4

VOLUME 24
NOVEMBER
2022
69721

Official Partner of the EMS

Scanning Microwave Microscopy

Combining STEM, EELS, EDXS

Scanning Microresonator

Coverslip Selection
 le.tab
look-up
t-based
; gradien
ded algae
n embed
ion of resi
Visualizat
TESCAN 3D FIB-SEM
TOMOGRAPHY
Go from data acquisition to visualization without the need for laborious manual data
segmentation. Visualize data in seconds with a combination of two-dimensional
intensity AND gradient look up table.

A single click imports the dataset and the step-by-step wizard guides you through
the pre-processing steps to create a complete, detailed 3D visualization.

At TESCAN we specialize in nano-scale tomography, designing instruments with

users in mind.

What can you do with TESCAN 3D FIB-SEM Tomography?

Find out more.

www.tescan.com
© alphaspirit - Adobe-Stock.com
Editorial
Editorial

Meet Us in 2023
With the new Omicron variants of Sars- excited to see what’s new in microscopy at
CoV-2, Covid-19 has lost its scare this year, IMC20. We look forward to meeting previous
with fewer people becoming severely ill and authors and partners and making new con-
dying as it progresses. As a result, in 2022, tacts. I hope you also have the most important
many scientists have again exchanged ideas events for 2023 on your calendar and that we
in person at trade shows and conferences. It will meet in person.
became particularly clear here that those con- Once again, we have compiled interesting
versations are not comparable with the digital articles for you from the world of microscopy
meeting world and, above all, that network- in this issue and hope that they will help you
ing is of great importance. This trend will in your work in research, development, and
certainly be noticed by many companies and application in the laboratory.
researchers in 2023, and the number of people
attending events will continue to rise. Enjoy reading this issue!
For our editorial team of Imaging and
Microscopy, 2023 will also be a year with
many events that we may personally attend.
We will start with the Microscopy Conference,
which will be next year at the end of Febru-
ary in Darmstadt, Germany. A special highlight
will be Focus on Microscopy, which will finally
take place live in Porto, Portugal in early April.
Also, the ELMI Meeting will be one of our stops
and we are looking forward to participating
in Noordwijkerhout, The Netherlands in early
June. Our long-standing collaboration with the
Royal Microscopy Society in the UK will then
take us to Manchester, UK in July. Followed by
Microscopy & Microanalysis in Minneapolis,
USA at the end of July.
The biggest event in 2023 for us will be the
International Microscopy Congress (IMC20) Dr. Birgit Foltas
in Busan, Korea in September. We are very Editor-in-chief

Imaging & Microscopy 4/2022 • 3

 Contents

EDITORIAL 3

NEWSTICKER 6

ANNOUNCEMENTS
Microscopy Conference 2023  8

Trends in Microscopy 2023  8

Focus on Microscopy 2023  9

© Romolo Tavani - Adobe-Stock.com

RMS IN FOCUS
Winners of 2022 RMS Award Revealed! 10

NEWS FROM EMS

EMS Newsletter #79, November 2022 11
© Image by Christoph Hohmann (MCQST)

COVER STORY
Mapping the Registry and Functional Properties
of Layered Materials Heterostructures 12
Using Conductive Atomic Force Microscopy
J. Kerfoot and V. V. Korolkov

SCANNING PROBE MICROSCOPY

Inverted Scanning Microwave Microscopy 14
Application and Perspective on Biological Samples
M. Farina et al.

PREVIEW:
ISSUE 1/2023

Coming out 16th March, 2023

Overcoming the Signal Overlap

A beginner‘s Guide to SMLM Colocalization
M. Bramkamp et al.

4 • Imaging & Microscopy 4/2022

 ELECTRON MICROSCOPY
Analytical Electron Microscopy of
Organic Matter from Asteroids 18
Insights into Prebiotic Chemistry by Studying Asteroidal Organic Matter
R. M. Stroud et al.

Lithium Detection with Secondary Ion Mass Spectrometry 20

Correlation of Surface Topography and Chemical Analysis
Gudrun Wilhelm et al.

LIGHT MICROSCOPY
Seeing the Unseen 23
Boosted Absorption Imaging and Spectroscopy Using a Scanning Microresonator
J. Noé et al.
COVER STORY
Mapping the Registry and
The Importance of Coverslips 26
Functional Properties of Layered
Tips & Tricks for Coverslip Selection
Materials Heterostructures
P. Lorentz
Using Conductive Atomic
Force Microscopy
IMAGE PROCESSING
QuPath: Taking Full Control
Through Workflows, Scripting, and Extensions 28 Manipulating the registry of layers within layered
My Favorite Image Analysis Tool, by Neubias Members materials heterostructures leads to changes in their
O. Burri functional properties both locally [1,2] and at scales
relevant to devices [2,3]. This motivates interest in
atomic force microscopy (AFM) as a tool capable
of resolving both inter-layer registry and a host of
WILEY ANALYTICAL SCIENCE AWARD 32 concomitant functional properties at nanometre
The Winners of the Award length scales. Here, we showcase conductive AFM
(CAFM) as a technique capable of determining the
registry of layers and extracting functional properties
SHOWCASE via spectroscopic measurements over individual
features.
Introduce Deep Learning to
your Materials R&D and QA/QC 34
12
PRODUCTS34

INDEX / IMPRESSUM INSIDE BACK COVER

4
VOLUME 24
NOVEMBER
2022
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,
learning, assessment and certification tools. Wiley continues to
share and deliver the answers to the world’s challenges
Scanning Microwave Microscopy

Combining STEM, EELS, EDXS helping you to further your mission.

Scanning Microresonator

Coverslip Selection
NEWSTICKER
Correlative Electron Microscopy Electron Microscopy
Monitoring Catalytic Reactions in Real Time Corrosion Has Turned Alhambra Gold Leaf Purple

photoemission electron microscopy Spain-based researchers have fi-

© TU Vienna
Using correlative microscopy, re-
searchers from Austria and Ger-
many have imaged a catalytic
conversion reaction, detecting an
unusual mechanism as reactive sites
interact and patterns form. Profes-
sor Günther Rupprechter from the
Institute of Materials Chemistry at
TU Wien, and colleagues at TU Wien
and FHI Berlin, used two variants of
- UV-PEEM and X-PEEM – as well
as low energy electron microscopy,
LEEM, to study the catalytic con-
version of hydrogen and oxygen to
water in real time. They observed the
reaction fronts on the crystal sur-
face forming remarkable geometric
patterns, and also discovered a new
propagation mechanism for these
fronts. As the researchers highlight,
a comprehensive understanding
of such processes is crucial for cli-
mate-relevant technologies such as
ecologically clean hydrogen-based
energy production.
More information:
http://bit.ly/IM-042022-c
nally solved the mystery of why
purple stains have appeared on
the intricate gilded tin plaster-
work of the historic Alhambra
palace in Andalusia, Spain. Us-
ing electron microscopy, Isabel
Guerra and Carolina Cardell from
the nearby University of Granada © 10.1126/sciadv.abn2541
discovered how an unusual cor-
rosion process has spontaneously Original publication:
converted the gold leaf into pure doi: 10.1126/sciadv.abn2541
gold nanospheres that were just More information:
the right size to appear as a pur- http://bit.ly/IM-042022-g
ple hue. Guerra and Cardell hope
their latest results will help histo-
rians and conservationists to pre-
vent further corrosion in ancient
gilded objects.

Camera Technology
AI-Designed Camera only Records Objects of Interest

Professor Aydogan Ozcan and col-
leagues at University of Califor-
nia Los Angeles have developed a
camera with 3D-printed plastic
lenses that can image certain ob-
jects whilst ‘ignoring’ everything
else in that shot. Designed by
artificial intelligence, the ‘priva-
cy-preserving’ camera takes pic-
tures of only relevant objects while
instantaneously preventing other
details from being captured. This
approach also massively reduces
camera’s data storage - Ozcan
reckons the camera could inspire
future imaging systems that con-
sume orders of magnitude less
computing and data transmission
power. The camera comprises suc-
cessive transmissive surfaces, each
composed of tens of thousands of
diffractive features at the scale of
the wavelength of light. The struc-
ture of these transmissive surfaces
has been optimized using deep
learning to modulate the phase © Ozcan group
of the transmitted optical fields Original publication:
such that the camera only images doi: 10.1186/s43593-022-00021-3
certain types/classes of desired ob- More information:
jects and erases the others. http://bit.ly/IM-042022-f

Combining Light, Electron and X-Ray Microscopy Event-Driven Software

Fossil Leaves Harbor Crystal Surprise Capturing Rare Mitochondrial Division in Real-Time

than 20 million years old from New open-source software based

© Mahdieh Malekhosseini/University of Bonn
Germany- and US-based re-
searchers have shown, for the
first time, that microstructures
in fossil leaves, known as ‘ghost
crystals’, originate from calcium
oxalate. Using light microscopy,
electron microscopy and X-ray
imaging, Professor Jes Rust from
the Institute of Geosciences, Uni-
versity of Bonn, and colleagues,
studied a fossilized leaf, more
the Rott fossil site near Bonn,
Germany. The common biomin-
eral, calcium oxalate, is formed
by very many living plants, but its
function has not yet been conclu-
sively clarified. Researchers have
suspected that the crystals serve
as calcium stores, and because
they are formed in the leaf and
can penetrate the leaf surface as
they grow, they probably repel
pests.
Original publication:
doi: 10.1038/s41598-022-20144-4
More information:
http://bit.ly/IM-042022-a
on neural networks can be used
with fluorescence microscopy to
capture biological events, such as
cell division, in real-time. The event-
driven Python plug-in, developed by
Switzerland-based biophysicists, has
been trained to detect the tell-signs
of the beginning of mitochondrial
and bacterial division, and then
trigger real-time control to opti-
mize data acquisition. As Manley
and colleagues highlight in Nature
Methods, the set-up adapts image
acquisition by capturing the onset
of mitochondrial and bacterial divi-
sion, at a slow imaging rate. Then, as
division proceeds, the imaging rate
© EPFL/Hillary Sanctuary
speeds up to match the dynamic
timescales of these events. The
scientists are making the control
framework available as an open-
source plug-in for the open micro-
scope software Micro-Manager.
Original publication:
doi: 10.1038/s41592-022-01589-x
More information:
http://bit.ly/IM-042022-e

6 • Imaging & Microscopy 4/2022

 Newsticker

High-Speed AFM 3D Microscopy Atomic Force Microscopy

Sheding New Light on How Crystals Form Endoscope Uses Bendable GRIN Lens Probing Proteins in Liquid

US-based researchers have created a flexible

needle-like endoscopic imaging probe that can
acquire 3D microscopic images of tissue. The
bendability is possible thanks to a new long and

© PNNL
US-based researchers at the Pacific Northwest
National Laboratory have used high-speed
atomic force microscopy to study the nucle-
ation of an aluminum hydroxide mineral on a
mica surface in water. PNNL Physical Sciences
Division Chemist, Ben Legg, and colleagues,
were able to characterize dynamically fluctuat-
ing nanostructures that precede the formation
of an aluminum hydroxide film, shedding new
light on how the mineral forms. The nucleation
and growth of minerals from solution is a critical
process in materials synthesis, biomineralization,
and geochemistry but the exact nature of these
processes can vary widely between systems.
flexible graded index (GRIN) lens developed by
Guigen Liu from Harvard Medical School, and col-
leagues. Incorporated into an endoscopy probe,
experiments indicate that the set-up’s imaging
properties are maintained even when the probe is
bent. GRIN lenses, commonly used in fluorescence
microscopy to image deep into tissue, are silica
glass rods with a continuously changing refrac-
tive index that focuses light coming through the
rod without requiring a separate focusing lens.
The researchers custom-designed a GRIN lens
500 microns in diameter and about 100 mm long.
The lens’ long, thin shape and its lack of a rigid
outer casing gives it the flexibility to bend about
10 degrees without breaking. The researchers
incorporated their new GRIN lens into an endo-
scopic imaging probe and tested it by performing
two-photon 3D fluorescence imaging through it.
© Berkley Lab
A new technique using infrared light from the
Advanced Light Source, US, has revealed how the
self-assembly of proteins is affected by environ-
mental conditions in a surrounding liquid. Most
proteins organize into hierarchical structures,
governed by dynamic non-covalent interactions,
such as hydrogen bonding, that are sensitive to
environmental conditions. However, the mo-
lecular complexity that ensues, calls for in vitro
characterization tools that are noninvasive and
provide nanoscale resolution. With this in mind,
researchers from Berkeley Lab and the Advanced
Light Source (ALS) combined AFM imaging and
synchrotron infrared nanospectroscopy to probe
soft matter in a liquid matrix at the nanoscale.

Original publication: Original publication: Original publication:

doi: 10.1126/sciadv.abn70 doi: 10.1364/OE.468827 doi: 10.1073/pnas.220001911
More information: More information: More information:
http://bit.ly/IM-042022-h http://bit.ly/IM-042022-d https://bit.ly/IM-042022-b

STOP Damaging Your Samples!

Get the all new and affordable CP-8000+ and see what you’ve been missing.
Traditional sample prep techniques can destroy the very features you are trying to study. Ion milling preserves delicate structure
while simplifying sample preparation. And ion milling is the easiest way to improve the imaging capability of the microscope you
already have. Give us a call and let us show you how the CP-8000+ can improve your image.

www.coxem.com

Imaging & Microscopy 4/2022 • 7

Announcement

Microscopy Conference
Darmstadt, Germany, February 26 – March 2, 2023

Modern electron microscopes offer a detailed each of the various main topics with plenary
view into the nanocosmos to better under- lectures, poster sessions and award ceremonies.
stand nature, materials and substances. Current In the accompanying industrial exhibition, man-
findings and trends in electron microscopy will ufacturers will show the latest developments in
be presented at the Microscopy Conference microscopy and microscopy technology with
MC2023 from 26 February to 2 March 2023 in accessories and consumables, preparation tools
© Comofoto - Adobe-Stock.com

Darmstadt, Germany. and image analysis systems.

At this largest and most important scientific
event in this field in Europe, the German Elec- The complete scientific program for MC2023
tron Microscopy Society (DGE) brings together is available at conference website.
leading experts from a wide range of disciplines
and aspiring young researchers for a compre-
hensive professional exchange. For five days,
the latest research results in the fields of in-
Technical university, Darmstadt

strumentation and methods as well as bio- and

material sciences will be discussed in Darmstadt
under the scientific direction of Prof. Dr. Ute
Kolb, Mainz, and Prof. Dr. Hans-Joachim Kleebe,
Darmstadt. Microscopy methods and their appli- Registration:
www.microscopy-conference.de
cations in the life sciences or materials sciences
will be presented in seven thematic sessions on

Trends in Microscopy
Münsingen, Germany, March 20-24, 2023

The “Trends in Microscopy” (TiM) for microscopy enthusiasts, with

is the biannual scientific meeting
of German BioImaging  – Society
for Microscopy and Image Analy-
sis e.V. (GerBI-GMB), with the next
TiM2023 planned for March next
year. Since 2020, TiM takes place
as a highly interactive and intense
microscopy “boot camp”. Inspired
by the French “MiFoBio School for
Microscopy”, the new TiM Ger-
BI-GMB Spring School mixes ple-
nary lectures with round-the-clock
practical sessions. Workshops
cover a variety of microscopes to
study a broad range of samples
as well as data analysis and man-
agement. The secluded and idyllic
Albgut-Altes Lager on the Swa-
bian Alb near Münsingen (usually
a highly requested wedding loca-
tion) transforms into a getaway
a fully functional S1 laboratory
for sample preparation, all kinds of
advanced microscopes, connected
to an image analysis server system
with several analysis workstations.  
The next TiM Spring School will
take place from March 20-24, 2023
in the same format. 45 excellent
workshops have already been se-
lected by the scientific program
committee and include MINFLUX
microscopy, expansion microscopy,
FRET experiments, tissue clearing,
light-sheet microscopy, big data
processing and visualization, image
handling in OMERO, ImageJ, Na-
pari, and many more. In addition,
we are happy to welcome  Stefan
Details and registration: Impressions of the
Hell, Erin Tranfield, Moritz Kreysing,
https://gerbi-gmb.de/ TiM2020: https://bit.ly/
Ulrike Endesfelder and Jan Huisken event/tim2023 video-TiM2020
for the TiM2023 plenary sessions.

8 • Imaging & Microscopy 4/2022


Focus On Microscopy 2023
A
fter the successful FOM2022 online
conference earlier this year, it is a
pleasure to announce FOM2023:
the next in a series of unique interdisciplin-
ary meetings on advanced and multi-di-
mensional light microscopy and image pro-
cessing. The conference will take place in
the week before Easter from Sunday, April
2 to Wednesday, April 5, 2023.
FOM2023 continues a long-standing (since
1988), yearly conference series on the latest in-
novations and developments in (optical) micros-
copy and their application in biology, medicine,
and the material sciences.
The FOM2023 conference will take place at
Centro de Congressos da Alfândega do Porto,
Rua Nova da Alfândega, Edifício da Alfândega,
Porto, Portugal.
The Porto conference will start on Sunday
morning, April 2, with tutorials, followed by par-
allel sessions, Flash poster presentations and a
plenary opening session with invited speakers
at the end of the afternoon. The first day will be
closed with a welcome reception. A technical ex-
hibition will be part of the Porto 2023 conference.
All details concerning registration, abstract
submission and deadlines are or will be available
on the conference website.
A wide range of microscopy and microscopy
related subjects will be addressed, ranging from
the physics of super-resolution, spatial image
formation to the advanced use of fluorescent
probes in living cells.
Porto, Portugal, April 2–5, 2023
Topics of the Upcoming FOM
Conference Include:
▪ Theory and practice of confocal and multi-
photon-excitation microscopy
▪ Super-resolution, nanoscopy imaging: from
PSF engineering (4pi, SIM, STED), fluorescence
activation / quenching, stochastic / centroid
(PALM, STORM, GSDIM, SOFI and related tech-
niques) to TIRF
▪ 3D and 4D live cell and tissue imaging
▪ Adaptive optics for microscopy
▪ Light sheet microscopy
▪ Phase / interference based microscopies
▪ OCT, holography, endoscopy
▪ Advanced fluorescence imaging / spectroscopy:
FRET, FRAP, FLIM, FCS, SOFI
▪ New fluorescent probes, proteins, quantum
dots, single molecule imaging
▪ Clearing and expansion techniques
▪ Coherent non-linear microscopies: SHG, THG,
SFG, CARS
▪ Multi-dimensional fluorescence and Raman
spectroscopy imaging
▪ Correlative microscopies e.g. light and elec-
tron
▪ Laser manipulation and tracking, photo-acti-
vation
More information and registration:
www.FocusOnMicroscopy.org
Announcement
© CC BY-NC-ND“ Associação de Turismo do Porto e Norte, AR
▪ Fast acquisition, automated and high-content
microscopy
▪ 3D image processing and visualization for
multidimensional data.
For information on program schedule, confer-
ence venue, and background on the present and
previous FOM conferences and exhibition please
visit the conference website.
Welcoming you to Porto for the
FOM2023 Conference and Exhibition,
the FOM2023 Organizers:
▪ Paula Sampaio, University of Porto, Portugal
▪ Hans Gerritsen, University of Utrecht, The
Netherlands
▪ Fred Brakenhoff, Honorary member
The FOM2023 Conference
Incorporates the:
▪ 36th International Conference on 3D Image
Processing in Microscopy
▪ 35th International Conference on Confocal
Microscopy
Contact the organizers:
info2023@FocusOnMicroscopy.org
Imaging & Microscopy 4/2022 • 9
RMS in Focus
Winners of 2022 RMS Awards Revealed!
T
he Royal Microscopical Society (RMS)
recently announced the winners of
its full range of awards recognizing
individuals making a special contribution
across microscopy, cytometry and imaging.
RMS President, Professor Grace Burke said: “It is
always a privilege to announce our award-win-
ners and give formal recognition to the work of
some of our leaders in microscopy. My warmest
congratulations to all of the 2022 winners. It
has been a pleasure to learn about their impres-
sive achievements and important contributions
to the microscopy community and beyond.”
This year’s awardees include:
Science Section Awards
Celebrating outstanding scientific achievements
across all areas of microscopy and flow cytome-
try. Selected by each RMS Science Section
▪ Alan Agar Award for Electron Microscopy: Dr.
Erin Tranfield, Instituto Gulbenkian de Ciência
in Oeiras, Portugal
▪ Award for Light Microscopy: Professor Chris-
tian Eggeling, University of Oxford, Fried-
rich-Schiller University Jena, and Leibniz In-
stitute of Photonic Technology Jena, Germany
▪ Award for Innovation in Applied Microsco-
py for Engineering and Physical Sciences:
Dr. Natalie Reznikov, McGill University, Canada.
▪ AFM and SPM Award: Dr. Alice Pyne, University
of Sheffield, UK
▪ Award for Life Sciences: Dr. Anjali Kusumbe,
University of Oxford, UK
▪ Early Career Award: Katherine Paine, University
of York, UK
▪ Award for Flow Cytometry: Chris Hall, Flow
Cytometry Facility Deputy Manager, The
Babraham Institute, Cambridgeshire, UK
▪ Award for Data Analysis in Imaging (Joint
Award): Dr. Peter Bankhead, University of Edin-
burgh; Dr. Robert Haase, TU Dresden, Germany
Scientific Achievement Award-Winners
Celebrating and marking outstanding scientific
achievements in any area of microscopy or flow
cytometry for established, mid-career researchers
▪ Dr. Andrea Centrone, National Institute of
Technology (NIST), USA
mmc2023:
www.mmc-series.org.uk
10 • Imaging & Microscopy 4/2022
RMS President, Professor Grace Burke
▪ Dr. Roland Nitschke, Albert-Ludwigs-Universität,
Freiburg, Germany
▪ Dr. Lothar Schermelleh, University of Oxford, UK
▪ Dr Hari Shroff, Janelia Research Campus, Vir-
ginia, USA
▪ Mr. Wim Hagen, European Molecular Biology
Laboratory, Heidelberg, Germany
▪ Dr. Ardan Patwardhan, EMBL-EBI, UK
RMS President‘s Award
Recognizing exceptional voluntary contributions
to the work of the RMS
▪ Mr. Steve Couzens, University Hospital of
Wales, UK
RMS Vice Presidents‘ Award
Recognizing the critical contributions to mi-
croscopy research, technique development or
education through this award to a scientist,
engineer or laboratory research staff
▪ Ms. Xiangli Zhong, University of Manchester
Chris Hawes Award for Outreach
and Education
Celebrating a substantial contribution to the
field of education, or to outreach and public en-
gagement, over the course of someone‘s career
▪ Dr. Elisabeth Bik, Harbers Bik LLC
RMS EDI & A Policy:
https://bit.ly/RMS-EDIA
The Pearse Prize
Established in 1982 to honor the work of Pro-
fessor AGE Pearse. A prestigious award  recog-
nizing significant contributions to histochemisty
and life sciences either through the development
of a new technique or through the application of
existing methods
▪ Professor Michael P. Sheetz, University of Texas
Medical Branch in Galveston, USA
RMS Launches EDIA Policy
A Working Document setting out how the RMS
is striving to become a more inclusive Society is
now available to download.
This has been produced by a working group
recently set up to look into the RMS’s Equity,
Diversity, Inclusion & Accessibility (EDI&A) policies
and to review all its activities.
The RMS is committed to being a welcom-
ing, inclusive Society and encourages diversity
across all activities and in the membership of its
committees and groups.
The Society welcomes discussion and scru-
tiny of its proposals and aims to be transparent
and open in all its policies and actions.
Save the Date!
Microscience Microscopy Congress 2023
(mmc2023) incorporating EMAG 2023 takes
place at Manchester Central from 4 – 6 July
2023. The flagship RMS event is returning as
an in-person event for the first time since 2019,
showcasing the very best in microscopy, imag-
ing and cytometry.
Contact
Allison Winton
Chief Executive
Royal Microscopy Society
London, UK
awinton@rms.org.uk
More on all award-winners:
https://bit.ly/RMS-Awards-revealed

José Maria Valpuesta,
EMS President
Virginie Serin,
EMS Secretary
Dear EMS members,
Last month, the second 2022 EMS Extension
(16th Multinational Congress on Microscopy
(MCM) took place in Brno, Czech Republic, on
September 4-9, 2022, gathering a substantial
number of international participants. The con-
gress was of very high quality with a substantial
number of excellent scientific contributions, in a
very exciting and pleasant atmosphere.
At the meeting, two activities were orga-
nized by the EMS. The Executive Board met on
Tuesday September 6, 2022 to discuss several
urgent matters and to prepare the General
Assembly Meeting of the following day. Fur-
ther, the General Assembly was attended by
94 EMS participants, including the EMS board
and EMS members (70 members on-site +  24
members online). Regular matters that in-
cluded the presidential report, budget, updates
on the EMC 2024, Sponsored Events, OPAs and
other activity prospects were discussed and
approved.
EMS web page:
https://bit.ly/EuroMicr
EMS Newsletter #79
A total of 16 scholarships of 300 € have been
awarded to students from 7 different countries
scholarships, for their participation in different
events, but mainly at the EMS extensions PICO
and MCM16.
For the next year, many good applications for
sponsored events for the first half of the year
(January till June 2023) have been received and
are now under review by the Board.
In the coming weeks, material for the 2022
EMS Yearbook will be gathered at the secretarial
office, including reports from the EMS Exten-
sions, the EMS sponsored events and the EMS
lectures, EMS scholarships, etc.
If you have organized or are aware of a par-
ticular event in the past year which you feel is
relevant for the European Microscopy Commu-
nity to be reported in the Yearbook (awards,
special anniversaries, ...), please send us a short
text with one or two pictures, so that it can be
included. In case we receive too many requests,
All EMS Events:
https://bit.ly/EMS-spons-Events
News from EMS
November 2022
some of them will be placed on the correspond-
ing pages of the EMS website
At the beginning of December, the call for 2022
EMS Outstanding Paper Award will be opened
to all members and we look forward to receiving
many excellent nominations. The awards will be
announced at IMC 2023 in Busan (South Korea).
Finally, we invite you to visit the EMS web-
page, which is daily updated to efficiently pro-
vide you with information from our community.
Any suggestions and ideas for future newslet-
ters are welcomed.
Contact
Prof. Dr. Virginie Serin
EMS Secretary
sec@eurmicsoc.org
Prof. Dr. José Maria Valpuesta
EMS President
jmv@cnb.csic.es
Suggestions and ideas
for future newsletters:
sec@eurmmicsoc.org
Imaging & Microscopy 4/2022 • 11
Cover Story
Mapping the Registry and Functional Properties
of Layered Materials Heterostructures
Using Conductive Atomic Force Microscopy
M
anipulating the registry of layers
within layered materials hetero-
structures leads to changes in
their functional properties both locally
[1,2] and at scales relevant to devices
[2,3]. This motivates interest in atomic
force microscopy (AFM) as a tool capable
of resolving both inter-layer registry and
a host of concomitant functional proper-
ties at nanometre length scales. Here, we
showcase conductive AFM (CAFM) as a
technique capable of determining the reg-
istry of layers and extracting functional
properties via spectroscopic measurements
over individual features.
Introduction
When two rigid layers with a fixed spatial pe-
riodicity are overlaid, a moiré pattern may be
observed. The symmetry of such features is
dependent upon the symmetry of the overlaid
layers and the periodicity is dependent upon
the mismatch in period of the two features
and the angle between them. Moiré patterns
can be observed in many situations including
in overlaid fabrics and installations such as
bridges but most recently moiré patterns have
received significant interest for their influence
over the functional properties of layered ma-
terials heterostructures, namely the field of
twistronics [3,4]. One prominent example of
where moiré patterns have been explored in
layered materials heterostructures is the case
of single layer graphene (SLG) on hexagonal
Fig. 2: Using contact mode AFM, a current map (a) of SLG on hBN was measured with VTS = 1
V and SPMech ~ 260 nN with Multi75E cantilevers, showing a uniform hexagonal moiré pattern
in the current map, as shown in Figure 1. To measure the moiré period, a line profile was taken
at an angle close to the fast scan direction (b), with an extracted mean spacing of 2.20 nm be-
tween local current maxima.
12 • Imaging & Microscopy 4/2022
James Kerfoot1 and Vladimir V. Korolkov1
Fig. 1: Using the Park Systems FX40 automatic AFM (a), CAFM measurements were performed
in contact mode to map the morphology of moiré patterns. A heterostructure of single layer
graphene (SLG) (b) on hexagonal boron nitride (hBN) (c) was produced by stacking mechanically
exfoliated flakes. The similar lattice constants of hBN and SLG give rise to a moiré pattern, as
depicted for a twisted heterostructure (d). With an electrode applied to the substrate and the
tip-sample interaction controlled via contact mode (e), current maps acquired under constant
voltage may be measured (f) which reveal a hexagonal moiré pattern due to the spatially modu-
lated stacking registry of hBN and SLG.
boron nitride (hBN). Using hBN as a substrate ic stamps. After removing polymer residue
[5] and encapsulant [6] of SLG enables the through solvent immersion and then mechan-
fabrication of devices in which the effects of ically by performing contact mode AFM over
contaminant induced doping is suppressed, the area of interest, samples were measured
yielding state-of-the-art performance charac- using CAFM on the Park FX40 automatic
teristics such as mobility. For SLG on hBN, a AFM with Multi75E probes (Fig. 1e and f).
hexagonal moiré period is seen owing to the
hexagonal symmetry and small, ~ 2%, spatial
mismatch between hBN and SLG (Fig: 1 b-d). Conductive Atomic Force Microscopy
Samples were first fabricated by exfoliat-
ing both hBN (HQ graphene) and graphene Conductive AFM utilizes the same setup as
on thermally grown SiO2 before flakes were contact mode AFM (with associated me-
sequentially picked up and transferred onto chanical setpoint: SPMech) to maintain the tip
pre-deposited contacts using polymer- in contact with the surface under constant
mechanical load via a feedback loop which
also allows the topography to be extracted.
By applying an electrical contact to the sam-
ple and using a conductive probe, the current
may be measured at the tip sample junction
and mapped spatially with sub-nanome-
tre resolution. Such current maps of SLG/
hBN (Fig.  1f  and  2a) reveal a hexagonal
moiré pattern with areas of higher current
observed for the network of domain bound-
aries between domains compared to the
central regions where the lattices stack in a
more parallel fashion [7]. When using CAFM,
structures are immediately observed in the
periodic, structures are seen in the current
channel without significant modification of
setpoints. The contrast of such features in

AFM images may be improved by optimizing
SPMech, the voltage applied between the tip
and sample (VTS), increasing the scan rate to
suppress thermal drift and changing the state
of the tip by applying short and controlled
pulses of higher voltage. Upon optimization
of the aforementioned acquisition parame-
ters, current maps showing nanometre scale
corrugations (Fig.  2a and 2b) may be ac-
quired, with extracted line profiles (Fig. 2c)
showing remarkable conformity.
In general, for layered materials hetero-
structures, the intra-layer forces arising from
bonding within layers are stronger than inter-
layer forces arising from van der Waals inter-
actions. For this reason, layered materials
heterostructures typically exhibit moiré pat-
terns as the stronger intra-layer interactions
yield comparatively rigid layers which are
not disrupted even when small relaxations
may lead to favorable inter-layer adhesion
energies [8]. In some special instances how-
ever, layered materials heterostructures may
be formed between layers of the same or sim-
ilar lattice constant and marginal twist angle
(typically <  2°) such that inter-layer inter-
actions are sufficient to induce changes in
the intra-layer structure, known as atomic
reconstruction and demonstrated for the first
time in marginally twisted bilayer MoS2 by
Weston et al [9,10].
We fabricated such samples using the
so called ‘tear and stack’ method [11], by
first exfoliating 1L-MoS2 (SPI supplies) on
polydimethylsiloxane (PDMS) using scotch
tape. The flake was then brought partially
into contact with a freshly cleaved highly
oriented pyrolytic graphite (HOPG) surface
and retracted, such that the flake broke, with
part left on the HOPG before the remaining
1L-MoS2 on PDMS was aligned to the first
flake and stacked on top. By breaking and
restacking the same flake, the twist angle
between two layers can be controlled deter-
ministically (0° was used here). Before mea-
suring using CAFM, samples were annealed
to 150°C for 5 minutes in air before cool-
ing to room temperature and scanned using
contact mode AFM to promote the removal
of contamination from the top MoS2 surface
and buried interfaces.
The cleaned 2L-MoS2 (0°) on HOPG
was measured using CAFM (Fig.  3b) with
VTS = 0.5 V and SPMech ~65 nN, with regular
triangular domains observed in both the cur-
rent and lateral force channels (Fig. 3 d and e)
but not in topography (Fig.  3c). Contrast
between current of AB/BA domains with
stacking registries (offset by half a unit cell)
was in good agreement with observations in
the literature [9,12]. In addition to the cur-
rent map, contrast was measured in the lateral
force image (the deflection of the cantilever
parallel to the scan direction and perpendicu-
lar to the cantilever) which implies differences
Fig. 3: By deterministically breaking and re-stacking a single mechanically exfoliated 1L-MoS2
(a) on HOPG, parallel stacked (0°) registries of 2L-MoS2 can be studied using CAFM (b). While
no contrast is observed in the topography channel (c), regular triangular patterns are observed
in both the current map (d) and lateral force map (e) due to differing stacking registries.
in the mechanical interaction between the tip
and 2L-MoS2 across different domains.
Comparing different areas of the same
2L-MoS2 (0°) on HOPG sample, areas of com-
paratively regular triangular domain mor-
phology (Fig.  3d) are observed in addition
to more distorted structures (Fig. 4a), which
we attribute to uneven distributions of strain
induced mechanically during sample fabri-
cation. In addition to measuring the mor-
phology of such domains, CAFM allows the
current-voltage characteristics of the junc-
tion between the tip and sample to be mea-
sured from isolated locations. By perform-
ing arrays of point current-voltage measure-
ments across a pre-mapped region, current
voltage maps from specific domains may be
measured, as shown in Figure 4b, to isolate
the properties of specific registries and gain
insights into how local variations affect the
performance of completed devices.
Conclusions
Stacking layered materials to form hetero-
structures may induce structural features
such as moiré patterns and atomic recon-
struction, which influences functional prop-
erties at both ‘locally and at device scales’.
Here, we have shown that CAFM enables such
Cover Story
Fig. 4: For the same parallel
stacked 2L-MoS2/HOPG sample
shown in Figure 3, a larger area
current map was measured, re-
vealing less regular distorted
domains (a). By positioning the
probe over specific domains,
current-voltage curves may be
measured (b), revealing differing
characteristics over regions of
high and low current contrast.
features in electrically conductive samples to
be measured with nanometre scale resolu-
tion and highly relevant functional proper-
ties measured via mapping and spectroscopy.
The advantage of CAFM is that it provides
information on the electrical properties of
materials at nanometre length scales which
are complementary to device characteriza-
tion techniques. Such insights are valuable
both in exploring the fundamental properties
of twisted layered materials heterostructures
and in the optimization of devices such as
transistors, memristors, photodetectors and
light emitting diodes.
Affiliation
1Park
Systems UK Ltd,
MediCity Nottingham, Nottingham, UK
Contact
James Kerfoot
Park Systems UK Ltd
MediCity Nottingham
Nottingham, UK
pse@parksystems.com
References:
https://bit.ly/IM0422-ParkSystems
Imaging & Microscopy 4/2022 • 13
Scanning Probe Microscopy

Inverted Scanning Microwave Microscopy

Application and Perspective on Biological Samples
Siti Nur Afifa Azman1, Eleonora Pavoni1, Marco Farina1

S
canning Microwave Microscopy (SMM)
is prominent for providing imaging of
sub-surface structures and allowing
local quantitative characterization of the
sample. A novel technique known as Invert-
ed Scanning Microwave Microscopy (iSMM)
is the improvement developed recently to
broaden the application beyond the current
focus on surface physics and semiconduc-
tor technology. With a simple metal probe,
the iSMM can be converted from existing
atomic force microscopes (AFM) or scan- Fig. 1: Schematic of (A) a conventional AFM-based SMM and (B) an inverted SMM.
ning tunneling microscopes (STM) outper-
forming the conventional SMM in terms of
bandwidth, sensitivity, and dynamic range.
The iSMM was primarily used to analyze bi-
ological samples as it can operate in liquid.

The scanning microwave microscope (SMM)

[1] is an instrument in the family of scan-
ning probe microscopes (SPM) [2], a fam-
ily including the well-known atomic force
microscope (AFM) and scanning tunneling
microscope (STM). In SMM a probe acting as
an antenna is raster scanned near a surface,
and during the scan, the local reflection co-
efficient of a microwave signal is recorded,
providing information about surface and
sub-surface impedance. A fundamental ad-
vantage of the SMM is its ability to quan-
titatively characterize the electromagnetic
properties of a sample by utilizing the near-
field electromagnetic interaction between a
nanoprobe and the sample itself. In some
implementations, a vector network ana-
lyzer (VNA) is used as both source and de-
tector of a microwave signal, radiated, and
sensed through the conductive probe. Usu-
ally, SMM works in tandem with some other
SPM technique, e.g., AFM or STM, providing
a mechanism to control and keep constant
the distance between probe and sample.
The use of SPM-based SMM microscopy
has recently gained more attention in bio-
logical and biomedical fields thanks to the
capability of this technique to measure elec-
tromagnetic parameters, strongly related to
physio-pathological conditions. However,
feeding an SPM probe in an extreme envi-
ronment, such as physiological buffers used to
keep cells healthy, has proven extremely chal-
lenging. A new setup called inverted-SMM
(iSMM), introduced by the authors in 2019
[3] overcomes most limitations of the origi-
nal SMM, related to the physiological envi-
Fig. 2: Simultaneous (A) AFM and (B) iSMM |S11| images of dried Jurkat cells.
ronments: the structure of the inverted-SMM
is low-cost, easily available, and compatible
with the physiological environment, which
also enables the application of SMM to the
biological living system. The idea is to move
the feeding from the probe to the sample
holder; in the iSMM, the sample holder is a
transmission line over which reflection and
transmission are measured while the SPM
probe, AC-grounded, only perturbs the line
through the sample. Consequently, iSMM
can be created by any existing SPM, by sim-
ply providing the appropriate sample-holder,
and of course, software syncing the measure-
ment on the transmission line and the SPM
scan. It is important to underline that the
proposed system is broadband, enabling fre-
quency spectroscopy, time domain analyses,
and microwave tomography. By far, the SMM
has been used to characterize living biological
cells despite the challenges operating in phys-
iological buffers [4,5]. Besides that, it has also
been used on subcellular organelles such as
mitochondria [6] that are responsible for cell
respiration and energy production. The iSMM
has proved to be able to overcome the limita-
tions of in-liquid operation by demonstrating
the first successful microwave imaging of live
cells in a physiological buffer [3].
Instrument Development
For a few years, research activity has been
based on a homemade STM-assisted SMM
built by mixing some features of systems
by Imtiaz [7], and the one developed by
Keysight [8]. Here in particular, a standard
tunneling microscope was combined, whose
feedback circuit was used to keep the probe
at a given distance from the sample, with a
microwave signal, in a reflectometric set-
up. However, in this instrument there is no
resonator, unlike in the Keysight instrument
and other available devices; consequent-
ly, the microscope could record data in the
whole frequency range allowed by the VNA.
In detail, this system exploits and controls a
commercial STM Microscope, a Solver P47
by NT-MDT, and an Agilent Vector Network
Analyzer PNA E8361, featuring 67 GHz
bandwidth and 120 dB of dynamic range. As
an example, this technique was applied to
image mitochondria [9], to assess dried can-
cer cells, and purposely treated to ascertain
the presence of incorporated fullerene [10].
System sensitivity has been improved by
exploiting the correlation of the images ob-
tained at several close frequencies and using
an expedient, the time-domain reflectometry

14 • Imaging & Microscopy 4/2022


[11-13], which can be understood in terms
of “spread-spectrum” modulation of the mi-
crowave signal using the tip / sample inter-
action; the information, spread across a fre-
quency spectrum, is recovered by collapsing
it in a single time instant by an inverse Fou-
rier transform. STM-assisted SMM provided
very high-quality images, with reduced arti-
facts due to topography “cross-talk,” namely
a replica of the topography due to changes
in the probe capacitance during scanning.
However, STM is extremely challenging
when dealing with poorly conductive sam-
ples such as biological ones, and even more
difficult when it is to be used in liquid.
Conventional SMM as shown in Figure 1
A) is generally obtained from an AFM (or an
STM) where the microwave signal is injected
and sensed by a reflectometric system: the
ratio between the reflected and the injected
signals, the so-called reflection coefficient
(S11), can be used to determine the spread-
ing impedance or dielectric permittivity of the
sample, after proper calibrations and analy-
sis. Such a one-port reflection measurement
usually has a dynamic range of 40-60 dB as
limited by directional couplers. In the iSMM
configuration as shown in Figure 1(B), the
conductive scanning probe (AFM or STM) is
always grounded, and the microwave signal
is injected through a transmission line (e.g.,
coplanar waveguide, slot line) that becomes,
in this way, the sample holder. The input and
output of the transmission line are connected
to a VNA, so that both reflected and transmit-
ted signals (S11 and S21, respectively) can be
measured [3,14,15]. Such a two-ports mea-
surement usually has a dynamic range of
120−140 dB, which makes it easier to sense
Fig. 3: Simultaneous (A) AFM and (B) iSMM |S21| images of a live L6 cell in saline solution.
Fig. 4: (A) AFM topography, (B) iSMM |S21| capacitance, and (V) iSMM |S21| dielectric
constant images of a dried L6 cell.
Scanning Probe Microscopy
the tiny perturbance caused by the grounded
probe when it scans across the sample.
iSMM Characterization of
Jurkat Cells and L6 Cells
Initially, the iSMM was demonstrated on
dried Jurkat cells as well as both dried and
alive L6 cells [3]. Figure 2 shows a compar-
ison of the AFM and iSMM S11 images of
dried Jurkat cells. Meanwhile, Figure 3 com-
pares the AFM and iSMM S21 images of live
L6 cells in a saline solution. The iSMM S11
and S21 signals were filtered at 4 GHz and
3.4 GHz respectively. The iSMM S11 image of
the dried Jurkat cells exhibits equal quality
to that of the AFM while the iSMM S21 of the
live L6 cell displays among the best quality
formed by transmission coefficient mea-
sured in liquid condition by two-port SMM.
In this work, a calibration procedure for
transmission mode measurement [16] was
applied on iSMM S21 of dried L6 cells. Fig-
ure 4 illustrates the effects of the calibration
presenting the AFM topography image, the
iSMM S21 capacitance image corrupted by
sample topography, and the iSMM S21 dielec-
tric constant image with the topography effect
removed at 6.2 GHz of the dried L6 cell. As
expected, ridges appeared near the periph- Filters, objectives, lenses, and more:
ery of the dried cell but the dielectric con- precision components for life science
stant of 2.8 ± 0.7 was uniform across the cell. applications
Essentially, this value is comparable to that of
• One-stop-shop with over 34.000
lipid bilayers in electrolyte solution [17] but is
different products
lower than that of dried E. coli bacteria [18].
Later, the quantitative characterization • Nearly 2 Million components in
in reflection mode measurement on iSMM stock for fast shipping
was performed on dried Jurkat cells [19].
• Support & manufacturing from
prototype to scale production
• Enabling applications including
OCT, ophthalmology, fluorescence
imaging, qPCR and more
EO - Your Optics Partner for Life
Sciences & Medical Devices
Find out more at:
www.
edmundoptics.eu/
life-sciences
EU/UK: +44 (0) 1904 788600
GERMANY: +49 (0) 6131 5700 0
FRANCE: +33 (0) 820 207 555
sales@edmundoptics.eu
Scanning Probe Microscopy
Fig. 5: (A) AFM topography, (B) iSMM |S11|, (C) iSMM φ11, and (D) dielectric constant image of dried Jurkat cell.
Fig. 6: (A) AFM topography, (B) iSMM |S11|, (C) iSMM |S21|, (D) time-gated iSMM |S11|, and
(E) time-gated iSMM |S21| images of the same mitochondrion in a glucose isotonic solution.
Figure 5 shows the AFM topography, the
raw iSMM S11 in magnitude, and the phase
acquired simultaneously at 4 GHz. The fig-
ure displays a good contrast between the
region with and without the sample, reveal-
ing additional characteristics related to elec-
trical properties that vary in the superficial
and sub-surface regions. The raw iSMM S11
image was calibrated following the algorithm
already described [20]. Figure 5 (D) shows
the extracted dielectric constant image of the
dried Jurkat cell which was around 2.6 ± 0.3
and homogeneous over the cell. This value
is congruent with previous data obtained by
conventional SMM on dried L6 cells [21].
iSMM Characterization of Mitochondria
in Living Environment
The iSMM latest work was performed on mi-
tochondrion fully immersed in liquid oper-
ating in a non-contact mode that minimizes
damage to the sample [22]. Figure 6 (A), Fig-
ure 6 (B), and Figure 6 (C) show the AFM to-
pography image with iSMM images S11 and
S21 acquired simultaneously on the same
mitochondrion with a diameter of approx-
imately 1 µm. The iSMM signals were fil-
tered and averaged over the frequency band
of 1.6-1.8 GHz. Visibly, the |S11| and |S21|
16 • Imaging & Microscopy 4/2022
images are of comparable quality, and both
reveal details that are not present in the AFM
image. Since the mitochondrion is non-con-
ductive, the contrast is readily apparent from
the surrounding CPW electrode. Different
than most SMMs, the iSMM is capable of
broadband measurements. So, it enables the
S11 and S21 signals measured from 1.6 GHz to
1.8 GHz by the iSMM to be transformed into
the time domain by inverse Fourier transfor-
mation. Subsequently, the unwanted signals
can be gated out to further improve the SNR
[13,20]. Finally, Figure 6 (D) and Figure 6 (E)
show the time-gated iSMM S11 and S21 im-
ages revealing finer details. The interaction
impedance between the iSMM probe and
the mitochondrion can be obtained from the
S11 and S21 measurements. In turn, the local
variation of the dielectric properties of the
mitochondrion can be extracted as has been
done for a live cell by the SMM [3].
Summary
The iSMM is capable of performing non-in-
vasive and label-free imaging of intracellular
structures of biological samples. iSMM can
be easily obtained by any existing scanning
probe technique, by simply using a suitable
sample-holder, providing an opportunity
to exploit this technique by most laborato-
ries. The iSMM images of the Jurkat cells,
L6 cells, and mitochondrion show good sen-
sitivity and quality displaying details that
cannot be seen in the AFM topography. The
quantitative characterization in transmission
and reflection modes measurements were
conducted for dried Jurkat cells and L6 cells,
respectively, by implementing a calibration
algorithm developed for conventional SMM.
The dielectric constant of Jurkat cells was de-
termined to be around 2.6 ± 0.3 meanwhile
the L6 cells showed around 2.8 ± 0.7. The
time domain analysis improves the iSMM
qualitatively and provides more insight into
the sample such as the mitochondrion.
Acknowledgments
We would like to acknowledge our research
group and all the people who contributed
to the scientific results herein reported. Part
of this work received funding from the Eu-
ropean project “Nanomaterials enabling
smart energy harvesting for next-gen-
eration Internet-of-Things” (NANO-EH)
(grant agreement No. 951761) (FETPRO-
ACT-EIC-05-2019). We would also like to
acknowledge Dr. Francesco Bigelli and Dr.
Paolo Scalmati from SOMACIS, Italy, for
their help in the realization of prototypes of
the sample holder.
Affiliation
1Department of Information Engineering,
Marche Polytechnic University, Ancona, Italy
Contact
Prof. Dr. Marco Farina
Department of Information Engineering
Marche Polytechnic University
Ancona, Italy
m.farina@staff.univpm.it
References:
https://bit.ly/IM-Farina
 speed
sen
siti
vity

re
s
olu
tio
n
3D Raman image of a pharmaceutical ointment.

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

Raman • AFM • SNOM • RISE www.witec.de

MADE IN GERMANY
Electron Microscopy
Analytical Electron Microscopy
of Organic Matter from Asteroids
Insights into Prebiotic Chemistry by Studying Asteroidal Organic Matter
Rhonda M. Stroud1, Bradley T. De Gregorio2, Conel M. O’D. Alexander3
C
arbonaceous asteroids contain rem-
nant organic building blocks from
the dawn of our solar system, some
in near original composition, and some sig-
nificantly altered over time due to heat-
ing and interaction with aqueous fluids
and minerals [1]. Laboratory imaging and
spectroscopy of the organic matter can
provide insight into its thermal and aque-
ous processing, and the variety of prebiot-
ic chemistry delivered to early Earth by as-
teroid and meteorite impacts. Meteorites
collected on Earth are the primary sources
of asteroidal organic matter for laboratory
study, but surface samples from asteroid
Ryugu are now available from the Japa-
nese Hayabusa2 mission [1] and samples
from Bennu are expected to be delivered
to Earth by the NASA OSIRIS-Rex space-
craft in 2023. The returned asteroid sam-
ples are especially valuable for investigat-
ing the primordial organic building blocks
of the Solar System because they have
well-established origins and limited expo-
sure to terrestrial contaminants.
Analysis Protocols for Organic Matter in
Meteorites
To make the best use of the highly valuable,
limited quantities of returned asteroid sam-
ples, analysis protocols for the organic mat-
ter must first be established through study of
18 • Imaging & Microscopy 4/2022
meteoritic organics. Acid-insoluble organic
matter (IOM) occurs in primitive meteorites
as discrete micron-to-sub-micron particles,
as clusters of particles, as coatings on in-
organic particles, and as vein deposits ex-
tending several microns. For measurement
of its bulk properties, the IOM is extracted
from its host meteorite through a series of
chemical dissolution steps [2]. The resultant
powder can be prepared by ultramicrotomy
Fig. 1: Annular dark-field scanning trans-
mission electron microscope (STEM) image of
organic matter extracted from a primitive me-
teorite by acid dissolution. Common micro-
structures of the organic matter include round
nanoglobules (NG) and fluffy intergranular
coatings.
© Romolo Tavani - Adobe-Stock.com
for scanning transmission electron micros-
copy (STEM) to study the nanoscale varia-
tion of the IOM in elemental composition
and C functional chemistry.
Using STEM, EELS and EDXS
to Characterize Meteorites
In our study, we used STEM imaging (Fig. 1),
electron energy loss spectroscopy (EELS) and
energy dispersive x-ray spectroscopy (EDXS)
to characterize IOM from meteorites with dis-
tinct thermal and aqueous alteration histories.
We prepared ultrathin sections of the extract-
ed IOM by S embedding meteorite particles
and then microtome sectioning them to thick-
nesses of 30 nm - 70 nm, and finally placing
the sections over holes in non-continuous C
support films. The STEM analyses were per-
formed with the Nion UltraSTEM 200-X at
the Naval Research Laboratory (NRL), oper-
ated at 60  kV with probe currents ranging
from 25 pA to 100 pA, with a nominal probe
size of 1.5 Å. The NRL UltraSTEM includes a
Gatan Enfinium ER energy loss spectrometer,
and a Bruker windowless, 0.7 sr SDD EDXS
system. In order to measure the C-K edge
fine structure and correlate it with elemental
composition variations, we recorded simul-
taneous EELS-EDXS spectrum images (SIs)
with Digital Micrograph in a single pass at a
spatial resolution of ~25 nm/px and 0.02 eV/
ch energy dispersion, with dwell times up to
 Electron Microscopy

Fig. 2: Simultaneous STEM-EELS-EDXS spectrum imaging (A) reveals variations in the C:N:O
ratios and C functional chemistry at the few nanometer scale, as illustrated with an RGB over-
lay of C, N, and O EDXS maps (B) and C-K edge near edge fine structure (fig. 3).

© agsandrew - stock.adobe.com

Fig. 3: (Left) Extracted C-K edge EEL spectra from ROIs 1-4 (fig. 2A), after power-law back-
ground subtraction and 5-pixel adjacent averaging for noise reduction. Variations in the C bond-
ing include regions of nanodiamond (1); graphite (2); and aromatic (C=C), amide (C=N), ketone
(C=O) and carboxyl (O=C-O) (3&4) functional groups. (Right) Extracted spectra from the stand-
alone EDXS SI of the corresponding ROIs permit quantitative determination of C:N:O variation
and validate the functional group assignments.

8 s/px and 16× sub-pixel scanning to spread Affiliations Access all articles by
the beam dose and reduce local damage (fig. 1Arizona State University, Tempe, AZ, USA scanning the QR-code
2U.S. Naval Research Laboratory, Washing-
2). Stand-alone EDXS SIs (2—4 nm/px) were

subsequently obtained with the Bruker Espirit
EDXS system for quantitative determination
of relative C, N, and O abundances. The func-
Meet
ton, DC, USA
3Earth and Planets Laboratory, Carnegie In-
stitution of Washington, Washington, DC,

the Greatest
tional chemistry assignments based on EELS USA
fine structure are validated by the variations

Minds in
in N and O x-ray intensity, e.g., increased O
counts in the EDXS corresponds to higher Contact
level of ketone (C=O) and carboxyl (O=C-O) Rhonda M. Stroud, Ph.D.
groups. Applications of these methods to Ha-
yabusa2 samples is underway and will help
determine which class of meteorite is most
Arizona State University
School of Earth and Space Exploration
Tempe, Arizona, USA
Microscopy
similar to Ryugu. Rhonda.Stroud@asu.edu

More on Geosciences: References: Wiley Analytical Science

https://bit.ly/WAS-Geoscience https://bit.ly/IM-Stroud

ARTICLE COLLECTION

analyticalscience.wiley.com
Electron Microscopy

Lithium Detection with

Secondary Ion Mass Spectrometry
Correlation of Surface Topography and Chemical Analysis
Gudrun Wilhelm1, Ute Golla-Schindler1, Timo Bernthaler1, Gerhard Schneider1

S
econdary ion mass spectrometry
(SIMS) allows the analysis of light
elements, especially lithium. We
used three different detectors to combine
secondary electron images with element
mappings that correlate surface topog-
raphy and chemical analysis, gaining new
insights into the aging phenomena of lith-
ium-ion batteries. Chemical compounds
were identified by measuring standard
samples and comparing their mass spectra
to those of aged anodes.

Introduction

The use of electric cars, bicycles and scoot-

ers is increasing and requires batteries with
high-performance and a long lifetime. In
developing these batteries, one essential
topic to understand is the aging process. If
lithium-ion batteries are aged, lithium en-
richment occurs on the anode surface. This
is proportional to the loss of functional
working lithium, which decreases the ca-
pacity of the battery. Nevertheless, the exact
structures and chemical composition remain
elusive. We expect that the combination of
imaging with secondary electrons and sec-
ondary ion mass spectrometry (SIMS) with
the correlative visualization of lithium will
deliver new insights.

Materials and Methods
The investigations were performed using a
Zeiss crossbeam 540 equipped with a Gem-
ini II column, a Schottky field emission
electron gun, an Inlens detector, an Oxford
Ultim Extreme EDS detector, and a focused
ion beam using gallium ions. A Zeiss time
of flight detector and a Hiden quadrupole
detector were attached to enable the SIMS
analysis. The third detector was a magnet-
ic sector detector which was attached to a
Zeiss Orion NanoFab working with helium
or neon ions. The lithium detection was
demonstrated using three different NMC/
graphite battery systems with reduced ca-
pacity (< 80%) and higher > 900 charge and
discharge cycles.
Fig.: 1: Surface topography of cycled anode foils with high lateral resolution; the graphite plates
are covered with (a) an encrustation, (b) small grains, (c) large precipitates consisting of spheri-
cal particles and fibers in the µm-range; (d) cycled anode surface analyzed with EDS; the pre-
sented point and field spectrum show differences in oxygen, fluorine and phosphorous content;
oxygen and fluorine prefer the same surface structures in the phase mappings.
Results homogeneous distribution which indicates
locally different aging conditions and pro-
The detection of secondary electrons with cesses. The chemical composition has been
scanning electron microscopy (SEM) vi- analyzed using Energy Dispersive Spectros-
sualizes the surface topography of cycled copy (EDS, fig. 1d). The EDS spectrum detects
anode foils with high lateral resolution the elements carbon, oxygen, fluorine, sodi-
(fig. 1a, b, c): the anode graphite plates are um and phosphorous. Aside from carbon, the
covered with (a) thin encrustations (a few nm highest detected amounts were oxygen and
thick), (b) nanoparticles (about 10-100 nm), fluorine. It is evident that the EDS field and
(c) large precipitates such as spherical par- point spectra are different: The field spectrum
ticles (about 100-500 nm), and large fibers has higher amounts of oxygen, fluorine and
in the µm-range. These structures have in- phosphorous. The phase mapping reveals

20 • Imaging & Microscopy 4/2022

that the measuring point of the
EDS point spectrum is placed in
a region of low oxygen and flu-
orine content, and that oxygen
and fluorine are both part of the
nanoparticles. This proves that
the inhomogeneous distribution
is proportional to a locally dif-
ferent elemental composition.
SIMS can detect a high lithium
signal (m/z 6 or 7) which allows
a lithium mapping in correlation
with a secondary electron image
(fig.  2a,  b). Lithium covers the
complete surface and is part of
all surface structures: the encrus-
tation, the nanoparticles, and
small and large fibers. High sig-
nal can be detected for particles
with high oxygen concentration
as the electronegativity of oxygen
improves the detection of lithium.
Lithium has a different bonding
partner leading to different sur-
face structures. Exemplarily, the
mass to charge ratios 33 and 55
(fig. 2c, d) are shown. M/z 33 is
part of the large fiber structures
whereas m/z 55 is enriched in the
small fiber structures. The inter-
pretation of the mass to charge
ratios must be done carefully.
M/z 33 can be interpreted as the
positive ions Li2Li3+, OLi2+ and
Li2F+. M/z 55 can be interpreted
as manganese. Copper, cobalt,
and nickel are present in the
same surface structures as man-
ganese. These elements indicate
the decomposition of the cath-
ode material (Mn, Co, Ni) and the
leaching of anode current col-
lector (Cu). Neither the encrusta-
tion nor the nanoparticles con-
tain m/z  33 and m/z  55. Only
m/z 6, 7 and 14 could be detected
in the positive ion mass spectrum.
The negative ion mass spectrum
delivers m/z  16 and m/z  19 for
them which can be correlated to
oxygen and fluorine.
The anode side of a separator
foil has been measured using the
Fig. 2: Surface topography (a) of a cycled anode foil correlated with Zeiss Orion NanoFab [1], which
SIMS element mappings (b-d); (b) lithium covers the complete surface allows a higher lateral resolution
and is part of all surface structures; (c) m/z 33 and (d) m/z 55 (man- than conventional SIMS. The
ganese) prefer different surface structures indicating different chemical lateral resolution depends on the
compounds. size of the ion probe and is there-

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
you need 3Dimaging, low pressure, EDS, or even EBSD, the EM-30 delivers the performance
and features advanced users expect, at a price that entry-level users can afford. Call your
local agent to arrange a demonstration, or visit our website for more information.

www.coxem.com

Imaging & Microscopy 4/2022 • 21

Electron Microscopy
Fig. 3: Surface topography of a cycled sep-
arator foil (anode side); correlated with SIMS
element mappings; Precipitates contain lithi-
um and fluorine and a small amount of oxy-
gen; nanoparticles contain lithium, fluorine,
silicon, and oxygen; semi-quantitative inter-
pretation of the secondary ion mass spec-
trometry measurement.
fore significantly improved for the NanoFab
(fig.  3). Spherical particles and nanoparti-
cles can be recognized. The spherical parti-
cles show a high signal for (b) m/z 6 (lithium),
(c) m/z 19 (fluorine) and (e) m/z 16 (oxygen).
The nanoparticles contain the same elements
and additionally (d) silicon (m/z 28). The
mass spectrometry results can be interpreted
semi-quantitatively using the mean counts per
pixel. This demonstrates the different chem-
ical compositions of the spherical particles
and the nanoparticles. The SIMS mass spec-
trum consists of element and molecule peaks.
The element peaks represent single isotopes,
and the molecule peaks consist of several iso-
topes. Precise interpretation of molecule peaks
is possible by comparing them with the peak
22 • Imaging & Microscopy 4/2022
Fig. 4: Comparison of (a) LiF mass spectrum with (b) cycled anode mass spectrum; the peaks of
m/z 6, 7, 14, 32 and 33 can be correlated to the cycled anode mass spectrum; the correct inter-
pretation of m/z 33 requires further standard sample measurements.
spectrum of standard samples. This has been
done in the next step and allows the determi-
nation of chemical compounds of the surface
structures. Figure 4a shows the mass spectrum
(positive ions) of the chemical compound LiF.
Several peaks could be found: m/z 6, 7, 14 and
a peak series around m/z 32 and 33. These are
the main peaks which can be interpreted as
Li (6 and 7) and Li2 (14). The group might be
seen as Li2Li3+ or OLi2+ or Li2F+. The lithium
isotopes 6 and 7 lead to several m/z ratios. This
mass spectrum can be compared to the mass
spectrum (positive ions) of the cycled anode
(fig. 4b). The main peaks show a good correla-
tion whereas the peaks with minor intensity
might be not visible due to the low LiF con-
tent on the cycled anode. The same must be
done for the mass spectra of the negative ions.
There the main peaks could be correlated as
well. This procedure proves that LiF is precip-
itated on top of the cycled anode. Comparing
this result with the SIMS mapping in figure
2 reveals that m/z 33 (and m/z 6, 7 and 14)
are part of the large fiber structures (fig. 3c).
Therefore, the large fiber structures may con-
tain LiF or may consist of LIF. Measuring stan-
dard samples can be used as a fingerprint-
ing technique and opens new avenues for the
interpretation of SIMS results.
Conclusion
The inhomogeneous surface topography
showing encrustation, nanoparticles and large
precipitates could be visualized with second-
More on battery research:
https://bit.ly/WAS-BR
ary electron images and analyzed with EDS
and SIMS. The lithium analysis with SIMS re-
veals that all structures contain lithium with
varying bonding partners such as oxygen,
fluorine and silicon in nanoparticles, lithium,
fluorine and oxygen in spherical particles,
and manganese in small fiber structures. The
preparation of standard samples, for example
LiF, enables mass spectra interpretation to de-
fine the exact chemical compounds.
Acknowledgments
We thank Hiden GmbH for the quadru-
pole mass spectrometer and Graham Cooke
for the helpful discussion, we thank Peter
Gnauck, Fouzia Khanom, Antonio Casares
and Carl Zeiss for the SIMS measurements
with the Orion, we thank Hubert Schulz for
the SIMS measurement with the time of
flight detector, we thank the IMFAA collab-
orators for their help and project LiMaPro-
Met for financial support.
Affiliation
1Materials Research Institute (IMFAA), Aalen
University, Aalen, Germany
Contact
Gudrun Wilhelm
Materials Research Institute
Aalen University
Aalen, Germany
gudrun.wilhelm@hs-aalen.de
References:
https://bit.ly/IM-Wilhelm

Seeing the Unseen
Boosted Absorption Imaging and Spectroscopy Using a Scanning Microresonator
Jonathan Noé1,2, Michael Förg1,2, Manuel Nutz1,2, Florian Steiner2, Rute Fernandes2, Ines Amersdorffer2, David Hunger3,4, Thomas Hümmer1,2
O
ptical detection of nanoscale ob-
jects without relying on fluores-
cence is a current challenge due
to their extremely weak interaction with
light. Resonator-enhanced absorption mi-
croscopy is a novel tool to heavily boost
the light-matter interaction. It combines
highly sensitive, spatially resolved imaging
with spectroscopy, offering a competitive
alternative to established methods for the
detection and analysis of nanoscale sys-
tems in nanotechnology, material design,
and the life sciences.
Current Limitations of Microscopy for
Nanomaterials
Characterizing nanoscale objects is pivotal
in the fields of nanotechnology and the life
sciences. Currently, the most used meth-
ods to detect and characterize the optical
properties of nanoscale materials are flu-
orescence and Raman scattering. Raman
scattering, however, requires very high laser
intensities to characterize nanoscale matter,
which often damages or destroys the sam-
ple. Fluorescence measurements, on the oth-
er hand, are limited to only a small fraction
of all nanoscale materials that show intrin-
sic fluorescence. Alternatively, detection by
labeling with fluorescent dyes is restricted to
nanoscale objects that are viable for label-
ing and does not provide information about
the inherent optical properties of the sam-
ple. Furthermore, dyes are subject to pho-
tobleaching and therefore limit the time for
investigation.
Since light absorption is an inherent
property of all materials, absorption micros-
copy is a promising alternative for the char-
acterization of nanomaterials. However,
measuring the absorption signal of indi-
vidual nanoobjects is challenging, since the
absorption cross-section scales linearly with
the volume of the object. Only a handful of
imaging techniques have demonstrated the
ability to image such weak absorption sig-
nals and only on a laboratory scale, where
the required sensitivity is achieved by var-
ious elaborate noise rejection techniques
[1-3]. As an alternative to these approaches,
straightforward highly sensitive absorption
measurements can be accomplished by sig-
nal enhancement via optical microresona-
tors.
Light Microscopy
© Image by Christoph Hohmann (MCQST)
Resonator-Enhanced
Absorption Microscopy
Optical resonators consist of two opposing
highly reflective mirrors, between which
light forms a standing wave. In other words,
light travels back and forth up to a million
times before exiting the resonator. As a re-
sult, the interaction between light and mat-
ter within this resonator can be amplified
by many orders of magnitude. Pioneered in
quantum optics this technology is known
for its ultra-sensitive sensing capability
since the many roundtrips of the light in
the resonator enhance various photophysi-
cal processes (e.g., absorption, fluorescence,
scattering, dispersion) [4]. Nonetheless, the
large resonator mode of conventional Fab-
ry-Perot cavities and the lack of control over
its position relative to the sample, make
them unsuitable for imaging.
This limitation was overcome by the inven-
tion of microscopic mirrors [5]. These atomi-
cally smooth concave micromirrors are typi-
cally fabricated by laser ablation on the end
facet of an optical fiber which is subsequently
coated with a highly reflective dielectric coat-
ing. Combined with a planar macroscopic sam-
Imaging & Microscopy 4/2022 • 23
Light Microscopy

Fig. 1: (a) Specifications and schematic

drawing of the resonator-enhanced absorption
microscope formed by a concave micromirror
on the end facet of an optical fiber (top) and
a macroscopic sample mirror (bottom). Weak-
ly absorbing samples are deposited on top of
the macroscopic mirror. (b) Scanning electron
microscopy image of the fiber end facet after
laser treatment. (c) Photograph of the micro-
mirror and its reflection in the macroscopic
mirror while forming a resonator.

Fig. 2: Image of an unstained ultra-thin (~ 80 nm) section of a human kidney biopsy in (a) a brightfield, (b) a phase contrast light microscope,
and (c) a resonator microscope. Scale bars, 20 µm. (Samples provided by Prof. Dr. med. Stefan Porubsky, Institute for Pathology, University Medi-
cal Center of the Johannes Gutenberg University, Mainz)

ple mirror at a distance of only a few microm-
eters, a resonator is formed. This yields an
almost diffraction-limited mode waist on the
surface of the planar mirror (Fig. 1) and thereby
offers the spatial resolution required for imag-
ing [6,7]. Scanning the micromirror over the
macroscopic mirror, an image of the sample
can be obtained, where light has interacted
with the sample several thousands of times
within each pixel [7]. Due to the many round
trips of the light in the resonator, even weakly
absorbing nanoscale objects on the sample
mirror lead to an easily detectable reduction
in the resonator transmission, making even
minuscule absorption (0.0001%) visible.
The power of this new technique is illus-
trated in Figure 2 showing the comparison
between images of an unstained ultra-thin (~
80 nm) section from a human kidney biopsy
acquired by brightfield (Fig. 2a), phase con-
trast (Fig. 2b), and resonator microscopy
(Fig. 2c). The strong light-matter interaction
achieved with the microresonator leads to
a significantly increased contrast, making
weakly absorbing biological structures vis-
ible. This level of contrast is usually only
achieved in a conventional light microscope
by imaging a semi-thin (100-times thicker)
section that has additionally been treated
with a contrast agent.
Highly Sensitive Imaging and Spectroscopy
in the Resonator Microscope
The potential and versatility of resonator
microscopy have been demonstrated on
various nanoscale objects, ranging from the
ultrathin sections in Figure 2 over macro-
molecules and atomically thin semicon-
ductors down to atomistic defects. Carbon
nanotubes (Fig. 3a) for example are an im-
portant building block of future nanoscale
optoelectronic devices. Their absorption in
the near-infrared could be imaged for the
first time on a single tube level by resona-
tor-enhanced microscopy [6].

Optical Filters
For microscopy applications

High-end quality · Custom-specific www.ahf.de

24 • Imaging & Microscopy 4/2022


Fig. 3: (a) Extinction map of single carbon nanotubes dispersed directly on top of the macroscopic mirror of the resonator, scale bar: 20 µm. (b)
Extinction spectra of different defect densities in a two-dimensional semiconductor. Inset: Extinction map showing areas of different defect densi-
ties. (c) Extinction spectra of a two-dimensional semiconductor heterostructure at two orthogonal illumination polarizations. Inset: Extinction map
for one polarization direction.
By tuning the illumination
wavelength inside the resonator
microscope, it is possible to mea-
sure the full absorption spectrum
of a weakly absorbing material.
Since only light meeting the res-
onance condition can enter the
resonator, the microscope acts as
a very sharp spectral filter.
Figure 3b shows an exam-
ple of the absorption spectra
of a transition metal dichalco-
genide monolayer, where parts
were treated with different doses
of an ion beam to create atomic
defects in the structure (marked
areas) [8]. Even very low defect
concentrations could be imaged
(Fig. 3b, inset) and spectrally
characterized with the resona-
tor microscope.
Not only the wavelength of
the illumination light but also
its polarization can be freely
chosen. An example of the use
of ultrasensitive imaging com-
bined with polarization control
is shown in Figure 3c [9]. Polar-
ization-dependent non-fluores-
cent excitations in a two-dimen-
sional semiconductor heterobi-
layer, which are connected to
crystal strain in the structure,
are resolved spectroscopically
and by imaging (Fig. 3c, inset).
Conclusion
Smaller and smaller building
blocks in nanotechnology, mate-
rial design, and the life sciences
create the demand for better de-
tection and characterization of
nanoscale matter. Resonator-en-
hanced imaging and spectros-
Light Microscopy
copy allow to access one of the Center for Quantum Science Contact
most fundamental properties of and Technology (MCQST), Lud- Dr. Jonathan Noé (CEO)
matter, namely absorption, even wig-Maximilians-Universität, Qlibri GmbH
for extremely weakly absorbing Munich, Germany Munich, Germany
3 Physikalisches Institut, Karl-
materials. With resulting sensi- noe@qlibri.eu
tivity orders of magnitude below sruhe Institute of Technology,
the shot noise limit of conven- Karlsruhe, Germany
4Institut für Quantenmaterial-
tional absorption microscopes, References:
many nanoscopic objects can ien und Technologien, Eggen- https://bit.ly/IM-Noe
be optically detected for the first stein-Leopoldshafen, Germany
time. The potential of this tech-
nique was already demonstrated
by the visualization and char-
acterization of macromolecules,
atomically thin semiconductors,
and thin films utilizing imaging,
spectroscopy, and polarization
contrast.
Acknowledgments
This work was supported by
the Bundesministerium für
Wirtschaft und Klimaschutz
(BMWK, Federal Ministry for
Economic Affairs and Climate
Action), and Europäischer So-
zialfonds (ESF, European Social
Fund) within an EXIST Transfer
of Research (Qlibri, Grant No.
03EFPBY231), the European
Commission within the program
Horizon 2020 “Fiber Nano-En-
gineering” (FINE, Grant No.
101034604), and the Bunde-
sministerium für Bildung und
Forschung (BMBF, Federal Min-
istry of education and research)
within a GO-BIO Initial project
(HAL2, Grant No. 16LW0124).
Affiliations
1Qlibri GmbH, Munich, Germany
2Fakultät für Physik, Center for
Nanoscience (CeNS), Munich
Imaging & Microscopy 4/2022 • 25
Light Microscopy
The Importance of Coverslips
Tips & Tricks for Coverslip Selection
S
ample preparation is often underesti-
mated, and microscopy users are not
aware of its importance. Choosing
the right coverslip is one critical aspect
that has a major impact on image quality.
Once upon a time, there was a young stu-
dent in a microscopy lecture. At the end of
the talk, the lecturer asked the students what
they thought was the most important part of
a light microscope. Answers such as Köhler
illumination, the objective, or the camera
were given. However, these were not the an-
swers the lecturer was looking for.
He then said that all the technical proper-
ties of the microscope were well designed and
that if something was not working properly,
a service engineer could fix it. The only thing
that is really in our hands is the quality of the
sample. If the sample is not well prepared,
even the best microscope cannot produce a
good image. Therefore, it is very important
that the sample is appropriately prepared.
More than 15 years later, it is clear what
the lecturer meant. As a facility manager, the
student of yesteryear now often sees sam-
ples that are not suitable for the microscope
of choice.
Fig. 2: Engravings on an objective. The value
after the slash in the red circle shows the re-
quired coverslip thickness, which is 0.17mm
in this case.
26 • Imaging & Microscopy 4/2022
Pascal Lorentz
Fig. 1: Sample. The sample is mounted between a slide and a coverslip.The slide is usually
standardized with a size of 76 mm x 26 mm and a thickness of around 1mm. The coverslip on
the other hand comes in various dimensions and thicknesses.
Sample preparation is an extensive topic the objective is designed for an infinite tube
and cannot be covered in its entirety here. length and coverslips with a thickness of
This tips & tricks article will therefore focus 0.17 mm. Using a coverslip with an incorrect
on the selection of the right coverslip. thickness will result in spherical aberrations
and thus a blurred image (Fig. 3).
Sometimes objectives are also available
Coverslips with a correction collar. As shown in Figure
4, the objective is then specified for a range
In most cases, the specimen is prepared be- of coverslip thicknesses. In this case, the cor-
tween an object holder (slide) and a thin rection collar allows compensation for a cov-
coverslip (Fig. 1). The dimensions of a slide erslip thickness of 0 - 2 mm. The correction
are usually 26 mm x 76 mm with a thickness collar can then be used to compensate for an
of about 1 mm. Coverslips on the other hand incorrect coverslip or spherical aberrations due
do exist in a variety of sizes and thicknesses. to other refractive index mismatches. The cor-
Users often just take what they find in their rect position of the correction collar is usu-
lab, not knowing the role of the coverslip. ally determined experimentally. The collar is
At the microscope, the image is usually cap- adjusted while the focus of the microscope is
tured through the coverslip. Therefore, the readjusted. The correct position is found once
coverslip is part of the optical configuration the image reaches the best possible quality.
and must match the desired properties. Coverslips have a round or rectangular
Many different engravings can be found shape and are available in various thick-
on microscope objectives. One of them is nesses. The thickness is often indicated with
usually “∞/0.17” (Fig. 2). This means that the # sign followed by a number. Coverslips
Fig. 3: Image quality. Left: Coverslip with correct thickness and or well-adjusted correction collar.
Right: Coverslip with the wrong thickness or correction collar not properly adjusted.

Fig. 4: Engravings on an objective. The value
after the slash in the red circle shows the re-
quired coverslip thickness, which in this case
is 0- 2mm. This means that the objective can
be adjusted to the coverslip thickness with a
correction collar (red square).
specified as #1 are widely used and one might
think that #1 stands for the best possible qual-
ity. In fact, the number represents a thickness
range. #1 stands for a thickness of 0.13 - 0.16
mm (Tab. 1). As mentioned earlier, microscope
objectives without correction collar require
coverslips with a thickness of 0.17mm, which
corresponds to #1.5 coverslips. As indicated
in Table 1, the thickness can vary greatly
depending on the brand and the quality of
the coverslips. It is therefore recommended
to choose coverslips with a small, specified
range such as 0.170 mm ± 0.005 mm.
If it is planned to use the coverslips for
fluorescence microscopy, care should also be
taken to ensure that the coverslips are made
of glass without autofluorescence.
Tab. 1: Coverslips are classified with different
numbers corresponding to a certain thickness
range. #1.5 coverslips are usually the covers-
lips of choice.
Coverslip # Thickness
00 0.06 – 0.08 mm
0 0.085 – 0.115 mm
1 0.13 – 0.16 mm
1½ 0.16 – 0.19 mm
2 0.19 – 0.23 mm
3 0.28 – 0.32 mm
4 0.38 – 0.42 mm
5 0.50 – 0.60 mm
Sources for Coverslips:
https://www.marienfeld-superior.com/cover-glasses.html
https://www.assistent.eu/en/portfolio/microscopy-and-staining
Light Microscopy
nobrainer.
Fig. 5: Interferences. Stripe-like pattern due
to a bent coverslip. The image has been ac-
quired with a point scanning confocal in the
far-red channel. The stripes become more
prominent at longer wavelengths.
Besides the thickness, another import-
ant aspect is the shape of the coverslip. Even
though the shape does not directly affect the
image quality, it can still cause problems. Lasers for Neuroscience
Users love large coverslips that cover the Perfect solution for 2-photon
entire slide, especially if they have multiple Microscopy and Optogenetics
sections mounted. However, larger and espe-
cially squared coverslips tend to bend. The
longer the coverslips are, the more they will
bend if the mounting medium is not evenly
FemtoFiber ultra 920 & 1050,
distributed or if a single piece of tissue is cov-
ered with it. Bent coverslips can then cause vario 1030 HP
interference and produce a stripe-like pattern
(Fig. 5). Therefore, round and smaller covers- • Fully turn-key with integrated
lips should ideally be chosen as they are more AOM and GDD
rigid. When mounting multiple samples on the
same slide, it is advisable to use multiple cov-
• No noise-stress for animals
erslips instead of a single large one. thanks to fully air-cooled design
• Compact laser design saving
Conclusion valuable table space
• Low cost of ownership using
As described, the coverslip is part of the
robust & reliable fiber-laser
optical calculation and should therefore be
selected with care. technology
▪ Thickness: 0.17mm = #1.5 (use brands
with low variability)
▪ Size: Round and small coverslips are pref-
erable to large, squared ones.
▪ Type: Use glass without autofluorescence
for fluorescence microscopy. learn more...
▪ Objective: If available, adjust the correc-
tion collar.
Contact
Pascal Lorentz
Head of Microscopy Core Facility
Director of Nikon Center of Excellence
for translational research
Department of Biomedicine
University of Basel
Basel, Switzerland
pascal.lorentz@unibas.ch
References: https://
bit.ly/IM-Lorentz
toptica.com/lasers4neuroscience
Image Processing
QuPath: Taking Full Control Through
Workflows, Scripting, and Extensions
My Favorite Image Analysis Tool, by Neubias Members
Q
uPath is an open source software
targeting the quantitative analy-
sis of whole slide images (WSI). Its
graphical user interface (GUI) makes it very
approachable for biologists and non-pro-
grammers to perform complex analysis
workflows. For improved reproducibility,
access to scripting through the Workflow
tab enables the creation of workflows as
scripts with little to no user-written code.
Should this level of control prove insuffi-
cient, access to the QuPath Application
Programming Interface (API) is possible via
scripting using the Apache Groovy Language
and the built-in script editor. Finally, the
modular, extension-based nature in which
QuPath is built allows for the creation of
custom extensions that can integrate into
the QuPath GUI to extend its functionality.
This short article comes as a follow up to
Dr. Laura Murphy‘s “The QuPath Commu-
nity“ [1], showcasing the attractiveness for
QuPath in terms of automation, scripting
and expandability.
QuPath: Beyond the GUI
QuPath (“QUantitative PATHology“) [2] fills
a niche that had been missing in the open
source ecosystem for image analysis: Pro-
vide ways to visualize and analyze 2D mul-
tichannel whole slide images (> 30‘000 pix-
els in x,y). The overarching philosophy of
QuPath is to build functionality around the
user, which means applying best practices in
user interface creation and code architecture
to make a tool that is both responsive and
intuitive, despite the large dataset sizes.
The workflows in QuPath typically
include detecting tissue or cells, classify-
ing them based on the presence of markers,
and measuring intensity, distance, or shape
features. A more complete overview of the
analysis capabilities can be read directly
via the main QuPath documentation [3]. If
the built-in capabilities of QuPath are not
enough, the possibility to send data back
and forth between QuPath and ImageJ (and
the massive number of plugins available)
covers a very broad range of bioimage anal-
ysis needs with “just a few tens of clicks“
(Fig. 1).
28 • Imaging & Microscopy 4/2022
Olivier Burri
Automation and batch processing are
built directly into QuPath. Unlike ImageJ/
Fiji [4,5], which handles each image inde-
pendently, QuPath utilizes the concept of
Projects to group images belonging to an
experiment, for ease of navigation on the
one hand, but also for batch processing.
So how do we ramp up realistically for an
experiment, from the single image analysis,
after a first manual “proof of concept“ step
has been achieved, to the automated, repro-
ducible analysis of many images, represent-
ing the multiple conditions and replicates? Olivier Burri
is a BioImage Analyst for the BioImaging
From Workflow to Script, „Run For Project“ and Optics Platform (BIOP) at the Ecole
Polytechnique Fédérale de Lausanne in
The QuPath Analysis pane has a tab named Switzerland. He joined the BIOP after finish-
“Workflow“, which keeps a record of the ing his Masters in Computational Systems
commands that were run on the currently Biology. He is developing bioimage analysis
open image. This is similar to ImageJ‘s mac- pipelines for the platform’s users and on
ro recorder. Generally, the first record in the tech watch for testing and introducing new
„Command history“ is the Set image type softwares and state of the art workflows. His
command, which QuPath needs in order to responsibilities also include training users in
offer tailored algorithms. One such exam- lightsheet microscopy.
ple is Cell detection which will modify its
user interface and parameters depending on
whether the image is of type Brightfield or
Fluorescence. If the image type is not set on
import, a popup will appear each time an
image is opened for the first time. To au-
tomate this step, once the image type has
been set manually, the Worklow tab can be
used by clicking on Create script. This opens
QuPath‘s built-in script editor, and prints the Java) name auto-filling, which helps avoid ty-
line of code needed to set the image type pos and can help search for desired methods.
programmatically (for example setImage- Output logging spits out the result of „print“
Type(‘FLUORESCENCE’); ) statements at the bottom of the script editor,
Clicking the Run menu opens up Run for as well as whatever other logs might appear
project. The new dialog will allow for the from your code, to help you keep track of
selection of all images in the project that will your script‘s progress, or for debugging (you
be affected by the script. Finally, pressing OK can also erase this log at each new Run). Fi-
will apply the script to each selected image nally, time-logging (activated by clicking on
sequentially. Run>Log script time) can help you visualize at
what time a script started and finished, help-
ful for estimating runtimes in batch.
QuPath‘s Script Editor
QuPath has its own script editor, capable of Complex Workflows
autocompletion, output logging, time logging, with Minimum Scripting
and syntax highlighting. Autocompletion,
(called with the keys CTRL+Spacebar, or CM- Typically QuPath workflows can embrace
D+Spacebar) enables method („function“ in the complexity of modern microscopy data.

Consider the LuCa-7color6
dataset [6], made available by
PerkinElmer. It is a fluorescence
image of a tissue section with 8
channels (DAPI, six Opal dyes
targeting a specific cell type
marker, and an autofluores-
cence channel).
Suppose we wish to create
a workflow to count the total
number of cells positive for each
Opal dye (which can have com-
plex expression patterns of one or
more dyes within each cell), and
obtain the distance of these cells
to particular tissue regions e.g.
tumor, differentiated by the size
of their nuclei. In QuPath, such
a workflow might look like this:
▪ 1. Discriminate regions of
cells with large nuclei and
small nuclei using machine
learning (a pixel classifier)
▪ 2. Detect cells in each region
type with different „Cell de-
tection“ settings (to account
for the nuclei size difference)
▪ 3. Classify each cell as posi-
tive for any combination of
up to 4 stainings individually
and then jointly.
▪ 4. Determine the coverage of
another signal via machine
learning as a third type of
region
▪ 5. Measure the distance of
each cell to each region.
All these steps can be execut-
ed through the QuPath GUI as
illustrated in the excellent vid-
eo tutorial by Dr. Sara McAr-
dle, from the La Jolla Institute
for Immunology [7] (and from
which the script associated with
this article is based). After the
necessary manual steps are
completed (Creating the tissue
and cell classifiers), the Work-
flow tab (Fig. 2) can then gen-
erate a script that can be run
on any other image or in batch
(Fig. 3). The script is available as
a supplementary file [8].
Note that to create this script,
no code had to be written: it is
sufficient to remove unneces-
sary lines from the workflow
(like some unused cell detection
runs) to get a working code.
While this script is not
“self-contained“ (it will not
run unless the user has created
all the classifiers beforehand),
it does allow for a one-click
solution to batch analysing
Image Processing
Fig. 1: QuPath GUI. A powerful graphical user interface allows for the creation of complex workflows. There
is very little that cannot be done interactively either directly through QuPath or using the ImageJ connection.
all subsequent images. More- The most common example ist for QuPath“ series of Groovy
over, any updates in the classi- of a QuPath script that needs to scripts by Zaidi et al. [12], that
fiers will be automatically taken be written from scratch, unlike aim at transferring the existing
into account when the script is those based on workflow record- StarDist nuclear segmentation
re-run. ing, is the use of the StarDist models to other image modal-
[10] extension [11]. As no GUI ities.
is available for this extension, it In addition to calling exist-
Going beyond „Macros“, can only be called from a script. ing libraries through their APIs,
the Groovy Language and Having the extension available Groovy can help automate sim-
QuPath‘s Guts through scripting adds to its ple tasks in QuPath thanks to its
flexibility, as shown by the cre- many convenient methods for
QuPath is fully written in Java ation of the “Universal StarD- working with lists of objects.
and opted for Groovy as its
scripting language. Formal-
ly called The Apache Groovy
Language [9], it was born in
2007, and has seen consider-
able development since then
(Groovy 4.0 was released early
2022). It is a language built for
the Java platform that greatly
simplifies interacting with Java
libraries and contains powerful
productivity shortcuts to most
common operations, especially
on lists. It is loosely typed, like
Python, but Java code runs di-
rectly if copy-pasted into the
Groovy interpreter.
Unlike the ImageJ macro
language, however, Groovy
has access to the entirety of
any Java library or plugin that
has been loaded and isn‘t lim-
ited to a subset of commands.
In QuPath, this means access
to the core of QuPath itself,
along with any loaded exten-
sions and/or jar files in the Fig. 2: The QuPath Workflow tab, which keeps a history of the com-
QuPath user Directory ‚exten- mands that the user has used on the currently open image, and enables
sions‘ folder. one-click script creation.
Imaging & Microscopy 4/2022 • 29
Image Processing

Fig. 3: Using the Workflow tab to create a complex script for batch analysis does not necessarily require writing code. Choosing the appropriate
lines (highlighted) in the Command history can be more than enough.

Finding the largest cell in the current image one selection criterion. One way to over- value. Groovy provides many convenience
and displaying it to the user only takes two come this limitation, if we had to select mul- methods for iterating lists. To print out the
lines (Fig. 4A). tiple conditions, such as “of class “Tumor“ mean intensity of DAPI in the nuclei of
max is a Groovy method that operates with an area above 1000 pixels square“, cells, one can make use of the each method
on the list provided by getDetectionObjects(), is to use Groovy‘s findAll method instead (Fig. 5D).
which is an internal QuPath method belong- (Fig. 5B). If instead of printing, the goal is to collect
ing to the QP Java class [13]. Groovy‘s max Similarily, one can use a closure to help and store the values in a new list, the collect
method takes a closure, or piece of code, as compute the average circularity of all anno- method avoids the need to create a new list
an argument. tations (Fig. 5C). Here again, sum takes a clo- and add the values in a loop (Fig. 5E). This
If read in plain English, that line of code sure, which should explain what value should results in dapiMeanList containing the val-
translates to: „From the detection objects be summed up. That value, inside the closure, ues of each cell‘s DAPI average intensity in
list, return the object that has the maximum is the circularity metric, which is retrieved by the nucleus.
value defined by the area of its ROI (Region RoiTools.getCircularity(), which takes the ROI The code examples above are hardly
of interest). The unusual it element refers to of the annotation as an input argument. intended as a way to get started in script-
a single element in the object list (You can Very often, it is necessary to iterate ing in QuPath, but more as examples to
read it as “iterator“). This enables the request through a list of objects to either extract showcase the capabilities of scripts in effi-
of arbitrary metrics for the max calculation, some information from it or add information ciently accessing QuPath objects and their
without having to code a loop. to it, typically a new custom measurement features. Very complete and elegant online
For comparison, writing such a method
in modern Java can become quite verbose
(Fig. 4B).
As mentioned when introducing Groovy,
note that you can copy paste the Java code
above into QuPath‘s script editor and it will run!
Many methods in Groovy take closures,
such as findAll. Using the Workflow tab,
obtaining the list of “Tumor“ objects needs
just two line (Fig. 5A), of which only one is
written by the user.
While straightforward, it has the lim-
itation that a list can only be created with
pre-selected objects and there can only be Fig. 4: Groovy code to find the largest objects based on its area, and Java equivalent.

30 • Imaging & Microscopy 4/2022

 Image Processing

Table 1
Extension Repository
ABBA https://github.com/BIOP/
qupath-extension-abba
Cellpose https://github.com/BIOP/
qupath-extension-cellpose
OMERO https://github.com/qupath/
qupath-extension-omero
OMERO https://github.com/BIOP/
RAW qupath-extension-biop-omero
StarDist https://github.com/qupath/
qupath-extension-stardist
Warpy https://github.com/BIOP/
qupath-extension-warpy
Fig. 5: Illustrative code showcasing the strength of the Groovy language
to manipulate list type data, and the use of closures, used throughout
the article.
Description
Support for importing Aligning
Big Atlases And Brains (ABBA)
into QuPath
A wrapper that bridges the
Cellpose algorithm with QuPath
in 2D
Support for accessing images
hosted on an OMERO server
through OMERO’s web API.
Support for accessing images
hosted on an OMERO server
through the OMERO Gateway
API
Support for running the 2D
version of StarDist nucleus
detection developed by Uwe
Schmidt and Martin Weigert.
Support for non-linear deforma-
tion and registration in QuPath,
by using the ImgLib2 library

resources exist, such as the QuPath docu-
mentation itself, Michael S. Nelson‘s home-
page [14] contains a trove of information
on how to get started in QuPath scripting,
and the QuPath JavaDoc [15] which has
been referenced multiple times, for exploring
the different classes themselves. For those
interested in getting started with Groovy
itself, the incredibly high-quality posts of
Mr. Haki [16] featuring the “Groovy good-
ness“ tag were my personal starting point, as
well as the official Apache Groovy learning
resources [9].
Throughout the examples above, calls to
seemingly disconnected methods were made
(getAnnotationObjects(), selectObjectsByClas-
sification(), ...), so where do these come from?
Because QuPath was built with the under-
standing that the bulk processing will be
done through scripts and that no GUI will
be as flexible to create workflows as scripts,
many “static“ classes exist to help the user
create and manipulate QuPath objects. The
first entry points are typically the QP and
QPEx [17] classes that ease the manipulation
of annotations, detections, projects, classifi-
cations, and much more. Moreover, specific
classes exist to ease the automatic creation of
annotations and detections (the PathObjects
[18] class), to communicate with ImageJ (the
IJTools [19] class), to inform or prompt data
from the user (Dialogs [20] class) or to take
image snapshots (the SVGTools [21] class).
Each method in these classes is well docu-
mented in QuPath‘s JavaDoc and serves as an
entry point to QuPath‘s core functionalities,
so when looking to do something in QuPath,
search for class names that end with an “s“!
Even further Down the
Rabbit Hole: Building QuPath
Extensions
A lot of work was spent refactoring QuPath
during version 0.3.0 in order to support a
more modular framework, with extensions.
Thanks to this effort, self-contained jar files,
in much the same way as ImageJ plugins,
can be created and imported into QuPath
in order to bring in new functionality. The
StarDist extension is a prime example. Sim-
ply dragging and dropping the jar file en-
ables QuPath to call upon StarDist for cell
segmentation. Other such extensions that
currently exist are listed in table 1.
While not particularly simple to create
(there is not yet a formal documentation on
how to code an extension from scratch, the
extensions above can serve as starting points
for developers to create their own.
Hopefully this short overview of Groovy
and the scripting possibilities within QuPath
All NEUBIAS articles:
https://bit.ly/Neubias-IP
has motivated you to try building your own
scripts and has given you hints on how to
get started and more importantly, where to
look for information and guidance.
The resources provided in this short arti-
cle should offer a good foundation to dive
deeper as needed, and of course the image.
sc forum [22] remains the premier commu-
nication channel to discuss code, scripts and
image analysis in QuPath.
Acknowledgements
A great thank you to Pete Bankhead for all
the time spent explaining the inner workings
of QuPath and for actively and passively
helping me improve my Java skills through-
out our interactions. Thank you to Anjalie
Schläppi, Arne Seitz, Nicolas Chiaruttini,
Romain Guiet, Remy Dornier and Kota Mi-
ura for proofreading and their suggestions.
Contact
Oliver Burri
Ecole Polytechnique Fédérale
de Lausanne
Lausanne, Switzerland
olivier.burri@epfl.ch
Long version and references:
https://bit.ly/IM-Burri

Imaging & Microscopy 4/2022 • 31

Wiley Analytical Science Award 2023

The Winners of the Award

W
e are pleased to present the winners of the Wiley Analytical Science Awards 2023. Thanks to all participants and also to our
customers and readers for voting. Congratulations to all our award winners for their great products. View the complete award
ceremony on-demand: https://bit.ly/WASAward2023-Winners

Category A:
Spectroscopy & Microscopy MPX-1040: a Turnkey, Portable
and Flexible Multiphoton
Microscope
DriveAFM - High-Performance
Atomic Force Microscopy
The DriveAFM is a fully-motorized atomic
1 The MPX-1040 is a turnkey, compact,
and easy-to-use multimodal microscope,
combining different imaging techniques: 2
and 3-photon, higher harmonics, and widefield microscopy of samples
force microscope developed for applications in ranging from single cells up to living animals. Confocal resolution and
materials science, physics and chemistry and life sciences. It is a high penetration depth makes it a versatile 3D microscope, even for
the first fully motorized tip-scanning AFM with photothermal ex- label-free markers such as collagen in the tissue.Key features are the
citation that can be used as a benchtop system or can also be in- fully integrated, high-power, tunable fs-fiber laser and the

corporated with inverted optical microscopes for combined AFM
and optical microscopy. Its 100 x 100 x 20 µm3 scanner resolves
µm-sized structures and atomic lattices. It features photothermal
cantilever excitation (PTE), which ensures stable operation not
only but particularly in liquid environments. PTE
is the basis for fast off-resonance imaging
and temporally resolved mass measure-
ments at the nanogram level, overcomes
the ‘forest of peaks’ found with piezo-
based cantilever excitation in liquids and
provides a cantilever response across the
whole frequency spectrum. The device is
capable of characterizing the nanomechni-
cal and nanoelectrical (e.g. KPFM, PFM, SMM)
properties of many different samples.
Nanosurf AG | www.nanosurf.com
2
free-moving scan head, providing imaging flexibility even
for non-experts. The microscope consists only of two
compact parts: a small air-cooled controller unit with
an integrated PC and a compact imaging scan head, both
connected through a flexible umbilical cord. Key features are
the fully integrated high-power wavelength tunable femtosecond fiber
laser engine, resonant-galvo-galvo scanning, up to 4 highly sensitive
PMTs, high-performance wide-field imaging, and a motorized objective
turret.
Prospective Instruments LK OG | www.p-inst.com
Thanks to Wiley to let us participate in
this award and of course to the voters
which put us on the second place.

For Nanosurf as a company it

is really an honor to receive this
award.

Antimicrobial Coating
Thank you very
picoStudio, Drobot Dean - stock.adobe.com

By testing different mixtures of metal and metal oxide nanoparticles,

characterization by high resolution optical microscopy, SEM EDX, ICPOES, much for this
ICPMS, efficient antimicrobial mixtures were developed that are bound to
beautiful opportunity
the polymer surfaces by sol gel methodology, resulting in an antibacte-
rial coating with possible application in medical textiles, medical polymer to present our

3
surfaces, bandages, catheters and others. Chemometrics and mathematical
modeling were applied to optimize the formulae and find the most effective, innovation.
which managed to destroy golden bacteria resistant to antibiotics, such as MRSA.

University of Zagreb | www.ttf.unizg.hr

©

32 • Imaging & Microscopy 4/2022

 Wiley Analytical Science Award 2023

Waters Andrew+ Pipetting Robot Category B:

The Waters Andrew+ Pipetting Robot provides fully automated liquid han- Separation,
dling for applications like PCR Prep and plasmid DNA purification. Its flexible
architecture enables scientists to effortlessly transition from laborious man- Lab Automation
ual pipetting procedures to error-free, fully-robotized lab workflows, without & Equipment

1
any knowledge of programming, laboratory robotics, or automation engineering.
The Andrew+ robot uses single and multichannel electronic pipettes to dispense liq-
uid volumes from 0.2 µL up to 10 mL. The device can communicate wirelessly
via Bluetooth with its electronic stand which is connected to the cloud us-
ing Wi-Fi or Ethernet, so users can remotely monitor experiments from their Thanks for this
computer or tablet. The robot also executes protocols on its connected devices en-
abling guided pipetting, shaking, rapid heating/cooling, magnetic bead separation, distinguished honor.
or micro-elution for solid phase extraction. It gives users complete control over their
workflow through the compliant-ready OneLab software.

Andrew Alliance S.A. | www.andrewalliance.com

Waters MaxPeak Premier

Protein 250 Å SEC Analytical Columns
2
Wiley
Analytical Waters’ analytical columns address several needs within biopharma, biotech,
Science and contract research organizations as well as immunodiagnostic kit devel-
Award opers who need reliable, size-based analyses of mAbs, anti-drug conjugates,
2023 and other proteins that range in size from 10,000 to 650,000 Daltons. These
columns use the company’s proprietary MaxPeak column hardware technol-
ogy that significantly reduces undesirable ionic interactions and comes with its
bridged-ethyl hybrid SEC particles containing hydroxy-terminated polyethylene ox-
ide that reduce undesirable hydrophobic interactions. The columns use readily available phosphate buffer
saline (PBS) without having to invest time and resources developing an appropriate SEC eluent formula-
tion required by many traditional SEC columns. This synergistic coupling allows separations scientists to
use generic or platform-type SEC analytical methods for many different protein types.

Waters Corporation | www.waters.com

To receive this award in this area

validates this work and that this is
resonating with our customers.

3
CellScrew

The CellScrew Innovation is a patented single-use cell cultivation system for adherent
cell cultures. Such cells are used, for example, in the production of vaccines and cell and
gene therapeutics. Compared to conventional systems, the device provides a significant
increase in efficiency in the manufacture of upscale biopharmaceutical products that can
then be produced either in larger volumes or at less cost. Simultaneously, the cell cultivation can This prize gives me
be realized for the first time with CO2-neutral materials thanks to an innovative manufacturing pro-
cess. The CellScrew is the first cell culture vessel worldwide that is made out of a plant-based plastic hope for the planet
(PLA). Instead of just using the inner surface of the bottle as a growth surface, this new system has as we also tackle the
an Archimedes screw in the inside of the bottle. The culture medium can be drawn along the screw by
rotating the tilted container. There is then a 10 to 20 times larger growth surface within the dimen- sustainable issue of
sions of a standard roller bottle.
single use plastics.
Green Elephant Biotech GmbH | www.greenelephantbiotech.com

Imaging & Microscopy 4/2022 • 33

Products
Products

SHOWCASE |

Introduce Deep Learning to your Materials R&D and QA / QC

In today’s fast-moving environment, reducing the projects turnaround
time, meeting productivity targets, while reducing operating cost can
be challenging without a smart automated approach. To avoid product
recalls, meet increased customer demands and get products right the
first time, a thorough investigation of the materials microstructure is
key. Defect analysis, inclusion analysis, grain sized analysis are only a few
of the limitless applications.

MIPAR Image Analysis combines customized algorithms and powerful

deep learning systems to produce technology able to perform sophis-
ticated structure analysis processes. Whether is titanium, copper, steel,
aluminium, or ceramics, MIPAR software allows for automated micro-
graph analysis that streamlines the data analytics, improves data quality,
and offers new layers of information. Automation reduces operator error
and improves professional productivity.

Did you know that more and more companies are using deep learning
to double check the material quality provided by suppliers? Be ahead of
your customers by introducing these capabilities in house. Improve your
customer satisfaction, avoid rework, while reducing the operational cost.
Key Features
▪ Replace manual analysis, increase efficiency
▪ Meet increased customer demand without compromising accuracy
▪ Be in line with suppliers and internal R&D departments
▪ Recover software investment quickly

Mipar Software LLC

info@mipar.us Learn More About
www.mipar.us Deep Learning:

New Generation of Quality Monitoring Slides Supercontinuum White Light Laser

for Fluorescence Microscopes
AHF analysentechnik offers the
second generation of Argolight’s
quality monitoring slides! The
slides contain a glass with differ-
ent fluorescent reference patterns
and are designed to regularly
check and monitor the quality
and performance of fluorescence
imaging systems. The new gener-
ation is equipped with ArgoGlass
AG03 which improve the fluores-
cence intensity of the fluorescent
reference patterns two to three
times compared to glasses of the
prior generation. Thanks to the
new glass design it’s possible to
use the slides in a wider range
of wavelengths, especially in the
red. The new glass shape circle
also avoids issues with oil objec-
tives. Argolight has also updated
its software ‘Daybook’ for image
analysis and data management.
It comes along with improved al-
gorithms, optional windows that
make it easy to determine if the
imaging system is within speci-
fication and more. The Daybook
Data Manager will be able to es-
tablish statistics and easily deter-
mine tolerance thresholds.
AHF analysentechnik
www.ahf.de
To imitate Superman who can see
across the full electromagnetic
spectrum, the SuperK laser from
NKT Photonics helps to see things
that are usually invisible to the na-
ked eye. In a hyperspectral imaging
setup, you can analyze and sort
general waste and different plastic
types, classify textiles, determine high brightness light that can be
mineral composition, detect food focused into a small spot or narrow
adulteration and many more things. line - all across the SWIR range. It is
High-brightness light sources such made for industrial use, rugged and
as the SuperK lasers offer a higher compact, and runs for over 10,000
resolution and better signal-to- hours without maintenance.
noise ratio resulting in higher
throughput. The light source covers NKT Photonics
the 1700 – 2500 nm spectrum and www.nktphotonics.com

34 • Imaging & Microscopy 4/2022

 Index / Imprint

Aalen University 20 Green Elephant Biotech 32 Qlibri 23

AHF Analysentechnik 24, 34 Karlsruhe Institute of Technology 23 Royal Microscopical Society (RMS) 10

Andrew Alliance 32 Ludwig-Maximilians-Universität 23

Tescan Inside Front Cover
Arizona State University 18 Marche Polytechnic University 14
Toptica 27
Carnegie Institution of Washington 18 Microscopy Conference 8
Trends in Microscopy 8
Coolled  25 MIPAR Image Analysis Software 34
University of Basel 26
Coxem  7, 21 Mipare Software 34
University of Zagreb 32
Ecole Polytechnique Fédérale Lausanne (EPFL) 28 Nanosurf  32
US Naval Research Laboratory 18
Edmund Optics 15 NKT Photonics 34, Outside Back Cover

European Microscopy Society 11 Park Systems  Cover, 12 Waters 32

Focus on Microscopy 9 Prospective Instruments  32 Witec 17

Imprint Segment Sales Manager

Laboratoty & Biotechnology
Subscription 2022
Four issues 40.00 €, single copy 15.30 € ca. plus postage.
Published by Vanessa Winde Pupils and students receive a discount of 50 % at sight of a
Wiley-VCH GmbH Ph.: +49 6201 606-721 valid certificate. Subscription orders can be revoked within
A Company of John Wiley & Sons, Inc E-mail: vwinde@wiley.com 1 week in writing. Dispatch complaints are possible only within
4 weeks after publishing date. Subscription cancellations are
Managing Directors Media Consultant
Sabine Haag, Dr. Guido F. Herrmann accepted 6 weeks before end of year.
Dr. Stefanie Krauth
Ph.: +49 6201 606-728 Bank account
Publishing Director, Chem & Lab
Dr. Heiko Baumgartner E-mail: stefanie.krauth@wiley.com J.P. Morgan AG, Frankfurt
Konto-Nr. 6161517443
Regional Commercial Director Layout & Lithography BLZ: 501 108 00
Dr. Katja Habermüller Ramona Scheirich BIC: CHAS DE FX
E-mail: ramona.scheirich@wiley.com IBAN: DE55501108006161517443
Editor-in-chief
Dr. Birgit Foltas Specially identified contributions are the responsibility of the
Ph.: +49 6201 606-760 Production Manager author. Manuscripts should be addressed to the editorial office.
E-mail: bfoltas@wiley.com Jörg Stenger We assume no liability for unsolicited, submitted manuscripts.
Ph.: +49 6201 606-742 Reproduction, including excerpts, is permitted only with the
Editorial Team E-mail: joerg.stenger@wiley.com permission of the editorial office and with citation of the source.
Dr. Cecilia Kruszynski de Assis
Ph.: +49 (0) 3047031 105 Sales Administrator The publishing house is granted the exclusive right, with regard
E-mail: ckruszynski@wiley.com Kerstin Kunkel to space, time and content to use the works/ editorial contribu-
Ph.: +49 6201 606-731 · Fax: -790 tions in unchanged or edited form for any and all purposes any
Róisín Murtagh
number of times itself, or to transfer the rights for the use of
Ph.: +49 (0) 6201 606 042 E-mail: kerstin.kunkel@wiley.com
other organizations in which it holds partnership interests, as
E-mail: rmurtagh@wiley.com
well as to third parties. This right of use relates to print as well
Reprints as electronic media, including the Internet, as well as data-
Dr. Martin Graf-Utzmann
Dr. Stefanie Krauth bases / data carriers of any kind.
Ph.: +49 6201 606-766
E-mail: martin.graf-utzmann@wiley.com Ph.: +49 6201 606-728
E-mail: stefanie.krauth@wiley.com All names, designations or signs in this issue, whether referred
Dr. Christina Poggel to and/or shown, could be trade names of the respective owner.
Ph.: +49 6201 606-625 Advisory Board
E-mail: christina.poggel@wiley.com Prof. A. Diaspro, Italian Institute of Technology Printed in Germany
Dr. C. Durkan, Univ. of Cambridge, UK
Isabel Brenneisen Dr. M. Dürrenberger, Univ. of Basel, Switzerland
Ph.: +49 6201 606-716 Dr. R. Fleck, King’s Collage London, UK ISSN 1439-4243
E-mail: isabel.brenneisen@wiley.com Prof. M. Gu, Swinburne Univ., Australia www.imaging-git.com
Prof. B. Hecht, Univ. of Wuerzburg, Germany
Corinna Herbst Prof. M. Hegner, Trinity College Dublin, Ireland
Ph.: +49 6201 606-752 Prof. F.-J. Kao, National Yang-Ming University, Taipei, Taiwan
E-mail: corinna.herbst@wiley.com Prof. N. Kruse, Washington State University, WA, USA
Prof. D. Nicastro, Thermo Fisher, The Netherlands
Freelancer Dr. J. Rietdorf, Centre for Technological Development
Dr. Martin Friedrich in Healthcare (CDTS), Brasil
Ph.: +49 178 4596064 Dr. D. Spitzer, ISL, France
E-mail: git-redaktion.friedrich@outlook.de Prof. G. A. Stanciu, Univ. of Bucharest, Romania
Prof. G. Valdré, Univ. of Bologna, Italy
Wiley GIT Reader Service Dr. Roger Wepf, EMBL, Heidelberg, Germany
65341 Eltville / Germany Dr. T. Zimmermann, ORG, Barcelona, Spain
Phone: +49 6123 9238 246
Fax: +49 6123 9238 244
Circulation
E-Mail: WileyGIT@vuservice.de
Our service is available for you from 18,000 copies, 4 times per annum
Monday to Friday 8 am – 5 pm CET Advertising price list from January 1st 2022

Imaging & Microscopy 4/2022 • 35

 Confocal microscope image of a mice. Credits: Leica Microsystems

SuperK
CHROMATUNE
400 - 1000 nm
Use your SuperK CHROMATUNE
for Bioimaging, Fluorescence imaging
& spectroscopy Single molecule imaging,
Microscopy, Endoscopy and OCT at any
wavelength.

Excite at any wavelength in the 400

to 1000 nm range. Fiber delivered and
diffraction limited. Zero maintenance with
a lifetime of thousands of hours and two
years warranty.

nktphotonics.com