Issue-PDF I m122

1
VOLUME 24
MARCH
2022
69721
Official Partner of the EMS
‘Apochromatic’ Objectives
In Situ Mapping Techniques

Bioimage Analysis
ISTEM Imaging
Editorial
© fotoliaxrender - Adobe-Stock.com
Imaging the “Fastest Organisms on Earth”
Breathtaking innovations in microscopy in- be found near underwater volcanoes.. M.
strumentation have revolutionized our view villosus uses a molecular machine, called
of microorganisms. The first images of mi- the archaellum, to swim. The archaellum
croorganisms only served to satisfy human consists of an ATP-powered intracellular
curiosity. However, in the early 1800s, peo- motor that drives the rotation of an extra-
ple were primarily suspicious about optical cellular filament composed of multiple copies
instruments giving insight into new worlds: of proteins. The filament is composed of
“Microscopes and telescopes rather confuse two alternating archaellins, suggesting that
the human mind.” This quotation is derived the architecture and assembly of archaella
from Johann Wolfgang von Goethe’s last is more complex than previously thought.
novel “Wilhelm Meister’s Travels”, that was [1] “M. villosus swims at a speed of about
published in 1821. Microscopes were seen 500 body lengths per second,” Dr. Lavinia Thomas Matzelle
primarily in terms of their importance to the Gambelli from LSI says. “Considering that Scientific Editor
morality of the observer rather than in their the tiny cell is only about one micrometer
fundamental scientific value. 200 years af- in size, this means half a millimeter in one
ter Goethe’s novel, the scientific impact of second.” Thus, if “an M. villosus cell was the References
new instrumental approaches is in focus. size of a cheetah, it would swim at approx- [1] Lavinia Gambelli, Michail N. Isupov, Re-
Recent studies are teaching us more about imately 3,000 kilometers per hour, mak- becca Conners, Mathew McLaren, Annett
fascinating microorganisms and how the ing it one of the fastest organisms on the Bellack, Vicki Gold, Reinhard Rachel,
findings might have an impact on human planet.” In addition to this amazing com- Bertram Daum: An archaellum filament
health and technology, such as the develop- parison, the new findings show the structure composed of two alternating subunits,
ment of new pharmaceuticals, drug delivery of the archaellum in unprecedented detail. Nature Communications 13 (2022) doi:
systems and robotic devices and, in a more A deep knowledge of the molecular struc- 10.1038/s41467-022-28337-1
general perspective, to new approaches to ture and the propulsion mechanism is fun- [2]
Pool R.: Microbiologists investigate
solving the most challenging problems of damental for finding exciting new applica- ‘smallest propeller on earth’, Wiley An-
our time. tions. “In the future, it might even be pos- alytical Science (9 February 2022)
Researchers from the University of sible to develop microscopic robotic devices
Exeter’s Living Systems Institute (LSI) and for drug delivery based on the tiny propellers
from the University of Regensburg imaged used by archaea,” says Dr. Bertram Daum.
the detailed structure of the spiral-shaped Indeed, this study may contribute to prom-
filament propellers rotated by single-cell ising new approaches in the development of
archea. The recently published cryo-elec- micromachines.
tron microscopy study focuses on Metha-
nocaldococcus villosus, a species that can Enjoy reading this issue!
Imaging & Microscopy 1/2022 • 3

Contents
EDITORIAL 3
NEWSTICKER 6
ANNOUNCEMENTS
21st ELMI Meeting 8
th
16 Multinational Congress on Microscopy 8
© Smileus - Adobe-Stock.com
RMS IN FOCUS
RMS Meetings Provide Great Start to 2022 10
NEWS FROM EMS

EMS Newsletter #76, March 2022 11
COVER STORY
A New Confocal Experience 12
Integration of Advanced Deconvolution Technology Improves LSM and Airyscan Imaging
A. Bergter and H. Reuter
LIGHT MICROSCOPY
Ernst Abbe’s Research Program (1878-1886) 14
How Did He Succeed in Creating the ‘Apochromatic’ Objectives?
H. Kubbinga
© rgpilch - Adobe-Stock.com
CORRELATIVE MICROSCOPY
Mapping Techniques for Optoelectronic Devices 18
Advanced Multi-Functional and In Situ Mapping Techniques
W. C. Tsoi
The winner of PREVIEW: ISSUE 2/2022

Picture Puzzle Issue 4/2021 is Coming out 23th May, 2022
Dr. José M. Vila Fungueiriño, Correlative Microscopy in
from Galicia Additive Manufacturing
Finding and Investigating Failures

in 3D-Printed Metal Parts
T. Schubert et al.
4 • Imaging & Microscopy 1/2022

IMAGE PROCESSING
Napari 21
My Favorite Image Analysis Tool, by Neubias Members
R. D´Antuono
ELECTRON MICROSCOPY
Accuracy of Position Determination from ISTEM Images 24
A Simulation Study for BaTiO3
D. Marquardt et al.
Imaging the Attack on Intracellular Bacteria at the Nanoscale 26 Drosophila egg chambers stained for F-actin (Phalloidin,
magenta) and DE-Cadherin (cyan). Sample courtesy of
Imaging Intracellular Bacteria by Electron Microscopy
T. Jacobs, AG Luschnig, WWU Münster, together with T.
S. Rey and M. G. Ryadnov Zobel, Münster Imaging Network, Germany
SCANNING PROBE MICROSCOPY COVER STORY

Into the Depths of Hydrogen Fuel Cells 28 A New Confocal Experience
Nanoelectric and Nanomechanical Atomic Force Microscopy to Elucidate the Structures
Integration of Advanced Deconvolution Tech-
and Properties of Fuel Cell Components
nology Improves LSM and Airyscan Imaging
T. Morawietz et al.
Laser Scanning Microscopes (LSM) are valued for

ADVERTORIAL delivering instant optical sections at high image
quality. What could improve such a versatile
Park Systems: instrument at its core without compromising
its flexibility and ease of use? With the latest
Thickness-Dependent Electrochemistry of 2D Materials 31 developments for Zeiss LSM 9 systems – LSM Plus and
Airyscan Joint Deconvolution – resolution, sensitivity,
and speed performance are enhanced, while the
cherished LSM characteristics remain untouched.
PRODUCTS 32
Laser Scanning Microscopes
SHOWCASE LSMs are the perfect tool to reveal structural

information from a wide range of fixed or living
Abberior’s TIMEBOW Lifetime Imaging Gives Time a Color 33 samples by uncovering details in the super-resolution
range, entering deep into tissue, and performing
advanced measurements (e.g., Fluorescence
Correlation Spectroscopy). Reflecting its impact on
microscopy and science, the prototype of the Zeiss
INDEX / IMPRESSUM INSIDE BACK COVER LSM 44 from 1982 is on display at the “Deutsche
Museum” in Munich. In the 40 years since its
introduction, LSM technology continued to advance,
expanding the experimental opportunities by
providing great flexibility to address complex imaging
experiments, and becoming easy to use. However,
the single spot image construction and the need
for an aperture (pinhole) to block out-of-focus light
inherently limited acquisition speed and sensitivity.
12
1
VOLUME 24
MARCH
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
helping you to further your mission.
‘Apochromatic’ Objectives
In Situ Mapping Techniques

Bioimage Analysis
ISTEM Imaging
NEWSTICKER
X-Ray Imaging Scanning Confocal Microscopy
Revealing the True Structure of Pollen Walls Tracking Chiral Molecules in Live Cells
used a raft of analysis methods to UK-based researchers have de-

scrutinize the exine structure of veloped a laser scanning con-
modern and fossil coniferous bi- focal microscope that can track
saccate pollen. They selected this left and right-handed molecules,
pollen as it has a longer evolution- also known as enantiomers of
ary history than that of flowering chiral molecules, within live cells.
plant pollen and could be compared The scientists reckon their circu-
very effectively with fossil material larly polarized luminescence laser
from the NHM collections. They scanning confocal microscope -
© Cojocaru, R. et al, Science Advances first carried out experiments on the CPL-LSCM - is the first of its kind © University of Durham
Researchers from the UK and France coniferous bisaccate pollen wall to track luminescent chiral mole- tween cell organelles and drugs,
have discovered that pollen survived using X-ray nanotomography and cules in cells and holds huge po- no direct techniques yet exist that
mass extinctions thanks, in part, to X-ray fluorescence nanoimaging tential for bioimaging. They expect can follow the behavior and cellu-
a never-before-seen nanofoam wall on beamline ID16A at the European the new type of light microscope lar interactions of these molecules
structure. The outer layer of pollen Synchrotron Radiation Facility. will allow researchers worldwide in chiral molecular systems.
grains - the exine - has been known to study fundamental interac-
to play a key role in the survival of Original publication: tions between cells, organelles Original publication:
terrestrial plant life, but its struc- doi: 10.1126/sciadv.abd0892 and drugs. While tracking chiral doi: 10.1038/s41467-022-28220-z
ture in different groups of plants More information: molecules within live cells is key More information:
has proven enigmatic. The scientists https://bit.ly/IM-012022-c to understanding interactions be- https://bit.ly/IM-012022-d
Atomic Force Microscopy Cryogenic Electron Microscopy

How Solvents Boost Solar Cell Performance Protein Machinery of Respiration Becomes Visible
are formed by combining two Oxygen and sugar are the basis
polymer solutions that solidify of life for animals, plants, fungi
into a film on an electrode, in the and many bacteria. The
form of interpenetrating networks. metabolic process called
Introducing solvent additives to respiration makes it pos-
the polymer solution can increase sible to convert food into
© ACS Appl. Polym. Mater. 2022, 4, 1, 169-178 the cell efficiency but the exact energy for the cells. Re-
mechanism has proven elusive. To searchers have now visualized
Using photoconductive atomic better understand this process, the for the first time with unparal- © University of Freiburg / CIBSS
force microscopy, researchers from scientists turned to photoconduc- leled precision how an assembly
Japan, have determined the role tive atomic force microscopy (PC- of protein machines, which also cryogenic electron microscope
of solvent additives in all-polymer AFM), which allows photocurrents supplies energy to humans, is analysis could aid in the devel-
blend solar cells. While all-polymer to be visualized with nanome- structured and functions. The opment of new drugs to treat tu-
blend solar cells hold great prom- ter-scale resolution. team studied two respiratory berculosis or diphtheria.
ise as the devices can be produced chain complexes fused into a su-
in large-scale flexible sheets, per- Original publication: percomplex in a group of bacteria Original publication:
formance has lagged behind that doi: 10.1021/acsapm.1c01173 called Actinobacteria. In addition doi: 10.1038/s41467-022-28179-x
of more traditional alternatives. More information: to providing a basic elucidation More information:
These polymer-based solar cells https://bit.ly/IM-012022-f of respiratory processes, the https://bit.ly/IM-012022-g
Cryo-Electron Microscopy
Investigating the ‘Smallest Propeller on Earth’
UK- and Germany-based research- volcanoes off Iceland, the research- they believe could contribute to the
ers have used cryo-electron micros- ers discovered that the organism’s filament’s flexibility and help it to
copy to expose new detail on the archaellum consists of thousands of propel the cell at high speed.
spiral-shaped filament propellers ro- copies of two alternating proteins,
tated by single-cell archea to move whereas previously investigated fil- Original publication:
at extraordinary fast speeds. Focus- aments showed only one protein. doi: 10.1038/s41467-022-28337-1
ing on Methanocaldococcus villosus, The researchers were also able to More information:
a species found near underwater identify structural elements that https://bit.ly/IM-012022-b © University of Exeter

Newsticker
3D STEM Tomography Rapid-FLIM

Self-Assembling Nanorings Form Hierarchical Composites Unexpected Role of Ion Channel in the Swelling of Nerve Cells
the mix. Analyses revealed that A team of researchers from Ger-

nanocomposite blends containing many has used rapid fluorescence
four or more components of vari- lifetime imaging to better under-
ous sizes and chemistries, including stand why brain tissue swells – a
complex polymers and nanoparti- condition known as cerebral oe-
cles, could accommodate impuri- dema that often follows a stroke
ties. The new findings could enable and can lead to a dangerous rise
the large-scale manufacturing of in inter-cranial pressure. They used
multifunctional nanocomposites combined rapid-fluorescence life- © HHU/Niklas J Gerkau, Institute of Neurobiology
that could, for example, enable ad- time imaging (FLIM) with new cor- becoming charged with sodium
© Berkely Lab vanced fiberoptics for high-speed rection algorithm schemes to study ions. However, the researchers also
US-based researchers have demon- broadband telecommunications, the changes that lead to the swelling discovered a new mechanism for
strated how ring-shaped nano- and multifunctional coatings for of nerve cells in real time. Neuronal this fatal sodium charging, in which
structures can self-assemble into buildings, automobiles, and aero- signals were captured at an unprec- the TRPV4 ion channel in nerve cells
hierarchically structured compos- space. edented temporal resolution. As part plays a key role.
ites with precision and efficiency. of their research, they recreated the
Their novel technique coaxes Original publication: conditions to which nerve cells are Original publication:
diverse blends of polymers and doi: 10.1021/acsnano.1c04606 and exposed during an ischaemic stroke doi: 10.1523/JNEUROS-
nanoparticles into spontaneously 10.1002/adma.202103563 in the laboratory. Initial analyses re- CI.0819-21.2021
forming tiny nested rings within More information: vealed that a breakdown in cellular More information:
minutes of adding an impurity to https://bit.ly/IM-012022-a energy supply leads to nerve cells https://bit.ly/IM-012022-e
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Announcement
21st ELMI Meeting

Turku, Finland, June 7-10, 2022
T
he 21st International European Light
Microscopy Initiative (ELMI) meeting
is coming back in 2022 as an in-per-
son meeting held in Turku, Finland in June
2022. ELMI 2022 will be a hybrid event,
and a subset of the program, including the
scientific presentation, will also be avail-
able online.
Turku is the oldest city in Finland, situated by

the beautiful archipelago of the Baltic Sea and
the home of the Euro-BioImaging headquarters.
ELMI 2022 will update you on the latest light
microscopy developments, research, techniques, mixing high-quality scientific talks and posters Consult the ELMI website for more information
and instrumentation. During the morning ses- with workshops and demos of the latest imaging regarding the meeting.
sions, international leaders will give plenary technologies, not forgetting social activities. This The organizers hope many of you will join
lectures. During the afternoon sessions, com- year the ELMI gala diner will be held in Naantali, ELMI 2022 in Turku.
pany workshops will provide demos of the latest a nearby city part of the Finnish archipelago and
microscopy techniques and instruments. ELMI home of the Moomin island. As with previous
2022 will continue the successful tradition of ELMI meetings, there will be the traditional aca- More information:
bringing together microscopy users, develop- demia versus industry football game as well as a https://elmi2022.eu/
ers, core facility staff, and industry leaders by satellite meeting dedicated to core facility staff.
16th Multinational Congress on Microscopy

Brno, Czech Republic, September 4-9, 2022
T
he 16th Multinational Congress on Mi-
croscopy continues in the long-stand-
ing tradition of conferences on the
latest innovations in microscopy and its
applications in biology, medicine, and ma-
terial sciences. Congress organized by eight
national microscopy societies of the MCM
consortium is scheduled for September 4th-
9th 2022 in Brno, the Czech Republic.
Brno is a beautiful city in Central Europe and

offers modern facilities for international confer-
ences, such as the Best Western Premier Hotel In-
ternational Brno – the congress venue. Moreover,
Brno is ideal place for microscopy events not only
because of the long tradition in the development
and production of microscopic instruments, but
also thanks to numerous universities and research
centres performing great science, usually in close
connection with microscopic techniques. The 16th
MCM will be a key microscopic event in Central
European region as part of EMS Extension. The
quality of scientific content will be ensured by and products in all fields of microscopy. Last but
leading scientists from across Europe. Affordable not the least, enjoyable atmosphere of microsco-
conference fees will support the accessibility for py-rich city with numerous historical monuments Registration:
students. The meeting will offer excellent op- and vibrant nightlife will stimulate also less for- www.16mcm.cz
portunity to present new scientific results, ideas mal personal meetings and collaborations.

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

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RMS in Focus
RMS Meetings Provide Great Start to 2022

It’s certainly been a busy start to the year at the ▪ RMS Honorary Fellows networking platform for expanding knowledge,
RMS, with a wide range of well-attended, virtual ▪ Summer Studentship Applications Open sharing new developments, and exchanging
meetings and courses taking place – and other Meanwhile, applications for the RMS’s fan- best practices in microscopy.
activities starting up in earnest. tastic Summer Studentship scheme are now be-
Our January events covered a wide range of ing invited. Up to six studentships of £2,000 are
microscopical methods, techniques and appli- offered every year, split evenly between physical Journal of Microscopy: Early Career
cations, drawing in attendees from across the sciences, biological sciences and interdisciplin- Special Issue – Call for Papers!
world. ary projects.
The Light Microscopy Facilities Meeting (UK- Applications for our Summer Studentships A Journal of Microscopy Special Issue is in the
LMF) and Flow Cytometry Facilities Meeting must include a significant microscopy compo- pipeline featuring recent microscopy develop-
each attracted around 300 participants over nent and should be submitted by a suitable host ments by Early Career Researchers. The issue will
two days, and more than 130 people logged on academic on behalf of a student. The students be titled A Lens on the Future: Next Generation
for EM-UK 2022. The latter event had initially are asked to complete a 500-word report about Microscopy by Next Generation Microscopists
been planned to take place at London’s Natural their project and a short video recording about and submissions are welcome from any micros-
History Museum - but was swiftly moved on- their experience for the RMS YouTube Channel. copy discipline.
line amid concerns over the Omicron Covid-19 The deadline for applications is 31 March
variant. Another virtual event - Facility Manag- 2022. Find out more and apply at RMS web page.
ers Training Course 2022 – took place in early
February and was fully-booked.
A huge ‘thank you’ to everyone who took Watch the International Microscopy
part in these events and helped make them such Lecture Series on YouTube
a success. We very much hope to see the return
of some in-person meetings later this year. The International Microscopy Lecture Series
As always, please check out the online events continues to go from strength to strength, with
calendar at RMS web page for all the latest in- talks from some of the biggest names in micros-
formation on upcoming RMS meetings, courses copy. If you missed any of the opening lectures,
and conferences. you can watch them in full on the RMS website
– via the Society’s YouTube channel.
Apply Now for an RMS Award!
Applications are now open for a wide range of

prestigious RMS awards covering all aspects of
microscopy.
The awards cover Scientific Achievement,
Research Support, Outreach and Education and
more. Visit RMS web page to find out more
about each of the awards listed below, including
eligibility criteria and how to apply. The applica-
tions deadline for each award is 22 April 2022. Author guidelines can be found on the Journal
The list of awards is as follows: website and papers should be submitted by 6
▪ 2022 RMS President’s Award (recognises an May 2022.
exceptional voluntary contribution to the Each virtual lecture is uploaded around a week If you have any questions, please contact
work of the RMS) after its initial broadcast, which means there’s the Journal Editorial Office Manager, Jill Hobbs:
▪ 2022 RMS Vice-Presidents’ Award (recognises no need for anyone to miss out! Recordings of journaladmin@rms.org.uk.
the ‘unsung heroes’ of microscopy by making each of our speakers in informal conversation
an award to an engineer, technician or labora- are also available. These are uploaded in ad-
tory research support scientist) vance of each lecture. Contact
▪ 2023 RMS Section Awards (awarded by each The International Microscopy Lecture Series Allison Winton
of the RMS Scientific Sections) is a joint collaboration by the RMS and a num- Chief Executive
▪ 2022 Chris Hawes Award for Outreach and ber of other microscopical societies. Supported Royal Microscopy Society
Education award (recognises individuals who by the International Federation of Societies for London, UK
have made a substantial contribution to ed- Microscopy (IFSM), it is part of an international awinton@rms.org.uk
ucation, outreach and/or public engagement
over the course of their career)
▪ 2022 RMS Scientific Achievement Awards
(awarded in recognition of the outstand- RMS web page: RMS YouTube channel:
ing scientific achievements of estab- www.rms.org.uk https://bit.ly/RMS-YouTube
lished, mid-career researchers)

News from EMS
EMS Newsletter #76

José Maria Valpuesta, March 2022
EMS President
Virginie Serin, EMS Secretary
Dear EMS Members,
The new EMS year has started with many EMS-supported activities. The call ▪ Winterschool 2022
for nominations for the EMS Outstanding Paper Awards 2021. EMS has re- www.zmb.uzh.ch/en/teaching/Winterschool.html
ceived a significant amount of very high-quality papers, presenting a good
balance between the three different categories. The jury will be chaired ▪ Scandem 2022- The 72nd Annual Meeting of the Nordic
by Randi Holmestad, and headed by the President José-María Valpuesta. Microscopy Society
Both are non-voting members of the jury. The award-winning papers will https://events.tuni.fi/scandem2022/about-scandem/
be announced to the EMS members in May this year in our newsletter, and
the authors will receive their awards during the EMS extension, PICO 2022 ▪ Plant-microbe Microscopy Workshop
(8-12 th May 2022; Kateel Vaalsbroek, Netherlands). www.plant-microbe-microscopy.com
This year is special since EMS will have two extensions, the second being
the 16th Multinational Congress on Microscopy (MCM16) (4-9th Septem- ▪ QEM2021 (awarded in 2021, postponed to 2022)
ber 2022; Best Western Premier Hotel International Brno, Czech Republic). www.qem2021.com
At this extension, there will be a variety of opportunities to interact with
speakers, exhibitors, and other conference participants. Most important, This financial support from the EMS will ensure that these smaller events,
the General Assembly will be held during this meeting. often organized in the form of a school or course, can count on the pres-
The call for Scholarships has been launched at the beginning of January, ence of top scientists to lecture our coming generations of microscopists.
deadline for applications February 18 (for PICO), and April 1 (for MCM16). For the second half of the year (July to December 2022), calls for spon-
The EMS executive Board will have its first annual meeting end of May sored events will be launched at the end of February. Any suggestions and
this year. ideas for future newsletters are welcome at sec@eurmicsoc.org.
In the first half of 2022, the following events will also be financially
supported by EMS: Contact
Prof. Dr. Virginie Serin
▪ The Second Joint Meeting of the Microscopy Society of Ireland and EMS Secretary
the Scottish Microscopy Society sec@eurmicsoc.org
https://bit.ly/MSI-SMS
Prof. Dr. José Maria Valpuesta
▪ EMAS 2022 Workshop EMS President
www.microbeamanalysis.eu jmv@cnb.csic.es
EMS on Facebook and Twitter: Suggestions and ideas

EMS webpage:
https://bit.ly/EuroMicr-FB for future newsletters:
https://bit.ly/EuroMicr
https://bit.ly/EuroMicr-TW sec@eurmmicsoc.org

Cover Story
A New Confocal Experience

Integration of Advanced Deconvolution Technology Improves LSM and Airyscan Imaging
Annette Bergter1 and Hanna Reuter1
L
aser Scanning Microscopes (LSM) are a)
valued for delivering instant optical
sections at high image quality. What
could improve such a versatile instrument
at its core without compromising its flex-
ibility and ease of use? With the latest
developments for Zeiss LSM 9 systems —
LSM Plus and Airyscan Joint Deconvolution
— resolution, sensitivity, and speed perfor-
mance are enhanced, while the cherished
LSM characteristics remain untouched.
Laser Scanning Microscopes

b)
LSMs are the perfect tool to reveal structur-
al information from a wide range of fixed
or living samples by uncovering details in
the super-resolution range, entering deep
into tissue, and performing advanced mea-
surements (e.g., Fluorescence Correlation
Spectroscopy). Reflecting its impact on mi-
croscopy and science, the prototype of the
Zeiss LSM 44 from 1982 is on display at
the “Deutsche Museum” in Munich. In the
40 years since its introduction, LSM tech-
nology continued to advance, expanding
the experimental opportunities by providing
great flexibility to address complex imag-
ing experiments, and becoming easy to use. Fig.1: a) Live imaging of LLC-PK1 dividing cell (porcine kidney), expressing H2B-mCherry (red)
However, the single spot image construction and α-Tubulin-mEGFP (cyan). Comparing without (left) and with LSM Plus (right). b) GATTA 4C
and the need for an aperture (pinhole) to NIR cells with labelled nucleus in blue, mitochondria in green (Tom20 with Alexa Fluor 488) actin
block out-of-focus light inherently limited in yellow (with Alexa Fluor 647) and microtubules (magenta) α-Tubulin (with Alexa Fluor 750).
acquisition speed and sensitivity.
than a standard confocal setting [1,2,3]. LSM Plus

Sensitivity and Resolution: Airyscan, the Wiener Filter deconvolution (DCV, a mathe-
New Standard for LSM Systems matical method to reverse optical distortion LSM Plus retains the above-mentioned ad-
[4]), became the robust way to the final im- vantages of the Wiener Filter: fast linear pro-
Pushing the boundaries for new findings of- age — while avoiding artefacts and omitting cessing, including the auto-function, leading
ten means imaging fainter signals at faster complex computational methods. This fast to quantitative results. LSM Plus is used with
speeds. To achieve this, it is paramount to and linear DCV method gave way to the au- an open pinhole (1 Airy Unit), collecting the
preserve all emission light while eliminating to-function, which suggests the best setting same light as in standard LSM imaging. All
noise such as laser light reflection. When a right after image acquisition. Airyscan’s LSM images, sampled from 0.6x to 1.2x Ny-
new interpretation of the confocal principle Multiplex modes, which speed imaging up quist, can be processed with LSM Plus, no-
was needed to further improve LSM imaging, to 10 times, also are rooted in the advan- ticeably increasing signal to noise (SNR) and
Zeiss launched the Airyscan detector (2014). tages of the Wiener Filter DCV to reliably resolution. This push in performance makes
Airyscan improves LSM imaging by directly deliver quantitative super-resolution images the difference when sensitivity and resolution
capturing the optical section with 32 circu- [5]. This continuing work with Wiener Filter are crucial for a successful experiment. Live
larly arranged detection elements instead of DCV led to improved adaptation of the sim- cell imaging is one such case in which reso-
using a light-clipping pinhole. Each element ulated PSF and processing integration. So, lution, speed and acquisition duration may
acts as a small pinhole, contributing to su- it was logical to use the time-tested Wiener be limited by phototoxicity. Imaging of living
per-resolution information (120 nm), while Filter to improve all LSM imaging modali- samples is easier with LSM Plus, which keeps
the entire detector area collects more light ties. The result: LSM Plus. laser power low while capturing the required

Cover Story
Fig. 3: Cos7 cells stained for mitochondrial outer membrane protein Tom20 (green, Alexa Fluor-488) and mitochondrial inner membrane protein
ATP5a (magenta, Alexa Fluor-647), imaged with LSM 900 on Zeiss Celldiscoverer 7. From left to right: confocal GaAsP detectors with left: LSM
data, and right: LSM Plus; followed by Airyscan 2 detector, left: Airyscan processing, right: Airyscan jDCV. Sample courtesy of Zhang Y, Universi-
ty of Science and Technology of China.
structural information (fig. 1a). Recently, a or multiphoton imaging — while the original sample. All 32 datasets are deconvolved and
growing number of fluorescent labels emit- LSM files remain untouched. Because LSM joined into one image. Such multi-view de-
ting light in the near infrared (NIR) range Plus is based on the Wiener Filter DCV, it convolution is therefore termed Joint Decon-
have been developed, providing numerous is possible to progressively push resolution volution (jDCV). The new Airyscan jDCV is an
advantages for sensitive live cell imaging and down to 120 nm by closing the pinhole. accelerated joint Richardson-Lucy algorithm.
deeper tissue penetration. These labels can be With the cost of lost emission light known, This iterative method reaches sample-depen-
added to multi-color experiments to separate this method can be an inexpensive way to dent resolutions down to 90 nm (fig. 2). Ease
more structures simultaneously. Currently, achieve super-resolution for bright samples. of use, reliability and quantitative results re-
however, available PMT detector sensitivity main paramount for Airyscan imaging. Ac-
is not ideal for NIR labels, degrading SNR in cordingly, this advanced DCV method utilizes
comparison to dyes of the visible spectrum. Airyscan jDCV the unique multi-view data input while keep-
In addition, the long wavelength will reduce ing user interaction to a minimum. Standard
achievable resolution, as described by Ernst Deconvolution algorithms have grown in settings are provided to guide first-time users
Abbe’s famous formula [6]. To overcome popularity, driven by the rapid improvement to the ideal number of iterations. jDCV is an
these pitfalls, LSM Plus delivers enhanced of computational power. Multiple DCV meth- integrated addition to Airyscan imaging that
sensitivity and improved resolution, making ods have been developed in concert with new gives easy access to advanced resolution. New
it beneficial to include NIR labels (fig. 1b). microscopy techniques. Airyscan, like Struc- discoveries can be made, without switching to
LSM Plus is fully integrated into the LSM 9 tured Illumination Microscopy (SIM) or Light a different microscopy technology.
operating software, ensuring a smooth work- Sheet Multiview imaging, is collecting multi-
flow from acquisition setup to the final im- phase (multi-view) data, providing a new cat-
age. Users can easily decide whether to use egory of raw data for DCV. Simply speaking, Summary
LSM Plus to improve multi-color or spectral each of Airyscan’s 32 individual detection
imaging, time series, large volume tile scans, elements has a slightly different view on the LSM Plus and Airyscan jDCV are new de-
convolution-based methods to improve
LSM imaging at its core. Collectively, they
a) improve sensitivity, speed, and resolution
— beyond former LSM or Airyscan perfor-
mance — for a new confocal experience (fig.
3). The full integration into the ZEN micros-
copy software makes them readily available
to drive scientific discoveries.
Affiliation
1Zeiss Microscopy, Jena, Germany
b)
Contact
Dr. Annette Bergter
Marketing Manager, Business Sector Life Sciences
Carl Zeiss Microscopy GmbH
Jena, Germany
annette.bergter@zeiss.com
www.zeiss.com/lsm
Fig. 2: a) GATTA SIM nanoruler imaged with Airyscan SR (GATTA-SIM 120B, left) and Airy-
scan jDCV (GATTA-SIM 90B, right). B) Synaptonemal complexes immunostained for the chro-
mosome axis protein SYCP3 (green) and transverse filament component SYCP1 (magenta), im- References:
aged with Airyscan jDCV. The intensity profile indicates a resolved distance below 100 nm for https://bit.ly/Zeiss-0122
SYCP3 (green). Sample courtesy of S. Sandhu and N. Hunter, University of California, Davis.

Light Microscopy
Ernst Abbe’s Research Program (1878-1886)

How Did He Succeed in Creating the ‘Apochromatic’ Objectives?
Henk Kubbinga
U
p to 1886, imaging in microsco-
py was still seriously hampered by
chromatic aberration. The construc-
tion of microscopes as such was a matter
of trial and error, that is, with respect to
the glass. In his report on the first world
expo of scientific instruments, held in
1876 in London, Ernst Abbe stressed the
importance of a research program to be
launched, a program aiming at the devel-
opment of new glasses by systematically
varying their chemical composition. In
other words: the production of new glass-
es with new properties. The success was
almost immediate.
Introduction
In the history of microscopy Ernst Abbe

(1840-1905) stands out both as a vision-
ary and a creator, a ‘Macher’, in his mother
tongue. As of 1866 he was what we would
call Director Research & Development of Carl
Zeiss, in Jena. The idea of CEO Carl Zeiss had
been to analyze all the physics of the micro- Fig. 1: Ernst Abbe about 1876 (lithography by Peter Halm).
scope in view of improving it on a scientific
base. Within a decade of extended trial and
error, Abbe came to realize that a further re- reviewed and evaluated. Moreover, as to the combined with a more pronounced disper-
duction of chromatic aberration could only be resulting material, apart from the densities, sion, did not show up. Undeniably, howev-
achieved by new kinds of glass. In the report the refraction indexes should be measured er, Schott had demonstrated the viability of
he wrote on the world expo in London he with the utmost accuracy, not so much for the experimental approach: his Li-glass was
stressed the lack of respect for glass as a raw white light, but for the monochromatic rays something entirely new. In due course Schott
material: as if it was just sand, helpful only as that had recently become individually avail- was invited to come over to Jena to discuss
ballast material to stabilize ships. Moreover, he able, i.a. thanks to the new gas discharge the possibilities, theoretical and practical. As
wrote, the monopoly position of the main pro- tubes. A special instrument, the ‘refractome- the consequence of a memorandum of under-
ducers—Charles Feil (Paris) and Chance Broth- ter’, was devised for the purpose (fig. 2). standing, dated 1881, Schott settled in Jena
ers (London)—should be broken. To be true, to do what remained to be done: namely the
glass was a commodity abused by the political production of small amounts of countless
authorities to increase their income through Zeiss, Abbe, Schott new glasses by systematically varying the in-
taxation: the production was carefully mon- gredients and their quantities, and, in so do-
itored, on the spot, by civil officers, while the A next step would be the systematic varia- ing, improve the production techniques. Stir-
number of window panes, of private houses, tion of the composition of glasses, with an- ring the melt, to homogenize it, for instance,
counted for the individual’s real estate dues. It other novelty in mind: the Periodic System had always been a problem, but Schott solved
can’t surprise that innovation was rare. of the elements (Mendeleev, 1869). Among it by introducing claypipe stirrers. Once fin-
the readership of Abbe’s report was, in all ished, the samples were transformed into an
probability, one Otto Schott, an experienced appropriate prism and a standard lens, both
Glass, New Glasses; Refraction Indexes table glass maker, who, at home in 1879, set the same for all glasses. With the brandnew
out to make a lighter glass by exchanging Pb ‘refractometer’ the refraction indexes for
Glass makers only paid attention to the spe- (flint) or Ca (crown) for Li. He, Schott, sent a some well-defined wavelengths were subse-
cific weight of the glasses they produced, sample of the new glass to Jena kindly asking quently measured: NaD, Kα, and the first three
mostly crown and/or flint. In other words, Abbe to measure the refraction indexes for lines of the hydrogen spectrum, lines known
inhomogeneities like striae, inclusions and the various wavelengths. Abbe was enthused as Hα, Hß, and Hγ. These 5 lines corresponded
other imperfections were not their problem. by Schott’s initiative, even though the results (approximately) with lines from the Fraun-
Abbe, in reaction, argued, that the whole pro- were slightly disappointing to the extent that hofer spectrum, namely with D, A, C, F, and
duction process of glass should be carefully the hoped for decrease in refraction index, G, respectively. Importantly, the dispersions,

Light Microscopy
Fig. 3: Cross-section
of an apochromatic
objective dated 1921.
Apochromatic Objectives (1886)
By combining lenses of the new kinds of

glass on this principle, chromatic aber-
ration was importantly reduced, so much
so that Abbe called the new generation of
microscope objectives ‘apochromatic’, in
the sense of ‘extremely achromatic’. Inter-
estingly, construction drawings of the first
such ‘apochromatic’ objectives survive in
the Archive of Carl Zeiss, Jena, drawings
that specify all numerical details though
the calculations in the background are no
longer there. We would like to discuss one
such objective design, developed in 1885-
1886, and reconstruct the way in which
Abbe had worked to determine the optimal
focal distances. In figure 3 we reproduced
Fig. 2: Abbe-refractometer (model 1893) for prismatic pieces of glass. The prism is mounted on a a cross-section of an apochromatic objec-
turn table, T. The telescope, on the left, is fixed on a second turn table with a scale division, to be tive, model 1921, and in figure 4 the cor-
read out (in tenths of an arcsecond) with the two vertical scopes, on the left and on the right. The responding construction drawing, dated
telescope could be moved through the spectrum produced by white light. The light of monochromatic 9 March 1886 and signed, in the header,
beam sources (in the upper right) may be directed at the prism with a condenser (lower left). The v.A., that is, by Abbe, in person. We see a
direction of the refracted beam may be found with the first telescope (engraving by Max Hunger). combination of a singlet, a doublet and a
triplet lens. From figure 4 we read out the
particular kinds of glass (O152, S8, OO, S34,
whose irregularities had driven whole gener- sions. In defining a lens, the most effective S37, and S46) used and the radii of curvature
ations of opticians into despair, were defined procedure, then, consisted in following the of the lens-surfaces in question. Only one
anew and calculated: nA – nC, nC – nD, etc. path of paraxial rays of monochromatic light focal distance is mentioned in the header,
From the notebooks that survive Schott and on their way through the standard lens and namely f = 16.0 mm; it is the focal dis-
Abbe’s frantic search for mathematical dis- charting the focal point as a function of the tance on the object side of the single front
persion laws can be read out. It was in vain, height of the incoming ray. By plotting that lens, made out of glass O152. From the few
unfortunately. The optician’s fingerspitz- height against the focal distance, Abbe ob- published data on the course of the focal
engefühl—that is: their heaped up experience tained curves for the various wavelengths: point for the various wavelengths we gath-
of decades—remained indispensable. The use it allowed him to define an optimal height ered that the optimal paraxial height for
of a singlet in combination with a doublet for each glass, such that the focal points of a incoming rays of three wavelengths (yel-
and triplet, for instance, was already standard majority of the principal wavelengths came low-green, D; red, A; blue, G) was at 5/9th
as was their feeling for handling the disper- closest to each other. of the lens’ radius.
Optical Filters
For microscopy applications
Customized designs · OEM solutions www.ahf.de · info@ahf.de

Light Microscopy
Construction & Calculation sinß2 = 0,0767/0,6507 = 0,1178

of Focal Distances and, to conclude, ß2 = 6,77° and its
complement (90 – 6,77) = 83°23’,
Now, with the help of millime- with a tangens value of 8,42.
ter paper—about 1880 another The position of the point of im-
novelty, not only in the domain pact B2 on the flat lens surface,
of optics—the focal distance on that is the distance S1B2, is read
the object side of the front lens out from the millimeter paper
can be obtained by constructing and appears to be 37 mm. With
the light path for a ray of yel- the tangens value of 8,42 for
low-green light of the NaD-line, the complement of ß2 we final-
its refraction index for O152 being ly calculate the focal distance
n = 1,5368. Importantly, we use f1 as 8,42×37 = 311,54 mm. In
a magnified version (×20) of the other words: the focal point F1
front lens, drawn on a sheet of of the front lens is situated at
A3-millimeterpaper, in view of 311,54 mm from point S1 on the
increasing the accuracy of our surface of the lens. The scale of
procedure. In figure 5, the ray the drawing is 20 : 1, so the focal
to be followed comes from the distance of the real front lens is
right. At B1, the point of impact, 311,54/20 = 15,58 mm, an out-
the radius of curvature M1B1 come that, given the inaccura-
(fig.4: 8,0 cm) is drawn and ex- cies involved, fits in nicely with
tended to R1. This allows us to Abbe’s outcome of 16.0 mm.
read out, thanks to the millime- In much the same way, mutatis
ter paper, tgα1, and subsequently mutandis, any lens may be ad-
calculate α1 and sinα1; making dressed of any kind of glass, pro-
use of nD = sinα1/sinß1 = 1,5368, vided that the required physical
we then calculate sinß1. In this data are available (optimal height
way we find α1 = 14,12° and and refraction indexes). Crucial-
ß1 = 9,14°. We then construct ly, there is no higher mathemat-
the broken ray at B1—through ics in the game: any sufficiently
tgß1—and find the point of im- trained employee in an optical
pact B2. At B2 we draw the line laboratory, then and now, is able
Fig. 4: Apochromatic objective device for a standard microscope (without parallel to the main axis, read to do the job. By the way, the
immersion) with long tubus, dated 9 March 1886 (Archive Carl Zeiss out tgα2, calculate α2 and, then, procedure shows, between the
AG, BACZ 19.886, p.27.r). The focal distance of the front lens is indicat- sinα2 (= 0,0767). The refraction lines of the argument, that the
ed in the header to be 16,0 mm, the (numerical) aperture being 0.3. On concerns the transition glass-air, focal distance is reckoned from
the right we see i.a. in the first column the radii of curvature (in cm). The hence nD = 1/1,5368 = 0,6507, the surface of the lens.
radii of the frontlens surfaces are indicated (in red) as 8,0 and ∞, resp. such that sinα2/sinß2 = 0,6507, or
Acknowledgment
Courtesy Carl Zeiss AG and Otto

Schott AG, Jena.
Contact
Prof. Dr. habil. Henk Kubbinga
(retired)
European Physical Society-History
of Physics Group
European Society for the History
of Science
Int. Academy of the History of Science
University of Groningen
Groningen, Netherlands
h.kubbinga@home.nl
More on light
microscopy: https:
//bit.ly/WAS-LM
Online article:
Fig. 5: Construction of the path of a paraxial ray of NaD-light through the front lens of the objective pictured https://bit.ly/
in figure 3 and 4 in view of finding its focal distance f1 (= F1S1) on the object side. The height of the incom- IM-Kubbinga
ing ray corresponds to 5/9× the radius. For details of the construction & calculation, see the text.

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
Correlative Microscopy
Mapping Techniques for Optoelectronic Devices
Advanced Multi-Functional and In Situ Mapping Techniques
Wing Chung Tsoi
O
ptoelectronic devices like solar cells, monly used can study the distribution of the To demonstrate the power of such
light emitting diodes, photodetec- PC and EL in the devices, respectively. The multi-functional mapping technique, the
tors, etc, are important to our dai- important questions are: Could these tech- authors applied the technique to study the
ly life. Critically, the microstructures and niques be combined together to probe the performance of perovskite solar cells (PSCs).
stability of the semiconducting layers in same sample location? Furthermore, can the PSCs are solar cells based on perovskite
the devices can affect their performance combined techniques be used to study sam- semiconductors, which have shown promis-
significantly. Therefore, imaging/micro- ples with controlled environments? ing power conversion efficiency (PCE) com-
scopic techniques which can provide more parable to silicon solar cells, but with sig-
insights in these aspects are important. nificant potential to be much lower cost and
Multi-Functional Optical Mapping for new applications. Here, the example of
the study on mesoporous-carbon PSCs (with
Introduction To answer the first question, the author have MAPbI3 perovskite) is shown which although
recently designed and added the PC mapping has lower PCE than other device architecture,
Optical imaging techniques and optical and the EL mapping to a commercial Raman has much better device stability and can be
mapping techniques have been applying system which allow the measurements of fabricated in low-cost [1]. The device struc-
to study semiconductor layers and devices, Raman, PL, PC and EL mapping at the same ture is shown in figure 1.
which can be used to probe their proper- sample locations. Furthermore, some of these As shown in figure 1, there were micron
ties with micron-meter resolution or even mappings can be performed simultaneously domains on the devices. From the PL peak
higher resolution. However, only one kind (e.g. PL-PC mapping). It is important to note position mapping, it showed the PL peak
of optical imaging or optical mapping tech- that the addition of the PC and EL mapping to position red shift at the domains. The PC
nique is normally applied at a time to gain the system is very simple, which means that and EL mapping showed that at these
one specific information. For example, Ra- any labs should be able to modify their Raman domains, the PC and the EL intensity is also
man spectroscopy (RS)/mapping is used to systems to perform the multi-functinal map- higher than the surrounding regions. Fur-
gain information on the chemical/structural ping. The capability to measure the different thermore, the Raman mapping of the TiO2
properties of semiconductor layers. Photo- properties at the same locations is important interlayer showed lower Raman intensity at
luminescence (PL) spectroscopy is used to to correlate the chemical/structural properties the domains. By combining this informa-
probe the photo-physical/photo-chemical with the photophysical/photo-chemical prop- tion obtained, we proposed all these phe-
properties of semiconductor layers. While erties and the optoelectronic properties. This nomena can be explained by a better contact
photocurrent (PC) mapping and electrolumi- is particularly useful when the samples are of the perovskite layer with the TiO2 layer at
nescence (EL) mapping which is less com- inhomogeneous in micron-meter scale. the domain regions, due to better infiltra-

nobrainer.
Lasers for Neuroscience
Perfect solution for 2-photon

Fig.1: PL peak position mapping, PC mapping, EL mapping and Raman intensity mapping
(TiO2) of a meso-porous PSC [1]. Microscopy and Optogenetics
tion of the perovskite solution to the TiO2 to perform optical imaging/mapping studies
layer at this regions. As the perovskite infil- of the films/devices with the environmental
trated better into the TiO2, the Raman sig- factors ex situ, it will be important to study
nal from TiO2 is reduced due to the laser the devices in situ to gain deeper under-
excitation light being absorbed by the per- standing on the degradation dynamics, etc. FemtoFiber ultra 920 & 1050,
ovskite first before it hits the TiO2 (Further- To demonstrate the usefulness of in situ op-
more, the Raman signal from TiO2 can also tical spectroscopy, the auhtors applied this vario 1030 HP
be absorbed by the perovskite). Due to the technique to study degradation of PSCs with
better electrical contact, charges are easier humidity (and temperature) in situ [2]. As an • Fully turn-key with integrated
to be extracted out from the devices or be example, the effect of humidity (and time) AOM and GDD
injected into the device, hence higher PC and on the Raman signal from the perovskite • No noise-stress for animals
EL intensity, respectively. The faster charge semiconductor in a solar cell with standard thanks to fully air-cooled design
extraction at the domains will also quench device architecture is shown in figure 2a-
the higher energy PL emission from band-to- c. Figure 2a showed that the intensity of • Highest stability and reliability for
band charge recombination, hence resulted the Raman peak at 110 cm-1 (I110cm-1) and long term experiments
in a red shift of the PL peak position. Impor- 168 cm-1 (I168cm-1) started to increase after • Compact fiber laser technology,
tantly, for larger mapping area (not shown < 10 min at 90% RH and these peaks grow saving valuable table space
here), such domains with better performance until 35 min when they saturate. The results
contributed to only ≈ 4% of the device area, match well with the formation of dihydrated
which can be used to understand the poor perovskite without forming PbI2, since the
PCE for the devices. This multi-functional authors did not observe a Raman signal at-
mapping technique also can be used to tributable to PbI2 at 96 cm-1.
understand the PCE for MAPbI3 perovskite For more quantitative study, I168 cm-1 ver-
with AVAI additive or using two-step fabri- sus I145cm-1 (the signal from TiO2) (i.e. I168cm-1/
cation process [1]. I145cm-1) in figure 2a is plotted in figure 2c. Schedule a free live
As shown in figure 2c, at 90% RH, I168cm-1/ or virtual demo!
I145cm-1 increases at a rate of ≈ 0.012 min-1
In situ Optical Spectroscopy in the first 15 min (I168cm-1/I145cm-1 ≈ 0.95 at
43% RH), and then increases at a faster rate
Regarding to study optoelectronic devices (0.036 min-1) up to 35 min. After 35 min,
with controlled environments, environmen- I168cm-1/I145cm-1 tends to saturate (≈1.96). As
tal factors, e.g. light, oxygen, temperature shown in figure 2b and inset in figure 2c,
and humidity could affect the device per- I168cm-1/I145cm-1 remains at a similar magni-
formance and stability significantly. For tude ≈1.29 when the device is dried from
example, it is well-known that perovskite 90% RH to 50% RH. However, I168cm-1/I145cm-1
semiconductors can be degraded by these drops significantly at ≤ 40% RH (≈1.04), and
environmental factors. While it is common continues to drop with 20 min drying time,
toptica.com/lasers4neuroscience
Fig. 2: In situ Raman spectra of the device:
(a) with increased humidity and time,
(b) with decreased humidity (inset shows
Raman spectra at the interlayer (spiro-OMe-
TAD), and interlayer/Au regions after dried,
respectively), and (c) I168cm-1/I145cm-1 with
increased humidity and time. Inset shows
I168cm-1/I145cm-1 versus decreased humidity
[2].
becoming similar to the spectrum before the advanced multi-functional in situ mapping. The coupling of multi-functional optical
humidity was raised (≈1.00). These results are For example, apply the multi-functional mapping with in situ optical spectroscopy
consistent with and support the reversible (at mapping to map the semiconductor layers/ will provide even more powerful way to un-
least semi-reversible) dihydration process of devices as a function of humidity levels (and derstand the performance of optoelectronic
the perovskite. Importantly, the two Raman time) to study the effect of humidity to the devices.
peaks were still observable in the interlayer properties of different micron domains on
(spiro-OMeTAD)/Au region (but not at the the devices. Technically, it should not be
interlayer region) with the drying timescale difficult to combine the two techniques. An
used (≈30 min, inset of figure 2b). This result environmental chamber which can be cou- Contact
suggested that the dihydrated perovskite spe- pled to the scanning stage of the microscope Dr. Wing Chung Tsoi
cies still remain at the Au electrode (consis- should be sufficient enough. SPECIFIC, Department of Materials Science
tent with higher degree of moisture trapping and Engineering
at this region) which can lead to more severe Faculty of Science and Engineering
device degradation. This kind of in situ study Conclusion Swansea University
provide significantly more information than Swansea, UK
ex situ method. Multi-functional mapping technique which w.c.tsoi@swansea.ac.uk
includes Raman, PL, PC and EL mapping www.swansea.ac.uk
was developed based on a commercial Ra-
Multi-functional In Situ Optical Mapping man system in a simple way, which provide
in-depth understanding on the PCE of PSCs.
For future development, it will be powerful In situ optical spectroscopy with controlled References:
to combine the multi-functional mapping environments was successfully applied to https://bit.ly/IM-Tsoi
techniques with the in situ spectroscopy for study the degradation dynamic for PSCs.

Image Processing
Napari
My Favorite Image Analysis Tool, by Neubias Members
Rocco D’Antuono
M
y favorite bioimage analysis tool (fig. 1). A typical microscopy data set is the
is “napari” [1]. Originally devel- z-stack: a series of images of a sample cap-
oped as a multidimensional viewer tured at different z positions; by visualizing
written in Python, it is now equipped with the different slices, it is possible to exam-
many useful functions for analyzing im- ine the morphology and intensity through-
age data [2]. I discovered it some years out the sample. Napari offers the 3D render-
ago thanks to my colleague Donald Bell ing from the traditional slice viewing with
who told me about a tweet mentioning just a single click. A second click allows the
this new software package. Inspired by his creation of lateral views of the sample. In
comments about this promising software, other software, such as ImageJ, this is possi-
I looked further to examine its potentiality ble through reslicing (which creates a resam-
and I decided to adopt it as a viewer for pled version of the data set), 3D viewer that
my bioimage analysis projects. As I used often has issues depending on the machine,
it often for my projects, I became more or BigData Viewer plugin (an additional Rocco D‘Antuono
and more positively impressed, so I decided viewer based on “lazy import” with its own
to promote it together with the Neubias navigation keyboard commands). The con- studied Physics in the Laboratory for Ad-
Academy committee, and start to get my- venience of using napari instead of several vanced BioSpectroscopy (LABS, Milan, Italy)
self into plugin development. This is an ex- ImageJ viewers is determined by the inte- under the supervision of Prof. Giuseppe
ample about how your future years can be gration of multiple features such as the 3D Chirico. In 2011 he joined the FIRC Insti-
well influenced by a simple conversation rendering, lateral views, transposition of the tute for Molecular Oncology (IFOM, Milan,
with experienced colleagues. Sometimes it axes, and grid view in the same GUI. The end Italy) as image analyst and microscopist
would be better to chat only about sport, user can then access all those functionalities specialised in live-cell imaging, TIRF, and
instead of getting more work for the years without having to write a single line of code. confocal microscopy. In 2014 he moved to
to come! Though napari was developed initially as a the Institute for Research in Biomedicine
3D viewer, it now is equipped with many (IRB, Bellinzona, Switzerland), running the
excellent features which will be explained microscopy and intravital facility; there he
Reasons below. had the opportunity to work also with flow
cytometry and help in the setup of the mass
The analysis of image data sets may require spec service. In January 2018 he joined the
the use of many separated software to iden- Installation Crick Advanced Light Microscopy facility
tify objects of interest, such as cells or vesi- (CALM, London, UK) as senior microscopist,
cles, and measure them. For example, ilastik For inexperienced users, the installation of supporting microscopy projects with the use
and ImageJ can be used to classify pixels or napari is possible as a bundled app [4], while of advanced fluorescence techniques and
objects in batch mode, calling ilastik in an coding users interested to software version- image analysis. He promotes the training in
ImageJ macro [3]. ing can get it as a python package [5]. For bioimage analysis through the activity of
The reason why I prefer napari for the practical usage, I created a GitHub repos- Neubias Academy.
visualization and analysis is that it allows itory with links to valuable resources and
several levels of interaction with the user, example scripts [6].
from a pure click-only GUI usage to an
in-depth customization of the interface,
all in a single Python ecosystem. Further- Napari Viewer
more, the vast collection of python pack-
ages makes it easier to implement any sort The viewer has a simple graphic interface
of bioimage analysis workflow, includ- offering an easy-to-use environment. It has
ing simple operations such as a 2D image three panels: the menu, the image canvas
segmentation by intensity thresholding on (where the image is shown; for example the
cell fluorescence, or a complicated 3D seg- mouse embryo in fig. 1), and the layers (in or with the use of several plugins for data
mentation and nearest neighbor analysis fig.1 the selected layer is called “Embryo”). import.
of the cell positions, to study how the dis- Layers are like those traditionally called Additionally, it is possible to use an IPy-
tance between cells of different populations “channels” in ImageJ, but can be handled thon console to interactively execute com-
affects their interaction. like the layers in Adobe Photoshop. Mul- mands and run scripts [7]. For instance, the
The first striking feature of napari is tidimensional image data sets (time-lapse, current layers can be grabbed as objects with
its 3D viewer. Switching between differ- positions, channels etc.) can be imported as the command “viewer.layers”, and it is pos-
ent views is astonishingly easy and smooth layers directly with drag & drop, via menu, sible to operate on them with command like

Image Processing
2D / 3D Segmentation, Parent-child

Relation, Tracking, Registration,
Lazy Import, Bioformats
Though its history since it became public

is not so long, the potentiality of napari
has been hugely extended by developers
who have adopted already existing python
packages, or have implemented new meth-
ods as plugins, to run a variety of tasks
such as: the segmentation of 2D/3D data-
sets (napari-pyclesperanto-assistant [9])
and the parent-child relation of segment-
ed objects (napari-zelda [10]), cell tracking
Fig. 1: Screenshot of napari lateral views and 3D view. Sample: mouse embryo. Courtesy of and lineage analysis (napari-btrack-reader
Fabrice Prin (The Francis Crick Institute). [11]), the registration of different data sets
(affinder [12]), the lazy import that allows
the handling of big data (napari-omero
[13]), and to ensure the compatibility with
Interactivity with microscopy file formats (napari-biofor-
Python / Jupyter Notebooks mats [14]). Currently, as of Jan 2022, the
total number of public plugins counts up
Jupyter notebooks editor or the JupyterLab al- to 142.
low the execution of bioimage analysis work- Powered by scikit-image, scipy, numpy
flows by running scripts step-by-step, execut- and other python packages, napari is a very
ing one or multiple code cells at the time. convenient tool to segment and visualize
There are superb webinars to learn how to results in 2D/3D. For example, a 2D image
use Jupyter notebooks for bioimage analysis segmentation and visualization can be done
with python language [8]. efficiently using less than 15 lines of code
In my view, the success of napari is not (fig. 5). Python has been a great tool for
only due to its excellent 3D viewer but also image processing and analysis with its rich
to the integration of this notebook coding variety of libraries ranging from numerical
capability, which has facilitated beginners computation to deep learning. With such a
Fig. 2: Script execution within the napari IP- in adopting python as a scripting language. powerful backend, napari allows us to freely
ython console. The script is available at [6]. As the code “composed” in a Jupyter note- wrangle image data without losing the visual
book is linked directly to the powerful mul- understanding of what is going on with
tidimensional viewer of napari, coding can them, classifying it as an excellent multidi-
“viewer.layers[1].visible=False” (this instruc- become intuitive by seeing the output of mensional viewer.
tion will hide the “Edges” layer in fig. 2). scripts immediately and visually. As useful starting point, a webinar about
Furthermore, it is possible to call an existing The few lines in fig. 4 are an example how to use napari was organized by Neubias
script, which will be executed within the IPy- script, alternative to the GUI commands, to Academy in 2020 and its recording is pub-
thon console, and the results will be shown load image data sets into napari. licly accessible [16].
in the current viewer. The example in figure
2 shows how to call a script that processes
an immunohistochemistry image to obtain
the edges (or eventually the area) of the DAB
staining. The script is available in the GitHub
repository associated with this article.
Annotation
The manual annotation of cells and organ-

elles, such as the delineation of their bound-
aries, is possible in napari by using the graph-
ical tools available in the different layer types:
points, polygons, and labels. The created an-
notations, which are stored in the labels layer,
can be visualized in 2D or 3D with different
rendering modes and opacity (fig. 3). This can
be incredibly helpful to inspect the accuracy
of the annotations, or the labeling obtained by
segmentation through some plugin or script. Fig. 3: napari viewer rendering in 3D the manually created annotations of two cell nuclei.

Image Processing
Plugin Management
Napari is equipped with an helpful installa-

tion tool which shows the list of the napari
plugins that are available online and their
most recent version, so the users can always
keep their distribution up-to-date. Alterna- Fig. 4: A small code to open a napari viewer and load an image.
tively, in case of specific need, it is possible
to install plugins manually from a local spe-
cific folder or from a URL.
Plugin Development
The customization of user interface is facili-

tated by magicgui [17] package, that can be
used as a library to quickly build interac-
tive GUI modules (such as a file browser or
dropdown menu etc.), the cookie-cutter [18]
template to start developing a plugin, and
the useful workshop documentation made
available by developers [19].
Plugin Listing and Promotion
Usually, python packages are listed and

hosted on the Python Package Index [20].
Napari plugins also have to be uploaded and
described on this website. In addition to this
standard publication, a specific website has Fig. 5: Less than 15 lines of code are sufficient to segment cells in 2D/3D with python and vi-
been dedicated to promote and better publi- sualize the result in napari. Minimally adapted from scikit-image documentation (scikit-image
cize software in the napari community, also watershed [15]), the scripts in fig. 4 and fig.5 are available at [6].
with the possibility to customize the presen-
tation of the listed projects and group them
by category (napari hub: [21]).
Developing coding skills may take some image.sc and other media. Finally, I am very
time but in the context of life science, like proud of being part of the Neubias commu-
An Active and Growing Community for civil rights and chemical formulae, it may nity (and of the NeubiasAcademy), to which
soon become a must for researchers to learn I owe a great part of my knowledge and
The community revolving around napari is how to “speak” and communicate ideas via progress in bioimage analysis.
still growing but quite a few achievements computer codes, allowing scientific investi-
have been reached. In fact, good efforts have gation to become more efficient, more repro-
been put by napari developers to schedule ducible, and more reliable. Towards this
appointments where end users and early direction, napari is an excellent partner for
developers can get help from experts, or your research.
discuss the future developments of this soft-
ware. For example, a first napari hackathon
has just been held on 18 January 2022, Acknowledgements
while community meetings take place every Contact
week [22]. Beyond the common image.sc I would like to thank Kota Miura for the Rocco D’Antuono
forum, napari developers and users have a proofreading and useful suggestions to im- Crick Advanced Light Microscopy STP
dedicated online chat forum [23], and work- prove the current article. Another acknowl- The Francis Crick Institute
shop series [24]. edgement is due to the napari community London, UK
and developers for making available a huge rocco.dantuono@crick.ac.uk
amount of documentation, and being very https://roccodant.github.io
Conclusion engaged with the life science end-users on www.linkedin.com/in/rocco-dantuono
To conclude, I consider napari as a software

worth to be known by every life science
researcher. The expertise to use it and cus-
tomize its GUI (through python) will be an All Neubias articles: References:
indispensable component of every bioimage https://bit.ly/Neubias-IP https://bit.ly/IM-DAntuono
analyst repertoire.

Electron Microscopy
Accuracy of Position Determination

from ISTEM Images
A Simulation Study for BaTiO3
Dennis Marquardt1, Marco Schowalter1, Florian F. Krause1, Andreas Rosenauer1
I
maging scanning transmission electron intensity maxima or minima and further
microscopy (ISTEM) is a novel mode, refines them by parabolian fits in a least
which combines the illumination of STEM squares sense to the projected intensities.
and the imaging of CTEM to benefit from Then the deviation (length of the distance
the respective advantages. The improved vector) is displayed as thickness-defocus
resolution due to partial incoherence as well map (fig. 2b-e).
as its ability to image light and heavy atoms A region around of about +-6 nm (indi-
makes it very attractive for measurement of cated by the red rectangle) around the zero
atomic displacements in perovskites giving defocus could be identified to yield reli-
rise to their ferroelectric properties. Here able results for the Ti and O atomic column
the authors report on a simulation study positions. Images outside this region do not
of accuracy of position determination from show a clear contrast of the atoms due to the
ISTEM images in BaTiO3 (BTO). strong defocus.
In order to study the influence of aberra-
tions on position determination the devia-
Introduction tions were averaged within this region. The
averaging process then yields one data point
ISTEM [1] images are acquired by scanning in figures 3b-c.
the specimen with a convergent beam. During
the time the probe is scanned over the field
of view of the camera, an image is acquired Results
in TEM mode. Since the probe is small, only
a small part of the camera is illuminated for From figures 2b-e one can see that the Ti, O
a single scan point, as shown in figure 1. The and Ba positions are accurately found with-
full image is created by the incoherent sum- in the identified defocus window. Only O1
mation of these small parts for all scan points Fig. 1: Acquisition process of ISTEM images positions (fig. 2d)) are about 7 pm off. This
yielding partial incoherence and an increased with BTO crystal structure and a simulated corresponds approximately to the distance
resolution compared to CTEM [1]. Accord- image. between the Ba and O1 columns. Such a
ing to the reciprocity theorem [2], the ISTEM small distance cannot be resolved using cur-
mode can be made equivalent to bright field rent microscopes and therefore a common
(BF) [3] or annular bright field (ABF) STEM Ti and O2 atoms along the [001] direction. extremum is formed somewhere in between
[4] by adjusting the shape of the condenser In the ISTEM image in figure 1, these atom the two columns. Since Ba is much heavier
aperture, which should have the shape of the columns exhibit a zigzag structure around a than O the extrema are found close to the
detector in STEM. horizontal line and thus the corresponding Ba position so that the deviations for this
ISTEM images are independent of aber- displacements along [001] have to be mea- position are small (fig. 2b). In the follow-
rations of the condenser system, with the sured. ing, only positions of Ti and O2 atoms are
exception of shape and size of the condenser In practice, the surface of the specimen considered.
aperture. The resolution of ISTEM improves is not perfectly perpendicular to the elec- Thickness-defocus tableaus were com-
with a larger diameter of the condenser aper- tron beam yielding a variation of defo- puted and evaluated using the automated
ture, but this is accompanied by a reduction cus over the image region. Additionally, routine resulting in deviation maps (like
of image contrast [5]. In contrast to STEM the specimen thickness cannot be selected maps shown in fig. 2b-e) for a series of
images, ISTEM does not exhibit scanning arti- specifically. Therefore, the authors simu- beam tilts. Again, the deviations were aver-
facts such as described in reference [6]. There- lated ISTEM images as a function of spec- aged in the thickness-defocus window and
fore, ISTEM is a very attractive imaging tech- imen thickness and defocus for BaTiO3 in displayed as a function of beam tilt in fig-
nique for measuring atomic column positions [110] direction (fig. 1 bottom left) using ure 3a. An increase of the mean deviation
in perovskite-type materials. the STEMsim software [7]. Figure 2a shows with beam tilt is found. However, for the
ISTEM images for some thicknesses and beam tilt a clear definition of the true atom
defoci selected from the full tableau (for a column position is difficult and the devi-
Methods spherical aberration of the objective lens of ation is not a good quantity for reflect-
20 µm). Atom column positions were deter- ing the accuracy of position determina-
For BTO the ferroelectric polarization can be mined in each image in the full tableau by tion. Therefore, figure 3b shows the dis-
switched by means of defined shifts of the an automated software routine, that finds tance between Ti and O2 atoms along the

Electron Microscopy
[001] direction. The solid and dashed lines

show the true distance and an error inter-
val of one pixel (~3.3 pm). The direction
of the beam tilt here was chosen in such a
way that the x direction corresponds to the
direction in which the Ti and O atoms are
displaced to each other. From figure 3b the
authors conclude that for beam tilts of up
to 2 mrad the Ti-O distance can be mea-
sured reliably.
In a similar manner, thickness-defocus
tableaus for different values of the spheri-
cal aberration constant Cs of the objective
lens were simulated and evaluated. It was
found that the choice of Cs is not critical
at least for aberration corrected machines
(for more details the reader is referred to
reference [8]).
Figure 3c shows the Ti-O distance as a
function of the amplitude of twofold astig-
matism of the objective lens for different
angles with respect to the [001] direction
of BTO. The angle is 0° when the elonga-
tion of the ellipse is parallel to the [001]
direction. For amplitudes up to about 3 nm
the Ti-O distance can be reliably measured,
although a scattering of the distances for
the different angles of astigmatism is
already visible. The scattering increases
for larger amplitudes.
All simulations were carried out using a
radius of the condenser aperture of 9 mrad,
which corresponds to the optimum radius
for a condenser lens with spherical aber-
ration of 1.2 mm. In principle, the resolu-
tion of ISTEM can be improved using larger
condenser apertures, but with the draw-
back of a reduction of contrast. The authors
found that in simulations (neglecting count-
ing noise) the size of the condenser aper- Fig. 3: a) Mean deviation of O2 and Ti columns
ture is not so critical for the determination from their true positions as function of the
of the Ti-O distance, besides for an aper- beam tilt. Projected distance between Ti and O
ture radius of about 17 mrad. For this radius as function of b) beam tilt and c) astigmatism
a change in contrast (fig. 3d) is observed, amplitude for different angles of the astigma-
which leads to greater difficulties in deter- Fig. 2: a) Thickness-defocus tableau of tism. d) ISTEM images for different radii of the
mining the atom positions. Since the con- ISTEM images. b)-e) Deviation of measured condenser aperture. A contrast change can be
trast decreases quickly with the radius of the atomic column position from true position. observed for a radius of 17 mrad.
aperture and as the influence of counting
noise increases with decreasing contrast, a
diameter of approx. 9 mrad is recommended
as a compromise. More details and further parameter found is two-fold astigmatism of Contact
investigations on ISTEM can be found in the objective lens, which should be below Dennis Marquardt
reference [8]. 3 nm. Institute for Solid-State Physics
University Bremen
Bremen, Germany
Conclusion Acknowledgement marquardt@ifp.uni-bremen.de
www.uni-bremen.de/en
From the evaluation of the image series This work was supported by the Deutsche
upper limits on the magnitude of lens aber- Forschungsgesellschaft (DFG) under Con-
rations could be determined. It turned out tract No. RO2057/14-1
that relative positions can be measured re-
liably for beam tilts up to at least 2 mrad Affiliations References:
and spherical aberration of the objective 1Institute for Solid State Physics, University https://bit.ly/IM-Marquardt
lens at least up to 50 µm. The most critical of Bremen, Bremen, Germany

Electron Microscopy
© rgpilch - Adobe-Stock.com
Imaging the Attack on
Intracellular Bacteria at the Nanoscale
Imaging Intracellular Bacteria by Electron Microscopy
Stephanie Rey1 and Maxim G. Ryadnov1,2
T
he emergence of resistant bacteria Introduction host defense responses by hiding inside host
stimulates the search for novel ma- cells, and even those cells that specialize in
terials against which antimicrobial Conventional antibiotics lose their effica- killing the pathogens, e.g. macrophages [5-
resistance is less likely. An increasing em- cy at an alarming rate, while traditional 6]. In addition, host cell membranes pres-
phasis is made on agents able to target paradigms of antimicrobial discovery fall ent a significant barrier for antibiotics to
bacterial cells residing in human and an- short of providing sustainable solutions. overcome preventing them from reaching
imal cells. Biometrology team at the Na- Novel molecular classes which can lead to bacteria. Here, the authors describe an intra-
tional Physical Laboratory (NPL) developed pre-clinical agents capable of hitting multi- cellular imaging model comprising infected
virus-like particles which safely enter in- ple targets in a bacterial cell are of particu- macrophages and an electron microscopy
fected host cells and destroy pathogens lar interest but also of a persistent challenge methodology allowing to monitor the tar-
inside them. To understand such targeting [1-2]. The challenge is compounded by the geting of intracellular bacteria by virus-like
remains challenging and requires the de- poor efficacy of antibiotics to target intra- particles in single cells [7].
velopment of an accurate imaging model cellular bacteria which remains one of the
at the nanoscale. Herein the authors dis- most fundamental tasks in the development
cuss such a model and the results enabled of antimicrobial treatments [3]. Although an Infection Model
by the model in tracking the antimicrobial effective means to rapidly kill bacteria on
action inside host cells. contact exist [4], many bacteria can evade The infected macrophage model is based on
alveolar macrophages. These cells are im-
portant components of the immune system
and programmed to recognize, ingest, and
destruct bacteria. The macrophages were in-
fected with Escherichia coli, one of the most
common Gram-negative bacteria, which can
aggressively grow in macrophages and effec-
tively evade their destruction mechanisms.
Single-Cell Resolution Electron

Microscopy Analysis
Fig. 1: Single cell analysis of infected host cells. Left, identification of intact (yellow) versus ne- The infected cell model and cell responses to
crotic (blue) host cells in presence of the bacteria. Middle, an electron micrograph of an infected antimicrobial treatments were analyzed with
host cell highlighting intracellular bacteria (black square). Right, higher magnification of the a single-cell resolution using room tempera-
middle image area (black rectangle). Dividing bacteria are in red and single bacteria in green. ture ultramicrotomy and conventional elec-
False colors are used to highlight cell differences. tron microscopy techniques [7–8]. Electron

Electron Microscopy
microscopy can highlight nanoscale defects

on bacteria membranes caused by the antimi-
crobial agents. Ruptured membranes of intra-
cellular bacteria were detected and character-
ized in detail. The effects were correlated with
cytotoxicity measured by biochemical assays
and fluorescence microscopy which accessed
larger cell numbers. Such a correlative ap-
proach was necessary to address two practical
questions. First, can the virus-like particles go
through host cell membrane and distribute in
the cytoplasm? Secondly, are these particles Fig. 2: Morphology of intracellular bacteria after antimicrobial treatment. Left, a micrograph of in-
toxic for the host cells? Analyses by light fected cells treated with virus-like particles highlighting bacterial cells with an intact membrane
microscopy helped answer the first question, (green) and a resin-like morphology (red). Middle and right, example of bacterial clumping in re-
while cell proliferation and viability assays sponse to virus-like particles, accompanied by the change of morphology of individual bacterial cells.
shed light on toxicity.
and how this activity impacts on the shape particles on bacteria infected macrophages
Stable Infection Model Quantification and integrity of individual bacterial cells. was introduced. The methodology comprises
The efficacy of the agents to disrupt bacte- a stable host-cell infection model, electron
For defining a model of infection and study- ria was assessed by the single-cell electron microscopy analysis and correlative orthog-
ing the effect of an intracellular antimicro- microscopy analysis using infected host cells onal measurements by optical microscopy
bial agent, it was important first to separate without and with antimicrobial treatments. and biochemical assays applied to correlate
the efficacy of antimicrobial agents in out- Two main parameters were assessed: the nanoscale defects in bacteria caused by vi-
competing the cytotoxic action of bacteria effect of antimicrobial agents on the bacte- rus-like particles with a reduced cytotoxicity
internalized by the host cells. Therefore, an ria and the protective effect the antimicrobial in macrophages as a result of antimicrobial
active stable infection model was estab- agents provide to host cells judged by the treatment. The developed methodology is ap-
lished. Since bacterial uptake and infection decrease in the number of necrotic host cells. plicable to other material types, e.g. intracel-
need to be tracked from early timepoints to The effect of different agents was compared lular delivery and targeting of gene delivery
several hours [6,7,9,10], the evolution of the to reveal that the designed virus-like particles agents, as the conditions for analysis and cell
infection by quantifying the number of bac- cause drastic changes in the cell morphology processing are largely the same.
teria per host cell as well as per unit area and behavior of intracellular bacteria.
was studied. The latter was done to mitigate Specifically, intracellular bacteria, which
the variability of bacteria counts per cell. underwent treatment by the virus-like parti- Acknowledgements
The number of necrotic host cells killed by cles were drastically different from bacteria,
infection was measured over time to com- which were spared the treatments (fig. 1 and The authors acknowledge funding from the
plement the results. 2). The bacteria disintegrated and deformed UK’s Department for Business, Energy and
For the single-cell analysis, the infected into electron dense, raisin-like morpholo- Industrial Strategy and UKRI’s Industrial
host cells were chemically fixed at set time gies (fig. 2). In addition, the damaged bacte- Strategy Challenge Fund. The authors ac-
points, processed, microtomed and imaged ria appeared to clump together (fig. 2 mid- knowledge the Electron microscopy imaging
using transmission electron microscope. Figure dle and right panel) – common phenome- centre of the University of Sussex, funded
1 corresponds to 70 nm microtomed cross-sec- non for bacteria trying to resist antimicrobial by the School of Life Sciences, the Wellcome
tions of host cells analyzed at 150 min post host responses or mitigate the effects of Trust (095605/Z/11/A, 208348/Z/17/Z) and
infection. membrane-disrupting agents. The clump- the RM Phillips Trust for their support & as-
The cytotoxic action of bacteria led to the ing observed in this study is attributed to sistance in this work.
necrosis of macrophages (fig. 1 left panel). The the action of virus-like particles, which was
bacteria inside the cytoplasm were identified in contrast to untreated cells which remain Affiliation
and counted (fig. 1 middle panel), with divid- intact, proliferating and did not cluster. As a 1National Physical Laboratory,
ing bacteria indicating an active intracellular consequence of this action significant reduc- Teddington, UK
2Department of Physics, King’s College
infection (fig. 1 right panel). No morphologi- tions in the number of necrotic host cells
cal changes or signs of rupture were observed were observed indicating a high recovery London, London, UK
in bacterial cells within the window of infec- rate of host macrophages when the virus-
tion during the analysis (fig. 1 right panel). like particles were used [7].
It was possible to identify a stability trade Contact
off at a specific time point reflected by pro- Dr Stephanie Rey
found increases in the number of necrotic host Conclusion National Physical Laboratory
cells and the number of intracellular bacteria. Teddington, UK
An analytical single-cell methodology to www.npl.co.uk/biometrology
monitor the antimicrobial effects of virus-like Stephanie.rey@npl.co.uk
Effect of the Antimicrobial Agent
on the Intracellular Bacteria
With the established infection model, the au- More on biometrology: References:
thors focused on measuring the membrane www.npl.co.uk/biometrology https://bit.ly/IM-Rey
disrupting activity of antimicrobial agents

Scanning Probe Microscopy
Into the Depths of Hydrogen Fuel Cells

Nanoelectric and Nanomechanical Atomic Force Microscopy to Elucidate
the Structures and Properties of Fuel Cell Components
Tobias Morawietz1,3, Jan-Frederik Heger2, K. Andreas Friedrich3,4, Hanno Käß1
I
n addition to battery technology, which
is currently in the focus of attention,
fuel cells operated by green hydrogen
are an essential component of the energy
transition. Measurement methods based
on atomic force microscopy (AFM) offer
the possibility to measure nanoelectric and
nanomechanical properties and thus con-
tribute to the investigation of the struc-
ture of fuel cell components and accord-
ingly to their improvement.
Polymer Electrolyte Fuel Cell
This article presents specifically studies

on polymer electrolyte fuel cells (PEMFC),
which are used in vehicles and in domestic
energy systems. The heart of a PEMFC is
the membrane electrode assembly (MEA),
which consists of a proton-conducting
© Renate Hiesgen
membrane and electrodes. The electro-
chemical reactions that take place at the
electrodes provide cell voltage and cur-
rents in an external circuit for consumers.
The electrodes consist of catalyst (usually
finely dispersed platinum nanoparticles on
carbon substrate (Pt/C)), ionomer (ion-con- Atomic Force Microscope Measurements surface (snap-in, point 1). Then the force is
ducting polymer) and they have a pore increased to a value specified by the operator
structure. The ionomer typically consists of The AFM, in contrast, can differentiate the (“Peak Force” point 2) (specified in V or in
a hydrophobic main chain (C-F2)n and side areas with ionomer from the surrounding nm when entering the sensitivity in nm/V).
chains with a terminal hydrophilic sulfon- material at room conditions and elevated The force is selected so that the tip penetrates
ic acid end group. Therefore, the ionomer humidities. The contrast here is provided by sufficiently deep into the sample to be able
spontaneously forms two different phases the differences in nanomechanical proper- to evaluate the nanomechanical properties.
in contact to water. ties and electronic conductivity. In a sim- The path between point 1 and point 2 cor-
The main chain with the stable C-F plified description, the AFM works similar responds to the deformation of the sample.
bonds forms crystalline areas with high sta- to a record player. An extremely fine tip is After that, the force is reduced, resulting in
bility and chemical resistance. The hydro- moved over the surface and images it. Struc- a linear decrease of the force (point 3). This
philic sulfonic acid chains accumulate tures of a few nanometres can be resolved decrease can be evaluated using the DMT,
when water is absorbed into the material, and the material-specific properties are re- Sneddon or Hertz model to determine the
resulting in ion-conducting regions. The corded simultaneously. The measurement stiffness value of the sample. Before leav-
structures of the ionomer have dimensions of the nanomechanical properties is made ing the sample completely, the tip is held
in the nanometre range and can therefore possible via force-distance curves at each back by adhesion forces (point 4) until these
not be analyzed with conventional meth- individual measuring point, which typical- break off and the tip oscillates freely again
ods. Electron beam-based methods are ly provides 1024 force-distance curves in a (point 5). At the same time, an electronic
problematic in this respect, as the con- measuring line of one micrometre. current can be measured when a voltage is
trast between ionomer and carbon is too Figure 1 shows a measured force-dis- applied between the sample and the AFM
low. By exchanging the protons with Cs+, tance curve on a thin ionomer layer. A scan tip. For this purpose, a fast lock-in amplifier
measurements of the ionomer on PEMFC cycle can be described as follows: The AFM is used, which integrates the current signal
electrodes with the transmission electron tip is brought close to the surface and is over different time constants. In this com-
microscope (TEM) were made possible [1], drawn by attractive forces, such as capil- bination, the AFM is able to elucidate even
which then, however, no longer depict the lary forces, by a water film present on the complex compositions - as present in tech-
original structure. surface, or by intermolecular forces with the nical electrodes.

Measurement Data
and Simulations
To describe the properties of

the ionomer in the electrodes,
the nanostructure of the poly-
mer electrolyte was investi-
gated on different size scales.
The imaging of the smallest
structural units of the ionomer Fig. 2: Ionomer bundles on mica.
was achieved by separating the
ionomer from a highly diluted
solution with 5∙10-4 wt.% on tion, adhesion and electronical
mica. This allows the struc- conductivity. In the topogra-
ture of the individual ionomer phy, the size of the agglomer-
bundles to be studied. Fig- ates and the pore structure can
ure 2 shows the structures of be analyzed. With the AFM it
an Aquivion ionomer with an Fig. 1: Force-distance curve on a thin ionomer layer measured with AFM. is also possible to measure the
equivalent weight of 790 g/eq. depth of each pore. The iono-
The equivalent weight describes mer shows a higher adhesion
the molar mass per sulfonic around the Pt/C agglomerates electronically conductive elec- than the catalyst and can there-
acid group. The height of the has been demonstrated with trode surface can alternatively fore be assigned to the bright-
ionomer units was determined TEM and AFM [1,3]. The thick- be measured with the AFM. In ly depicted areas in the mea-
to be 2.1 nm, which corre- ness of the ionomer layers is rel- this case, the particles not cov- surement. In the deformation
sponds to the minimum height evant for oxygen diffusion and ered with ionomer are detected. measurement, the ionomer can
of the layers that can occur in ionic conductivity. The analysis For this purpose, a voltage is be seen as a brighter area due
fuel cell electrodes. Depending of the ultra-thin ionomerlayers applied between the sample and to the higher deformability of
on the substrate, this height can in the electrodes via AFM must the AFM tip coated with plat- polymers compared to platinum
vary [2]. The behavior of these be carried out using tips with inum, gold or doped diamond. and carbon. The measurement
primary units on different sub- a radius of a few nanometres, The current then flows locally of the electronic current shows
strates is relevant for the func- since the thickness of the iono- in a range of around 25 nm. the areas not covered with ion-
tion of the hydrogen fuel cells. mer layers is typically less than Figure 3 shows the surface of omer. The measured values are
Thin layers are formed by ag- 20 nm. In addition to the direct an electrode with its topogra- also plotted along the red line
glomeration, whose existence detection of the ionomer via phy and the spatially resolved to illustrate the differences be-
in the electrodes of the PEMFC nanomechanical properties, the values of stiffness, deforma- tween catalyst and ionomer. In
Fig. 3: Measurements of the surface of a PEMFC electrode with a) height, b) values of c-e plotted along the marked line, c) adhesion,
d) deformation and e) electronic current.

Fig. 4: Interface between membrane (left) and electrode on a cross-section measured with the AFM: a) height, b) adhesion, c) deformation and d)
electronic current. The course of the boundary is marked in red.
Fig. 5: a) Model created from the AFM measurement data and b) simulation of the conductive paths.
addition to surface measurements, it is also topography. These include, in particular, Affiliations

1Faculty
possible to analyze cross-sections with an mechanical properties and local conductiv- of Applied Natural Sciences, Ener-
atomic force microscope. Figure 4 shows the ity, which can be recorded by AFM with a gy and Building Technology, Esslingen Uni-
interface between membrane and electrode. spatial resolution in the range of nanome- versity of Applied Sciences, Germany
2Central Scientific Facility Orientation and
The membrane can be clearly distinguished tres. These data can then also be read out
from the electrode by a higher adhesion and and processed for modelling and simulation. Undergraduate Studies, Esslingen University
deformation. This was demonstrated here using the exam- of Applied Sciences, Germany
3German Aerospace Centre, Institute for Tech-
AFM scans with topography and mechan- ple of fuel cell MEAs.
ical properties can be imported into MAT- nical Thermodynamics, Stuttgart, Germany
4University of Stuttgart, Institute for Build-
LAB for further processing. In the exam-
ple shown here (fig. 5), the image was seg- Acknowledgement ing Energetics, Thermotechnology and En-
mented into catalyst and ionomer on the ergy Storage, Stuttgart, Germany
basis of the material-sensitive properties. The authors would like to thank Renate
Threshold values of adhesion and defor- Hiesgen (†) for her important contribution Contact
mation were used for this. The segmented to the development of the work present- Prof. Dr. Hanno Käß
image is decomposed into layers based on ed here. The authors would like to express Faculty of Applied Natural Sciences
the contour lines. The images obtained in their gratitude for the financial support Energy and Building Technology
this way are read into the 3D simulation within the framework of the EU project Esslingen University of Applied Sciences
software GeoDict in order to simulate ionic FURTHER-FC. The FURTHER-FC project has Esslingen, Germany
conductive paths, for example. received funding from the Fuel Cells and ORCID: 0000-0001-9133-5962
Hydrogen 2 Joint Undertaking under grant Hanno.Kaess@hs-esslingen.de
agreement No. 875025. This Joint Under-
Conclusion taking receives support from the European
Union’s Horizon 2020 Research and Inno- References:
Modern AFM methodology can provide vation programme, Hydrogen Europe and https://bit.ly/IM-Morawietz-1
a range of other information beyond pure Hydrogen Europe Research.

Advertorial
Thickness-Dependent
Electrochemistry of 2D Materials
Using 2D Transition Metal Dichalcogenides for Electrochemical Applications
Marc Brunet Cabré1, Aislan Esmeraldo Paiva1, Matej Velický2,3,4, Paula E. Colavita1, Kim McKelvey1,5
Introduction
Two-dimensional (2D) transition metal

dichalcogenides (TMDCs) display unique
electronic and chemical properties [1],
which facilitate their use for electrochem-
ical applications such as electrodes for bat-
teries [2-4]. The preparation of 2D-TMDCs
can result in heterogeneous morphologies
with flakes of various sizes and layer num-
bers that affect their electrochemistry [2].
Atomic force microscopy (AFM) combined
with local electrochemistry measurements
via scanning electrochemical cell micros-
copy (SECCM) allows correlating the elec-
trochemistry with the layer number of
2D-TMDCs. SECCM achieves a resolution
of hundreds of nanometers by limiting the
measurement area to a droplet at the end
of a nanopipette serving as electrochemical
cell [6-8]. Fig. 1. (A) Schematic of the Park NX10 experimental setup, with SECCM probe and ex-
In the publication [9], SECCM and AFM changeable AFM probe head (B) Schematic of the SECCM mapping on TMDC surface and (C)
on Park Systems’ NX10 (fig. 1A) were used to nanodroplet-based electrochemical cell at the end of the SECCM probe, where the electro-
compare the electrochemical response from a chemical reaction takes place. Images are reproduced with permission. [9] Copyright 2021,
[Ru(NH3)6]3+/2+ redox couple on mono-, bi-, Electrochimica Acta
and trilayers of the four different TMDCs
(fig. 1B-C). For SECCM, nanopipettes with
300 – 600 nm radius were filled with the
redox couple solution, an Ag/AgCl electrode
was inserted and the nanopipette was posi-
tioned on the surface. Here, linear sweep
voltammograms (LSV) were recorded on a
predefined grid by sweeping the potential
between +0.2 V to -0.8 V at a speed of 1 V/s.
Results
Prior to SECCM, optical microscopy of the

NX10 AFM was used to identify suitable
measurement areas that feature TMDCs
flakes with different layer numbers (1L-3L)
(fig. 2A). To resolve the exact layer num-
ber of the TMDC at the SECCM measure-
ment spots, AFM stepheight profiles were
obtained after SECCM (fig. 2B-D). Salt res-
idues from the SECCM allowed identifying
the measurement spots and gave an esti-
mate of the contact area during SECCM. Fig. 2. (A) Optical image of MoSe2 with differing layer number. (B) AFM image of region in
LSVs on 1L, 2L, and 3L WS2 in figure red rectangle in (A). The AFM was taken after the SECCM measurements and revealed the
3A-C clearly show that the electrochemi- electrolyte residues at the contact areas of contact of the droplets. (C) and (D) line profiles of
cal behavior depends on the number of 1L/2L and 2L/3L boundaries, respectively, taken from areas shown by black lines in (B). Im-
TMDC layers. The [Ru(NH3)6]3+ reduction is ages are reproduced with permission. [9] Copyright 2021, Electrochimica Acta

Advertorial
Advertorial
performed on MoS2, MoSe2, and WSe2 to

find that all investigated TMDC materials
show a slowing down of the kinetics with
the increasing number of layers [9].
Conclusion
To investigate the electrochemical response

of 2D TMDC materials in dependence of
their layer number, the AFM sample height
was correlated to a SECCM grid measure-
ment with a redox couple using a Park NX10
AFM. The electrochemical response on the
different thicknesses changed by featuring
increased reaction rates on decreasing layer
numbers on all tested TMDCs [9].
Affiliations
1Trinity College Dublin, Ireland
2University of Manchester, United Kingdom
3Cornell University, United States
4J. Heyrovský Institute of Physical
Chemistry, Czech Republic

5Victoria University of Wellington,
New Zealand
Fig. 3: (A), (B), and (C) LSVs of [Ru(NH3)6]3+ reduction on 1L, 2L, and 3L WS2 respectively
from the points shown in Figure 3 D (black traces). Simulated response to median in red and
simulated response to 25th and 75th percentiles of kmax in blue. (D) Color-coded layer number Contact
assignment in AFM image. (E) kmax values determined from simulation for different layer thick- Park Systems Europe GmbH
ness. Images are reproduced with permission. [9] Copyright 2021, Electrochimica Acta Mannheim, Germany
more facile on monolayers than on bi- and face were modelled. This allowed an esti-
trilayers as shown by the onset potential. mate of the apparent reaction rate kmax for
To quantify the observed electrochemical each layer number: An increase in layer References:
response, the diffusional transport and the number decreases the reaction rate (fig. 3E). https://bit.ly/IM-PS0122
electron transfer kinetics at the sample sur- In the publication, the same analysis was
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Index / Imprint
Abberior Instruments 32, 33 European Microscopy Society 11 Royal Microscopical Society (RMS) 10
Åbo Akademi University 8 King’s College London 26 Swansea University 18
Academy of Sciences 8 National Physical Laboratory 26 Tescan Orsay Holding 9
AHF Analysentechnik 15 NKT Photonics 32, Outside Back Cover The Francis Crick Institute 21
Atik Cameras 34 Olympus Europa Inside Front Cover, 34 Toptica Photonics 19
Carl Zeiss Microscopy Cover, 12, 34 Park Systems Europe 31, 33 University Bremen 24
CoolLED 34 Prior Scientific 7 University of Groningen 14
Esslingen University of Applied Sciences 28 Rigaku 34 Witec 17, 33
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