scoresby VIC 3173Ph: +61 (0)499 802 040

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Method 3 (Mah-Lee Ng, et al, 2004, Emerging Infectious Diseases) Method 5 (Clarissa Villinger et al, 2015, Viruses)

Method 4 (Vero-cells on connective tissue support, infected with cowpox virus)

VIRUS ISOLATION � NEGATIVE STAINING � TEM IMAGING CELL CULTURE � VIRUS INFECTION � EM SAMPLE PREPARATION � FIB/SEM TOMOGRAPHY � DATA PROCESSING

The infected cells with 100 mL of SARS-CoV for 1 h were cubated in 37°C incubator with 5% carbon dioxide. Then they were finished by traditional SEM Sample Preparation(including double xation, dehydration, critical point drying, coating). After that the cells were transferred to the scanning electron microscope(Thermo Scientic XL30 FESEM) for the imaging.

Human foreskin fibroblasts (HFFs) were seeded on carbon coated and glow discharged sapphire discs. After cell attachment for 24 h they were infected with TB40-BAC4 with a multiplicity of infection (MOI) of 1 and incubated for three or five days.EM sample preparation was conducted by high-pressure freezing, freeze substitution and Epon embedding as described earlier.FIB/SEM tomography was conducted with a Helios Nanolab 600 FIB/SEM.Slice and view was performed using the software module Auto Slice & View.G1 The open source software IMOD was used for automatic alignment of the images. Manual improvement of the alignment and segmentation was then conducted using Avizo 6.3

The epon sample contains Vero cell culture cells which were cultivated on top of a connective tissue support (collagen fibres with few collagen producing cells in it). The Vero cells were infected with cowpox virus and replicate efficiently within the host cell cytoplasm. Cells develop virus factories which are large protein aggregates surrounded by many immature and mature virions. The sample was chemically fixed by a combination of paraformaldehyde and glutaraldehyde. The postprocessing and embedding was done according to the Ellisman-protocol provided by FEI. As resin we have used epon hard mixture according to Horstmann & Denk.

Method 2 (Peng Zhou et al, 2020, Nature)

SAMPLE COLLECTION � VIRUS ISOLATION � VIRUS AMPLIFICATION � TEM SAMPLE PREP � TEM IMAGING

The samples from patient oral swabs/anal swabs/blood/BALF methods were centrifuged to get the supernatant, The Vero E6 and Huh7 cells were infected and incubated used for virus isolation,The virus is amplied in later passage cell, examined by qRT–PCR methods and are collected and nished by traditional TEM Sample Preparation(including double xation, dehydration, embedding, ultra-section, staining). After that the sections are transferred to the transmission electron microscope(Thermo Scientic TecnaiG2 20) for the imaging and data collection. Vero cells were grown to 70% con uency on sterile glass coverslips in 24-well tissue culture plates.

VOLUME SCOPE SEM

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Method 7 (Daniel Wrapp et al, 2020, Science)

Method 8 (Yun-Tao Liu et al, 2020, Cell Discovery)

PROTEIN EXPRESSION � PROTEIN PURIFICAITON � PLUNGE FREEZE � SPA CRYOEM � DATA PROCESSING

CELL CULTURE � VIRUS INFECTION � EM SAMPLE PREPARATION � FIB/SEM TOMOGRAPHY � DATA PROCESSING

According to the reported genome sequence of 2019-nCoV, Its modified plasmid are cloned into the mammalian expression vector p αH/pHCMV3 and expressed in the FreeStyle 293F cells (Thermo Fisher) using polyethylenimine. The recombinant protein was purified by StrepTactin resin (IBA) or Protein A resin (Pierce)and additional purification by size-exclusion chromatograph. The purified S protein was ash-frozen in liquid ethane cooled by LN with Vitrobot and imaged in a Titan Krios. After collecting and processing 3207 micrograph movies, the 3.5Å-resolution prefusion Structure of the 2019-nCoV S pretein with a single RBD in the up conformation.

The dissociated cells were plated over the grids at a density of 40,000 cells/ml in 35mm diameter Petri dish. At 6 DIV, neurons were infectedwith either PRV443 (PRV Becker EGFP-VP26) or PRV86(PRV Bartha EGFP-VP26) at 10 MOI. The grids were frozen by Vitrobot IV, inserted into the Cryo Light Microscope and CryoEM for correlative CryoLM/CryoET Imaging. CryoET data were collected using FEI Tecnai F20 TEM equipped with 4K × 4K 4-ports readout CCD camera. Three-dimensional reconstructions weremaneuvered with IMOD. And Segmentation and surface rendering of the tomogram was done by volume tracer and color zone in UCSF Chimera.

Method 6 (Cuang Liu et al, 2020, bioRxiv)

VIRUS ISOLATION � VIRUS AMP. � EM PLUNGE FREEZE � CRYOEM IMAGING � DATA PROCESSING

The virus particles are isolated in some culture cell using bronchoalveolar lavage uid (BALF) sample from the patient, then supernatant from later passage cell inactivated and utra-centrifuged to sediment virus particles. After that they are immediately frozen in the liquid ethane and transferred to the CryoEM (Thermo Scientific Glacios/ Titan Krios) for the imaging by a Falcon 3/Falcon 4 direct electron detector and data collection.

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MULTISCALE 3D IMAGING SOLUTIONS FOR LI-ION BATTERIES

INTRODUCTION

One of the biggest challenges to understand Li-ion battery technology is its intrinsic 3D multiscale nature. The battery needs to be characterized from cell level (mm length scale) to particle level (nm length scale) in three dimensions. In this application note we present a multiscale 3D imaging and analysis workflow that enables quantitative understanding of the structure-performance relationship in Li-ion batteries.

CHARACTERIZATION AT THE CELL LEVEL

Micro computed tomography (microCT)is the enabling technology that allows for imaging large volumes at the mm scale with µm resolution. Researchers and engineers are interested in using this technique to characterize batteries at the cell level. Figure 2 shows a 18650 battery cell imaged in 3D with the Thermo Scientific™ HeliScan microCT to better understand the cell structure

The increasing demand for electric vehicles and consumer electronics in recent years has caused Li-ion batteries to attract significant attention due to their high energy and power density compared to other commercial rechargeable battery technologies. In order to further advance Li-ion battery technology for better performance and to increase safety, a fundamental understanding from materials chemistry to battery structure is essential.

evolution during cycling. The quantitative analysis of the current collector position before and after cycling indicates the electrode volume expanding during cycling. These insights can be used to correlate with battery degradation phenomena.

CHARACTERIZATION AT THE ELECTRODE LEVEL

At the electrode level, the Thermo Scientific™ Helios PFIB DualBeam enables researchers and engineers to generate a representative volume (>100 µm field of view) with nm resolution with high-throughput materials milling.

The 3D volume can then be further used in microstructure

quantification for structure-performance analysis as well as a 3D template for modeling and

performance prediction. Figure 2 shows the cross-sectional image of a nickel manganese cobalt (NMC) cathode and a graphite anode at 100 µm and 120 µm horizontal field of view respectively.

Figure 3 shows a 3D reconstruction of a SiO/C anode by using low kV energy dispersive spectroscopy (EDS) mapping. The silicon particles in the anode can be seen to have a CMC coating, which is applied to make them more resistant to cycling degradation. This 3D

reconstruction was obtained using a Thermo Scientific Scios DualBeam.

ELEMENTAL ANALYSIS

One challenge of using EDS for elemental analysis is to detect

Figure 1. 3D imaging analysis on an 18650 Li-ion cell. Blue represents the fresh cell and yellow represents the cycled cell; (a) 3D reconstruction of the Cu current collector in the 18650 cell (fresh state); (b) Overlay of the Cu current collector of fresh and cycled battery in 3D; (c) 2D image (XY plane) of the fresh and cycled cell with Cu current collector extracted and overlay at center area; (d) Quantitative comparison of the Cu current collector position between fresh and cycled cell

Figure 2. 3D imaging of Li-ion battery electrode with Helios G4 PFIB DualBeam. (a) Cross-sectional view of the NMC cathode; the field of view is 100 µm; (b) 3D rendering of the graphite anode, 125 µm field of view.

Figure 3. 3D EDS mapping of SiO/C anode.

Figure 4. (a) 3D lithium (7Li isotope) distribution within the NMC cathode; 3D reconstruction volume of 40 um x 26 um x 1.25 um (x*y*z). (b) Enlarged EDS map of a 2D slice showing Li distribution within NMC particles.

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Find out more at thermofisher.com/EM-Sales

lithium within the electrode/particle, which is critical for electrode materials property analysis. With a time-of-flight secondary ion mass spectrometer (TOF-SIMS) in the DualBeam system, direct imaging of lithium in the cathode structure can be achieved. Figure 4 shows the 3D reconstruction of the lithium distribution within an NMC cathode.

DualBeam systems also provide detailed chemical and elemental analysis at the electrode particle level. Figure 5 shows the analysis of a single NMC particle in 3D. Images at different depth are taken in the Scios DualBeam. By milling the particle and taking images

at different depth of the particle, high resolution SEM images of the microstructure within the particle are collected. The different contrast present in the primary particles may indicate chemical composition or crystallographic differences and can be further analyzed by EDS, secondary ion mass spectrometry and electron backscatter diffraction techniques available in the DualBeam.

CONCLUSION

Understanding the structure-performance relationship in Li-ion batteries at different stages in the lifecycle requires imaging and

analysis at multiple length scales and in 3D. Geometric parameters such as volume fraction, surface area, particle size distribution, and tortuosity are typically assessed using a combination of microCT and focused ion beam scanning electron microscope techniques. Thermo Fisher Scientific provides a complete workflow of microCT and DualBeam systems with advanced accessories and software suites that provide a complete understanding of the battery at multiple scales and in 3D, ultimately enabling researchers and engineers to develop better performing, safer batteries with longer lifetimes.

Figure 5. Imaging analysis of Li-ion battery electrode particle via DualBeam. Cross-sectional view of the individual NMC particle at different depth.

RELATED PRODUCTS

HeliScan microCT Scios 2 DualBeam Helios G4 PFIB DualBeam

Helios Hydra DualBeam

TRANSMISSION ELECTRON MICROSCOPY IN THE PHYSICAL SCIENCESWorkshop organisation and text by Magnus Garbrecht

ONLY A FEW WEEKS BEFORE COVID19 social restrictions hit Australian universities and moved all teaching and seminars to remote and online, we were lucky to host another two days of lectures and labs about “Transmission Electron Microscopy (TEM) in the Physical Sciences” with Nestor Zaluzec (Argonne National Lab, United States) and Matthew Weyland (Monash

University, Australia), following last year’s great success. A parallel workshop focussing on biomedical- and cryo-applications was held at Sydney Microscopy & Microanalysis (SMM) by cryo TEM expert William Close.

The workshop was booked out in no time after it was announced, and hosted participants from electron microscopy facilities at institutions across the

entire greater Sydney area (The University of Sydney, The University of New South Wales, The University of Technology Sydney, Macquarie University, Western Sydney University, The University of Wollongong).

The workshop started off with a full day of lectures delivered by Nestor and Matthew taking turns on both beginners’ and advanced topics such as TEM

Two day lecture/lab series by Nestor Zaluzec and Matthew WeylandHeld 24–25 February 2020Sydney Microscopy & Microanalysis, The University of Sydney

Nestor Zaluzec, ANL, USA.

Figure 1: PACBED pattern of SrTiO3 (a) and image of the segmented DF4 detector (b) as used for e.g. iDPC imaging mode in STEM, recorded by Matthew Weyland. Illustration of the change in Signal/Background in EELS (c) and EDS (d) as a function of thickness in a NiO specimen as demonstrated by Nestor Zaluzec during the workshop. All data recorded at the FEI Themis-Z 60-300.Matthew Weyland, Monash, AUS.

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instrumentation and its underlying physics. This included the imaging process and relevant contrast mechanisms, TEM imaging and diffraction modes and a large variety of scanning TEM techniques. Spectroscopies were also discussed, such as electron energy-loss spectroscopy (EELS) and energy dispersive x-ray spectroscopy (EDS) in addition to concepts and details about hardware and accessories, such as detectors, cameras, and spectrometers, as well as data analysis methods.

The second day was split into parallel sessions in smaller groups. For early stage TEM users Nestor and Mathew demonstrated basics

imaging and spectroscopies at the JEOL 2100 and JEOL 2200 microscopes, with technical support by SMM’s Hongwei Liu. Advanced users had signed up for the sophisticated sessions held at SMM’s FEI Themis-Z 60-300, with the topics of “Practical STEM imaging.

Setting up STEM detectors and camera lengths for different imaging conditions, setting amplifiers to ensure no lost information, calibrating detectors directly, tilting crystals efficiently and measuring local crystal thickness via position averaged convergent beam electron diffraction (PACBED).” – Matthew, and “Practical Hyperspectral

Imaging and Analysis using XEDS & EELS: Setting up conditions for optimized XEDS and EELS, recognising and avoiding artifacts. Practical tips on data collection and measuring parameters for quantification. Understanding the limits of XEDS vs EELS, and when to choose one vs the other.” – Nestor, with instrument support by Magnus Garbrecht. Examples of data collected during the workshop are shown in figure 1.

With the overwhelming attendance outcome and feedback from workshop participants, we are looking forward to hosting another edition in 2021 when hopefully Covid19 restrictions are a thing of the past.

MULTI-LINE LASERS SIMPLIFY BIOMEDICAL IMAGING INSTRUMENTATION

INTRODUCTION

Over the last decade, the fluorescence-based life science industry has already been transitioning from bulky gas-laser sources into solid-state lasers with a smaller footprint, longer lifetime, and lower maintenance requirements. The development of compact, reliable solid-state lasers was an initial enabling technology for commercialization and expansion to new markets and applications. While some applications are able to utilize the advancements in light emitting diodes (LED) and super-continuum white-light sources; the high-resolution, high-speed techniques still rely on the high-brightness and wavelength precision of lasers.

Currently, many researchers and manufacturers align and integrate individual laser sources for each

desired wavelength on the optical bench or in the instrument. These assemblies require additional optics for each laser line, all of which need to be aligned with high precision and typically into a fiber delivery system. This design often requires the time and cost of installation and service by a technician from the instrument manufacturer or, the time of a researcher spent aligning optics instead of collecting new data. Laser combiners and laser light engines have simplified some of these assemblies substantially. However, they do not eliminate the need for alignment (and re-alignment) over time. Laser combiners can also contribute to the bulkiness of a manufactured solution and can be sensitive to thermomechanical stress causing misalignment.

As new techniques are

developed for clinical applications, ease-of-use and the ability to commercialize the instrumentation become increasingly important. While maintaining the highest quality and performance, laser manufacturers must deliver reliable, simple, and cost-effective solutions for both commercial systems and laboratory instrumentation for fundamental research.

The use of multi-line lasers as an alternative to conventional laser combiners or laser engines solves many of these common pain-points in fluorescence microscopy applications. A “multi-line laser” is several individual laser wavelengths built into one laser platform, with permanent and stable fixation of all beam alignment optics included the same package. The Cobolt Skyra™ is a totally customizable, permanently aligned multi-line

The introduction of multi-line lasers to fluorescence instrumentation provides a compact, easy-to-use, and service-free solution for integrating up to four laser wavelengths with reliable, stable performance. In this white paper we explore the advantages of a permanently aligned multi-line laser with fully integrated electronics.

Figure 1a: Left – typical power stability out of the SM/PM fiber at each wavelength across the temperature range 20-50oC.Figure 1b: Right – Cobolt Skyra™ multi-line laser with integrated electronics (Dimensions: 70 x 144 x 38mm).

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laser solution offering up to four individual wavelengths, ranging from 405nm to 660nm, in a single laser output.

The availability of a compact, easy-to-use, and reliable high-performance multi-line laser will assist with the commercialization of new fluorescence-based-instrumentation and further expand existing technologies into laboratories with a lower barrier

of entry, for both the instrument manufacturer and end-user.

MULTI-LINE LASER TECHNOLOGY

The Cobolt Skyra™ multi-line laser is unique in its’ design and manufacturing. It is built using patent-pending alignment techniques and utilizing Cobolt’s proprietary HTCure™ technology. The lasers are built on a single,

temperature-controlled platform for stable operation and protection from thermomechanical misalignment. All the optical elements, including components for beam combining, beam-shaping and alignment, are precision-mounted and the entire package is hermetically sealed. The temperature-stabilized and compact package (meaning short beam paths) provide stable

Figure 2a (left): Vertical and horizontal beam pointing (urad) over 18 hours continuous laser operation and temperature cycling between 20oC to 50oC.Figure 2b (right): Beam profile of typical 4-line Cobolt Skyra™ demonstrating Gaussian beam overlap.

Figure 3a (left and centre): An example of an images taken in a single-molecule localization microscopy (SMLM) setup from Department of Biotechnology & Biophysics at Julius-Maximilian-University of Würzburg.The 3-color image shows african green monkey kidney cell (COS7) with nucleus (blue), microtubules (red/magenta) and the actin sceleton (green/cyan) staining. Recording time 4s per channel at 2048x2048px field of view.Figure 3b (right): Cobolt Skyra™ laser is shown in use in the Department of Biotechnology & Biophysics at Julius-Maximilian-University of Würzburg.1

beam-pointing and robustness in varying environmental conditions (Figure 1a). The Cobolt Skyra™ can be coupled with single-mode, polarization-maintaining fiber coupling directly on the laser head. The output power stability in figure 1a below is measured through the SM/PM fiber, from 20 to 500C.

Cobolt’s HTCure™ technology was an integral part of developing a compact and reliable multi-line laser source for fluorescence microscopy techniques. It eliminated the need to align lasers in the field, by maintaining alignment under various ambient operating conditions, and keeping the laser lines focused into a fiber delivery system. In addition, the control electronics of the multi-line laser are integrated directly into the laser head, for a simple, clean, and easily integrated solution (Figure 1b).

Different fluorescence microscopy techniques, applications, and day-to-day experiments can utilize multiple combinations of performance and capabilities within the experimental design. Most of these demands can now be met simultaneously with one standard or customized variation of a multi-line laser source. As standard on Cobolt Skyra™, the modulation and control of each wavelength is independent from the others. The controls are compatible with digital and/or analog inputs, as well as software commands via USB. Fast and deep digital modulation up to 5 MHz modulation frequency is possible and 500 kHz in analog modulation.

In addition to the inherent flexibility of multi-line lasers in the laboratory or commercial instrument, custom wavelength combinations are also available, with or without direct fiber coupling. By including both direct-diode and diode pumped solid state laser technology on the multi-line laser platform, a wide range of wavelengths are available. The Cobolt Skyra™ can include up to four wavelengths, within the range of 405nm to 660nm with

beam position overlap <50 um at the exit and pointing stability <10 urad/oC over a temperature range of 20 oC to 50 oC (Figure 2). The output beams of the Cobolt Skyra™ can be collinear and coupled into single mode fibers for convenient launching into microscope set-ups or tailored to form stacked light sheets at a precisely defined location in front of the laser for direct alignment to an external target., (For example, a flow cell in a cytometer.)

IN THE LAB

Some of the earliest users of the Cobolt Skyra™ in academia have utilized the technology to equip laboratories with a powerful tool for multiple types of microscopy techniques. One such laboratory is that of Prof. Dr. Markus Sauer at the Department of Biotechnology and Biophysics at Julius-Maximilian-University of Würzburg. Researchers in Prof. Dr. Markus Sauer’s lab are focusing on single molecule sensitive fluorescence spectroscopy and imaging techniques, including super-resolution microscopy and its applications in biomedical sciences. The Cobolt Skyra™ laser has, for example, been used in a single-molecule localization microscopy (SMLM) setup to gain new insights into the organization of proteins within a cell. The set-up in their laboratory system provides images with spatial resolution nearing the molecular level, from which quantitative biological data can be extracted (Figure 3a, 3b). The Cobolt Skyra™ was an economical, high-performing, and easy-to-use solution in their instrumentation, helping to move research along at a faster pace with consistent and reliable results1.

Another interesting technique for use of multi-line lasers is in the field of cancer diagnostics and the progression towards increasing fluorescence instrumentation in clinical settings. The barrier of developing suitable instrumentation and achieving

clinical certification is high, but a critical step is creating advanced instrumentation that can be commercialized and accessible. The first challenge is to develop the technique, but it is often followed by a second challenge of making that new technology dependable and user-friendly.

A team from Dr. Jonathan Liu’s laboratory at the University of Washington has recently developed a cutting-edge open-top light-sheet microscope for fast, non-destructive, slide-free, 3D pathology2. The technique rapidly images 3D biological samples, without slicing the tissue-sample as in traditional pathology techniques. A unique application for this technology is applied to prostate needle-core biopsies and cancer diagnosis. Furthermore, Dr. Liu and his team have continued to drive their technology towards commercialization3. The use of an easy to control, compact, and permanently aligned multi-line laser assisted in the simplifying of the optical assembly in their innovative instrument design3.

OUTLOOK

Fluorescence imaging is a key technique in both biomedical research and clinical diagnosis. Fluorescence microscopes for high-resolution and high-throughput multi-fluorophore imaging typically rely on the use of several individual laser sources at different wavelengths, within the same instrument. Traditionally these lasers have been coupled into the microscopes through laser combiners, which add bulk, cost, and alignment complexity.

Multi-line laser solutions are an attractive alternative to laser combiners to simplify fluorescence imaging instrumentation and furthermore aid in the process of commercialization for new, cutting-edge imaging systems for clinical use. Multi-line lasers enable smaller and more cost-efficient instruments which are much easier to manufacture and maintain.

AMMS JUNE 2020AMMS JUNE 202034 35

This development supports the strive for bringing more advanced laser-based instrumentation into research and clinical settings for improved medical diagnostics and further development of new analytical techniques.

REFERENCES

[1] M. Sauer and M. Heilemann, Chem. Rev., 117, 7478−7509 (2017); doi:10.1021/acs.chemrev.6b00667.

[2] Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).

[3] Glaser, A., Reder, N., Liu, J., Buckley, S., True, L. https://lightspeedmicro.com/ January 2019.

About the Author

Melissa Haahr – Melissa is the product manager for Cobolt’s lasers for Life Sciences. She joined the company in 2014 with knowledge in analytical instrumentation and analysis techniques, as well as technical sales experience. Melissa holds a Master’s degree in physical chemistry from University of Oregon and Bachelor’s degrees in Chemistry and Environmental Science from Alfred University.

About the company

Cobolt AB is now a part of HÜBNER Photonics. At HÜBNER Photonics, we not only make gamechanging lasers, but also rethink all kinds of other technologies including terahertz imaging and high-frequency radar. Since 2015, our portfolio has been further reinforced by the acquisition of Cobolt AB, a world leading manufacturer of high performance lasers for analytical instrumentation. Together, we unite proven corporate values with innovative ideas and top-notch technologies for the whole electromagnetic spectrum.

NEXT GENERATION PHENOM XL G2 DESKTOP SEM BRINGS ADVANCED AUTOMATION TO THE FOREFRONTWHILE TRADITIONAL SEMS REMAIN RELATIVELY LARGE and difficult to operate, Thermo Scientific Phenom desktop SEMs provide an alternative being smaller, faster and now, even easier to use.

Building on the performance of its predecessor, the next-generation Phenom XL G2 Desktop Scanning Electron Microscope (SEM) delivers new automation solutions for quality control (QC), enabling more detailed failure analyses than is possible using optical microscopes. Manual, repetitive tasks can be automated to allow a high volume of samples to be quickly processed and to discover potential failures early to guide rapid adjustment to production processes. Particles, pores, fibres or large SEM images

can be automatically characterised and foreign contaminants automatically identified and evaluated for chemical composition.

Users can obtain high-quality images in just 40 seconds—three times faster than other desktop SEM systems on the market. Offering an improved resolution of 10 nanometres, the system enables more resolving power and the ability to explore large samples of up to 100 by 100 millimetres.

A key feature of the Phenom XL G2 is its easy-to-use, intuitive user interface (UI) combining two screens into one for viewing images and performing analysis. With one simple click on the impurity, users can see what elements are present using the live energy-dispersive X-ray (EDS)

analysis. The optical navigation camera makes it possible to view the entire sample while the user is in SEM mode.

Requiring little lab space, the Phenom XL G2’s compact size allows users to place the microscope exactly where it’s needed—whether that’s in the lab or on the production line for real-time analyses. The latest Phenom ParticleX series is designed to provide technical cleanliness and additive manufacturing companies faster QC analyses of materials. Consisting of a high-performance Phenom XL Desktop SEM with automation software packages, the Phenom ParticleX provides in-house analysis and validation of produced goods against industry-approved standards, up to ten times faster than outsourcing.

PRESS RELEASE

If you would like further information or a free quote, please contact:

ATA Scientific Pty Ltd

+61 2 9541 3500

enquiries@atascientific.com.au

www.atascientific.com.au

https://www.atascientific.com.au/products/phenom-xl-desktop-scanning-electron-microscope/

AMMS JUNE 2020AMMS JUNE 202036 37

SELF-ALIGNING MICROSCOPE SMASHES LIMITS OF SUPER-RESOLUTION MICROSCOPY

UNIVERSITY OF NEW SOUTH WALES (UNSW) medical researchers have achieved unprecedented resolution capabilities in single-molecule microscopy to detect interactions between individual molecules within intact cells.

The 2014 Nobel Prize in Chemistry was awarded for the development of super-resolution fluorescence microscopy technology that afforded microscopists the first molecular view inside cells, a capability that has provided new molecular perspectives on complex biological systems and processes.

Now the limit of detection of single-molecule microscopes has been smashed again, and the details are published in the current issue of Science Advances.

While individual molecules could be observed and tracked with super-resolution microscopy already, interactions between these molecules occur at a scale at least four times smaller than that resolved by existing single-molecule microscopes.

“The reason why the localisation precision of single-molecule microscopes is around 20-30 nanometre normally is because the microscope actually moves while we’re detecting that signal. This leads to an uncertainty. With the existing super-resolution

instruments, we can’t tell whether or not one protein is bound to another protein because the distance between them is shorter than the uncertainty of their positions,” says Scientia

Professor Katharina Gaus, research team leader and Head of UNSW Medicine’s European Molecular Biology Laboratory (EMBL) Australia Node in Single Molecule Science.

PRESS RELEASE

An ultra-precise microscope that surpasses the limitations of Nobel Prize-winning super-resolution microscopy will let scientists directly measure distances between individual molecules.

To circumvent this problem, the team built autonomous feedback loops inside a single-molecule microscope that detects and re-aligns the optical path and stage.

“It doesn’t matter what you do to this microscope, it basically finds its way back with precision under a nanometre. 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,” says Professor Gaus.

With the design and methods outlined in the paper, the feedback system designed by the UNSW team is compatible with existing microscopes and affords maximum flexibility for sample preparation.

“It’s a really simple and elegant solution to a major imaging problem. 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.”

To demonstrate the utility of their ultra-precise feedback single-molecule microscope, the researchers used it to perform direct distance measurements between signalling proteins in T cells.

A popular hypothesis in cellular immunology is that these immune cells remain in a resting state when the T cell receptor is next to another molecule that acts as a brake.

Their high precision microscope was able to show that these two signalling molecules are in fact further separated from each other in activated T cells, releasing the brake and switching on T cell receptor signalling.

“Conventional microscopy techniques would not be able to

accurately measure such a small change as the distance between these signalling molecules in resting T cells and in activated T cells only differed by 4–7 nanometres,” says Professor Gaus.

“This also shows how sensitive these signalling machineries are to spatial segregation. In order to identify regulatory processes like these, we need to perform precise distance measurements, and that is what this microscope enables. These results illustrate the potential of this technology for discoveries that could not be made by any other means.”

Postdoctoral researcher, Dr Simao Pereira Coelho, together with PhD student Jongho Baek – who has since been awarded his PhD degree – led the design, development, and building of this system. Dr Baek also received the Dean’s Award for Outstanding PhD Thesis for this work.

A T cell showing precise localisation of T cell receptors (pink) and CD45 (green).

“It doesn’t matter what you do to this microscope, it basically finds its way back with precision under a nanometre. 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.”

AMMS JUNE 2020AMMS JUNE 202038 39

TESCAN HAS INSTALLED THEIR 3000TH MICROSCOPE TO HELP SCIENTISTS DEVELOP FUEL CELLS AND SPECIAL PROSTHESES

THE OFFICIAL OPENING OF TESCAN AMBER X was marked with a presentation that took place in the Department of Microsystems Engineering (IMTEK) where the microscope is housed. The first two research projects that will utilise the capabilities of the microscope were also presented.

The first project lead by Prof. Stieglitz aims to develop advanced types of neuroprosthetics. These devices should soon replace the missing functions of the neural system in the human body with the use of sensory implants and prostheses.

Thanks to them the patient should receive information on hardness and surface of an object when touching it with a special prosthesis.

The other research project where the electron microscope will initially be used is searching for new technologies for fuel cells with the goal being to develop innovative methods of electric energy production. This work will be performed by Dr. Vierrath and his team.

“These two applications show how universal TESCAN AMBER X is for scientific research. I am very pleased that our microscope will assist with such ambitious and noble projects,” said Sven Gosda,

General Manager of TESCAN GmbH.

Last year TESCAN produced nearly 300 electron microscopes in Brno with the overwhelming majority of them destined for international clients.

“We are clearly an export company, supplying microscopes worldwide. We are happy to have installed our jubilee microscope in Germany, a market where we are planning to grow in the long-term,” claimed Maroš Karabinoš, TESCAN’s Global Marketing

Director in the official presentation. TESCAN has been enjoying consistent growth in Germany and has demonstrated its commitment to the local market by acquiring its existing distributor in 2018 and establishing a local subsidiary, TESCAN GmbH.

While this is the University of Freiburg’s first plasma focused ion beam-scanning electron microscope (FIB-SEM), they already have a TESCAN VEGA3 SEM. This has been in operation since 2016.

PRESS RELEASE

The jubilee electron microscope with production No. 3000 was installed at the University of Freiburg, Germany. The system is a TESCAN AMBER X, which is amongst the latest models from the Brno-based manufacturer. It combines ultra high-resolution imaging with a plasma ion beam for high-speed sample surface milling.

TESCAN AMBER X Xe plasma FIB-SEM installed at the University of Freiburg.

For more details about the TESCAN AMBER X and other microscopy solutions, please visit www.axt.com.au

AMMS JUNE 2020AMMS JUNE 202040 41

HIROX RELEASE NEW DIGITAL MICROSCOPE WITH INCREASED RESOLUTION

FIB STUB 3.0 FROM DENSSOLUTIONS SIMPLIFIES TEM LAMELLA ON MEMS NANO-CHIPS

THE HRX-01 INCLUDES A LIST OF new features that will help you perform more details inspections. With a higher resolution CMOS camera, you will be able to capture higher resolution images as well as impressive full HDR (4K) video. Also available for the HRX-01 is a newly designed motorised stand/stage with 80mm Z-travel and tilt angle sensor for even greater versatility.

Operation of the instrument has been streamlined with a new user interface. This also enables changes of magnification via the software and motorised lenses. Some lenses also allow users to change lighting to suit your specific application.

As with its predecessors, the Hirox HRX-01 offers the widest range of lenses and accessories allowing you to tailor the system to your requirements.

Combined with a powerful software package, 3-dimensional imaging and measurements are all easily achieved. The HRX-01 will soon be bolstered with the availability of the nano point scanner (NPS) non-contact profilometer option that gives it true metrology capabilities. This

option was first introduced on the HRX-01’s predecessor, the RH-2000 and incorporates a

white light confocal profilometer that offers ultrafast profiling with submicron Z precision.

TEM MICROSCOPISTS KNOW THAT SAMPLE PREPARATION is key to good imaging and analysis. FIB lamellae preparation is a common and complicated method, especially when preparing lamella onto micro electrical mechanical systems (MEMS)-based Nano-chips. The latest version solves many limitations from previous versions, improving reliability and allowing you to produce TEM lamellae more successfully.

FIB Stub 3.0 features an additional flat side for placing the samples that ensures a conventional geometry and the very same and the well-known process used by any FIB operator when making and lifting out the lamella. The new geometry improves imaging quality, even during low kV milling and polishing stages. Additionally, more effective grounding pads reduce charging effects.

A new dedicated pocket and a smart clamping mechanism drastically simplifies and speeds up the Nano-Chip loading and unloading, increasing its user-friendliness. The new design reduces the risk of breaking the membrane when handling the Nano-Chips. In addition, the need to use sticky tapes to fix or to ground the Nano-Chip is eliminated, making the process much cleaner.

Safety is also significantly increased as FIB Stub 3.0 brings the position of the sample and the Nano-Chip to a similar eucentric height. This minimises the possibility of crashing into the pole piece, Gas Injection System or the manipulator during the operation.

FIB Stub 3.0 is compatible with many dual beam FIB-SEMs and will benefit those working with double tilt Heating and/or Biasing Nano-Chips. Furthermore, it is a

beneficial addition for researchers planning to work with in situ heating samples or electronic devices like non-volatile memory based on resistive switching or phase change materials, solid state batteries, solar cells, etc.

PRESS RELEASE PRESS RELEASE

Hirox, the pioneers of digital microscopy continue to set the pace with the release of the HRX-01. Their latest microscope has increased levels of automation and resolution and is applicable across a wide range of industries from materials science, through life science and forensics, to failure analysis, QC and non-destructive testing. Common to other Hirox digital microscopes, it continues to push the limits of optical microscopy and closes the gap on electron microscopy.

DENSsolutions introduces the 3rd generation of the FIB stub which enables researchers to prepare a lamella and place it directly on the Nano-Chip, all inside the FIB. The new FIB Stub 3.0 incorporates many improvements that make FIB sample preparation easier, safer and quicker, while eliminating the need to break the vacuum of your FIB chamber.

The Hirox HRX-01 is now available for sale through AXT and joins their vast portfolio of microscopy products. For more details,

please visit www.axt.com.au

The new system was developed in conjunction with collaborators from EMAT (University of Antwerp) and AEM (University of Darmstadt) and customers from Germany,

UK, Singapore, Spain, Sweden, etc. FIB Stub 3.0 is now available for sale in Australia and NZ from AXT. For more information please contact info@axt.com.au

AMMS JUNE 2020AMMS JUNE 202042 43

NEW POSSIBILITIES IN LAB-BASED TIME-RESOLVED MICRO-CT IMAGING – WEBINAR REPORT

PREPARING FOR THE HYDROGEN ECONOMY

THE POTENTIAL FOR THIS TECHNOLOGY WAS echoed by the huge interest in the recent webinar hosted by AXT and TESCAN XRE in April. The webinar was presented by Frederick Coppens from TESCAN XRE, formed following TESCAN’s acquisition of if Belgium’s XRE NV, which in turn was spun out of Ghent University in Belgium.

For anyone who is not familiar with micro-computed tomography (microCT) as an imaging technique, Frederick explains the basis of how the technique works. He then contrasts it to dynamic CT, including the added ability to observe processes as they happen. While more conventional micro-CT systems can perform time-lapse studies, they can often miss dynamic or rapid processes.

In contrast, a truly dynamic system with high temporal resolution can scan continuously ensuring the most important events are never missed.

The ability to perform time-resolved studies relies on a seamless integration between hardware components (X-ray optics), system architecture and software.

One of their systems for instance features a unique gantry-type

architecture that is very conducive to in situ experiments keeping the sample stationary and enabling the easy incorporation of test rigs into the chamber.

This must all be mated to software that handles data acquisition, visualisation, reconstruction and analysis.

Frederick demonstrated the

applicability of dynamic CT using a number of different examples and systems including:• Metal foam compression• Additive manufacturing• Drainage in sandstone• Germinating seeds• Baking• Bone fracture• Pharmaceuticals.

WHEN STEELS ARE EXPOSED TO HYDROGEN, they can become brittle, leading to catastrophic failures. In normal conditions this is not too much of an issue as hydrogen levels in the atmosphere are quite low.

However, as we move towards a more hydrogen-fuelled future, with pipelines and tanks needed to transport and store hydrogen fuel, this problem becomes far more important. Indeed, it has been considered as one of the major obstacles for the realisation of the hydrogen economy. Dr Yi-Sheng Chen and Prof. Julie Cairney from the University of Sydney (USyd), working in partnership with CITIC Metals, set out to understand hydrogen embrittlement at the atomic level.

Hydrogen embrittlement involves complicated interactions of hydrogen with defects in the metal at many length scales. However,

understanding these interactions is limited by the fact that it is experimentally challenging to measure the precise location of hydrogen atoms at the atomic scale: hydrogen being the smallest and lightest of the elements.

Although macroscale techniques can identify hydrogen retention in metals, they do not allow the measurement of the relative contributions of different types of microstructural features.

Dr Chen used the state-of-the-art cryogenic atom probe in the Microscopy Australia facility at USyd to directly observe hydrogen (in the form of deuterium) at specific microstructural features in steels.

This technique was first prototyped by Prof. Roger Wepf of the University of Queensland, then developed and implemented in partnership with instrument developer Microscopy Solutions in

USyd. This development allowed the team to find that hydrogen accumulates at dislocations and at the grain boundaries as the prelude of the hydrogen-induced degradation. These observations provide the critical information to validate models of how hydrogen causes embrittlement.

In addition to these observations, Dr Chen also found the first direct evidence that clusters of niobium carbide within the steel crystals trap hydrogen in such a way that it cannot readily move to the dislocations and grain boundaries and cause embrittlement. These carbides could be the key to the informed design of embrittlement-resistant steels.

REFERENCE

Yi-Sheng Chen et al., Science, 10 Jan 2020 Vol. 367, Issue 6474, pp. 171-175

Micro-CT with its ability to view non-destructively inside a component in 3-dimensions is quite possibly the most exciting imaging technique available right now, however it comes with its own limitations and challenges. The recent development of time-resolved or 4D-CT has overcome some of these limitations in a lab-based system bridging the gap with synchrotrons. This technology has wide applicability across a wide variety of fields including materials, life and earth sciences.

If you missed this webinar, you can view the recording at https://www.axt.com.au/time-resolved-3d-imaging-in-the-lab-webinar/

Article link: https://science.sciencemag.org/content/367/6474/171

2-D sliced image of 3-D cryo-APT data. Hydrogen atoms (white) are found to accumulate at microstructural defects of steels that is visualised by the colour-graded carbon concentrations associated with the defects.

AMMS JUNE 2020AMMS JUNE 202044 45

INTRODUCTION

Sample vitrification is a critical step in achieving high-resolution imaging and reconstruction for all protein samples. Membrane proteins require special considerations for solubilization and purification to preserves their functional state.

A range of approaches has been applied to enable structure determination for G protein-coupled receptors (GPCRs).

The most common to date has been solubilization in detergents; this application note is specifically related to cryo-EM grid preparation for GPCR complexes solubilized in detergent [1, 2].

Optimized sample preparation conditions, which ensure sample and transmission electron microscopy (TEM) grid homogeneity combined with high particle density, allow for efficient imaging by conventional defocus TEM data collection and subsequent single particle image analysis (SPA).

THE PROTEIN SAMPLE SHOULD BE OPTIMIZED BEFORE MOVING TO CRYO-EM

As with all structure determination projects, the

sample for imaging should be optimized for purity, homogeneity, stoichiometry, stability and concentration, with the quality of

protein assessed by size exclusion chromatography (SEC), Western blot, Coomassie stain and negative stain single particle analysis prior to moving to cryo-EM [3].

An example of a GPCR complex suitable for imaging is illustrated in Figure 2. This step has proven to be most critical in achieving the best possible resolution from a single particle cryo-EM experiment.• Assuming it is possible to

achieve a stable complex as determined by a monodispersed SEC elution profile, in combination with SDS-PAGE analysis (both by Western blotting with appropriate antibodies as well as Coomassie Blue staining), the next step would be to prepare a negatively stained specimen.

• To standardize preparations, assume that 1 AU280 nm = 1 mg/mL (as measured by a Thermo Scientific NanoDrop Spectrophotometer) then dilute the sample in the buffer that the GPCR is stable in (without any detergent) to a concentration of 0.1-0.2 mg/

mL immediately before grid preparation.

• For GPCRs we have found that the stain that yields the best results is uranyl formate.

• For protein preparations confirmed to contain intact and homogeneous complexes by image analysis of the negatively stained specimen, the next step is vitrification. We found best results come from GPCR preparations where more than 60% of particles represent complexes of interest.

VITRIFICATION OF DETERGENT-SOLUBILIZED GPCRS

List of prerequisites and things to check• Thermo Scientific L120C,

Glacios or Krios Cryo-TEM• Thermo Scientific Vitrobot Mk

IV System• Plasma discharger (Quorum

GloCube Plus https://www. quorumtech.com/products/glow-discharge-for-tem-and- surface-modification, or Ion Bombardier https://www.shinkuu. co.jp/plasma-treatment-device/pib-10/)

• 200 or 300 mesh Quantifoil R1.2/1.3 Cu grids or 300 mesh Ultrafoil R1.2/1.3 Au mesh

by Matthew Belousoff, Patrick M. SextonMonash Institute of Pharmaceutical Sciences, Monash University

GRID PREPARATION FOR DETERGENT- SOLUBILIZED GPCR SAMPLES

Figure 2. Assessing protein quality and suitability for grid preparation. a) Coomassie stain of two SDS-PAGE gels, one after FLAG affinity chromatography (left) and one after SEC (right). By Coomassie staining, only the components of the GPCR complex are visible. b) Typical SEC chromatogram during the purification of a GPCR; the center of the peak is collected and pooled and subjected to a freeze thaw cycle to ensure complex stability c) SEC chromatogram after a freeze thaw cycle, showing a monodispersed, Gaussian distributed elution profile. d) A representative micrograph from a Thermo Scientific Talos L120C TEM. The specimen is negatively stained with uranyl formate at a concentration of 0.08 mg/mL (assuming 1 AU280=1 mg/mL). e) 2D class averages from the same negative stain images. In this case 65% of selected particles were clearly GPCR complexes.

Figure 1. Example of a high-resolution structure of a GPCR determined by single particle cryo-EM on a Thermo Scientific Krios Cryo-TEM. Left: Density map colored by local resolution of a GPCR. Right: FSC curve of the final 3D reconstruction showing a nominal resolution of 2.2 Å (0.143 FSC, gold standard).

AMMS JUNE 2020AMMS JUNE 202046 47

» Grids are prewashed with acetone, usually a day before vitrification

• Dilution buffer (same used for SEC)

• GPCR sample at concentration greater than 3 mg/mL

Plasma discharge conditionsWe have noted that the

conditions used to plasma discharge the TEM grids prior to sample application and vitrification have a profound effect on the resulting quality of the specimen embedding in vitreous ice. In general, longer plasma discharge times and optimized plasma current have yielded the best results.• For the GloCube Plus

» 15-20 mA plasma current with the polarity making the air positively charged

» Discharge time: 60-90 seconds

• For the Ion Bombardier » 9 mA plasma current with

the polarity making the air positively charged

» Discharge time: 90 seconds

Sparse screening of sample concentration and blotting conditions

It is necessary to screen for the optimal blotting conditions that yields the thinnest possible ice with good particle distribution in the grid holes.

This is achieved by a sparse screening of both sample concentration and blot time or sample concentration and blot force.

We have found that grids that have highly dense, monodispersed particles give the best results as shown in Figure 3b.• It should be noted that the

buffer has lower surface tension due to the presence of detergent, requiring much higher than “usual” blotting forces on the Vitrobot System.

• To save sample and reduce the time needed to screen grids, generally only blot

time or blot force (along with sample concentration) should be varied in any individual screening session.

• An example of a sparse screening strategy can be seen in Table 1.

• Sample is “flash” thawed at 30°C, swiftly mixed by pipetting, and immediately placed on ice until application to a TEM grid.

• Vitrobot System with fresh, dry filter papers is pre-cooled to 4°C, with the humidity set to 100% (during sample application, humidifier should be disabled avoiding wetting of sample or filter paper).

• If dilutions need to be made, they are prepared with matched buffer that was used for the final purification step (usually SEC buffer). Typically, only one or two dilutions are made (i.e. 7 mg/mL and 3 mg/mL).

• 3 µL of sample is applied to the grid.

Screening for optimal vitrification conditions

Once a series of grids has been vitrified, optimal grids are identified by screening on either a 200 kV Thermo Scientific Arctica or Glacios Cryo-TEM, prior to SPA data collection on a 300 kV Thermo Scientific Krios Cryo-TEM. As both the Arctica and Glacios Cryo-TEM have a grid autoloader, Thermo Scientific EPU

Software can automatically collect an atlas for each grid in the cassette, as shown in Figure 3a.• Grids that appear to have

consistent, thin ice are further investigated. Figure 3a shows a TEM grid with optimal grid squares, featuring both thin ice and many areas to collect data.

• Grids that appear consistent with high quality data collection are highlighted and further screened at high magnification to identify regions suitable for

imaging particles. These are homogenous and feature regions with a uniform (and highly dense) distribution of particles (see Figure 3b).

Best 3D reconstructions have been achieved with Quantifoil UltraFoil gold grids due to the minimal beam induced motion that they impart. However, blot conditions that have been found for the holey carbon grids for the same specimen are not necessarily transferable to gold grids.

CONCLUDING REMARKS

As active GPCRs are relatively small molecules to be analyzed by cryo-EM, thin ice is key for optimal signal-to-noise ratio and successful image analysis.

Optimally thin ice can be promoted by highly dense packing of particles, which we found still allows for automated imaging even at low defocus.

Final note: The quality of data recovered from single particle imaging is critically dependent on the quality of the protein used for vitrification.

TROUBLESHOOTING

Ice is too thick• Provided particle distribution

looks to be sufficiently dense, then more grids need to be prepared at higher blot force/ blot time.

• Promote thin ice by increasing protein concentration of sample and longer blot times.

Sample looks clumped/patchyThis is a common problem with

preparation of GPCR grids.• A first step would be to check

a series of sample dilutions. Smaller molecular weight GPCRs tend to need much higher concentration (even up to 8 mg/mL) and much higher blot forces (typically 18-20 on the Vitrobot System) to yield a monodispersed layer of

Blot time (sec) Blot force Drain time

(sec) Sample conc. (mg /mL)

10 20 0 4

10 20 0 4

7 20 0 2

Blot time (sec) Blot force Drain time

(sec) Sample conc. (mg /mL)

5-10 15-20 0-0.5 1-8

Table 1. Example of our Vitrobot settings for sparse screening of blotting conditions, with blot times and blot force settings higher than conventionally used.

Table 2. Some other blot conditions that have yielded good vitrification.

Protocol for preparation of negatively-stained grids

1. Preparation of uranyl formate (UF) (cat no# 24762-1 from Bioscientific) stain

– Bring water to a boil on hot plate, weigh out 37 mg of UF powder in a 5 mL beaker

– Add 5 mL boiling water to the beaker containing UF; stir for 5 min

– Add 5 µL of 5M NaOH into solution, stir for another 5 min

– Filter stain through a 0.2 µm filter and keep stain in dark

2. Continuous carbon grids (cat no# CF100H-Cu-50 from EMS) are glow-discharged for 30 sec

3. Keep all samples on ice. Dilute sample with detergent-free buffer to a concentration of 0.1-0.2 mg/mL immediately before applying on grid.

4. Add 3.5 µL of diluted sample onto grid and allow it to sit for 1 min

5. Remove excess fluid by touching the edge of the grid to a Whatman filter paper

6. Prepare 3 drops of UF stain (~10 µL each) on clean parafilm.

– Gently hold grid to first drop of stain and immediately remove excess fluid

– Repeat

– Lastly, allow grid to touch to third drop of stain for 30 sec and blot off. If grids look heavily stained, duration of third drop may be reduced.

7. Blot the final drop and leave the grid to dry

8. Grid is ready for EM

Figure 3. Inspection and screening for optimal vitrification. These optimal vitrification conditions led to the 3D reconstruction in Figure 1. The figure in (b) shows an example of densely packed particles that yielded a high-resolution structure (sub 2.5 Å). Slightly lower density can also yield high quality data but we do not recommend collecting on grids with sparse particle density in thin ice.

AMMS JUNE 2020 49

particles in ice.• If the problem still persists,

increasing blot time and adding a small drain time can be helpful (see table 1 after combining with table 2)

• After trying both points above, revisit glow discharging. Try a longer plasma discharge regime (90-120 seconds) at both higher and lower plasma currents.

Sample concentration too low• If possible, increase sample

concentration.• Adjusting glow discharge

conditions can be helpful to “encourage” the GPCRs into

the grid holes.• Try different buffer conditions,

eg. changing from Tris to HEPES or MOPS and adjusting the ionic strength of the sample.

• Double application of the sample to the grid prior to plunging. This can be done either manually, prior to placing the sample in the Vitrobot System, or by blotting on the Vitrobot System without the ethane container mounted on the robot. In the latter, one would need to ensure the humidity is appropriately high around the sample to avoid drying.

REFERENCES

[1] Zhao et al., Activation of the GLP-1 receptor by a non-peptidic agonist, Nature 2020.

[2] Liang et al., Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins, ACS Pharmacology & Translational Science.

[3] Booth et al., Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition, Journal of Visualized Experiments.

WHAT IS APEER?

APEER is an open and free digital microscopy platform that enables creation and customization of image processing workflows for specific jobs or extended tasks. The platform connects Microscopists, Image Analysts and Data Scientists with the aim to address current and future microscopy challenges such as fragmented software solutions, incompatible file formats, data storage and processing on APEER, microscopy software developers can easily package their code and share it with other users in the form of modules and workflows. A module is the central element that is designed to perform a specific task and development is possible in nearly every programming language. Users can then combine several modules to project-specific workflows. As APEER runs in a cloud environment, the processing can be easily scaled up on demand for computational intensive modules.

SHOWCASING APEER IN NANOPARTICLE RESEARCH

Introduction Engineered nanoparticles

have a wide range of industrial applications including catalyst research, electronics, inks and pigments, coatings, cosmetics, filtration, energy materials, pharmaceuticals and biomedical applications. With nanotechnology applications likely to develop rapidly in these and other industries, advancing nanoparticles

research will play an important role in improving the quality of life.

Challenges1. Separation and classification of

individual nanoparticles from an agglomeration of particles

2. Automation of analysis of what can typically be hundreds of nanoparticles in a single micrograph

Conventional image segmentation methods use the watershed approach after initial

thresholding by greyscale or red green blue (RGB) values. However, in the case of separating agglomerated particles, the problem is more challenging, and measurement of nanoparticle size is still a highly manual process which is often not repeatable or reliable.

TaskA Zeiss GeminiSEM 500 was

used to acquire a high resolution micrograph of particles from the

Manoj Mathew1, Kirstin Elgass2, Alisa Stratulat3, Matthew Andrew4, Sreenivas Bhattiprolu4

1ZEISS Research Microscopy Solutions, Singapore2ZEISS Research Microscopy Solutions, Melbourne3ZEISS Microscopy, Cambridge4Carl Zeiss X-ray Microscopy, Inc., Pleasanton

A ZEISS INITIATIVE FOR AN OPEN DIGITAL MICROSCOPY PLATFORM

www.APEER.com

Figure 1. (a) Standard analysis workflow, (b) Intellesis pixel classification approach

Figure 2. (a) Original image of particles obtained at 2 kV beam energy, using the Inlens SE detector, (b) Image of separated particles using machine learning and subsequent processing.

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sparks ferrocerium collected on a silicon substrate (Fig. 2a).

These were imaged using Secondary Electron (SE) imaging, a technique which is extremely sensitive surface topography.

It is this variation in surface topography which (during visual inspection) can be used to separate otherwise overlapping particles.

ApproachIn this study we use machine

learning trainable image segmentation with Zeiss ZEN Intellesis.

Instead of performing standard image processing operations on the 2D image, it classifies images on local and non-local greyscale, gradient and texture features, effectively creating a 33-dimensional hyper-image.

APPER SolutionAn end-to-end automated

workflow is proposed that combines image acquisition, machine learning advanced image segmentation, particle analysis, and report generation - all on a digital solutions platform, APEER.

APEER enables the combination of easy-to-use modules into workflows that can be personalized to address the most challenging research problems.

ConclusionThe size and size distribution

of nanoparticles strongly affects quality, properties and applications materials.

In this application, accurate and automated measurement of

nanoparticles was made possible by a combination of advanced microscopy and image analysis techniques in a single end-to-end workflow, using APEER.

The described workflow can be used to optimize synthesis processes of nanoparticles and better understand the relationship between size nanoparticles and material properties.

This allows researchers to expand the use of these materials to novel industrial applications.

The advantages of APEEROne of the principal challenges

associated with complex image processing and analysis workflows is modularity. Single software packages, while offering powerful analytical capabilities, do not provide an end-to-end complete solution for every application. As such, multiple disparate packages must be used (or even created from scratch) making workflow replication at an industrial scale extremely challenging.

APEER provides a platform where such custom or

disconnected components can be defined, developed and connected to create complete workflows, ready to be shared for use. Detailed analytical workflows can be made without the requirement of a deep expertise in computer vision. Using the information achieved through image segmentation, a new platform, APEER, is now available to allow the integration of customized modules in a full workflow, to gain flexibility and save research time.

The Vision Behind APEERAPEER as a vision goes beyond a

simple processing tool and has the potential to become an ecosystem for community-developed solutions for complex image analysis solutions.

For further information please contact:Carl Zeiss Pty Ltd

Microinfo.au@zeiss.com

www.zeiss.com.au

Figure 3. APEER interface for nanoparticle segmentation and analysis, showing how modules can be connected to create a full workflow.

Figure 4. The APEER Vision of bringing together a diverse range of experts in their respective fields to create unique image analysis solutions for everyone.

LIGHT SHEET FLUORESCENCE MICROSCOPY (LSFM) WITH its unique illumination principle allows fast and gentle imaging of whole living model organisms, tissues, and developing cells. The high stability of ZEISS Lightsheet 7 enables researchers to observe living samples over extended periods of time – even days –with less phototoxicity than ever before. The new light sheet microscope can also be used to image very large optically cleared specimens in toto, and with subcellular resolution. The dedicated optics, sample chambers, and sample holders of ZEISS Lightsheet 7 can be adjusted to the refractive index of the chosen clearing method to observe large samples, even whole mouse brains.

New Light Sheet Fluorescence Microscope Introduced

ZEISS LIGHTSHEET 7 ALLOWS MULTIVIEW IMAGING OF BOTH LIVING AND CLEARED SPECIMENS

Researchers can use the exceptionally stable ZEISS Lightsheet 7 to observe living samples over extended periods of time, or to image large optically cleared specimens.

Illumination principle of ZEISS Lightsheet 7

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For further information please contact:Carl Zeiss Pty Ltd

Microinfo.au@zeiss.com

www.zeiss.com.au

IMAGING OPTICALLY CLEARED SPECIMENS

Choosing an optical clearing method depends on the type of tissue, fluorescent labels, and the size of the sample itself. ZEISS Lightsheet 7 is designed to match a multitude of conditions. Specimens with a size up to 2 cm, with a refractive index between 1.33 and 1.58, and in almost any clearing solution can be accommodated. The stable turnkey system allows acquisition of overview images as well as of data with subcellular resolution. Researchers can now perform fast and gentle LSFM imaging of optically cleared organoids, spheroids, organs, brains, or other specimens.

OBSERVING LIVE SAMPLES – FAST AND GENTLY

ZEISS Lightsheet 7 now features the high-quantum efficiency of pco.edge sCMOS detectors

to enable observations of the fastest processes at the lowest illumination light levels. Scientists are able to obtain a real-life view of large samples without the adverse effects of excitation light on their biology. For vertically oriented specimens and highest frame rates, the CMOS detector ZEISS Axiocam 702 can be used. A special sample chamber provides heating, cooling, and CO2 to maintain the optimal environment for live cell experiments. Adding Multiview and triggering options to control external devices makes it possible to observe live processes in an almost unlimited range of organisms

MORE FLEXIBILITY FOR BROADER APPLICATION RANGE

ZEISS Lightsheet 7 brings LSFM imaging a step further to tackle a broad range of applications with best image quality. Newly designed optics and sample chambers allow users to adjust the optics

to the refractive index of their samples. The new sample holder makes mounting larger specimens easier. Smart software tools assist in defining imaging parameters, such as light sheet and sample positions, the right zoom settings, tiles and positions as well as data processing parameters. All these new features go hand in hand with the reliable ZEISS combination of cylindrical lens optics and laser scanning to generate the illumination light sheet. The patented Pivot Scan technology delivers artifact-free optical sections for best possible image quality.

Image of a mouse kidney with ZEISS Lightsheet 7 detection optics 5× / 0.16foc. 3D whole organ imaging and computational image analysis help to gain a better understanding of the mechanisms of various kidney diseases, e.g. diabetic nephropathy. © Sample courtesy of U. Roostalu, Denmark.