Blaetterkatalog Sim0223

25 th
Anniversary
2
VOLUME 25
MAY
2023
69721
Official Partner of the EMS

© petunyia - stock.adobe.com
Light Sheet Microscopy
Virtual Microscopy
4D STEM
Atomic Force Microscopy

13 - 15 September 2023
ICFO - The Institute of Photonic Sciences, Barcelona
Conference Topics SPM Methods

• Polymers and Composites • Nanomechanical and electrical
• Nanoelectronics, photonic and characterization
photovoltaic applications • Characterization of soft materials
• Nanomaterials and Life Science in liquid environment
• Special Session: • Advanced imaging
Using SPM to accelerate the clean energy • High resolution imaging
transition • Automation in AFM
event.nanoscientific.org/eu/2023
Keynote Speakers
• Prof. Dr. F. Pelayo García de Arquer • Prof. Dr. Lukas Eng

ICFO. Spain Technical University Dresden, Germany
• Prof. Ricardo Garcia • Dr. Moshe Ben Shalom
Materials Science Institute of Madrid Tel Aviv University, Israel
CSIC, Spain • Dr. Andrei Kholkin
• Dr. Esther Alarcon Llado University of Aveiro, Portugal
AMOLF physics of functional complex • Prof. Frederic Dubreuil
matter, The Netherlands École centrale de Lyon (ECL), France
• Dr. Deepak Venkateshvaran • Prof. Edoardo Albisetti
University of Cambridge, United Kingdom Polytechnic University of Milan, Italy Submit your
• Prof. Gustau Catalan • Dr. Christopher Kley Abstract at
Catalan Institute of Nanoscience and Helmholtz-Zentrum Berlin for Materials
Nanotechnology ICN2, Spain and Energy (HZB), Germany #NSFE2023
• Dr. Elena Gubanova • Dr. Thomas Miller and win up to
TUM School of Natural Sciences, Munich, University College London, €500
Germany United Kingdom AFM Scholarship!
Website I Registration I Abstract Submission: Deadline:
www.event.nanoscientific.org/eu/2023 June 1, 2023
Contact: info-eu@nanoscientific.org
SUPPORTED BY
REGISTER for #NSFE2023 TODAY

Editorial
Editorial
Combining Microscopy Techniques

with Machine Learning and AI
Five years ago, I was sitting in a conference
© Mahyar Dahmardeh
room in Cambridge, Massachusetts, watch-
ing Prof. Alán Aspuru-Guzik’s talk about
generative models for the inverse design of
molecules and materials and pondering the
possibilities of what the future of machine
learning could hold. I was studying new
photosensitizers for electronic/energy devic-
es, and the possibility of screening several
molecules at once before going to the lab
caught my attention. He discussed a gener-
al workflow for an automated, “self-driving”
approach to generate candidate molecules
for applications such as drug development
or renewable energy. In the heart of Biotech
startups, many questions emerged on how
this system could be used to discover poten-
tial drug candidates.
Years later, much progress has been made Abb.: Through a combination of highly advanced microscope technology in iSCAT and multiple
in different research fields thanks to artifi- machine learning techniques, the team from MPL and FAU continues to push the boundaries of
cial intelligence (AI) and machine learning. optical sensing.
Recently, Aspuru-Guzik and Christine Allen,
both from the University of Toronto, pub-
lished a study in Nature Communications [1] scope and assigned like barcodes using AI. It [3] Sigmund, F., Berezin, O., Beliakova, S. et
demonstrating how machine learning algo- can be used, for example, to identify electri- al.: Nat Biotechnol (2023) DOI: 10.1038/
rithms can predict experimental drug release cal synapses between nerve cells or receptors s41587-023-01713-y
from long-acting injectable systems. They that influence the interactions between can- [4] Dahmardeh, M., Mirzaalian Dastjerdi, H.,
also described how these trained models could cer and T-cells. Mazal, H. et al.: Nat Methods (2023) DOI:
guide the design of new long-acting inject- From the articles in this issue, you can 10.1038/s41592-023-01778-2
ables, decreasing the time and cost associated gain interesting insights into the world of
with drug formulation development. microscopy, including a study developed by
When combined with microscopy tech- Dahmardeh et al. [4] that employs interfer-
niques, machine learning can be used to ometric scattering microscopy (iSCAT) and
improve sensitivity and precision or even to AI to improve the spatiotemporal detection
solve multiple tasks at the same time. For of small proteins. By combining iSCAT with
example, Piñeda et al. [2] designed a method iForest and FastDVDnet, proteins of only 10
combining time-lapse microscopy and AI to kDa or less could be detected, pushing the
track cell motion at a spatiotemporal scale. boundaries of optical sensing, and opening
“The AI method uses the information in the doors to optical investigations of small traces
graph to adapt to different situations and of biomolecules and disease markers. Fur-
can solve multiple tasks in different experi- thermore, you can find other articles related
ments. For example, our AI can reconstruct to discoveries and achievements in micros-
the path that individual cells or molecules copy, like real-time imaging of cell secre-
take when moving to achieve a certain bio- tions or a new objective for light microscopy
logical function. This means that research- inspired by scallop eyes.
ers can test the effectiveness of different Inara Aguiar
medications and see how well they work as Enjoy the read!
potential cancer treatments,” said Piñeda. Inara Aguiar Inara is a science editor and writer with a
Pharmaceutical companies are already using Ph.D. in Inorganic Chemistry. After a postdoc
the new framework, which can help scien- in Computational Chemistry, she started
tists develop more efficient cancer technol- References working as a science editor in the fields of
ogies and treatments. Chemistry, Engineering, Bioengineering, and
Another interesting study reported by Sig- [1] Bannigan, P., Bao, Z., Hickman, R.J. et Biochemistry. She has been working as a
mund et al. [3] describes a new genetic reporter al.: Nat Commun (2023) DOI: 10.1038/ technical writer / editor for several scientific
system that can recognize structures and their s41467-022-35343-w publishers and recently joined Wiley Analytical
functions within the cells. The spherically sym- [2] Pineda, J., Midtvedt, B., Bachimanchi, Science as a freelance content creator and
metric and concentric barcodes (EMcapsulins) H. et al.: Nat Mach Intell (2023) DOI: microscopy news editor.
are easily identified under the electron micro- 10.1038/s42256-022-00595-0
Imaging & Microscopy 2/2023 • 3

Contents
EDITORIAL 3
NEWSTICKER 6
ANNOUNCEMENTS
SCANDEM 2023: 73rd Annual Meeting
of the Nordic Microscopy Society! 8
© Jürgen Mayer
Seeing is Believing – EMBO | EMBL Symposium 8
NEW BOOK
Light Sheet Fluorescence Microscopy 9
RMS IN FOCUS
mmc2023 (incorporating EMAG 2023): Book now! 10
NEWS FROM EMS

EMS Newsletter #81, May 2023 11
© TU Wien
COVER STORY
Raman Imaging of Polymer Materials 12
Comprehensive Insight into Plastics’ Chemistry and Structure
E. Vomhof et al.
LIGHT MICROSCOPY
© University of Antwerpi
Challenges in Building a Simple Conic-Based

Light Sheet Microscope 14
Combining Positive Axicon Lens with Further Aspherical Lenses to Obtain a Highly Localized Light Sheet
S. Saghafi et al.
Light Sheet Microscopy 17

Optical Sectioning with High Speed and Low Phototoxicity
J. Sanderson
Going Digital in Pathology 20

Introducing Virtual Microscopy and What Questions to Ask
K. Eser
Turnkey Multiphoton Microscopy for 3D Samples 23

Label-Free and 4D Imaging in Life-Science and Medicine
© Franceschi
S. Kiderlen and L. Krainer
PREVIEW:
ISSUE 3/2023
Coming out 21th August, 2023

Pushing the Boundaries of Optical Sensing
Combining State-of-the-Art Microscopy Technology in iSCAT and
Multiple Machine Learning Techniques
V. Sandoghdar
4 • Imaging & Microscopy 2/2023

CORRELATIVE MICROSCOPY
Correlative Microscopy of a Catalytic Reaction 26
Zooming in on Chemical Patterns in Hydrogen Oxidation on Rhodium
P. Winkler and G. Rupprechter
ELECTRON AND ION MICROSCOPY

4D Scanning Transmission Electron Microscopy (STEM) with
Event-Based Electron Detection 30
How to Overcome the Bottleneck in Scanning Speed
D. Jannis et al.
Can AI Do your Ptychography? 32

A Machine Learning Approach to Real-Time Phase Imaging with 4D STEM
S. van Aert et al.
SCANNING PROBE MICROSCOPY

Imaging the Surface Ions of Pristine Muscovite Mica 36
Using Non-Contact Atomic Force Microscopy in Ultra-High Vacuum
G. Franceschi et al. COVER STORY
Raman Imaging of
Polymer Materials
IMAGE PROCESSING
Comprehensive Insight into
Jupyter Notebooks for Generating and Distributing Plastics’ Chemistry and Structure
Bioimage Analysis Workflows 38
My Favorite Image Analysis Tool, by Neubias Members Raman imaging is a powerful tool for the chemical
analysis of polymers and composite materials, as it
R. Camacho can non-destructively investigate phase separation
and defects, even beneath the sample surface.
Combining the technique with complementary
methods helps researchers achieve a comprehensive
ADVERTORIAL understanding of their plastic products.
Investigating Surface Catalytic Activities Using SECCM 41 Polymers are an important component of many
A. Klasen and A. Cerreta everyday products such as tires, plastic toys, clothing,
food packaging, glues and varnishes. Their widely
varying mechanical and chemical properties make
them popular in many industries and for diverse
applications. Thorough knowledge of the morphology
PRODUCTS42 and chemical composition of multi-component
polymeric materials on a sub-micrometer scale is
crucial for developing new materials, optimizing
INDEX / IMPRESSUM INSIDE BACK COVER their properties and assessing the quality of the final
products.
12
25 th
Anniversary
2
VOLUME 25
MAY
2023
69721
Official Partner of the EMS Welcome to the knowledge age. Wiley builds on its 200-year
heritage by partnering with universities, businesses, research
institutions, societies and individuals to develop digital content,
© petunyia - stock.adobe.com
learning, assessment and certification tools. Wiley continues to

share and deliver the answers to the world’s challenges
helping you to further your mission.
Virtual Microscopy
4D STEM
Atomic Force Microscopy

NEWSTICKER
In Situ Conductive AFM Real-Time Nanoplasmonic Imaging
Novel Correlative Microscopy Approach Unveils Catalyst Surface Features New System Allows Spatiotemporal Monitoring of Single-Cell Secretions
Researchers at the Helmholtz-Zen- Researchers from the BIOnanopho-

trum Berlin (HZB), and collaborators tonic Systems Laboratory (BIOS),
from the Fritz Haber Institute (FHI) EPFL, and the University of Ge-
of the Max Planck Society, have suc- neva have developed an optical
ceeded in analyzing electrocatalyt- imaging approach that gives a
ically active materials using in situ four-dimensional view of cell se-
conductive Atomic Force Microscopy cretions in both space and time. By
© M. Munz/HZB (CAFM). The new method enables si- placing individual cells into micro-
multaneous real-space imaging of local electrical, chemical-frictional, and scopic wells in a nanostructured
morphological properties evaluation of electrocatalysts in aqueous media gold-plated chip and inducing © BIOS EPFL
and under potential control. It can further help scientists evaluate pro- a phenomenon called plasmonic velopment as well as fundamental
cesses involved in battery electrodes, photocatalysis, or active biomaterials. resonance on the chip’s surface, studies.
they could map secretions as they
Original publication: are being produced. The research Original publication:
10.1021/jacs.2c12617 provides a detailed view of how doi: 10.1038/s41551-023-01017-1
More information: cells function and communicate More information:
https://bit.ly/IM-022023-a and can aid pharmaceutical de- https://bit.ly/IM-022023-b
Scattering-Type Scanning Near-Field Microscopy
© Mahyar Dahmardeh
Using Blue Light instead of red to measure electrons in semiconductors
Researchers at Brown University length gets shorter, this becomes

have developed a new micros- a lot harder to implement. As a
copy technique that employs result, nobody had ever done it
blue light to measure electrons with blue light until now.” The
in semiconductors. “There is a lot research, recently published in phenomena in various materials. Original publication:
of interest these days in studying Light: Science & Applications, is In the future, it can help scien- doi: 10.1038/s41377-023-01137-y
materials with nanoscale reso- the first to provide nanoscale im- tists to achieve more energy-ef- More information:
lution using optics,” said Prof. aging to solve this longstanding ficient semiconductors and elec- https://bit.ly/IM-022023-e
Daniel Mittleman. “As the wave- problem limiting the study of key tronics.
Meta-Optics for Microscopes Interferometric Scattering Microscopy and AI

Observing Tiny Structures such as Nanoparticles or Transistors Improving Protein Detection Methods
opment are opaque to this light, Researchers at the Max-Planck-In-

no usable imaging systems have stitute for the Science of Light
been reported until now. Professor (MPL) and Friedrich-Alexander-Uni-
Marcus Ossiander: “I asked myself versität (FAU) have combined one
whether the classical principle of of the most effective microscopy
optics could not be reversed. Can methods with AI to detect small
you use the absence of material in proteins. The new approach uses in-
© Lunghammer, TU Graz small areas as the basis of an op- terferometric scattering microscopy
Attosecond physics uses the tical element?” A team at Harvard (iSCAT) and multiple machine learn- © Mahyar Dahmardeh
light-matter interaction phenom- University has developed a new ing techniques to push boundaries Harald Köstler from FAU, employed
ena wherein attosecond (10−18 s) lens, which was successfully tested in optical sensing. The research, two machine-learning techniques—
photon pulses are applied to un- by researchers at TU Graz using published in Nature Methods, opens iForest and FastDVDnet—to detect
ravel dynamical processes in mat- this design principle. the door to optical investigations proteins at only 10 kDa or less.
ter with unprecedented time reso- of small traces of biomolecules
lution. Because it employs extreme Original publication: and disease markers. In order to Original publication:
ultraviolet light, which oscillates doi: 10.1126/science.adg6881 enhance the iSCAT sensitivity, the doi: 10.1038/s41592-023-01778-2
quickly, and all materials in the More information: MPL team led by Prof. Vahid San- More information:
construction kit of optics devel- https://bit.ly/IM-022023-c doghdar, in collaboration with Prof. https://bit.ly/IM-022023-d

Newsticker
Photoacoustic Imaging Cryo Transmission Electron Objectives for Light Microscopy

Label-Free, Bond-Selective Imaging of Tomography and Deep Learning Using a Mirror Instead of Lenses to Achieve
Biological Tissue Observing of Platinum Catalyst Layers
© Fabian Voigt et al.

2023 / UZH
Inspired by the anatomy of scallop eyes, neu-
roscientists at the University of Zurich have
© Guyue Hu, PARS Mechanism 2023 developed an innovative objective for light mi-
Researchers from the University of Hong Kong © INE EPFL croscopy. The new objective uses mirrors to pro-
recently reported near-infrared photoacous- EPFL researchers have revealed, for the first duce images, enabling high-resolution imaging
tic remote sensing microscopy for noncontact time, the nanoscale structure of catalyst layers. of tissues and organs in a much wider variety of
imaging of lipids. The new approach provides a The new method combines cryogenic transmis- immersion media than conventional microscope
broader detection bandwidth and improves the sion electron tomography and deep learning to lenses. Astronomical telescopes use mirrors in-
detection sensitivity and signal-to-noise ratio. enable observation of the platinum catalyst lay- stead of lenses to create images, allowing them
Photoacoustic imaging is a powerful technology ers, highlighting how they could be optimized to capture as much light as possible from plan-
that uses light and sound to create biomedical for fuel cell efficiency. Proton-exchange mem- ets, stars, and galaxies. The Schmidt telescope,
images. By scanning the sample and collecting brane fuel cell (PEMFC) is an electrochemical developed in the 1930s by Bernhard Schmidt
the corresponding photoacoustic signals, re- energy conversion technology for automotive and still used in many observatories today, uses
searchers can reconstruct 2D or 3D images of applications that use nanocatalysts to trigger a thin corrective lens combined with a large
biological tissues. Photoacoustic remote sensing electricity-producing reactions between hydro- spherical mirror. A single Schmidt objective can
imaging is a novel photoacoustic imaging mo- gen and oxygen. Because most PEMFC catalysts be compatible with different clearing techniques
dality that uses a different laser source to detect contain platinum, a scarce and expensive metal, because it uses a mirror instead of lenses. The
acoustic signals instead of ultrasonic transduc- there is a pressing global need to develop pow- spherical mirror focuses light at the same point,
ers used by conventional acoustics. erful catalysts with reduced platinum content. whether the sample is immersed in liquid or air.
Original publication: Original publication: Original publication:

doi: 10.1117/1.APN.2.2.026011 doi: 10.1038/s41929-023-00947-y doi: 10.1038/s41587-023-01717-8
More information: More information: More information:
https://bit.ly/IM-022023-f https://bit.ly/IM-022023-h https://bit.ly/IM-022023-g
Encoded Barcodes
Reporter Protein Readable by Standard Electron Microscopy Techniques
Researchers from the Technical University of incorporated metal-binding proteins into dif-
Munich (TUM) have developed a new genetic ferently-sized capsules. The spherically symmet-
reporter system that can recognize structures ric and concentric barcodes (EMcapsulins) are
and their functions within the cells. The gene easily identified under the electron microscope
reporters are protein capsules with a fitting size and assigned like barcodes using artificial intel-
to be resolved by an electron microscope. The ligence.
capsules are produced by the cells themselves,
and their genetic blueprints are attached to Original publication:
specific target genes. When the target genes be- doi: 10.1038/s41587-023-01713-y
come active, the production of reporter proteins More information:
starts. In this new approach, the researchers https://bit.ly/IM-022023-j © Andreas Heddergott / TUM; Barth van Rossum
Optical Filters
For Fluorescence Microscopy
Visit us at ELMI 2023 in Noordwijkerhout, NL
and MMC 2023 in Manchester, GB! Wide selection · Custom-specific designs www.ahf.de

Announcement
SCANDEM 2023: 73rd Annual Meeting

of the Nordic Microscopy Society!
Uppsala, Sweden, June 12-15, 2023
After a four years break, this year, we will again opments in microscopy focused on material nich, Xiaowei Zhuang - Harvard Med. School,
hold the SCANDEM meeting as a in-presence science and biology. This year the meeting will Michael Laue - Robert Koch Institute, Ute Kaiser -
conference (www.scandem2023.se). The meet- feature invited talks by leading international Ulm University and Frances Ross - MIT. SCANDEM
ing hereby continues the tradition of inviting and local researchers in microscopy and asso- 2023 is organized by Uppsala University and the
and gathering international researchers in the ciated areas, oral and poster presentations of Scandem Nordic Microscopy Society.
field of microscopy to share their latest findings submitted papers and abstracts, as well as an
and socialize at a Nordic location- this time the industrial exhibition, and workshops with vari-
Ångström laboratory in Uppsala, Sweden. ous focus themes.
SCANDEM is an important forum Plenary speakers will be Xiaoqing Pan
for leading international re- - UC Irvine, Wolfgang Baumeister - TU Mu-
search groups in industry and
academia to meet and discuss
the latest trends and devel-
More information
and registration:
www.scandem2023.se
© oleg7799 - Adobe-Stock.com
Seeing Is Believing – EMBO | EMBL Symposium

Heidelberg, Germany, October 4 – 7, 2023
What past participants

say about the symposium:
“I was happy to attend this amazing meeting full

of talks showing incredible combinations of the
most recent state-of the-art techniques, some
of which even looked like being from far future
or sci-fi.” – Marketa Schmidt Cernohorska, Max
Perutz Labs Vienna, Austria.
Registration closes 23 August 2023.

From its beginning in 2011, ‘Seeing is Believing’ complexes, organelles, cells, tissues, organs and
has embraced novel imaging technologies that whole organisms. The symposium will bring to-
open new windows for biological discovery, in- gether the leading developers of imaging meth-
cluding single-molecule and super-resolution, ods with cutting-edge applications that illustrate
light sheet and correlative light electron micros- how imaging can answer biological questions. It
copy. provides many opportunities for presentations,
The molecular processes of life are naturally discussions, and interactions between students,
dynamic in space and time from the atomic to postdocs, junior, and senior investigators.
the organismal level. The new imaging methods Session topics include: dynamic super-reso-
allow us to understand the mechanisms of life as lution imaging, probes & biosensors, organismal Registration:
well as disease and is revolutionising our ability to and intravital imaging, cross-scale imaging and www.embl.org/events
visualise the inner workings of macromolecular image analysis & modelling.

New Book
Light Sheet Fluorescence Microscopy

A Book by Emmanuel G. Reynaud and Pavel Tomancak
Destined to set the standard, the first book deal-

ing exclusively with this revolutionary and novel
imaging technology serves as an easy-to- un-
derstand introduction while offering numerous
tips and tricks. a2
0 % e:
Adopting a practical approach, the authors Get unt cod
o
disc AS23
who developed this actual technique provide W
a comprehensive, hands-on overview of the
basics of light sheet fluorescence microscopy,
instrumentation, applications, sample prepa- 1. Edition August 2023
ration, and data analysis. As a reflection of the 416 Pages, Softcover
uncompromisingly interdisciplinary nature of Practical Approach Book
the topic, they merge their expertise in physics, ISBN: 978-3-527-34135-1
biology, and computer science, giving valuable Wiley-VCH, Weinheim
insider tips taken from their work at major man-
ufacturers.
The result is in-depth information on hard- Order the book here:
ware and software solutions for a straightfor- https://bit.ly/Wiley-VCH-LSM
ward implementation of LSFM in the lab.
You Can Have It All In a Tabletop Package
A Full-featured SEM without the high cost!

At COXEM, we believe that electron microscopy doesn’t need to be expensive or complicated.
That’s why we developed the EM-30, a tabletop SEM that doesn’t skimp on features. Whether
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local agent to arrange a demonstration, or visit our website for more information.
www.coxem.com / overseas@coxem.com

RMS in Focus
mmc2023 (incorporating EMAG 2023):

Book now!
Manchester Central, UK, July 4-6, 2023
W
ith just two months to go before
mmc2023 (incorporating EMAG
2023) kicks off in Manchester,
UK, the excitement is well and truly build-
ing ahead of the RMS’s flagship event.
The Microscience Microscopy Congress (mmc)

is one of the biggest and best international
events in microscopy, imaging, and flow cytom-
etry - bringing together hundreds of people who
use microscopes for work, study and pleasure.
Alongside a huge, three-day conference, the
event boasts a world-class exhibition, workshops,
social networking opportunities and more.
mmc2023 is taking place at the superb Man-
chester Central Convention Complex, from 4 – 6
July, and this year’s Congress is shaping up to
be one of the best ever. A record number of ab-
stracts (more than 400) have already been sub-
mitted, and the event will also include a new and sessions covering every aspect of microscopy,
improved RMS Learning Zone, plus a multi-cat- imaging and flow cytometry, including recent
egory, International Scientific Imaging Competi- and emerging applications. The blockbuster
tion. Registration is still open, so now is the time programme will cover the full range of latest
to book your place at this fantastic event for the techniques, applications and hottest emerging
microscopy and imaging community. topics – plus an incredible cast of speakers and
supporting poster sessions.
In addition to the academic content, the pro- the conference. An eye-catching gallery of the
Exhibition gramme includes a number of sessions sure to shortlisted images – covering all microscopi-
be of interest to a wide range of scientific in- cal techniques - will be on display throughout
Up to 80 companies – including many of the dustries, including: the event. The RMS will also be bringing its ev-
biggest names in microscopy and imaging – er-popular Learning Zone to mmc, with experts
will be on hand to showcase their products and ▪ Spatial and Imaging Cytometry on hand to share their knowledge with visitors
give practical demonstrations. The exhibition ▪ Artificial Intelligence and provide demonstrations.
also provides important exposure for a number ▪ Public Health: The Impact of Microscopy
of smaller companies keen to share their latest ▪ Multimodal Microscopy
technological developments. ▪ Advances in label-free Imaging Register Now!
The exhibition runs for three days alongside ▪ Clinical Diagnostic Imaging
the conference, and is completely FREE for any- Registration is still open, and all the information
one to attend. Visitors can simply turn up and on rates, accommodation and transport can be
register for an exhibition-only ticket, giving Satellite Meetings and Bonus Features found on the official mmc-series website.
them access to everything on show – including
live demonstrations, expert advice, and com- mmc2023 (incorporating EMAG 2023) will bring We look forward to seeing you there!
pany workshops all under one roof. together a number of smaller meetings, allow-
ing you to meet with colleagues working in your Contact
field as well as with cross-disciplinary peers, all Allison Winton
Conference at the same event. Chief Executive
Other popular features include the RMS In- Royal Microscopical Society
Meanwhile the conference itself consists of ternational Scientific Imaging Competition, the London, UK
six parallel streams, with no fewer than 36 winners of which will be announced during awinton@rms.org.uk
Book mmc2023: mmc exhibition: mmc-series website:

www.mmc-series.org.uk www.mmc-series.org.uk/exhibition.html www.mmc-series.org.uk

News from EMS
EMS Newsletter #81

José Maria Valpuesta, May 2023
EMS President
Virginie Serin,
EMS Secretary
Dear EMS members, international events. More information can be Finally, this year the EMS honors its 25 years
found on our scholarships page. birthday, and different initiatives are being or-
In March this year, the EMS Executive Board had ▪ EMS Sponsored Events’ applications for EMS ganized to celebrate it. A small symposium will
its annual winter meeting in Budapest, Hungary. sponsorships of microscopy-related events to take place at IMC20 (Busan, Korea) to commem-
Some of the most important topics and deci- be held in the second half of 2023 (July 1 - orate our first 25 years of existence.
sions were discussed during this meeting and December 31). As usual, the EMS continues with its support
are mentioned in this newsletter. to the activities of our members, so please have
The call for nominations for the EMS Out- This financial support from the EMS ensures a look at our webpage for the various meetings
standing Paper Awards (OPA) 2022 has now that also these smaller events, often organized and workshops that will take place over the
been closed. The EMS has received a number of in the form of a school or course, can invite top next year.
high-quality papers which are currently under scientists to lecture our coming generations of Please do not forget to regularly visit the
evaluation. The award-winning papers will be microscopists. You can find more information EMS webpage and the social media pages on
announced to the EMS members in May this on EMS Sponsored Events by visiting our web- Facebook and Twitter which are daily updated
year in our newsletter. The OPA winners will site. to efficiently provide you information from our
receive their awards at the 20th International community.
Microscopy Congress to be held on 9 to 15 Sep- If you have any questions, please do not hes-
tember, 2023, Bexco, Busan, Korea. We hope to EMS Yearbook itate to contact the EMS Secretary. Any sugges-
see most of you there. tions and ideas for future newsletters are also
The 2022 EMS Yearbook will arrive in the fol- welcome.
lowing months. This edition will include re-
Two other EMS calls have taken place ports from EMS awards, EMC, EMS Sponsored
during the last two months: and Special events, EMS students reports from Contact
EMC2020, and many other information. We Prof. Dr. Virginie Serin
▪ EMS Scholarships to support young research- hope you will enjoy reading the reports on the EMS Secretary
ers for the participation at the next IMC to be various activities of our society and members Toulouse, France
held on Sept. 10-15, 2023 in Bexco, Busan, Ko- during the past year, especially those written by sec@eurmicsoc.org
rea; EMS aims to issue 20 travel grants for the students who received a scholarship to attend
attendance of young European colleagues to the EMS extension meetings: PICO and MCM16, Prof. Dr. José Maria Valpuesta
Busan (€ 800 each). The EMS provides valuable some of these contain truly inspiring notions EMS President
support for our young colleagues to present from our enthusiastic new generation of mi- Madrid, Spain
their results and learn from the specialists at croscopists. jmv@cnb.csic.es
EMS sponsored events: EMS scholarships:

EMS web page:
www.eurmicsoc.org/en/meeting-calendar/ www.eurmicsoc.org/funding/en/
https://bit.ly/EuroMicr
ems-sponsored-events scholarships

Cover Story
Raman Imaging of Polymer Materials

Comprehensive Insight into Plastics’ Chemistry and Structure
Matthias Finger, Miriam Böhmler, Damon Strom, Eleni Vomhof
R
aman imaging is a powerful tool for
the chemical analysis of polymers and
composite materials, as it can non-de- a) b)
structively investigate phase separation and
defects, even beneath the sample surface.
Combining the technique with comple-
mentary methods helps researchers achieve
a comprehensive understanding of their
plastic products.
Polymers are an important component of

many everyday products such as tires, plas-
tic toys, clothing, food packaging, glues and
varnishes. Their widely varying mechan-
ical and chemical properties make them
popular in many industries and for diverse
applications. Thorough knowledge of the
morphology and chemical composition of c) d)
multi-component polymeric materials on a
sub-micrometer scale is crucial for devel-
oping new materials, optimizing their prop-
erties and assessing the quality of the final
products.
Correlative Raman
Imaging for Polymer Research
Raman analysis can help manufactur-

ers of polymers and composite materials Fig. 1: Correlative Raman-AFM study of a polymer blend. A: Raman spectra of PMMA (red)
throughout the entire lifecycle of a product. and PS (blue). B: Raman image showing the phase separation in a blend of PMMA (red) and PS
Based on inelastic light scattering, Raman (blue). C: AFM topography image of the same sample area. D: Overlay of AFM topography and
spectroscopy identifies molecules by their Raman images (B and C), correlating structural and chemical information.
unique Raman spectra and characterizes
their chemical properties. Polymers can be
identified, orientation and crystallinity can Raman-AFM Study of a Polymer Blend depressions in the raised matrix, but also
be analyzed, and contaminations, defects small, round islands protruding from the
and stress states can be revealed [1]. In con- A polymer blend was investigated by correl- depressions (Fig. 1C). The maximum height
focal Raman imaging, a Raman spectrum ative Raman and AFM imaging. The mea- difference within the examined area was
is acquired at each pixel and the obtained surements were performed with a WITec 380 nm.
chemical information is color-coded in the alpha300 RA, which combines AFM and Combining chemical and topographic
resulting Raman image. 2D scans can either Raman imaging capabilities in one instru- information enables deeper insights into the
visualize the spatial distribution of polymer- ment. The sample remained in place during sample composition. The overlay of AFM
ic components on a sample surface or show all measurements, as switching between and Raman images reveals that the depres-
their layered structure in depth scans [1-4]. imaging modes required just a turn of the sions consist of PS, while all elevated areas
Additionally, 3D representations can be gen- objective turret. are formed by PMMA (Fig. 1D).
erated from stacks of 2D images [2]. Two components were identified by their
For achieving a thorough understanding Raman spectra (Fig. 1A): poly(methyl meth-
of polymer samples, correlative microscopy acrylate) (PMMA) and polystyrene (PS). The Raman Analysis of Layered
techniques offer many benefits. Atomic force Raman image shows their separation on the Plastic Packaging
microscopy (AFM) acquires topographic sample surface (Fig. 1B). Both compounds
information and determines local mechan- formed rounded islands of different sizes Plastic is a common packaging material in
ical characteristics of a sample with high within the other phase, respectively. the food industry. Packaging systems often
spatial resolution. AFM and Raman imag- For structural insight, the topography of consist of multiple layers of different poly-
ing thus yield complementary information the same sample area was recorded by AFM. mers, depending on the type of food or drink
about plastic samples [4]. The AFM image shows irregularly shaped contained and its associated requirements. In

Cover Story
order to ensure safe and tightly sealed food

packing, the material composition needs to a) b)
be monitored and defects and inclusions
must be located and characterized, which is
possible with Raman imaging. The following
measurements were performed with a WITec
alpha300 series Raman microscope.
First, a cross section through the top foil
of a cheese packaging was investigated. The
foil lid is designed to tightly seal the con-
tainer while fused to it. Additionally, it will
stick to the lower part of the container after
its initial opening, so it can be easily closed
again to keep the contents fresh. The Raman
image reveals the layer structure of the foil
(Fig. 2A) and the Raman spectra of the indi-
vidual components (Fig. 2B) were identified
automatically using the TrueMatch spec- Fig. 3: Raman depth scan of a plastic foil with a defect. A: Bright-field image of a bubble in the
tral database management software. The foil. B: Raman depth scan along the blue line in A. The white line marks the bubble’s border.
foil contains layers of the polymers poly- Polyester (red) and two different PE modifications (green, pink) are separated by a layer of glue
propylene (PP) and polyethylene (PE). They (blue), which is missing in the bubble. Sample courtesy of Dr. Dieter Franke, Siegwerk Druckfar-
are separated by a thin film of polyurethane ben AG & Co. KGaA, Siegburg, Germany
(PU), on top of which several different pig-
ments were detected. The pigments formed a
printing on the foil and included, for exam- Non-Destructive Defect ed in a plastic product without cutting it,
ple, the titanium dioxide polymorphs rutile Analysis in Plastic Foils which is crucial for ensuring the product
and anatase for white color. A second thin quality. The bright-field image of a second
film between two PE layers consists of eth- With a confocal Raman microscope, it is plastic foil showed bubble inclusions (Fig.
ylene-vinyl acetate (EVA), which is respon- also possible to non-destructively inves- 3A) and a Raman depth scan characterized
sible for the stickiness of the cover foil after tigate the layer composition of transpar- this defect below the surface (Fig. 3B). The
initial opening that enables the reclosing of ent polymer samples below their surfaces. foil consists of a polyester and PE, separat-
the packaging. Inclusions and defects can thus be detected by a glue layer. In the observed bubbles,
the glue layer is missing. Similar depth
scans at different positions also revealed
that the thickness of the glue layer varied
a) b) over the sample area.
Conclusion
The presented measurements demon-

strate the power of Raman microscopy for
characterizing plastic samples. AFM and
Raman imaging yielded complementa-
ry topographic and chemical information
for detailed insight into the structure and
composition of a two-component polymer
blend. Raman imaging characterized the
layer structure of a food packaging and a
depth scan revealed a sub-surface defect in
a plastic foil.
Contact
Fig. 2: Raman image of a layered plastic WITec GmbH
packaging foil. A: Raman image of a cross Ulm, Germany
section through the cover foil of a food info@witec.de
container, color coded according to the https://raman.oxinst.com
spectra in B. B: Raman spectra of the
identified components: the polymers PP
(red), PE (blue), EVA (cyan) and PU
(green); rutile, anatase (both pink) and
yellow, blue, purple and red pigments References:
(yellow, violet, brown, orange). https://bit.ly/IM-WITec0223

Light Microscopy
Challenges in Building a Simple

Conic-Based Light Sheet Microscope
Combining Positive Axicon Lens with Further Aspherical Lenses to Obtain a Highly Localized Light Sheet
Saiedeh Saghafi1, Meraaj Foroughipour1, Klaus Becker1,2, Inna Sabdyusheva-Litschauer1, Massih Foroughipour1, Eugenijus Kaniusas3, Hans Ulrich Dodt1,2
T
he laser beam profile is a most im-
portant factor in light interaction
with materials. Accordingly, con-
trolling devices reshaping an incident la-
ser beam via tailoring its amplitude and
phase have been developed. Meso-Aspher-
ic-Optic (MAO) characterizes the optical
properties of conical wavefields and of-
fers ways for building conical lenses with
high precision. MAO elements with com-
plex structures (e.g., Powell lenses and
axicons) reshape a Gaussian laser beam
into the specific distribution required by
the application. Here, we describe how
a well-structured positive axicon lens in
combination with further aspherical lens-
es enables us to obtain a highly localized
light sheet for static light-sheet micros-
copy. This affordable light sheet generator
system is perfect for revealing details in
small chemically cleared biological speci-
mens, as well as for looking deeply into a
limited region of larger transparent sam-
ples.
Introduction
Lasers have become a pivotal tool for most Fig.1: MAO conical elements; A) Symmetrical conic element (axicon), B) The effects of axicon
applications in various fields (e.g., biotech- on the object pattern, C) Axial conic element (Powell lens), D) Object, E) Image of the object (D)
nology, photonics, communications, de- using Powell lens while the conical axis is vertical to the x-axis, F) Image of the object (D) using
fense, and medicine) [1-3]. The laser beam Powell lens when the conical axis is parallel to the x-axis
profile is one of the most critical factors in
laser beam interaction with different ma-
terials making beam shaping an important tical elements (ROEs) are cost-effective while and symmetrical conic structures (e.g., Axi-
prospect in the laser industry. Consequently, providing high-precision results and a high con lenses, meaning axis-image and Powell
beam-controlling devices for modifying the degree of freedom for diverse applications lenses as shown in Fig.1) has been suggested
laser output to get the desired irradiance dis- [7-11]. However, we might encounter optical [15-21].
tribution via tailoring the amplitude/phase problems either due to fabrication errors or Axicons have the property of a point
are in high demand. Using diffractive or re- natural optical phenomena [12] while they source but creates a line of focused points
fractive optics, various elements for reshap- need regular calibration [13-14]. A funda- instead of one focal point, thus, there is
ing laser beams have been developed. Dif- mental laser beam accommodates the max- no definite focal length. The beam gradu-
fractive optical elements (DOEs) can convert imum possible energy in a distinct region ally becomes a ring through propagation
the fundamental laser beam into a beam with of space forming a bell-shaped distribution [16]. This transformation is homomorphic.
specific characteristics [4,5]. These elements approximated by a Gaussian function within An axicon is not an imaging lens but can
are generally light in weight, and small in the scalar wave equation. Meanwhile, state- be considered a beam-shaping element. In
size. However, they are highly expensive and of-the-art beam-shaping methods enable us- an ideal axicon, the length of the line of
a lack of freedom and flexibility in the usage ers to overcome many of the limitations that focused points depends on the wedge angle,
of DOEs in any other applications except the we encountered by remaining in the Gauss- the diameter of the incoming beam, and
one that was initially intended has been re- ian regime. To minimize these limitations the refractive index of the material. How-
ported [6]. On the other hand, refractive op- using MAO optical elements of specific axial ever, fabrication errors in conic-structured

Light Microscopy
THE FUTURE
material. However, fabrication errors in conic-structured lenses can cause massive alte
these parameters.
Material and Methods
lenses can cause massive alterations in these
parameters.
and beam widths varied by a factor of 1.5
- 5. Figure 3 compares the effects
Effects of two
of Well-Structured DEPENDS ON
Axicon on the Incident Beam
OPTICS
Conical(fan
commercially available axicons lenses of special structures can convert a Gaussian beam into various diffracti
angle:
170°), one with manufacturingbeamserrors
[22-25],(left:
opening a new horizon for their application in 3D microscopy by generat
sheet with controlled expansion [21,26]. When a radially symmetric Gaussian fie
Material and Methods Fig.3 A1 - D1) and one with a high-preci-
considered a bundle of parallel rays along the propagation axis strikes an axicon with the
sion structure (right: Fig.3 index
A2 - D2), on anthe rays will refract individually upon arrival at the conical surfac
n (Fig.1-A),
Effects of Well-Structured Axicon incident beam with Gaussian highlydistribution.
focused points with a gradual change towards becoming a ring (Fig.2).
on the Incident Beam The effects are shown at the focus (mini-
Conical lenses of special structures can mum width) and 5 mm away from the focus.
Effects of Non-Ideal Axicon on the Incident Gaussian Beam
convert a Gaussian beam into various dif-
fraction-limited beams [22-25], opening Static Light Sheet Microscopy As pointed
Setup outUsingbefore, the precision in the fabrication of the conic elements is a challe
and can affect the final results massively. An axicon with high precision is usually
a new horizon for their application in 3D Well-Structured Conic Lenses compared with the commercially available ones. Depending on the materials, met
microscopy by generating a light sheet with We built the setup precision according utilizedtoin their manufacturing, the prices could differ. In our laboratory, we u
controlled expansion [21,26]. When a radi- DE102010046133B4-patent variety using of200 milli-Among them, a few suffered from manufacturing errors that were not
axicons.
ally symmetric Gaussian field that is con- watts Sapphire laser (488 nm, by macroscopic
1.5 mm width, inspection. Nevertheless, a consistent major alteration in the beam shape
sidered a bundle of parallel rays along the Coherent Inc./Germany) withbecameGaussianapparent when replacing the reference well-structured axicon with another one o
distri-
characteristics. The beam profiles change and beam widths varied by a factor of 1.5-5
propagation axis strikes an axicon with the bution for fluorescence excitation. The beam
demonstrates the comparison between the effects of two commercially available ax
refractive index n (Fig.1A), the rays will re- is guided toward a beam-shapingangle: 170°),unitonede-with manufacturing error (left- Fig. 3-A1 to -D1) and one with a high
fract individually upon arrival at the conical scribed by Saghafi. et. al. [26] for gradual
structure al- 3-A2 to -D2), on an incident beam with Gaussian distribution. The
(right- Fig.
surface forming highly focused points with teration from Gaussian to ashown at the focus (minimum width) and 5mm away from the focus.
flattened-Gauss-
a gradual change towards becoming a ring ian beam. The semi-uniform beam is guided
(Fig.2). toward a 170 º axicon (Asphericon/Germany)
Static Light Sheet Microscopy Setup Using Well-Structured Conic Lenses
placed at a 50 mm distance (element-1). A
We built the setup according to DE102010046133B4-patent using 200 milliwatts Sapp
Effects of Non-Ideal Axicon on precision-aspherized-achromatic(488nm,lens of focal
1.5mm width, Coherent Inc./Germany) with Gaussian distribution for fluores
the Incident Gaussian Beam length F1 (element-2) is placed at a distance
excitation. The beam is guided toward a beam-shaping unit described by Saghafi. et.
As pointed out before, the precision in the of 2F1 from the flat surface offorthe
gradual
axicon pro- from Gaussian toNaEflattened-Gaussian
alteration W beam. The semi-uniform
fabrication of the conic elements is a chal- ducing a magnified axially guided towardbeam
symmetric a 170º axicon (Asphericon/Germany) placed at a 50mm distance (eleme
from the flat surface of the axicon120i Infinity

a magnified Corrected
lenging task and can affect the final results with optimized parameters.precision-aspherized-achromatic
By guiding this lens of focal length F1 (element-2) is placed at a distan
producing axially symmetric beam with
massively. An axicon with high precision is beam toward an achromatic-cylinder lens of By guiding this beam toward an achromatic-cylinder lens of foca
usually expensive compared with the com-
optimized parameters.
focal length F2 (element-3) placed
length 𝐹𝐹at
! (element-3)
Objectives
F1+F2, a placed at F1+F2, a controlled ellipticity is introduced to the beam
mercially available ones. Depending on the Finally,toa the
controlled ellipticity is introduced positive
beamachromatic-cylinder lens of focal F3 (element-4) was placed at a dist
materials, methods, and precision utilized in structure. Finally, a positive√2𝐹𝐹 ! from F2 and enables us to form
achromatic-cyl- Designed to reduce
a thin sheet of light the overall
as can be seenweight
in Figure 4. Th
detection
inder lens of focal F3 (element-4) unitplaced
includes a customized microscope equipped with corrected objectives for
their manufacturing, the prices could differ. was and size of an imaging system while
In our laboratory, we used a wide variety at a distance of from Fcompensating
2 and enables us
refractive index mismatches, a tube lens, a computer-controlled band-pas
maintaining optical performance:
wheel for blocking the fluorescence excitation light, and a scientific-grade CCD camera
of axicons. Among them, a few suffered to form a thin sheet of light sCMOS,
as can be seen in 6.5 µm pixel, UK).
5 Megapixel,
from manufacturing errors that were not Figure 4. The detection unit includes a cus- • Reduces system length by up to
detectable by macroscopic inspection. Nev- tomized microscope equipped with corrected 42% compared to conventional
ertheless, a consistent major alteration in objectives for compensating refractive index microscope systems
the beam shape and width became apparent mismatches, a tube lens, a computer-con-
when replacing the reference well-struc- trolled band-pass filter wheel for blocking • Provides easy integration into
tured axicon with another one of identical the fluorescence excitation light, and a sci- many machine vision systems
characteristics. The beam profiles changed • Designed for use with next
generation imaging sensors
Find out more:
www.
edmundoptics.eu/
imaging
Visit us at
June 27-30, 2023 | Booth B1.415
Contact us:
EU/UK: +44 (0) 1904 788600
GERMANY: +49 (0) 6131 5700 0
Fig.2: Beam propagation through Axicon; A) Theoretical analysis, B) Real Axicon (Asphericon/ FRANCE: +33 (0) 820 207 555
Germany), C) At short distance after the tip of the Axicon, D) at the middle of the line of fo- sales@edmundoptics.eu
cused points, E) Ring formation after the focus.
Light Microscopy
Fig.4: Left-side; Schematic drawing (top-view and side-view) of the

axicon-based design for the static light sheet microscope including 1)
Axicon, 2) a precision-aspherized-achromatic lens of focal length F1,
3) achromatic-cylinder lens of focal length F2, 4) positive achromat-
ic-cylinder lens of focal F3, 5) Container filled with liquid and the
sample. Right-side; The actual system.
Fig.3: Effects of two 170° axicons on a 5mm laser beam with Gauss-
ian distribution; A1-B1) 3D laser profile at XY-plane and transversal
2D-laser beam profile normal to the propagation axis at 5mm away
from the tip of an axicon with fabrication error, respectfully, C1-D1)
3D transversal laser profile and transversal 2D-laser beam profile Fig.5: The 3D-reconstructed images of a forelimb of Thy1 GFP mouse
normal to the propagation axis at the center of the line of focused obtained by the Axicon-based light-sheet microscopy demonstrating
points at 12mm away from the tip of an axicon with fabrication error, fine details of the muscles, cartilage, tendons, bones, and GFP-positive
respectfully, A2, B2, C2, and D2 have the same expressions as given neuronal structures neuronal structure (outer and inner). For detection,
in A1, B1, C1, and D1, just for a high-precision 170° axicon, respect- a 2X objective (NA 0.14, Olympus/Japan) equipped with a modulator
fully. for compensating the refractive index mismatch was used.
entific-grade CCD camera (Neo 5.5 sCMOS, structure as shown in Figure 5. These imag- Affiliation
1Bioelectronics Section, FKE, TU Wien,
5 Megapixel, 6.5 µm pixel, UK). es were obtained from single stacks of im-
ages, no stitching was done and no axially Vienna, Austria
2Brain Research Institute-CBR, Medical Uni-
swept light sheet technique was used [27,28].
Experimental Results It demonstrates that this affordable system versity of Vienna, TU Wien, Vienna, Austria
3Ins. of Biomedical Electronics, TU Wien, Vi-
provedies excellent spatial resolution due to
The setup was used for imaging chemically the long width of the thin part of the light enna, Austria
cleared tissues of the forelimb of a 2-week- sheet.
old Thy1 GFP mouse. We used the 3DISCO Contact
clearing method to make the sample trans- Dr. Saiedeh Saghafi
parent. The dehydration was done by as- Conclusion Bioelectronics Section,
cending tetrahydrofuran (THF) series and FKE, TU Wien
dibenzyl-ether (DBE) as the clearing solu- It can be concluded that Meso-aspheric opti- Vienna, Austria
tion. Dehydration lasted for one week. Using cal lenses can be highly useful elements for saiedeh.saghafi@tuwien.ac.at
Amira (Thermo Fisher Scientific, USA), 3D designing laser beam shaping devices that are
images were constructed from 1600 optical useful for many applications, such as non-in-
sections that were obtained by an optically vasive 3D imaging techniques. However, the References:
corrected 2X objective (NA 0.14, Olympus/ results depend highly on the presence of man- https://bit.ly/IM-Saghafi
Japan) revealing the fine details of tissue ufacturing errors limiting precision.

Light Microscopy

Optical Sectioning with High Speed and Low Phototoxicity
Jeremy Sanderson1
© Jürgen Mayer
F
luorescence labeling is an integral
technique for biomedical and life
science research, yet the light flux
required to visualize and capture images
of cellular structures is very large. Living
organisms and cell cultures cannot with-
stand high flux densities. The light sheet
microscope enables researchers to image
tissues very fast in three dimensions with
minimal photobleaching. For this reason,
it has been called “the smart and gen-
tle microscope” [1]. The light sheet
was developed in 2004 by Ernst
Stelzer’s group at EMBL to study
developing cells, small organisms,
and large volumes of tissue.
A 3-D rendering of a cleared adult mouse brain. Tyrosine hydroxylase-positive dopaminergic neurons labeled with Alexa 488, pseudo-colored in
cyan. The parvalbumin-positive interneurons, labeled with Rhodamine red-X are shown in yellow, and the choline acetyltransferase in cholinergic
neurons labeled with Alexa 647 is seen here in magenta. The image was taken using a Bruker Luxendo LCS SPIM table-top system for large
cleared samples. Scale is 1.0 mm. The file size was 828 Gb. Rendering was performed with Imaris Viewer 10.0.0 and Fiji 2.9.0. With thanks to
Dr. Jürgen Mayer, Applications Specialist at Bruker for his help in acquiring and presenting this image.

Light Microscopy
One of the reasons put forward as an advantage for lightsheet microscopy was the ability
to image whole embryos and organs, rather than preparing thin samples or growing cells
mounted on a coverslip.
Fig. 1: Architecture of the

light sheet microscope.
Optical Sectioning The distance where the thickness of the light be illuminated from two sides. Recently, var-
sheet remains reasonably constant is called ious light sheet microscope configurations
The confocal microscope is the workhorse of the ‘confocal parameter’, which determines have been developed that either use the same
fluorescence imaging, optically sectioning the axial resolution and optical sectioning objective for illumination and detection or
the sample to create blur-free images. It is ability. Significantly thinner light sheets can illuminate the sample and detect the image
perfectly good for many applications but is be generated by using interference methods from the same side, so-called ‘open top’ con-
not ideal for imaging delicate living samples. to create a lattice sheet from a non-diffrac- figurations, which allow much larger speci-
In a conventional fluorescence widefield or tive Bessel or Airy beam instead of a Gauss- mens to be investigated.
confocal microscope, the objective acts as ian beam. This focused beam is scanned
its own condenser with both illuminating across the field of view and a camera with
light and emitted signal traveling on-axis a global or rolling shutter is used to acquire Sample Imaging
through the objective. The light sheet micro- the image only from the vicinity of the illu-
scope also creates optical sections similar to mination beam at any instant. This approach One of the reasons put forward as an ad-
the confocal microscope but with the illumi- is called digitally scanned laser light sheet vantage for lightsheet microscopy was the
nation objective forming a sheet of light set microscopy. ability to image whole embryos and or-
orthogonally side-on to a separate imaging Since the illumination usually penetrates gans, rather than preparing thin samples
objective (Fig. 1). By uncoupling the illumi- the sample from one side, obstacles lying or growing cells mounted on a coverslip.
nation and detection axes, the light sheet in the path of the light sheet can disturb A range of mounting methods is possible
microscope only illuminates a single layer its quality by scattering or absorption of [3]. The classic method is to hold the sam-
of the sample at a time. This makes it much the light. This causes unevenly-illuminated ple within a chamber in a molded agarose
faster and less phototoxic than the classical datasets and striping artifacts [2]. The sim- block or extrude a low melting-point aga-
laser confocal microscope. plest solution is to rotate the sample, which rose cylinder into the sample chamber from
In its simplest configuration, the light is then sequentially illuminated from alter- Teflon FEP tubing with a refractive index
sheet is generated as a Gaussian beam with nate sides and the image fused into a sin- similar to water.
cylindrical optics. Extra lenses, slits, and gle dataset. Another approach is to add a Biological tissues are opaque and scat-
mirrors are used to shape and steer the beam. third objective lens so that the sample can ter light; they require some form of chem-

Light Microscopy
fore developed. Weiss et al. [4] is a useful

Manufacturer System(s) QR-Code for URL starting point for working with clearing
agents.
As with any other emergent technology, the
light sheet microscope was first home-built
3i Lattice LightSheet
in a few reference laboratories. OpenSPIM
[5] exists to create an open-source [6] and
community-driven group for the develop-
ment and improvement of open-source light
Single-Objective Light Sheet/
Applied Scientific Instrumentation sheet microscopy. A sophisticated wealth
iSPIM/diSPIM/ct-dSPIM/oSPIM
of material [7-10], including a full parts
list, costings, and construction guide, is
available with step-by-step instructions for
MuVi SPIM/InVi SPIM Lattice building a microscope.
Bruker
Pro/Luxendo LSM The introduction of the Zeiss Z1 in 2012
was the first commercially-available turn-
key system, taking 6 years to develop the
complex lenses to form the light sheet. Since
Evident/Olympus Alpha3 Light Sheet Microscope
then, a wide range of different light sheet
microscopes has appeared on the market
(Tab. 1).
Stellaris 5 and Stellaris 8
Leica Instruments
Digital LightSheet (DLS)
Summary
The optical sectioning confocal microscope

is widely used to create blur-free images.
LifeCanvas Technologies SmartSPIM/MegaSPIM
The light sheet microscope also creates op-
tical sections but only illuminates a single
layer of the sample at a time. It is there-
Airy Beam Light Sheet imaging
fore much faster and less phototoxic than
M Squared Lasers the classical laser confocal microscope.
system
The light sheet is generated as a Gaussian
beam with cylindrical optics in its simplest
configuration. Thinner lattice light sheets
MBF Bioscience ClearScope have evolved from the initial Gaussian
beam configuration, and open-top config-
urations allow large specimens to be inves-
tigated. Concomitant with the evolution of
the light sheet microscope has been the
Miltenyi Biotec UltraMicroscope II
development of various methods to clear
tissues and embryos. The light sheet mi-
croscope was first home-built. OpenSPIM
LS1 Live light sheet exists to create an open-source platform to
Viventis microscopy system aid the self-building of light sheet micro-
scope systems.
Affiliation
Zeiss Lattice Lightsheet 7/ 1Bioimaging Facility, MRC Harwell Institute,
Zeiss
Zeiss Lightsheet 7
Oxfordshire, UK
Table 1: Commercially-available light sheet microscopes (this list may not be exhaustive).
Contact
Jeremy Sanderson
Bioimaging Facility Manager
ical clearing to visualize components deep ▪ hydrogel-based methods to preserve MRC Harwell Institute
within the tissue itself. The field of tissue tissue morphology Oxfordshire, UK
clearing has grown significantly alongside j.sanderson@har.mrc.ac.uk
the development of Light Sheet Microscopy. The solvent-based methods achieve a high
Clearing reagents are principally divided into level of tissue transparency relatively quick-
three categories: ly within several days but cause substantial
tissue shrinkage and quenching of endoge- References:
▪ solvent-based methods nous fluorescence signals. Aqueous and hy- https://bit.ly/IM-Sanderson
▪ aqueous-based methods drogel-based clearing methods were there-

Light Microscopy
Fig. 1: Virtual microscopy offers users the

ability to digitally microscope specimens
independent of time and location.
© PreciPoint GmbH
Going Digital in Pathology
Introducing Virtual Microscopy and What Questions to Ask
Katharina Eser1
T
he pathology laboratory as an insti- Shortage of Pathologists Worldwide pose, microscopic preparations are digitized
tution is undergoing change. Even and can thus be viewed and processed on
with a time lag, this immanently Today cancer rates are increasing and at the a screen later irrespective of the location
important medical discipline is turning to same time, the number of people who can and/or workstation. These digital prepara-
digitalization: The laboratory is becoming treat and detect it is decreasing. Many parts tions can be stored in databases and shared
virtual. Part of this process is also virtual of the world are medically underserved, but with an infinite number of users. To gener-
microscopy, which supports the change to even in the richest countries, there is a short- ate a digital image of a specimen, an analog
digital pathology. Many pathologists still age of specialists like pathologists. The rea- microscope can be used, equipped with an
look through an analog microscope while sons for this range from too little education additional camera. However, development in
deciding whether the small section of tis- and advertising during medical school to the pathology is tending toward the use of dig-
sue that lies in front of them as a sectional emotional factor that working in a labora- ital microscopes. Depending on the model,
preparation is infused with tumor cells. In tory is isolating and contact with patients these can usually not only produce a live
other laboratories, this task is already per- is often limited to looking at their tissue. image of the specimen but also scan it. Dig-
formed by an automated system that inde- But there is also the fact that most diseases ital microscopes not only show a single field
pendently places the sectional preparations become more complex the longer they are of view but scan the entire specimen.
under a scanning microscope, which scans looked at. The amount of data that would Digitized microscopy slides can be called
the sample and finally, an artificial intel- be necessary to identify certain correlations virtual slides, scans, or whole slide images.
ligence identifies, marks, and counts the cannot be provided by human hands. There- These terms describe a fully digitized micros-
tumor cells. To take this step, not only do fore, the possibilities that the digitization of copy specimen. To produce digital images,
you need the right equipment, but you also a pathology laboratory brings are infinitely the instrument scans the entire specimen
need a new workflow in the lab and person- attractive. on the slide piece by piece. The software
nel trained to do it. This article will help to An important mainstay of pathology is merges the resulting high-resolution indi-
highlight the challenges along the way and viewing tissue samples under a microscope. vidual images into a complete image. This
the issues that arise. Virtual microscopy offers users the abil- process is called stitching. On the computer,
ity to digitally microscope specimens inde- users can navigate over the sample, magnify
pendent of time and location. For this pur- it and analyze it.

Light Microscopy
© PreciPoint GmbH
Table 1: Having a digital workflow makes the work in a pathology lab faster, more efficient, and makes room for innovations.
Quality of the Specimen Is Essential and resolution. A higher scanning speed Pathology Today Is Manual Work
leads to a loss of image quality. However,
As with all microscopic procedures, the since these devices operate autonomously, Currently, in most cases, samples that need
quality of the specimens also plays a ma- the time loss can also be adjusted by adjust- to be examined in the pathology laboratory
jor role in virtual microscopy. The speci- ing the working time of the scanner, for arrive with a submission slip on which it is
mens must be cut as evenly as possible, as example at night. noted by hand what is to be done with them.
the software automatically sets focal points To make full use of microscopic scans, This information is transferred to the lab-
during the scanning process. Excessive suitable image-viewing software is required. oratory information system by staff. After
height differences can lead to plane jumps Depending on the image format, only very a macroscopic examination of the tissue by
and blurred areas in the finished scan and specialized programs can process images for a pathologist, medical technicians prepare
cannot be corrected. The specimens must pathology slides. The so-called viewing soft- the samples for further examination. With
also be within the fixed scan area of the ware offers different possibilities to evaluate sometimes considerable manual effort, these
instrument. Specimens must be uniformly the images as well. With different annotation specimens are prepared, cut, fixed in kero-
stained to correctly represent all cell struc- tools, for example, lines and circles can be sene, and stained using various histochem-
tures. In addition, air pockets, overlaps, and drawn in or written notes can be attached. In ical and immunohistological techniques;
other contamination of the samples should addition, it is also possible to integrate arti- they are cut, mounted on a glass slide, and
be avoided. In special cases, the nature of ficial intelligence into such programs. With covered with glass. The specimens are then
the sample recedes into the background. For the help of integrated AI, an automatic eval- sorted into folders and submitted to pathol-
example, during surgery for tumors, sections uation of certain structures or cells becomes ogists for examination. In some cases, the
of the removed tissue are often made during possible. Ideally, the annotations and evalu- specimens are also scanned. The specimens
the procedure, the so-called frozen section. ations can be stored according to the images. must also be manually inserted and regis-
Then only certain regions of the sample are It is possible to integrate viewing software tered for this purpose. If there are quality
viewed under the microscope. into a cloud. That way scans can not only defects, the process must be repeated. This
The quality of the digital specimen is also be shared with other users via the web server workflow, which is only roughly outlined
dependent on the quality of the camera used. but can also be viewed directly on the plat- here, involves many manuals, and small-
A camera attachment on an analog micro- form. In addition, specific information about scale work steps with numerous sources of
scope usually does not provide high quality, the images can often be provided. In most error.
as these systems are not designed for digi- cloud services, image storage, image sharing, At the other end of the development
tization processes. Digital microscopes are and image viewing facilities are available. towards a fully digitalized pathology labora-
designed for this process and, in addition to Viewing the scans is possible with any end tory, the automatic scanning of large quan-
the scanning function, have a live view so device. It doesn’t matter whether it’s a large tities of sectional preparations and digital
that the specimen can be viewed live on the screen, smartphone, tablet, or laptop. How- provision for diagnostics in conjunction with
screen. Pure slide scanner devices offer the ever, the nature of the screen is decisive for clinical data as well as digital report text
user the possibility to choose between speed the reproduced image quality [1]. generation are on the horizon. The system

Light Microscopy
can prioritize and process orders after the

registration of incoming specimens, and it
handles quality control. In addition, artificial
intelligence is used to support histopatho-
logical diagnostics. Furthermore, the system
can integrate the analyzed image data and
molecular information into the workflows. In
the meantime, several research projects are
approaching the realization of this vision,
revealing the actual opportunities and chal-
lenges of such a theory.
Algorithms Open a Wide Range
© PreciPoint GmbH
of Possibilities
Although there are advantages to a digital

image, it does not solve the many questions
and requirements that users have. However, Fig. 2: Having a digital sample makes it possible to have algorithms take over the costly job of
digitization opens a wide range of possi- counting and annotating.
bilities in image analysis using algorithms.
Classical algorithms can detect and count
well-defined structures such as tumor cells. specialized for specific disease patterns; their is also, of course, the question of personnel
This enables the pathologist to quantify by task is to recognize and quantify predefined and workflow in the laboratory.
a concrete measured value. In doing so, the structures. An algorithm can only be as good
algorithm proceeds efficiently and without as the quality of the data sets with which it
bias. Factors such as stress or time pressure has been trained [4]. The greater the number Conclusion
as well as the influence of optical illusions, and the more variable the spectrum of struc-
which affect humans, do not occur here. tures sought, the better and more reliable the The transformation of a pathology labora-
There are now many products on the mar- evaluation. This is where biobanks, which tory into a digitized pathology laboratory
ket that can be used for different analysis are now being established worldwide, play can only be implemented step by step. At
approaches. These programs can find pre- an important role. These provide not only the beginning of this process is the docu-
defined structures quickly and efficiently many physical samples but also numerous mentation and visualization of all processes,
and quantify them reproducibly. There are samples that have already been digitized. The which must be analyzed according to vari-
numerous studies describing the application next step is algorithms that are trained spe- ous parameters such as personnel, machines,
of algorithms in histological preparations of cifically for the application needs of users. and degree of development, and at the IT
different organs and various diseases [3]. Here, too, an interesting range of products and process support level. From this, mean-
Typically, these algorithms are trained so is developing [2]. ingful planning for the transformation can
that experts mark defined structures in a his- The challenge is the question of what for- emerge. Part of this is virtual microscopy,
tological section. The algorithm is trained mat the data sets obtained are converted into the equipment that meets the requirements,
with a series of similar sections until it rec- and how they are then finally integrated into and the algorithms that support the work.
ognizes the marked structures on its own. the laboratory information system and the There are now many companies that special-
Common programs on the market are usually systems of the departments concerned. There ize in helping labs with this transformation.
This is a very sensible service because this
transformation is complex, it costs time and
money, and it also must be well supported in
terms of personnel to function.
Affiliation
1PreciPoint, Freising, Germany
Contact
Katharina Eser
PreciPoint GmbH
Freising, Germany
katharina.eser@precipoint.de
www.precipoint.com
© PreciPoint GmbH
References:
https://bit.ly/IM-Eser
Fig. 3: The correct sample preparation is key in virtual microscopy.

Light Microscopy
Turnkey Multiphoton Microscopy

for 3D Samples
Label-Free and 4D Imaging in Life-Science and Medicine
Stefanie Kiderlen1 and Lukas Krainer1
C
ompared to epi-fluorescence wide- key element here was the understanding even 4D in vivo imaging can be used to eval-
field (WF) and laser scanning confo- that cells behave differently in a 3D sur- uate samples on the macro-scale as shown
cal techniques, multiphoton micros- rounding than in standard 2D cell culture in figure 2. However, when it comes to sam-
copy (MP) is highly superior when it comes [2]. As a result, experiments are becoming ples in the mm-cm macro scale, additional
to imaging depth or the identification of more complex, i.e., using awake animals or sample processing techniques such as tissue
label-free biomarkers. Here, excitation in vitro 3D cell cultures. The future requires clearing can be applied to enhance imaging
with a femtosecond laser typically between a next-generation microscope that is agile depth and increase the informational 3D vol-
780-1300 nm produces nonlinear optical and adaptable with features that lower the umetric content of these samples, as shown
fluorescence. The intensity of the gener- user barrier, expand on diagnostics with in figures 1 and 2.
ated signal increases with the square of multiple modes, and save resources and
the laser peak power (for 2-photon) or the time while improving results. With this
third power (for 3-photon). This phenom- concept in mind, Prospective’s MPX was Tissue Clearing for Whole Organ Imaging
enon is confined to a very tight focal vol- engineered to be suitable for label-free as
ume, significantly reducing the absorption well as 3D/4D imaging from the macro to Tissue and 3D cell constructs are highly
cross-section from out-of-focus planes. the micro-scale. The MPX-multiphoton mi- inhomogeneous, making them challeng-
Moreover, using wavelengths in the NIR croscope is a turnkey, compact, and fully ing to image due to light scattering and
range leads to lower scattering in tissue integrated next-generation multimodal absorption. Increasing the optical trans-
and thus yields higher penetration depth microscope, combining different imaging lucency by tissue clearing techniques can
and lower photodamage compared to linear techniques in one easy-to-use and por- enhance the imaging depth by a factor
confocal techniques [1]. By engaging dif- table device: non-linear MP microscopy >10. However, tissue-clearing methods
ferent imaging modalities, a broad range of (two-photon, higher harmonics - SHG & suffer from some drawbacks: organic sol-
applications including 3D, label-free, deep THG) and linear WF microscopy epi-fluo- vent-based clearing methods shrink the
tissue, live-animal, whole organ, and whole rescence and fluorescence lifetime (FLIM) sample, whereas water-based methods of-
slide imaging can be addressed. Here, we to maximize informational content and ten don’t yield the same clearing effect [5].
present high-quality imaging of 3D and 4D to yield complementary data sets, rang- Moreover, only a few reagents are known
samples as well as a label-free and non-de- ing from single cells up to living animals for tissue clearing in living organisms, so
structive imaging of unique biomarkers to as shown in figure 1 [3]. Here, the MPX clearing protocols are mostly related to
understand structural and functional rela- addresses a broad variety of samples from fixed samples. 3D imaging in fixed sam-
tionships in healthy and diseased tissue with the micro to the macro scale, starting with ples provides the advantage that there are
time-saving, multimodal MP microscopy ex vivo 3D cellular models comprising so- many staining and processing methods to
delivering orthogonal informational content called spheroids and organoids. improve the image quality, signal intensity,
and enhanced imaging depth. However, to mimic a healthy 3D sur- or penetration depth, however, they only
rounding, these models are limited in size represent a snapshot of the tissue. Figure 2
because of arising hypoxia in the core when shows a whole cleared mouse brain stained
One Size Fits All: growing bigger than ~400 um in diameter for neurons (green). The penetration depth
From the Micro to the Macro Scale [4]. To overcome size limitations because of here was limited by the working distance
necrotic cores 3D models with a vasculariza- of the objective (4 mm) and could be in-
3D and 4D ex vivo and in vivo imaging tion system such as tumors grown on a CAM creased by tissue clearing by a factor of
have gained increasing importance. The membrane, 3D printed cell constructs, or >10.
Fig. 1: One size fits all: the MPX provides a high flexibility and working space for a multiple range of samples from the
macroscale down to the microscale: key applications are 3D and 4D, label-free, deep tissue, whole slide, whole organ, and
live-animal imaging.

Light Microscopy
Intravital 4D Deep Tissue Imaging with lower photodamage are major advan- intrinsic biomarkers. Here, timesaving,
tages of MP microscopy. depth-resolved, sample-saving and non-in-
To investigate not only structural but also vasive data acquisition is highly beneficial.
functional relationships, living tissue and Label-free biomarkers can originate from op-
cells must be imaged in 4D time series. How- Label-Free Biomarkers for tical or structural properties and phenotype
ever, as mentioned before, optical clearing Translational Diagnostics of the sample. Currently, the workflow for
methods are mostly used for fixed samples. diagnosis from a biopsy or resection is tis-
To do 4D timelapse deep tissue imaging of The identification of label-free biomarkers sue fixation followed by paraffin sectioning
living cells and animals, the right sample using multimodal MP imaging has rapidly and histological staining e.g., H&E staining.
must be chosen. In figure 3 we demonstrate spread throughout biomedical research in This is time-consuming, requires experienced
zebrafish 4D time-lapse imaging. Zebrafish the past few years. A few nonlinear optical technicians, and includes toxic staining re-
have long served as invaluable subjects in modalities are being extensively considered agents. The use of label-free biomarkers in
the fields of metabolic pathology, develop- for clinical purposes. For example, two-pho- pathology would provide a faster and easi-
mental biology, and neuroscience. Its status ton, three-photon, second-harmonic gener diagnosis. Here, we demonstrate SHG and
as a vertebrate and its sustained transparency eration (SHG), third-harmonic generation two-photon imaging as powerful diagnostic
in the larval stage makes it a highly practical (THG), coherent anti-stokes Raman spectros- modalities providing endogenous molecular
and expedient live model for research. Figure copy (CARS), stimulated Raman spectrosco- and chemical distinction for differentiating
3 shows 4D time-lapse neuronal tracking of py (SRS), and fluorescence lifetime imaging healthy from diseased tissue as shown in
retina cells and the migration of neurons in (FLIM) are all used in research [7,8]. They all figure 4. The combination of both modali-
the spinal cord of 4-day-old zebrafish larvae. have unique advantages and disadvantages. ties provides a label-free contrast originating
Another prominent example of 4D However, combining modalities to maximize from molecules such as NAHD, FAD, elastin,
in vivo imaging is the investigation of neu- informational content and yield complemen- proflavine, and hemoglobin, providing in-
ronal activity in mouse brain using standard tary data is necessary when it comes to na- trinsic endogenous fluorescence or structural
markers such as GCaMP as shown in fig- tive biological samples since they are highly properties from non-centrosymmetric mole-
ure 2. GCaMP is a fluorophore-tagged and heterogeneous [1]. As described above this cules like fibrous collagen [9]. Morphologi-
genetically modified calcium indicator that heterogeneity on the one hand making it cal and structural changes in the cell or the
responds to the efflux of calcium (Ca2+) [6]. challenging to image these samples, but on surrounding matrix e.g. the nucleus shape
Overall, higher penetration depths combined the other hand allows to identify label-free in cancerous tissue or the alignment and
amount of collagen fibers in wound healing,
fibrosis, and tumors can serve as valuable di-
agnostic tools.
Engage Imaging Speed and Resolution –

Epi-Fluorescence Guided MP Microscopy
Even though non-linear microscopy tech-

niques are highly beneficial for deep tis-
sue and label-free imaging they are scan-
ning-based techniques with a limited ac-
quisition speed making them slower than
epi-WF microscopy. However, engaging the
speed of WF and the confocal depth resolv-
ing properties of non-linear microscopy can
be used for epi-fluorescence WF-guided MP
microscopy. Here, a WF overview fast scan
serves as guidance to the region of interest
(ROI) which is then imaged in detail using
MP microscopy [3]. The capability to per-
form multimodal analysis, on the same re-
gion of interest without the inconvenience
of moving the sample to another system,
maximizes content, provides orthogonal
data, and saves time as shown in figure 4.
Conclusion and Outlook
High-quality imaging of 3D and 4D samples

as well as label-free and non-destructive
Fig. 2: Intravital MP mouse brain imaging of GCaMP-expressing neurons in the cortex and deep imaging of unique biomarkers is becoming
tissue imaging of a fixed and cleared mouse brain. a) Microscope setup of live mouse in-vivo more important in understanding cellular
brain imaging and b) close-up of the cortex at ~300 µm z-position in depth, c) whole cleared mechanisms and their structural and func-
mouse brain, d) volume scan of ~3.5 mm in depth, and e) one exemplary z-plane showing tional relationship. Engaging different im-
stained neurons (green). Scalebars: 100 µm aging modalities, a broad range of key ap-

Light Microscopy
Fig. 3: MP 3D volume scan (a) and timelapse imaging (b-k) of zebrafish larvae. a) 3D projection of a fixed zebrafish larvae expressing prox1:Ta-
gRFP and stained with Hoechst (cell nuclei – blue) and Phalloidin (actin – green). b-f) Migration of retina cells in a living zebrafish eye imaged by
time-lapse microscopy. 4-day-old zebrafish larvae expressing tp1:LifeAct-mCherry (red) with the respective close-ups. Scalebar: 50 µm (b) and 20
µm (c-f). g-k) Neuronal tracking of cells in the spinal cord in a living 4 day old zebrafish larvae alpha-catenin-YFP (green), tp1:mCherry-NLS
(red), and the SHG signal from the muscle (blue) with the respective close-ups. Scalebar: 100 µm (g) and 20 µm (h-k).
copy techniques such as WF-guided MP

microscopy can save time and provide addi-
tional data by delivering fast overview scans.
Future developments include improvements
for even higher imaging resolutions and
depth by implementing adaptive optics (AO)
techniques and three-phtonon microscopy.
Acknowledgments
We are thankful for the support from the fol-

lowing collaborators: Amelie Erben and Dr.
Stefanie Sudhop, UAS Munich; Dr. Kim Fer-
rari and Prof. Bruno Weber, University Zurich;
Dr. Sara Caviglia and Professor Stephan Neu-
hauss, University Zürich, Dr. Linda Waldherr,
University Graz, Dr. Branislav Zagrapan, LKFH
Feldkirch and Monika Puchalska, Nencki Insti-
tute of Experimental Biology Warsaw for pro-
viding samples, images and dedicating time
and resources to the experiments mentioned.
Fig. 4: Epi-fluorescence widefield guided multiphoton microscopy of a human label-free skin mela-
noma FFPE section. Fast WF overview scan (a) and H&E stained ground truth (b) for orientation Affiliation
1Prospective Instruments LK OG, Dornbirn,
and respective close-ups of healthy tissue (d,c and h,i), interface between healthy and diseased tis-
sue (e and j), and regions in the lesion (j,g and k,l). a) Endogenous fluorescence collected by epi-flu- Austria
orescence λex./λem. 390/432 nm (magenta), 475/515 nm (green), and 555/595 nm (orange). c-g)
Two-photon endogenous fluorescence collected at λex./λem 1040/542nm (green) and 1040/595 nm Contact
(red) and the SHG signal at 520 nm (blue). Scalebar: 1 mm (a&b) and 200 µm (c-l). Dr. Stefanie Kiderlen
Prospective Instruments LK OG
Dornbirn, Austria
plications including 3D imaging, label-free, ing depth, and save time in 3D in vivo and stefanie.kiderlen@p-inst.com
deep tissue, live-animal, whole organ, and ex vivo samples as well as large area tis-
whole slide imaging can be addressed. sue sections. Higher penetration depth can
Here, we demonstrated the use of mul- be achieved by tissue-clearing techniques to References:
timodal MP microscopy to gain orthogo- enhance optical translucency in fixed sam- https://bit.ly/IM-Kiderlen
nal informational content, enhance imag- ples. We showed that using different micros-

Correlative Microscopy
© TU Wien
Correlative Microscopy of a Catalytic Reaction
Zooming in on Chemical Patterns in Hydrogen Oxidation on Rhodium
Philipp Winkler1 and Günther Rupprechter1
S
tudying spatiotemporal phenome- history (i.e., it exhibits multistable kinetics) of reactants with active sites on the cata-
na can deepen the understanding of [4]. In addition, the surface can also undergo lyst surface [7], enables improving the exist-
catalytic reactions and thus enable periodic switches between these states of ing and tailored design of new catalytically
the designing of better catalysts. Often, catalytic activity at some constant exter- active materials.
only combining several microscopy tech- nal parameters (i.e., it displays self-sustain- Due to the inherently necessary lateral and
niques in a correlative approach allows for ing kinetic oscillations) [5]. As the switches temporal resolution for such studies, micros-
capturing the full picture. Using three dif- between the two states occur via the spread- copy techniques are ideally suited to tackle
ferent electron microscopies, the forma- ing of reaction fronts, this can result in the these questions. Often, however, a single tech-
tion of chemical patterns in catalytic H2 formation of chemical patterns on the cata- nique alone cannot provide all data required
oxidation on Rh was studied and different lyst surface [6]. An atomic-level understand- for full understanding and several micros-
types of patterns and an island-mediat- ing of the mechanisms behind these phe- copies are thus combined in a correlative
ed propagation mechanism for reaction nomena, which result from the interaction approach.
fronts were observed [1].
Introduction
Heterogeneous catalysis is one of the key

enablers of sustainable energy generation
and storage. For example, renewable energy
can be stored in the chemical bonds of H2
and released on demand via catalytic H2 ox-
idation in a fuel cell, forming only water as
a product [2]. A fundamental understanding
of this reaction is therefore crucial for the
efficient operation of fuel cells [3].
Hydrogen oxidation on Rh can be in two
distinct states of catalytic activity, depending
on the set of external parameters: In the inac-
tive state, the surface is covered by oxygen
and water formation is inhibited, while in the
active state, the surface is nearly adsorbate
free and water is formed. In some regions
of the parameter space, the system can even
be in both states of catalytic activity at the Fig. 1: The experimental approach. Three different electron microscopies, each providing differ-
same set of external parameters, where the ent types of information, were applied in a correlative way in order to study the formation of
actual state only depends on the system pre- chemical patterns in catalytic H2 oxidation on Rh.

Methods image brightness depends on the local diffrac- ever, had one common aspect: The observed
tive properties of the sample surface. These type of spatiotemporal behavior was always
For in situ studying pattern formation in properties are influenced by several factors, characteristic of each domain in its entirety.
catalytic H2 oxidation on Rh, three different including the local atomic structure or the Just recently, during further UV-PEEM
microscopies were combined, each of them presence of adsorbates on the sample surface. studies, a curious situation was observed:
yielding complementary pieces of informa- All three techniques were combined for in on the same sample at specific exter-
tion, as shown in Figure 1 [1]. In UV pho- situ studies of the oscillating catalytic H2 oxi- nal parameters some domains were in a
toemission electron microscopy (UV-PEEM), dation in the 10-7 mbar pressure range on indi- steady state of catalytic activity or exhib-
the sample is illuminated by UV-light and the vidual domains of a polycrystalline Rh foil. ited patterns spanning the whole domain,
resulting low-energy photoelectrons are used The local atomic structure of each of these while other domains appeared to be frag-
for imaging by an electron optical system. The domains (several 100 µm in size) was deter- mented into multiple areas oscillating inde-
local image brightness is based on the local mined beforehand, turning the sample into a pendently from each other or not at all. Due
surface coverage of adsorbed reactant mole- library of different surface structures [9]. All to the limited lateral resolution of the used
cules and can thus serve as an indicator for three microscopies were applied to the exact UV-PEEM, details were hard to discern and
the local catalytic activity of the surface (ki- same domains on the same sample under these observations warranted further anal-
netics by imaging [8]). When the sample is in- identical external parameters (temperature, ysis by experimental techniques with higher
stead illuminated by monochromatic X-rays reactant pressures), i.e., in a correlative way. spatial resolution.
using X-ray photoemission electron micros- Due to being hosted in a single experimental
copy (X-PEEM), similarly, photoelectrons are setup (the SMART instrument at UE49PGM
generated. In X-PEEM, the photoelectrons, beamline of the BESSY II synchrotron light Correlative X-PEEM and LEEM Studies
however, possess higher energy as they origi- source [10]), X-PEEM and LEEM were even
nate from the atomic core levels and therefore performed in a single experiment by switch- X-PEEM and LEEM, which are examples of
carry information on the surface composition ing between the two operating modes of the such techniques, were used to zoom in on
and identity. By selecting and using only pho- instrument, representing an example of cor- those domains displaying multiple uncor-
toelectrons of specific energy for imaging, di- relative microscopy in its purest form. related areas with a resolution down to sev-
rect local chemical information is encoded in eral nanometers. While the image contrast
the image. Due to the high brilliance of the in UV-PEEM and X-PEEM is a direct result
X-ray beam required for X-PEEM, such ex- UV-PEEM Studies of the surface chemistry and is thus easy to
periments can typically only be performed at understand, the diffractive properties of the
synchrotron light sources. In low-energy elec- UV-PEEM studies of the oscillating mode of surface responsible for the LEEM contrast
tron microscopy (LEEM), the sample is illumi- catalytic H2 oxidation on Rh in the past few are often hard to predict.
nated by low-energy electrons. The electrons years revealed a number of effects, which Therefore, in the first step, the oscillating
elastically backscattered at the sample surface influence the observed spatiotemporal struc- mode of catalytic H2 oxidation on Rh was
are then used for imaging, whereby the local tures [5, 11, 12]. All of these studies, how- observed using both X-PEEM and LEEM and
Fig. 2: Correlative microscopy of kinetic oscillations in catalytic H2 oxidation on Rh at constant T = 468 K, p(O2) = 5.0 x 10-7 mbar, p(H2) = 4.5 x
10-7 mbar. The oscillations were visualized by correlative X-PEEM (a) and LEEM (b). Two types of reaction fronts were identified using the chemi-
cal sensitivity of X-PEEM and allowed the understanding of the image contrast in LEEM (frames 1, 2: slow oxygen front; frames 3, 4: fast hydro-
gen front). Within the yellow dashed rectangles in (a), the image contrast is enhanced for better front visibility; the insets show line intensity pro-
files along the A-A’ lines.

Fig. 3: Chemical patterns in

catalytic H2 oxidation on Rh at
constant p(O2) = 5.0 x 10-7
mbar, p(H2) = 4.5 x 10-7 mbar,
observed by LEEM. At 428 K
(a) rotating double spirals could
be observed, while at 433 K (b)
broad reaction fronts propagat-
ed over the whole field of view,
and at 468 K (c) small islands
are formed, which collapse be-
fore being able to merge. The
QR codes lead to corresponding
videos, published under a CC
BY 4.0 license as Supplementa-
ry Material for Ref. [1].
by switching between these operating modes formed, which were consumed by hydrogen P08) and the Helmholtz-Center Berlin for
after each oscillation cycle (Fig. 2). Two differ- fronts before having the chance to merge Materials and Energy (HZB) by the alloca-
ent types of reaction fronts were observed and with other islands. tion of beamtime 212 10440 ST. The SMART
identified by selecting a proper energy win- These observations, which can be instrument was financially supported by the
dow for X-PEEM imaging: The slow reaction explained by considering the differing rel- Federal German Ministry of Education and
front (dark in X-PEEM, bright in LEEM) was ative velocities of oxygen and hydrogen Research (BMBF) (05 KS4WWB/4), as well
related to spreading oxygen coverage, while fronts at different temperatures, enabled us as by the Max Planck Society. We are grate-
the fast one (bright in X-PEEM, dark in LEEM) to rationalize the peculiarities observed in ful to J. Zeininger, M. Raab, Y. Suchorski,
is caused by diffusing and reacting hydrogen. the previous UV-PEEM experiments. M.J. Prieto, L.C. Ta˘nase, L. de Souza Caldas,
A. Tiwari, T. Schmidt, M. Stöger-Pollach, A.
Steiger-Thirsfeld, and B. Roldan Cuenya for
Chemical Patterns Conclusion contributing to the original work.
Due to the better suitability of LEEM for A correlative microscopy study of chemical
studying fast processes, the formed spatio- pattern formation in H2 oxidation on Rh Affiliation
1Institute of Materials Chemistry, TU Wien,
temporal patterns were then imaged primar- was performed using UV-PEEM, X-PEEM,
ily by LEEM. Visualizing the ongoing oscil- and LEEM, tackling important challenges Vienna, Austria
lations in a temperature range from 428 K in studying catalytic surface reactions. Due
to 468 K at constant p(O2) and p(H2) led to to their higher lateral resolution, X-PEEM
the observation of three different types of and LEEM allowed zooming in on process- Contact
patterns (Fig. 3): At 428 K, rotating double es just barely visible in UV-PEEM. Differ- Prof. Dr. Günther Rupprechter
spirals occurred; increasing the temperature ent types of spatiotemporal patterns and an Institute of Materials Chemistry
to 433 K resulted in the propagation of broad island-mediated propagation mechanism of TU Wien
reaction fronts over the whole field of view. oxygen fronts on the catalyst surface during Vienna, Austria
For oxygen fronts, an unusual island-me- kinetic transitions could be detected. guenther.rupprechter@tuwien.ac.at
diated front propagation mechanism could
be detected, where small oxygen islands
nucleate ahead of the main front and then Acknowledgment
grow, finally merging with the main front. References:
Upon increasing the temperature even more, This work was supported by the Austrian https://bit.ly/IM-Rupprechter
to 468 K, just small oxygen islands were Science Fund (FWF) (P 32772 N and F81

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Visualizat
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Electron and Ion Microscopy
4D Scanning Transmission Electron Microscopy

with Event-Based Electron Detection
How to Overcome the Bottleneck in Scanning Speed
Daen Jannis1,2, Christoph Hofer1,2, Chuang Gao1,2, Timothy Pennycook1,2, Johan Verbeeck1,2
4
D Scanning Transmission Electron
Microscopy (4D STEM) records a
diffraction pattern at each probe
position while scanning the electron beam
over the specimen where the probe posi-
tion dependence of the electron scattering
is recorded in detail with a Timepix3 cam-
era. This method offers a wealth of infor-
mation that can be used to perform ad-
vanced analyses that are impossible with
monolithic integrating detectors. Howev-
er, very slow scans have been the norm in
4D STEM due to the speed of available
cameras. Here, this speed bottleneck is
overcome by using an event-driven camera.
Introduction
Fig. 1: (a) The incoming probe is scanned over the specimen in a schematic setup, with the pix-
In Scanning Transmission Electron Micros- elated detector placed in the far field. The averaged diffraction pattern over the scan is indicated
copy (STEM), a probe formed by a beam of as PACBED. (b) Illustration showing the difference between frame and event-based detection.
electrons scans the specimen. Images are
formed by collecting the scattering as a
function of position. Conventional images high magnifications. To put the problem Experimental Setup
use detectors that integrate over an angu- in perspective, most cameras for 4D STEM
lar range of this scattering, providing an in recent years have had a frame rate of A schematic of the setup is shown in Fig-
intensity value at each probe position. For a few thousand frames per second. A fast ure 1(a). The signal driving the scan coils is
example, annular dark field (ADF) images, STEM scan will typically use a dwell time synchronized with the Timepix3 detector.
in which intensity is proportional to rough- of around µs. To match this speed, a cam- Since both the scan generator and detec-
ly the square of the atomic number, are era needs to provide a million frames per tor are synchronized to the same clock, the
formed using an annular detector that inte- second, which has yet to be achieved. How- time-of-arrival of each event can be used to
grates the signal from electrons scattered to ever, this time resolution can be realized by determine the probe position it came from.
higher angles. Many different detector ge- avoiding frame-by-frame acquisition in the Hence, every detected event has four coor-
ometries can be used, but by capturing the camera in the first place with event-driven dinates, probe position (2D), and scattering
scattering information in detail with a full operations. Each time an electron hits an angle (2D). From this event stream, one can
2D image at each probe position, 4D STEM event-driven camera the pixel location and determine virtual images such as ADF and
enables all these to be applied to the same time are read out immediately, avoiding the ABF by selecting the appropriate scattering
scan. In addition to conventional signals, time-consuming process of reading out the angle regions or calculating the center of
4D STEM is used in a variety of experiments whole pixel array. In fast scans, it is often mass at each probe position. If multiple scans
that would not be possible with convention- the case that relatively few electrons strike are acquired, the shifts between the con-
al monolithic integrating detectors, such as the detector and thus most pixels contain secutive scans are calculated and this cor-
structural phase mapping, strain mapping only zeros and their readout wastes time in rection can be applied directly to the event
in crystalline or amorphous materials, and a conventional framing camera. The event- stream. Besides the speed improvement, the
atomic resolution phase imaging [1]. driven Timepix3 camera used can detect event-driven operation provides natural data
However, a major drawback to 4D STEM single electrons with a time resolution of compression by avoiding the readout of pix-
has been the slow scan speeds imposed 1.56 ns, and easily provides µs 4D STEM els containing zero counts, especially in low-
by the relatively low frame rate of cam- [2]. Here, these datasets are used to perform dose experiments. For example, 10 scans with
eras compared to the probe dwell time in dose-efficient phase imaging at atomic res- 1024x1024 probe positions result in 86 Gb of
a fast STEM scan. Slow scans are problem- olution using integrated center-of-mass data with a 256x256 frame-based camera in
atic because they lead to high doses and (iCOM) [3] and single-sideband ptychogra- 1-bit mode. Larger bit depths will of course
beam damage in sensitive samples, and just phy (SSB) [4]. The results are compared with result in much larger file sizes. Note that file
as importantly, distortions in the images due the conventional ADF and annular bright size is independent of the incoming electron
to instabilities, such as sample drift at these field (ABF) images. current for a frame-based camera. For event-

based detection, the file size only depends on

the electron current and the acquisition time,
which for 3 pA and a 1 µs dwell time results
in only 1.6 Gb for the same scan and pixel ar-
ray dimensions. One important aspect which
should not be overlooked in these types of
detectors is that the maximum incoming
count rate is limited. This Timepix3 setup is
limited to a maximum of 40 million hits per
second, corresponding to a probe current of
around 3 pA, an order of magnitude smaller
than conventional STEM currents (50 pA).
This can be addressed by incorporating ad-
ditional Timepix3 chips and readouts. How-
ever, small electron currents are often desir-
able, especially for beam-sensitive materials
such as organic materials, which undergo Fig. 3: A 3x1024x1024 scan at 6 µs dwell time. The acceleration voltage used during the acquisition
structural changes at higher currents. There- is 60 kV and the specimen is a monolayer of WS2. The total dose for the experiment was 18,000 e-/A2.
fore, this type of detector is ideally suited for
low-dose experiments, especially as the 4D
STEM-based phase imaging modes provide at 60 kV, a 3-scan 1024x1024 probe position tron beam with a focused probe, where the
greater contrast and SNR for a given dose. dataset was collected at 6 µs from 2D WS2. efficiency of 4D STEM phase imaging meth-
The stability of the material allowed a higher ods can be most effective.
dose of 18,000 e-\A2, which is required for
Methods and Results decent signal-to-noise ratio phase images
due to its lower scattering power. The dif- Acknowledgments
In this study, iCOM and SSB were performed as ferent reconstructions are seen in Figure 3.
highly efficient phase contrast imaging meth- In the ADF images, only the tungsten atoms The authors acknowledge the European
ods using the Timepix3 detector. The iCOM are visible due to their high scattering power. Union’s Horizon 2020 Research and Inno-
algorithm tracks the center of mass, which is Both the tungsten and sulfide atoms can be vation Program under Grant Agreement
proportional to the projected electric field of visualized by iCOM and SSB making these No. 101017720 “eBEAM: Electron beams
the specimen, to reconstruct the electric po- imaging techniques suited to image simul- enhancing analytical microscopy” (J.V. and
tential, which in turn changes the phase of the taneously low and high Z atomic columns. D.J); G042920N “Coincident event detec-
electrons. The other reconstruction algorithm tion for advanced spectroscopy in transmis-
used in this study is the SSB ptychography, sion electron microscopy” (J.V. and D.J.);
which directly retrieves the phase of the object Summary n. 101094299 “The IMPRESS project which
from the diffraction patterns. has received funding from Horizon Europe
In Figure 2, the different reconstructed Event-driven hybrid pixelated detectors en- framework program for research and inno-
images from a silicate-1 zeolite are shown able 4D STEM experiments with microsecond vation.”(J.V.). No. 802123HDEM funding
using a dose per scan of 300 e-/A2. 10 scans dwell times. The limit with the event-driven from the European Research Council (ERC)
with 1024x1024 probe positions were col- Timepix3 camera described here is not the under the European Union’s Horizon 2020
lected at 1 µs dwell time. Notably, the iCOM detector speed, which has nanosecond time Research and Innovation Programme via
and SSB images have much better contrast resolution, but instead the bandwidth of the Grant Agreement No. 802123HDEM (C.G.,
than the ADF and ABF images, making them scan coils. Scanning at low dwell times is C.H., and T.J.P.); the European Union’s Hori-
ideal for low-dose experiments. To show essential for low-dose imaging to minimize zon 2020 Research Infrastructure — Integrat-
that the detector can also detect electrons the specimen damage imparted by the elec- ing Activities for Advanced Communities
under Grant Agreement No. 823717 — ES-
TEEM3; The FWO Projects G013122N “Ad-
vancing 4D STEM for atomic scale structure
property correlation in 2D materials” (C.H.).
Affiliation
1EMAT, University of Antwerp, Belgium
2NANOlab Center of Excellence, University
of Antwerp, Antwerp, Belgium
Contact
Dr. Daen Jannis
EMAT
University of Antwerp, Belgium
daen.jannis@uantwerpen.be
Fig. 2: A 10x1024x1024 scan at 1 µs dwell time. The acceleration voltage used during the ac- References:
quisition is 60 kV and the specimen is a silicate-1 zeolite which is known to be beam sensitive. https://bit.ly/IM-Jannis
The total dose for the entire acquisition is 3,000 e-/A2.

Electron andIon
Electron and IonMicroscopy
Microscopy
© University of Antwerp
Can AI Do your Ptychography?
A Machine Learning Approach to Real-Time Phase Imaging with 4D STEM
Thomas Friedrich1, Chu-Ping Yu1, Johan Verbeeck1, Sandra van Aert1
In this study, a method using deep learn- ptychographic methods for phase object within the specimen. The phase shift can then
ing for the reconstruction of phase images reconstructions gained popularity for their be calculated by simply dividing the electron
from 4D Scanning Transmission Electron super-resolution capabilities. exit wave by the electron probe function. The
Microscopy (4D STEM) data is presented. However, these methods are computation- resulting image can be interpreted as a de-
The process retrieves electron exit waves ally demanding and often iterative in nature, piction of the projected electrostatic potential
locally for each scan position, based on a which limits their use mainly to post-acqui- of the specimen. In STEM experiments, the
set of 3x3 convergent beam electron dif- sition scenarios. This may leave the micro- probe is typically focused and very small, so
fraction patterns (CBEDs) around each probe scope operator practically blind during low- this division only yields a patch of the full-
position, and subsequently uses the phase dose experiments and calls for efficient, phase object, which can be constructed by a
object approximation to solve for the phase real-time capable phase object reconstruc- complex summation of all patches resulting
object. We show that the method is capa- tion methods. from the many positions in a scan grid. In 4D
ble of super-resolution imaging and good In this study, convolutional neural net- STEM, this requires knowledge of the probe
noise robustness. The contrast depends on works (CNN) are used to reconstruct the com- function and exit waves at all probe positions,
the atomic column type and thickness. The plex electron exit wave for every probe posi- both of which are unknown, however. Here,
combination of these properties makes the tion and use them to compute local patches of the incident waves based on the aperture size
method unique among live imaging meth- the phase object, based only on CBEDs at the for well-adjusted aberration-corrected micro-
ods in 4D STEM. probe position and its immediate surround- scopes are estimated. The complex exit waves
ing. This process can be very fast and is inher- for all CBEDs are recovered by the developed
ently local, such that it can be performed live CNN.
Introduction during acquisition. A machine learning setup, This CNN was designed to predict the
taking into consideration the physics at play amplitude and phase of the exit wave at
The development of direct electron detectors and mathematical constraints, ensures that each probe position, based on a kernel of 3x3
(DED) opened new possibilities for Scanning predictions are consistent and faithful. adjacent CBEDs. The estimated probe func-
Transmission Electron Microscopy. Higher tion is used as an input and directly added
frame rates and almost perfect quantum ef- to the network’s output, which enables global
ficiency of the cameras allow a much more Methodology residual learning and practically means that
efficient collection of convergent beam the CNN learns to estimate how a specimen
electron diffraction patterns (CBEDs) in 4D When fast electrons interact with electrostatic alters a given incident wave function, rather
STEM experiments. This not only provides potentials, the wavelengths of the electrons than building it from scratch. The network is
abundant information but also poses chal- are temporarily altered, which creates phase trained on millions of CBEDs simulated by
lenges regarding the efficient extraction and differences. While traversing a specimen, the a multislice package with crystal structures
interpretation thereof. Several approaches phase shift of an electron accumulates result- from the materials project database at various
have been developed to reconstruct 2D im- ing into a direct relationship with the poten- low-index zone axis. Each piece of data con-
ages from such datasets (e.g., iDPC, iCOM, tials of all the atoms it interacted with along sists of a 3x3 kernel of CBEDs as features and
Ptychography), with some astonishing re- its path. In the phase object approximation the corresponding exit wave in real space at
sults, pushing the resolution and dose effi- (POA), the sample is assumed to be very thin the center of the kernel as a label. In the sim-
ciency of STEM to new levels. Particularly, such that the electron probe does not change ulations, we ensured the real-space step size

was at least 10% smaller than the Results and Reconstruction

probe radius, effectively main- Characteristics
taining some probe overlap in
real space. The resulting differ- To test whether the reconstruc-
ences originating from the dis- tion faithfully preserves the
placement of the exit waves from physical properties of the object,
neighboring positions are utilized datasets of CBEDs are simulated
to solve the inverse problem of by using a single atom as a scat-
retrieving the phase. terer for the entire periodic table
The composite loss function up to atomic number Z = 102. The
consists of terms accounting for reconstructed phase objects are
the exit waveforms in both real averaged over different ranges
and reciprocal space, as well around the potential center and
as the phase object itself. This compared to the ground truth in
enforces physically meaning- Figure 1. The phase values, al-
ful constraints on the CNN and though not quite exact, follow
increases the robustness of its the theoretical values closely. At
estimations. larger integration ranges, the plot
The trained CNN in con- in Figure 1c shows that the orbit-
junction with the phase object al contraction at atoms with fully
approximation and appropri- filled orbitals is captured by the
ate weighting and shifting of reconstruction. These single-at-
the retrieved phase patches pro- om datasets were never part of
vides a non-iterative, robust, and the training, and thus these re-
fast (>4k CBED/s) phase imaging sults show that the CNN does not Fig. 1: Phase response of the CNN compared to ground truth transmis-
method with good dose efficiency simply make up images of atoms sion functions for simulations of single atoms throughout the periodic
and a resolution that is in princi- but reflects, to some level of ac- table at infinite dose for (a) peak intensity, (b) mean over 0.6x0.6 Å
ple only limited by the CBED col- curacy, the physical properties of around the atomic position, and (c) mean over 1x1 Å pixels around the
lection angle. the observed materials. atomic position.
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Fig. 3: Reconstruction results of a USY-Zeolite dataset for 2 different

approaches. (a) Neural Network (b) SSB reconstruction and the Fourier
transforms in (c) and (d), respectively. The data preprocessing is de-
scribed in reference [1].
Fig. 2: Reconstructed images of an edge of an Au crystal using (a) Acknowledgments

SSB, (b) HAADF, and (c) CNN reconstruction. Line profiles across the
nanorod illustrate the thickness dependence of the corresponding sig- The Authors acknowledge funding from the
nals in panel (d). European Research Council (ERC) under the
European Union’s Horizon 2020 research and
In Figure 2, the reconstruction of an exper- dark field electrons, which directly contrib- innovation program (Grant Agreement No.
imental gold particle dataset is presented, ute to the high-frequency features. This infor- 770887 PICOMETRICS) and funding from
and compared with other imaging methods mation is missing in SSB, as it only utilizes the European Union’s Horizon 2020 research
that are known for their sensitivity to thick- the intensity variation inside the bright field, and innovation program under grant agree-
ness (high angle annular dark field-HAADF) and the effect is reflected by the higher fre- ment No. 823717 ESTEEM3. J.V. and S.V.A.
or other advantages, such as dose efficiency quency components in the result, indicated by acknowledge funding from the University
(single sideband ptychography-SSB). It is the Fourier transform (FT) of the images. The of Antwerp through a TOP BOF project. The
shown that the phase contrast of the CNN comparison of the FTs further illustrates again direct electron detector (Merlin, Medipix3,
reconstruction resembles the HAADF result, the presence of low frequencies in the CNN Quantum Detectors) was funded by the Her-
which scales with the predicted thickness. reconstruction and a contrast transfer with an cules fund from the Flemish Government.
This indicates that the phase value can be overall wider frequency range. This work was supported by the FWO and
used to estimate thickness variations, to FNRS within the 2Dto3D project of the EOS
some extent even beyond the limits of the program (grant number 30489208).
POA, since ≈10 nm would typically not be Conclusion
considered a PO. The presence of accurate Affiliation
1EMAT, NANOlab Center of Excellence, Uni-
thickness contrast further shows that the A new reconstruction method is proposed that
CNN can retrieve low-frequency compo- uses a CNN for exit wave reconstructions and versity of Antwerp, Antwerp, Belgium
nents of the phase signal, which are com- the POA to solve for phase objects, utilizing
monly lost in SSB. all the electrons in the region covered by the Contact
One of the most significant advantages of detector for dose-efficient high-resolution Sandra van Aert
using DEDs over annular detectors is the fact imaging. The reconstructed phase values can EMAT
that they collect most of the transmitted elec- be correlated with atoms’ projected potentials NANOlab Center of Excellence
trons in the beam. Methods that can exploit and the resulting image contrast is dependent University of Antwerp
this fact may yield a much stronger contrast. on atomic species and the specimen thickness. Antwerp, Belgium
This is proven true in the reconstruction of The reconstruction algorithm does not rely
a silicalite-1 zeolite sample, taken at a dose on iteration and works on only 9 CBEDs at
that does not even permit HAADF imaging. In a time, which makes it attractive for live im-
Figure 3, the reconstructed results of both the aging, potentially allowing users to see their References:
CNN and SSB show a strong contrast. The CNN image develop in real time with lower doses, https://bit.ly/IM-vanAert
reconstruction benefits furthermore from the high contrast, and improved resolution.

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Scanning Probe Microscopy
Imaging the Surface Ions of

Pristine Muscovite Mica
Using Non-Contact Atomic Force Microscopy in Ultra-High Vacuum
Giada Franceschi1, Martin Setvin1, 2, Ulrike Diebold1
A
luminosilicate minerals are ubiq-
uitous on Earth and play a crucial
role in many natural phenomena.
An accurate knowledge of their surface
structure is a prerequisite for a deeper
understanding of the underlying molec-
ular-level processes. Our team at the TU
Wien in Vienna (Austria) has succeeded in
imaging the intrinsic atomic-scale details
of muscovite mica – a common alumino-
silicate 2D mineral with a long history in
surface and interface research. Until now,
muscovite mica has only been investigated
when affected by air and in solution. Our
© Franceschi
work shows that it is now possible to mea-
sure mica (and related insulating minerals)
also in pristine, ultra-high vacuum con-
ditions through non-contact atomic force
microscopy.
Introduction
Mineral surface chemistry is at the core of

many natural phenomena, such as disso- bulk structure). Mica is a common 2D min- Insights into the Intrinsic Distribution of
lution and weathering, adsorption of toxic eral that has been at the center of hundreds the Surface K+ Ions of Mica
ions in groundwater, and ice nucleation on of studies in diverse disciplines, including
mineral dust, which regulates cloud forma- nanotribology, electronics, geology, biol- Images of UHV-cleaved mica obtained with
tion and weather patterns. To reach a com- ogy, and environmental science [1]. Its per- our setup are shown in Figures 1c and 1d
plete understanding of the molecular-level fect cleavage planes offer atomically flat, [3]. They represent the shift in resonance fre-
processes occurring at mineral surfaces, an micrometer-sized terraces, ideal for scan- quency obtained while scanning the sample
accurate knowledge of their surface atom- ning probe microscopies. To date, mica-fo- at a constant height of the tip. The images
ic structures is needed. This can be ideally cused and AFM-based studies are abundant show a flat surface (as expected) decorated
achieved with the surface science approach in air and solutions but there is a surprising by an array of dark dots (attractive regime).
– combining atomically resolved investiga- lack of studies in UHV. This is because the In our work [3], we assigned these dots to K+
tions of well-defined single-crystalline sam- most used cantilevers for UHV non-contact ions at the mica surface.
ples in pristine, ultra-high vacuum (UHV, (nc) AFM are relatively soft. They interact As seen from Figures 1a and 1b, mica
pressures below 10−9 mbar) with ab initio strongly with the charges formed at the mica is expected to cleave within the K layers
modeling. surface after cleaving, snapping into contact that alternate with aluminosilicate layers
However, minerals present some hur- before atomic contrast is achieved [2]. along the [001] direction. The aluminos-
dles compared to the metals and (conduc- In our study [3], we obtained the first ilicate layers are made of three sheets: in
tive) oxides most popular in surface science. atomically resolved images of UHV-cleaved the middle, octahedrally coordinated Al
Often, they are electrically very insulat- mica using nc-AFM. An essential part of the ions with OH groups; at the sides, tetra-
ing, preventing scanning tunneling micros- success laid in the choice of the AFM probe: hedrally coordinated Si and Al ions in a
copy; moreover, they develop strong sur- not a standard, Si-based cantilever, but the 3:1 ratio. The substitution of Si4+ for Al3+
face charges when cleaved in UHV, making qPlus sensor [4]. Its larger stiffness allows introduces a missing charge in the tetrahe-
atomically resolved atomic force microscopy the probe to oscillate with smaller ampli- dral sheets, which is compensated by the
(AFM) challenging. tudes, push through the strong electro- K+ ions. From electrostatic considerations,
These hurdles are exemplified by the sci- static fields induced by surface charges, and one expects each cleaved surface to retain
entific history of muscovite mica (simply become sensitive to the short-range forces 50% of the K+ ions initially present in the
“mica”, hereafter; Fig. 1a, b for views of its that yield atomic contrast. K layer. However, until now, the intrinsic –

Scanning Probe Microscopy
i.e., unaffected by the interaction with the

environment – distribution of these ions
had not been determined experimentally.
Early low-energy diffraction studies have
claimed a random distribution, whereas
theoretical papers typically assume fully
periodic arrangements to minimize com-
putational costs. Our images in Figs. 1c, d
show that the distribution of the K+ ions is
neither random nor fully periodic. Instead,
the ions arrange in short, alternating rows
and 120° kinks.
Insights into the Intrinsic Distribution

of the Subsurface Al3+ Ions
In addition to the ordering of the surface

K+ ions, the arrangement of the Al3+ ions
in the subsurface aluminosilicate layer of
mica has been another source of ambiguity:
As Al and Si have similar cross sections in
X-ray diffraction, it has been difficult to
ascertain the presence of short-range or-
dering in the subsurface as well. Striving to
understand better the short-range order of Fig. 1: (a) Side view of bulk mica. Cleaving occurs at the K layer, leaving half the K+ cations on
mica’s surface K+ ions and to possibly tie it each side. (b) Top view of the surface after cleaving. (c) Atomically resolved constant-height
to the arrangement of the subsurface Al3+ non-contact AFM image of mica after UHV cleaving, acquired with a qPlus sensor at 4.7 K.
ions, we analyzed the experimental K+ dis- Adapted from Franceschi et al. [3].
tributions using density functional theory
calculations and Monte Carlo simulations
[3]. As for every surface, the arrangement AFM mode. The tuning-fork-based AFM Conclusions and Outlook
of the surface atoms is such that the surface sensors (k = 2,000−3,500 N/m, f0 ≈ 45 kHz,
energy is minimized. Our analysis shows Q ≈ 5,0000) had a separate contact for tun- Our results unveil new insights about an
that the structure of the surface K+ ions is neling current attached to electrochemically oldie and favorite of surface science – mus-
created to minimize the electrostatic repul- etched W tips. Before each measurement, covite mica – using nc-AFM with a qPlus
sion among the surface ions, as well as the the tips were prepared on a clean Cu(110) sensor in UHV. They show that its surface
repulsion of the surface K+ ions with the single crystal by repeated indentation and K+ ions are arranged in short-range order
subsurface Al3+ ions. Specifically, K+ ions voltage pulses. The coarse approach was and that the arrangement of Al3+ ions in the
will prefer to sit closer to lower-charged done with a setpoint of −1 Hz. The con- subsurface aluminosilicate layer influences
Al3+ than to Si4+ sites. This shows the im- troller was switched off, and the tip grad- this order. The study paves the way to inves-
portant role of the subsurface Al3+ ions in ually approached in constant-height mode tigate other mineral surfaces (insulating and
determining the surface properties of mica until a contrast was visible while scanning possibly similarly charged) similarly, setting
– a role that is often neglected in the litera- in x and y. AFM images were acquired in the stage to unravel many mineral-mediat-
ture and could be a potential cause of data constant-height mode. At times, the abso- ed natural processes, such as ice nucleation,
misinterpretation. lute values of frequency shifts during acqui- CO2 sequestration, and ion dissolution.
sition were large (up to 100 Hz) and not
reproducible on different regions on the Affiliation
Methodology same sample or different samples. This is 1Institute of Applied Physics, TU Wien,
because cleaving can create domains of Vienna, Austria

2
The experiments were carried out in a UHV trapped charges that can cause long-range Department of Surface and Plasma Science,
setup with base pressure <1 × 10−10 mbar. electrostatic interactions between the sur- Charles University, Prague, Czech Republic
Natural muscovite mica single crystals face and the tip. The corresponding electro-
[(0001) oriented disks of grade V1; 10 mm static fields can be partially compensated Contact
diameter and 0.25 mm thickness; from Ted- by applying a bias voltage between the tip Giada Franceschi
Pella] were glued on Omicron-style stainless and the sample. Most measurements were Institute of Applied Physics
steel sample plates with UHV-compatible performed by applying a bias voltage such TU Wien
epoxy glue (Epotek). They were cleaved in that the surface was measured as close as Vienna, Austria
UHV at room temperature before each ex- possible to the lowest local contact poten- franceschi@iap.tuwien.ac.at
periment. tial difference (LCPD) between the tip and
The AFM measurements were performed the sample. The bias voltage reported in the
at 4.7 K using a commercial Omicron qPlus presented images was applied to the back References:
head and a differential cryogenic ampli- of the sample plate while having the tip on https://bit.ly/IM-Franceschi
fier. Frequency-modulated non-contact the ground.

Image Processing
Jupyter Notebooks for Generating and

Distributing Bioimage Analysis Workflows
My Favorite Image Analysis Tool, by Neubias Members
Rafael Camacho1
T
o secure their reproducibility, much into Python implementations. This push
effort is being placed into developing has been facilitated by the involvement
norms for reporting bioimage anal- of analysts and developers from computer
ysis (BIAS) workflows. One view that has science fields, such as Data Science, Com-
gained many advocates is reporting BIAS puter Vision, and Machine Learning, that
workflows containing all the steps used to use Python as their Lingua Franca. Adopt-
go from raw data to final numeric output ing Python allows a bioimage analyst to
or figures [1]. In this context, workflow stay within the same programming lan-
documentation via scripting in a program- guage from raw image data processing to
ming language is critical. final numerical output and figures.
A classic and popular example of such Rafael Camacho

scripting documentation is ImageJ macros. Jupyter Notebooks for Developing and
Once the workflow has been written as an Sharing Bioimage Analysis Workflows works as a Scientific Officer at the Centre
ImageJ macro, it can be shared with others for Cellular Imaging, Core Facilities, of Go-
via public repositories, such as GitHub [2] Python programs can be executed in many thenburg university, Sweden, since 2018. He
and Zenodo [3]. More recently, a new play- ways, including running a Python Script did his PhD in the department of Chemical
er has entered the field: Jupyter Notebooks. using a terminal or using a Python Shell. Physics of Lund University at the Single
While these Notebooks are not new in the However, with Data Science and BIAS, it is Molecule Spectroscopy Laboratory of Prof.
Python world and have matured over many highly appreciated to have a more interac- Ivan Scheblykin. In 2014, he moved to KU
years in Data Science, they are now starting tive programming environment that permits Leuven as a Postdoctoral researcher in the
to be used for BIAS. Jupyter Notebooks are quick inspection of intermediate results of laboratory of Prof. Johan Hofkens for the
easy to install, have great documentation, data processing, cleansing, mining, and vi- development of 3D super resolution imaging
and can be used from quick prototyping to sualization during the construction of anal- and bioimage analysis software tools. He is
the sharing of fully reproducible workflows. ysis workflows. Great examples of such an particularly interested in the development of
For these reasons and other attractive fea- environment are the MATLAB workspace in Smart Microscopy workflows that use image
tures explained below, Jupyter Notebooks the commercial sector, and Jupyter Note- analysis to continuously monitor the sam-
have become my favorite tool for gener- book and JupyterLab in the open-source ple, give real-time feedback to the imaging
ating and distributing bioimage analysis sector. software, and program the microscope to
workflows. change its parameters during experimental
acquisition.
Jupyter Notebook in Practice
Python as a Scripting Language for
Bioimage Analysis Here, I wish to demonstrate the usefulness
of the Jupyter Notebook by taking a simple
While the ImageJ macro language has analysis workflow as an example. During the
gained popularity, the language has sever- scripting of a BIAS workflow, we use various
al clear limitations in handling non-image algorithms for image analysis that are al-
data: Manipulating multidimensional arrays ready implemented and accessible. In Python,
and tables, statistics, the use of data mining such collections of implemented algorithms
algorithms on measured values, and train- are called packages or libraries. To use them the output of a computation including fig-
ing new deep learning models for image within the workflow we “import” the desired ures, and markdown cells with written docu-
processing (e.g., segmentation and denois- module from those packages at the beginning mentation formatted in Markdown [8]. Each
ing). Due to these limitations, it has become of the workflow’s code. By having the used cell can be run multiple times, or sequential-
common practice to combine ImageJ with modules explicitly declared in the code, rely. It is common practice to change the input
other software packages, such as Python, sults can be reproduced among different users parameters of a cell and run it “interactive-
MATLAB, R, Excel, etc., to construct a BIAS without ambiguity. For example, we might ly” until the desired computational outcome
workflow. use the input/output module of scikit-image e.g., segmentation is obtained.
To overcome the complexity caused by [4] to load a .tif-file into a variable and use One common BIAS task is the pre-process-
using multiple software packages, there has another module matplotlib [7] to generate a ing of an image via different filters, and then
been a strong trend to move many BIAS figure of the image we loaded (Fig. 1). segmenting the objects of interest. For exam-
algorithms [4], workflows [5], and user inter- A Notebook consists of three types of cells: ple, we can use a Gaussian filter to attenu-
faces [6] (multidimensional image-viewers) Code cells with live code, output cells with ate uneven illumination effects, and then run

Image Processing
Fig. 1: Using scikit-image to load a .tif-file and matplotlib to show the Fig. 2: Background removal by Gaussian filter and segmentation using
image. Otsu’s algorithm.
a classical segmentation algorithm such as the site, you will note the great support of Image.sc forum [12], scikit-image, napari,
intensity thresholding by Otsu’s algorithm. GitHub to render Jupyter Notebooks. and py-clEsperanto [13].
This can be implemented using the scikit-im-
age package as shown in Figure 2.
As a final step, we can measure the fea- Python and Jupyter Notebooks Jupyter Notebooks for
tures of the segmented objects and take Installation Distributing BIAS Workflows
advantage of the rich availability of Data
Science packages in Python to get statisti- After demonstrating the usefulness of Jupy- Direct Sharing of Jupyter Notebooks
cal summaries directly out of the measure- ter Notebooks, we would like to lead you to If you wish to share your Notebook as a
ment results. For statistics, we can use Pan- a quick guide for installing Python on your document with text and figures, and the re-
das [9], a Python library used for tabular local machine. As the installation procedure cipient does not need to run the code, then
data manipulation and analysis (Fig. 3). is not as simple as downloading an instal- you can simply “Save and Export Notebook
lation package and double-clicking the in- As…” a PDF file (Fig. 4). Further, if you wish
staller, we provided an easy installation the recipient to run the code, then you can
Jupyter Notebooks for Prototyping guide for Jupyter Notebooks in the online send the *.ipynb-file. However, you will also
Bioimage Analysis Workflows documentation [11]. have to instruct the recipient on what pack-
One of the challenges when migrating ages to install, in such a way that they can
Figure 4 shows an example of a simple BIAS into Python for bioimage analysis is where to run the Notebook locally.
workflow that follows the steps described in start, which packages to install, and finding
Figures 1-3. The Notebook includes interme- good minimal examples that allow research- Sharing of Jupyter Notebooks
diate steps that were not successful, so you ers with little Python knowledge to learn the together with an environment.yml file
get a feeling for the advantage of interac- language by applying it directly to their own To make it easier for a 3rd party to run your
tive analysis. This workflow is available in projects. To help you get started, we provide Jupyter Notebook it is advised to share the
the online documentation of this article as a list of key resources in our GitHub repos- “content” of your virtual environment via
a notebook file: Example.ipynb. If you visit itory [11], including but not limited to the an environment.yml-file. This file allows
Fig. 3: Quantification and simple statistical summary.

Image Processing
Fig. 4: Full export of a BIAS workflow

documented as a Jupyter Notebook.
others to quickly reproduce your environ- vironment and development environment, ommend that you look at some of the ex-
ment, with all the packages and versions. e.g., JupyterLab. Test that the setup works amples listed in this article and give Jupyter
Instructions to export your environment can on example data. Finally, you can give ac- Notebooks a try on your next BIAS challenge.
be found in the GitHub repository [11]. cess to the user via Remote Desktop or other
alternatives such as NoMachine.
Sharing of Jupyter Notebooks Acknowledgments
with the World b) Sharing via Colab.
To share a BIAS workflow with the com- Google Colab is a free Jupyter Notebook envi- I would like to thank Kota Miura for the
munity in a more reproducible way, e.g., ronment that runs in the cloud [14]. It allows invitation, proofreading, and suggestions
for a publication, then we recommend us- anyone to write and execute Python code in that made this article possible. I would
ing GitHub. In GitHub, you can upload your the web browser. In a very real way, you are like to thank my colleagues at the Centre
Notebook, together with the environment borrowing computer resources from Google, for Cellular Imaging (CCI), Core Facilities,
file in a public (or private) repository, where including GPUs. In this strategy, Notebooks The Sahlgrenska Academy, University of
it can be used by others. This allows you are stored in Google Drive or via GitHub. Then Gothenburg, and the National Microscopy
to keep an integral copy of the Notebook you can share the Notebook with others and Infrastructure, NMI (VR-RFI 2019-00022),
and environment used for a publication. let them run it in the cloud. Note that the vir- for their support during the preparation of
The GitHub repository [11] associated with tual machine instance used during develop- the manuscript. I would like to thank the
this article is an example of this strategy. If ment will not be shared. Therefore, you must Swedish Foundation for Strategic Research
you want to have further control over the include cells that install and load the libraries for their financial support via the Research
version related to a publication, including a needed for the Notebook to run. Infrastructure Fellows 2 grant (RIF21-0043).
DOI, then you can use Zenodo [3] in combi-
nation with the GitHub repository.
Closing Remarks
Sharing of Jupyter Notebooks by Giving
Access to Controlled Environments One of the biggest limitations when migrating Contact
If you are a bioimage analyst providing ser- to Python for BIAS is the learning curve asso- Dr. Rafael Camacho
vices to a broad community (for example, ciated with using a new computer language. Centre for Cellular Imaging
working at a core facility), then you might However, Python and Jupyter Notebooks Core Facilities
need to share your workflows in an executable have several key features that make this pro- The Sahlgrenska Academy
environment, in terms of computer resources cess easier than with other languages. Python University of Gothenburg
and software installation. For this purpose, we is very user-friendly because its English-like Gothenburg, Sweden
recommend 2 different solutions: a) giving syntax is concise and easy to read. In combi- rafael.camacho@gu.se
access to a BIAS server, and b) Google Colab. nation with Jupyter Notebooks, the language
becomes very hands-on, allowing immediate
a) Access to bioimage feedback on your progress. Because Python is
analysis server. very versatile you can find libraries for many
Let us take as an example a simple Windows purposes, and you can use the language to References:
server. As analysts, you can create a user solve small and complex tasks alike across https://bit.ly/IM-Camacho
account. Install the appropriate virtual en- many domains. Therefore, I can highly rec-

Advertorial
Investigating Surface
Catalytic Activities Using SECCM
Alexander Klasen and Andrea Cerreta
F
rom converters that oxidize harmful meniscus when the pipette is close enough to reduction of phosphate ions to phosphite ions
nitrogen oxides in vehicle exhaust to the sample surface. A small electrode within at -0.8 V and the start of water cleaving and
fuel cells converting hydrogen into the pipette, usually an AgCl-coated Ag wire, subsequent hydrogen formation at approx.
clean energy or metal oxides that facili- allows applying a potential between the pi- -1.3 V. Please note, that TiO2 is an n-type
tate the transition of bulk chemicals into pette and the sample. The meniscus then rep- semiconductor with a large bandgap that
target molecules, catalysts are employed resents a spatially confined electrochemical forms a rectifying Schottky diode with the Ti
in almost every aspect of our modern cell in which reactions can occur, e.g. at con- metal, resulting in unidirectional current flow.
economy. Metal oxides are often used as stant bias or current (Fig. 1). A series of elec- Similar experiments conducted with the same
heterogeneous catalysts, fixed on a solid trochemical cycles can be done by lifting and setup within one hour on a far more conduc-
surface with a large interface to either liq- approaching the pipette at different points on tive HOPG sample showed an overall smaller
uid or gaseous reactants or products [1]. a point grid. (Fig. 2). current flow since HOPG does not possess sur-
The performance of such systems depends TiO2 has gained much attention as a pho- face catalytic properties. The observed current
on several factors like the surface area, tocatalyst [7,8] since oxygen vacancies in its flow was attributed to mere ion migration that
chemical composition, or surface acidity/ lattice can play a twofold role. Oxygen vacan- has a higher magnitude at positive applied
basicity [2,3]. Scanning probe microsco- cies in the TiO2 bulk create energy states close bias, probably due to higher ion mobility of
py-based techniques are well established to the conduction band of the semiconduc- Na+, K+, and H+ ions compared to phosphate
to investigate surface parameters since tor and hence act as n-doping centers. How- ions, which possess a larger size and higher
the physical interaction with a nanome- ever, surface defects create energy levels deep charge density.
ter-sized probe allows studying properties within the band gap of a TiO2 crystal or layer These two examples show that SECCM
such as the topography, work function, or and can act as charge carrier traps that form is a unique and easy-to-use tool to investi-
adhesion at high resolution [4,5]. local catalytic centers [9-11]. gate the catalytic properties of surfaces on
For this study, a piece of Ti metal was a local scale.
In this article, we present measurements con- thermally treated to form a thin TiO2 surface
ducted with Scanning Electrochemistry Cell layer. A standard phosphate buffer contain- Contact
Microscopy (SECCM). First introduced by E. ing 137 mM NaCl, 2.7 mM KCl and 10 mM Park Systems Europe GmbH
Daviddi, P. R. Unwin et al. [6], SECCM uses Na2HPO3 was used as electrolyte. On the TiO2 Mannheim, Germany
an electrolyte-filled pipette with a nm-sized surface, we could identify locations with
aperture to probe electrochemical characteris- strong catalytic properties visualized by bright
tics locally. The pipette position can be finely colors indicating large current flow (Fig. 2A). References:
tuned via piezo scanners. An electrolyte drop- In one exemplary IV curve, two distinct bumps https://bit.ly/IM-PS0223
let is present at its aperture, which turns into a could be seen that mark the beginning of the
Fig.1: A: An electrolyte-filled micropipette approaches the target (A) and Fig. 2: SECCM measurements conducted on a 10x10 grid on A: Ti met-
forms a small meniscus with the surface in close proximity: the electro- al with a thin TiO2 top layer and B: on a freshly cleaved HOPG sur-
chemical cell (B and D). An applied bias between the pipette electrode face. Each pixel represents a single EC cell where bias was swept be-
and the sample lead to a spike in current, once the meniscus is formed. tween -1.5 and +1.5 V. Displayed are the absolute highest currents of
(C). The approach is stopped and EC can be conducted by applying vari- the respective IV curves. Exemplary IV curves of each sample are dis-
ous bias or current settings to the sample and pipette electrode. played on the right side.

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Index / Imprint
AHF Analysentechnik 7 Hübner Photonics 42 Quantum Design 42
Centro Nacional de Biotecnologia (CNB-CSIC) 11 Inara Aguiar 3 Royal Microscopical Society (RMS) 10
Charles University 36 Scandem Nordic Microscopy Society 8

MCO Congres Marseille 11
Coxem 9, 42 Technische Universität Wien 14, 26, 36
MRC Harwell Institute 17
CrestOptics 42 Tescan 29
NKT Photonics Outside Back Cover
Edmund Optics 15 University of Antwerp 30, 32
Park Systems Europe Inside Front Cover, 41
EMS Secretary 11 University of Gothenburg 38
European Molecular Biology PreciPoint 20 WITec Cover, 12, 42
Laboratory (EMBL) Heidelberg 8 Prospective Instruments 23 Carl Zeiss 42
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