VOLUME 22

MARCH 2020

1
69721

Official Partner of the EMS

Metabolic Imaging

Volumetric Microscopy

3D Visualization

TEM Training

The Moving Virus
The on-going coronavirus epidemic is the
dominant topic in almost all media these
days. First identified in December 2019 in
Wuhan, Hubei, China, the virus based illness
is caused by COVID-19. As end of Febru-
ary more than 80,000 cases have been con-
firmed, including nearly 3,000 deaths. Ini-
tially limited to China, the disease has now
spread to more than 50 countries. Epidemi-
ologists have found that the epidemic likely
has its origins in zoonotic developments at
a live animal market in Wuhan, and that the
infection propagated through human-to-hu-
man transmission due to the virus’s ability
to mutate. The infection is primarily spread
via respiratory droplets and at first sight
the symptoms are comparable to those of
the flu. However, the incubation period is
typically between two and fourteen days
and complications such as pneumonia and
acute respiratory distress syndrome may
occur. So far, there is neither a special an-
tiviral treatment nor a vaccine available.
China has taken rigorous measures to pre-
vent it spreading further. When the first cas-
es occurred in northern Italy at the end of
February, demonstrating that the virus was
also spreading in Europe, the Italian govern-
ment started to lock down some cities south
of Milan. These rigorous measures are de-
signed to mitigate the strong economic im-
pact of the spread. Facing severe economic
impact of the coronavirus epidemic, vac-
cine research is being propelled by several
organizations around the world. In partic-
ular, modern microscopy provides decisive
contributions. A promising approach is to
find out the structure of the virus protein
and how it functions at the molecular level
when infecting a cell. Researchers from the
University of Texas in Austin have already
succeeded in mapping a specific part of the
coronavirus protein, the “spike”, which in-
fects human cells using cryo-EM. “As soon
as we knew this was a coronavirus, we felt
we had to jump on it because we could be
one of the first ones to get this structure,”
says Jason McLellan from UT-Austin and
the National Institutes of Health. He and his
colleagues have studied other coronaviruses
and the methods for locking spike proteins
for many years. Cryo-EM analysis has re-
vealed that the analyzed COVID-19 spike
protein has two conformations, one before it
infects the host cell, and another during the
infection. The structural change of the spike
protein is triggered by binding, enabling the
membrane fusion. This knowledge of the
structure of the spike protein and the infec-
tion mechanism is now a valuable basis for
vaccine design. Interestingly, the analysis
confirmed the prediction that COVID-19 is
similar to the SARS coronavirus spike. In
contrast, COVID-19 spike protein’s affinity
for human angiotensin converting enzyme
2 (ACE2) is ten times higher than in the
case of SARS. ACE2 receptors are the en-
try points into human cells for some coro-
naviruses, including COVID-19. Apparently,
this is why the new coronavirus is spreading
much more easily from human to human.
Although a usable vaccine is still months
away, theoretically, the spike protein itself
“could be either the vaccine or variants of a
vaccine” McLellan said. When injecting this
specific protein, “humans would make an-
Editorial
© dottedyeti - Adobe-Stock.com
tibodies against the spike, and then if they
were ever exposed to the virus, the body
would be prepared.” This is indeed a promis-
ing example involving modern microscopy
for the benefit of mankind.
Find more information on interesting
approaches in modern microscopy at Wiley
Analytical Science, our new designed web
platform.
References
[1] Daniel Wrapp, Nianshuang Wang, Kizzme-
kia S. Corbett, Jory A. Goldsmith, Ching-
Lin Hsieh, Olubukola Abiona, Barney S.
Graham, Jason S. McLellan: Cryo-EM
structure of the 2019-nCoV spike in the
prefusion conformation, Science (2020)
doi: 10.1126/science.abb2507
[2] analyticalscience.wiley.com/publication/
imaging-and-microscopy
Enjoy reading this issue!
Thomas Matzelle
Scientific Editor
Imaging & Microscopy 1/2020 • 3
 Contents

EDITORIAL 3

NEWSTICKER 6

COFFEE BREAK 8

EVENT CALENDAR 10

ANNOUNCEMENT
EMC2020: Time to Submit, Time to Enter 12

RMS IN FOCUS
Microscience Microscopy Congress 2021 14
What Do You Want to See at mmc2021?

NEWS FROM EMS

EMS Newsletter #68 15

COVER STORY
Efficient Digital Documentation in the Lab 16
Simplifying Biomedical Routine Lab Work
M. Gögler

LIGHT MICROSCOPY
Intracellular Journey of Cell Surface Receptors 18
Imaging Intracellular Trafficking Using Photoconvertible Fluorescent Proteins
J. Rossy et al.

Biochemical Imaging by Stimulated Raman Scattering Microscopy 22

Metabolic Imaging and Super-Multiplexed Vibrational Detection
F. Hu and W. Min

Single-Shot Volumetric Microscopy 24

Imaging Entire Optically Sectioned Volumes at an Instant
F. Ströhl

Congratulation PREVIEW: ISSUE 2/2020

The winner of Read & Win Coming out 28th May, 2020
issue 4/2019 is Optical Coherence Tomography Imaging
T. O’Neill from Ireland through the Spectral Ranges
State of the Art, Advances and Limits
Bettina Heise and Ivan Zorin

4 • Imaging & Microscopy 1/2020

 Cool Lenses for Light Microscopy 26
Immersion Objectives and Media for Cryo-Fluorescence Imaging
T. P. Burg

Smart Objective Compensate Aberrations  28

Free Form Programmable Lens for Any Type of Microscopes
S. Bonora

IMAGE PROCESSING
UCSF Chimera 32
My Favorite Image Analysis Tool, by Neubias Members
L. Schütz COVER STORY
Efficient Digital
Managing Big Imaging Data 35 Documentation in the Lab
Cell Imaging and Processing with Lattice Light-Sheet Microscopy
Simplifying Biomedical Routine Lab Work
N. D. Condon et al.

Smart Microscopy from Zeiss is a new concept

for routine digital documentation of microsco-
SCANNING PROBE MICROSCOPY pic samples. The microscopes Axiolab 5 and Axi-
Scanning-Probe-Assisted Nanowire Circuitry 39 oscope 5 can be combined with the microscope
cameras Axiocam 202 mono and Axiocam 208
A Novel Technique for the Fabrication of Metal Nanoelectrodes
color, to form a Smart Microscopy system that
P. Ares et al. takes away a large share of the workload from
the users. It automatically adjusts many of the
required settings, thus digital documentation
of microscopic specimens becomes easier and
ELECTRON MICROSCOPY more efficient.

TEM Training in Core Facilities 42

Efficiency and Quality Are Key
Effect of Duration and Number of Trainees on Improvement
Efficiency and high-quality microscopic images
J. D. Riches are key in the lab. It takes time to acquire
detail-rich, true-color images of biomedical
specimens. Many steps are required: place the
sample on the microscope stage, focus the re-
X-RAY ANALYSIS gion of interest, switch to the computer, ad-
just settings such as white balance, exposure
Soft X-Ray Microscopy of Complex Samples 45 time and gain, then acquire an image, insert
From Tissue Culture to Tissue Components a scale bar, switch back to the microscope ...
C. Hempel et al. and so on. That’s what a typical documentation
workflow looks like. The same steps must be re-
peated again and again and the settings of the
PRODUCTS 48 microscope and software must be adjusted. The
INDEX / IMPRESSUM INSIDE BACK COVER constant switch between microscope and PC is
tiring and time-consuming.

16
Welcome to the knowledge age. Wiley builds on its 200-
VOLUME 22
MARCH 2020

1 year heritage by partnering with universities, businesses,

69721

Official Partner of the EMS 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.

Metabolic Imaging

Volumetric Microscopy

3D Visualization

TEM Training
NEWSTICKER
X-Ray Microscopy
Nanoparticles Can Change Cells
Nanoparticles easily enter into
cells, which is of great interest to
the medical world as these parti-
cles could be coated with active in-
gredients and used to destroy can-
cer cells. But despite the potential,
© Burcu Kepsutlu / HZB
Transmission Electron Microscopy
Transforming TEMs into High-Speed Atom-Scale Cameras
Researchers have retrofitted a
transmission electron microscope
so it can create high-quality
movies of super-fast processes
at the atomic and molecular
scale. Designed to be a cost-ef-
fective add-on to existing TEMs,
the retrofit promises to bring
fresh insights into next-gener-
ation computer chips, biological
tissue and more. The retrofit en-
ables TEMs of any age to make
high-quality movies on the scale
of picoseconds by using a rela-
tively simple ‘beam chopper’. The
beam chopper comprises a pair of
phase-matched modulating and
de-modulating travelling-wave
metallic comb striplines, or puls-
ers. These pulsers generate a
tunable RF wave that interrupts
the continuous electron beam,
so it can instead deliver precisely
timed pulses of electrons to cap-
ture frames of cyclic processes at
time intervals from one nanosec-
ond to 10 picoseconds. They ret-
rofitted the TEM by opening the
6 • Imaging & Microscopy 1/2020
much needs to be learned about
nanoparticle dynamics within cells
and the effects of different particle
sizes and coatings. In a world first,
Germany-based researchers have
revealed complete, 3D high-res-
olution images that show how
nanoparticles can change cells.
Studies at the Berlin-based syn-
chrotron, BESSY II, indicate that
nanoparticle-treated cells contain
fewer lipid droplets and multive-
sicular bodies, yet have more mito-
chondria and endosomes.
Original publication:
doi: 10.1021/acsnano.9b09264
More information:
http://bit.ly/IM-12020-c
© NIST
microscope column directly under
the electron source, inserting the
beam chopper and sealing the
microscope again. According to
the researchers, the system can
be used to image ferromagnets,
memristors, MEMS nanoelectro-
mechanical systems, and batter-
ies.
Original publication:
doi: 10.1063/1.5131758
More information:
http://bit.ly/IM-12020-g
Super-Resolution Live-Cell Imaging
Providing Unexpected Insights into the Dynamic Structure
of Mitochondria
As power plants and energy
stores, mitochondria are essen-
tial components of almost all
cells in plants, fungi and animals.
Until now, it has been assumed
that these functions underlie a
static structure of mitochon-
drial membranes. Researchers
have now discovered that the
inner membranes of mitochon- © Heinrich Heine University Düsseldorf
dria are by no means static, but uously change their structure dy-
rather constantly change their namically within seconds within
structure every few seconds in mitochondria. This showed that
living cells. One molecule of ATP the cristae membrane dynamics
is produced about 20,000 times requires a recently identified pro-
a day and then consumed again tein complex, the MICOS complex.
for energy utilization. This im- Malfunctions of the MICOS com-
mense synthesis capacity takes plex can lead to various serious
place in the inner membrane of diseases
the mitochondria, which has nu-
merous folds called cristae. The Original publication:
researchers succeeded for the doi: 10.15252/embr.201949776
first time in showing that cristae More information:
membranes in living cells contin- http://bit.ly/IM-12020-e
Camera Technology
Combining Lasers, Computers & THz Waves to Build Camera that Sees
‘Unseen’ Details
A team of physicists at the Univer-
sity of Sussex developed the first
nonlinear camera capable of cap-
turing high-resolution images of
the interior of solid objects using
terahertz (THz) radiation. They built
a new type of THz camera capable
of detecting THz electromagnetic
waves with unprecedented accu-
racy. Images produced using THz
radiation are called ‘hyperspectral’
because the image consists of pix- © University of Sussex
els, each one containing the elec- ples. THz imaging makes it possible
tromagnetic signature of the object to ‘see’ the molecular composition
in that point. Lying between micro- of objects and distinguish between
waves and infrared in the electro- different materials – such as sugar
magnetic spectrum, THz radiation and cocaine, for example.
easily penetrates materials like pa-
per, clothes and plastic in the same Original publication:
way X-rays do, but without being doi: 10.1364/OPTICA.381035
harmful. It is safe to use with even More information:
the most delicate biological sam- http://bit.ly/IM-12020-d

Tissue Clearing
Transparent Human Organs Allow 3D Maps at the Cellular Level
For the first time, researchers
managed to make intact human
organs transparent. Using micro-
scopic imaging they could revealed
underlying complex structures
of the see-through organs at the
cellular level. Resulting organ
maps can serve as templates for
3D-bioprinting technologies. In
the future, this could lead to the
creation of on demand artificial
organs for many patients in need.
After exhausting trials, the team
discovered that a detergent called
CHAPS could make small holes
throughout the entire stiff human
organs. CHAPS allows additional
solutions to travel deep into cen-
timeters-thick human organs and
Electron Diffraction
Sneaking up on Tiny Crystals
© Robert Bücker
Understanding the structure of pro-
teins, the building blocks of life, is
essential to obtain insight into their
biological function. Due to their
minute size and extreme fragility,
these structures are enormously
difficult to determine. Acquiring
data of sufficient resolution requires
immense doses of high energy X-ray
radiation, which unfortunately irre-
vocably damage the proteins prin-
More News:
http://analyticalscience.wiley.com
© Helmholtz Zentrum Munich / Ertürk lab
convert them into a transparent
structure. The researchers named
this new technology SHANEL
(Small-micelle-mediated Human
orgAN Efficient clearing and La-
beling).
Original publication:
doi: 10.1016/j.cell.2020.01.030
More information:
http://bit.ly/IM-12020-a
cipally being investigated. Now re-
searchers from the MPSD and DESY
in Hamburg have developed an in-
ventive new method which avoids
these pitfalls and uses accessible,
cost-effective technology.
Original publication:
doi: s41467-020-14793-0
More information:
http://bit.ly/IM-12020-f
Newsticker
Cryo-Electron Microscopy
Deciphering the Activation Chain of Protein Degradation
In a breakthrough for disease pre-
vention, Germany- and US-based
researchers have deciphered the
activation chain of protein destruc-
tion, so very important to cell func-
tion. Using cryo-electron micros-
copy the scientists analyzed how a
small protein label, called ubiquitin,
binds to a protein, marking it for © Kheewoong Baek, MPI of Biochemistry
degradation and thereby prevent- To enable this, the timing of protein
ing infection or disease. The latest activities is exquisitely controlled
structural insights of this cellular by ubiquitin that attaches to un-
process pave the way to the design wanted proteins, marking them for
of new drugs to tackle cancer, heart degradation.
disease and more. Proteins are mo-
lecular work horses in the cell that Original publication:
perform specific tasks - once a pro- doi: 10.1063/1.5126597
tein has fulfilled a task, its degra- More information:
dation ends unwanted processes. http://bit.ly/IM-12020-b
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Imaging & Microscopy 1/2020 • 7
 Coffee Break

Book Tip!
m
ob e.co
-Ad
S tock
-
SG
oto
©
Ph Correlative Imaging
Focusing on the Future

Crazy Science
Two Separate Droplets Coalesce into One

☛ Cameras shooting up to 25,000 frames a second have been

© Leeds University
used to capture the moment two droplets of liquid come together,
opening up research into new applications for 3D printing. With
one of the color cameras positioned below the droplets and the
other to the side, the synchronized system was able to record the
moment one of the droplets passed over the other, creating a
surface jet that formed less than 15 milliseconds – 15 thousandths of a second – after they coalesced.
http://bit.ly/IM-droplets

This new Wiley book brings a fresh point of

An Additional Moon for the Earth view to the current state of correlative ima-
ging and the future of the field. It provides
☛ In the skies above Earth, astronomers with the Catalina Sky Survey have contributions from international experts on
© DigiArt-Stock.Adobe.com

spotted what might be a new friend: an asteroid temporarily captured by correlative imaging, describing their vision
our planet’s gravity, what we call a minimoon. It’s named 2020 CD3, a of future developments in the field based
small chunk of likely carbonaceous rock between 1.9 and 3.5 meters (6.2 on where it is today. Starting with a brief
and 11.5 feet) in diameter. And here’s the kicker - the rock’s trajectory historical overview of how the field evolved,
indicates it’s been in orbit for around three years already. it presents the latest developments in micro-
 http://bit.ly/IM-minimoon scopy that facilitate the correlative workflow.
It also discusses the need for an ideal corre-
lative probe, applications in proteomic and
Shaving Doesn’t help to Avoid the Coronavirus elemental analysis, interpretation methods,
© KenImages-Adobe.Stock

and how correlative imaging can incorporate

☛ A 2017 workplace safety infographic compiled by the US Centers for force microscopy, soft x-ray tomography, and
Disease Control and Prevention (CDC) has run amok on social media volume electron microscopy techniques. Work
and in news reports, spawning claims that authorities are telling peo- on placing individual molecules within cells is
ple to shave off beards and moustaches to avoid the novel coronavirus. also featured.
Contrary to emerging claims, the CDC infographic, which covers an as-
tonishing amount of facial hair options (amongst them some controversial Correlative Imaging
ones), was not produced in relation to the current outbreak of COVID-19. Focusing on the Future
http://bit.ly/beard-coronavirus Editors: Verkade, Paul / Collinson, Lucy
RMS - Royal Microscopical Society
1. Edition November 2019
Fish Are also Picky when it Comes to Choosing a Partner 248 pages, hardcover
Wiley & Sons Ltd
☛ The owners of a pet have long been convinced that science ISBN: 978-1-119-08645-1
© Goethe University

has now confirmed that animals have personality too. A study

created at Goethe University shows that even with fish, perso-
nal characteristics, but also those of the potential partner, are More information:
decisive when choosing the „groom”. http://bit.ly/book-correlative-imaging
http://bit.ly/picky-fish

8 • Imaging & Microscopy 1/2020

 Coffee Break

Sudoku!
9 5 7
7 8 6 9
2 3 5
3
Web Tip!
4 9 1 3 More Microscopy on Wiley Analytical Science

5 Qur new web page is launched – Wiley Analytical Science. The “Micro-

2 1 9
scopy channel” combines all content from our magazines Imaging & Mi-
croscopy and Microscopy and Analysis and more. Wiley Analytical Science
www.crausworth.com

is a single subject-based website hosting professional and peer-reviewed

2 4 3 6 content from five well-established publication-based websites in Analyti-

cal Science. This new website will help you to easily access and navigate

8 9 7
the largest repository of validated information around the latest tech-
niques, equipment and news to support your professional success. Have a
look and give us a feedback via imaging-microscopy@wiley.com.

http://bit.ly/Wiley-WAS

Look closely is the task in this puzzle. We manipulated the right image. Find

!
all eight mistakes we have hidden for you. Send the image with the marked
errors to imaging-microscopy@wiley.com with subject line “Picture Puzzle”.
Among the correct submissions, the lot decides the winner of a small surprise
Closing date is May 8, 2020
Picture Puzzle

© wWeiss Lichtspiele - stock.adobe.com

Imaging & Microscopy 1/2020 • 9

EVENT CALENDAR
JUNE
HI
G

HL
IG H
ELMI Meeting

T
9-12 June 2020
Nordwijkerhout, The Netherlands
https://elmi2020.eu

© pixs:sell - stock.adobe.com AUGU

ST

HI
Microscopy & Microanalysis

GH
LIG H T
2-6 August 2020
JUNE Milwaukee, USA
HI
G
HL www.microscopy.org/MandM/2020

Frontiers in Bioimaging
IG H
© SeanPavonePhoto- stock.adobe.com
24-25 June 2020
T

London, UK
http://bit.ly/RMS-Frontiers2020

© LAFORET Aurélien - stock.adobe.com More Events on

http://bit.ly/WAS-conferences

Events 2020
Focus On Microscopy 5 - 8 April Osaka, Japan www.focusonmicroscopy.org
EBSD 21 - 22 April Sheffield, UK www.rms.org.uk/discover-engage/event-calendar/
ebsd-2020.html
EMBL Course:Fundamentals of Widefield 11 - 15 May Heidelberg, Germany www.embl.de/training/events/2020/MIC20-01/index.html
and Confocal Microscopy and Imaging
EMBL Course: Advanced Fluorescence 24 - 29 May Heidelberg, Germany www.embl.de/training/events/2020/MIC20-02/index.html
Imaging Techniques
CLEM Microscopy Workshop 8 -12 June Washington DC, USA https://nic.gwu.edu/clem-workshop
ELMI Meeting 9 - 12 June Nordwijkerhout, The https://elmi2020.eu
Netherlands
EM-STERMAT 14 - 17 June Wisla, Poland www.stereology.pl
EMBO Practical Course: Advanced Electron 16 - 26 June Heidelberg, Germany www.embl.de/training/events/2020/EMM20-01/index.html
Microscopy for Cell Biology
PhD/Postdoc course Advanced 17 - 21 June
  Maastricht, The Neth- https://gcb.mumc.nl/phdpostdoc-course-advanced-
Optical Microscopy 2019 erlands optical-microscopy-2019
Frontiers in Bioimaging 24 - 25 June
  London, UK http://bit.ly/RMS-Frontiers2020
15th International Conference on Mass Data 12 - 15 July
  New York, USA www.mda-signals.de
Analysis of Images and Signals in Artificial Intelligence
and Pattern Recognition MDA
Microscopy & Microanalysis 2 - 6 August
  Milwaukee, USA www.microscopy.org/MandM/2020
17th European Microscopy Congress 23 - 28 August
  Copenhagen, Denmark www.emc2020.eu
Materials Science and Engineering Congress 22 - 25 September
  Darmstadt, Germany www.mse-congress.de
International Conference on Nanoscopy 28 Sep - 1 Oct
  Jena, Germany www.icon-europe.org

10 • Imaging & Microscopy 1/2020

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Announcement
EMC2020: Time to Submit, Time to Enter
Copenhagen, Denmark, August 23-28, 2020
T
  he European Microscopy Congress
2020 (emc2020) will be Europe’s larg-
est event dedicated to microscopy and
imaging. It is also set to be the largest in the
event’s 64-year history with the organisers
expecting in excess of 2,000 submitted ab-
stracts and a vast exhibition packed with
every major manufacturer and supplier.
The international conference lies at the heart of
emc2020, with more than 40 sessions shared
across five symposia.
“These sessions and symposia provide a
wonderful platform to accommodate the broad
world of microscopy,” said Professor Klaus
Qvortrup, Chair of the emc2020 Executive
Board. “However, we are aware that there is
more than one way to organise subjects within
a conference setting, and that is why we have
six attention-grabbing, cross-cutting themes.
The tone of these will be set by our Plenary
Speakers, who all have outstanding reputations
in their fields.”
The Themes and Plenary Speakers are: Phase
Sensitive Methods with Photons and Electrons
- Professor John Rodenburg, UK; Imaging Quan-
tum Phenomena - Professor Roland Wiesendan-
ger, DE; Live and Fast Super-resolution - Profes-
sor Claus Ropers, DE; Artificial Intelligence in Big
Data Analysis, and Computational Microscopy -
Professor Moritz Helmstaedter, DE; Cutting Edge
Advanced Sample Preparation - Professor Caro-
lyn Larabell, US; and The Lab in the Microscope
- In situ, in vivo, in operando and Multimodal
Microscopy - Dr Frances Ross, US.
12 • Imaging & Microscopy 1/2020
The deadline for submitted abstracts for oral
presentation passed on 1st March, but abstracts
for late-breaking poster presentations are being
accepted until 15th June. Klaus explained why.
“Organising the scientific programme takes a
monumental effort from the Session Chairs and
the Congress Organisers. The logistics involved
mean that the March deadline is a necessity. But,
we are very aware that techniques and applica-
tions develop quickly. By welcoming late-break-
ing posters we can include an additional three
months of discovery in the programme. So, I say
to anyone who is thinking of submitting an ab-
stract to get on and do so.“
The Bella Center in Copenhagen is an ideal
venue for the congress. The permanent lecture
theatres flank the exhibition hall. This is a rare
luxury and it will promote seamless transitions
between conference sessions and time spent
with exhibitors.
“The layout of the venue is such a bonus,”
said Klaus. “Too many large-scale conferences
suffer from being disjointed. This will not be
the case for emc2020 where it will be so easy
to move from sessions, to the exhibition, to the
coffee areas, and back and forth with no time
lost in between. This facilitates and promotes
networking because that person that you need
to speak to will never be far away.”
The venue also makes it easy to host those
value-added features that can make a congress
stand out. Short-listed entries to the emc2020
Scientific Imaging Competition will be on dis-
play throughout the event and will be inte-
grated within the main hall. The images provide
a colourful and thought-provoking backdrop for
socialising and further networking. If you are
creative in your microscopy, there is still time to
submit an entry.
“The imaging competition is a great opportu-
nity to demonstrate your skills, and I am antic-
ipating some breathtaking images,” said Klaus.
“As always, the exhibitors are being very gener-
ous and there will be some great prizes for the
winners. I am a big fan of these competitions,
and I would encourage anyone with the desire
to produce a striking image to do so and to sub-
mit it before 1st June.”
Of course, if you are planning to attend
emc2020, you need to register. And there is
good reason to do so sooner rather than later:
The Early-Bird rates end on 6th July.
“There are great early discounts of up to
20 %, and there are very attractive rates, partic-
ularly for Young Scientists,” said Klaus. “If you
are going to go to one major conference this
year, you should make sure it is emc2020, and I
look forward to seeing you there.”
More information on emc2020:
www.emc2020.eu
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RMS in Focus
Microscience Microscopy Congress 2021
What Do You Want to See at mmc2021?
I
s there a scientific session that you would
like to see at the Microscience Microsco-
py Congress 2021 (mmc2021)? Or, rather
than making a suggestion, you could chair
a session at this ever-popular international
event.
Planning for mmc2021 is well underway. The
Manchester Central Convention Complex is
booked for July next year, and a vision is be-
ginning to emerge. The Scientific Programme is
being designed to accommodate microscopy in
its broadest sense. Five Plenary Speakers have
been confirmed. They are Dr Aydogan Ozcan
- University of California, USA, Professor Buzz
Baum - University College London, UK, Professor
Joanne Etheridge - Monash University, Australia,
Professor Janet Iwasa -University of Utah, USA,
Dr Olga Ovchinnikovo - Oak Ridge National Lab-
oratory, USA.
These much-respected speakers will help set
the tone of the conference, but there is still time
to influence the sessions that will appear in the
final programme.
“We pride ourselves in responding to feed-
back and the wishes of past and future del-
egates,” said Allison Winton, CEO of the Royal
Microscopical Society (RMS), the organisers
of mmc2021. “Our approach is that this event
belongs to our members and to the wider mi-
croscopy community. As a result, we provide
as many opportunities as possible for them to
influence the science that is presented, and ele-
14 • Imaging & Microscopy 1/2020
ments of the exhibition. This year is no different
and we welcome suggestions for sessions. These
may reflect emerging techniques and applica-
tions, or the evolution of established ones.”
This is not a new approach by the RMS.
Sessions that were suggested during the plan-
ning stages appeared in the programme for
mmc2019, and they proved popular.
“In addition to suggesting a session, I would
also encourage anyone who has reached a stage
in their career who feels that their next step is
to Chair a session at an international conference
to step forward,” said Allison. “We are always
looking to introduce fresh thoughts and ideas
to the planning phase, and if it will be your first-
time chairing, you will work alongside an expe-
rienced co-Chair. It is the perfect introduction to
a large-scale event.”
It is not just the conference that is receiving
external input. The exhibition will be Europe’s
largest dedicated to microscopy and imaging,
and it is expected that one hundred companies
will be represented. These range from the major
manufacturers to consumable suppliers.
“We take advice from our Corporate Advisory
Board which has a large and vibrant member-
ship,” said Allison. “It provides invaluable input
to all elements of the exhibition. Its members
exhibit regularly all over the world, and it is
great that they bring new ideas and suggestions
to the table.”
The international conference and exhibition
will always be the heart of a Microscience event,
but there are many other features that come
together to make for a memorable Congress.
Some will be familiar, and some will be new –
something that appeals to Allison. She said, “We
are always on the look-out for ways to keep
things fresh. One area that always bears fruit
is the inclusion of existing meetings within the
Congress. We are keen to speak with organisers
and other societies who would like to benefit
from being part of a much larger event. If this is
an attractive proposition, please do not hesitate
to make contact.”
Details of how to submit ideas for a session
or how to put yourself, or a colleague forward
to chair a session are available on the mmc2021
website.
The Microscience Microscopy Congress 2021
will be held in Manchester from 5 – 8 July 2021
at Manchester Central. The RMS looks forward
to welcoming you.
Contact
Allison Winton
Royal Microscopical Society
London, UK
awinton@rms.org.uk
More on mmc:
www.mmc-series.org.uk
 NewsNews
fromfrom
EMSEMS

Josef Zweck, EMS President

EMS Newsletter #68
March 2020

Virginie Serin, EMS Secretary

Dear EMS Members,

The new EMS year has started with the call for
nominations for the EMS Outstanding Paper
Awards 2020. EMS has received a considerable
amount of very high-quality papers presenting
good balance between the categories. These all
with very strong recommendations so the jury
will again have a hard task in selecting the win-
ners. The decision will be announced to the EMS
members in August this year, and the respective
authors will receive their awards during the
17th European Microscopy Congress (emc2020)
in Copenhagen, Denmark.
Although emc2020 is organized under the
sponsorship of the International Federation of
Societies for Microscopy (IFSM), EMS has strong
ties/ relations with the organizing bodies. EMS
provides a number of scholarships for early
stage career EMS members to attend this event.
The meeting takes place from 23-28 August,
2020 at the European Microscopy Congress in
Copenhagen, and promises to be the interna-
tional microscopy highlight for 2020.
The EMS executive Board will have its meeting
followed by the General Council and the annual
General Assembly. During the General Assembly,
the Outstanding Paper Awards will be delivered,
the next EMC announced (voted at the General
council), and the EMS executive board proposal
of a new board voted. EMC2020 awards, spon-
sored by JEOL, is also an important event of
emc2020.
In the first half of 2020, the following four
events are financially supported by EMS:
▪ Coiled coils, Myosin, Titin and striated muscle:
A reflection on the contributions of John Tri-
nick and Gerald Offer, 10 January 2020, Leeds
– United Kingdom
▪ Gordon Research Conference (GRC) on Liquid
Phase Electron Microscopy, 26 to 30 January
2020, Renaissance Tuscany Il Ciocco – Lucca
– Italy
▪ Symposium on Recent Advances in Microsco-
py Characterization of Photonic and Optoelec-
tronic Materials, 23 and 24 June 2020. London
– United Kingdom
▪ The XVIIth International Conference on Elec-
tron Microscopy EM’2020 and the 11th Inter-
national Conference on Image Analysis and
Stereology in Materials STERMAT’2020, 14 to
17 June 2020. Wisla – Poland.
This financial support from the EMS ensures
that also these smaller events, often organized
in the form of a school or course, can invite top
scientists to lecture our coming generations of
microscopists.
For the second half of the year (July to Decem-
ber 2020), calls for sponsored events is being
launched end of February.
In the coming months, you will receive the
2020 EMS Yearbook, which is now under con-
struction. This edition will include reports from
past EMS sponsored events, EMS Extensions and
their EMS lecturers, EMS scholarships, and many
other information.
We welcome our new Corporate Member Gam-
madata Instruments AB.
We look forward to seeing many of you in Co-
penhagen at emc2020.
Contact
Prof. Dr. Virginie Serin
EMS Secretary
CEMES- CNRS & Université Toulouse
Toulouse, France
virginie.serin@cemes.fr

EMS on Facebook and Twitter: Suggestions and ideas

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

Imaging & Microscopy 1/2020 • 15

Cover Story

Efficient Digital Documentation in the Lab

Simplifying Biomedical Routine Lab Work
Michael Gögler

S
mart Microscopy from Zeiss is a new
concept for routine digital documen-
tation of microscopic samples. The
microscopes Axiolab 5 and Axioscope 5 can
be combined with the microscope cameras
Axiocam 202 mono and Axiocam 208 col-
or, to form a Smart Microscopy system that
takes away a large share of the workload
from the users. It automatically adjusts
many of the required settings, thus digital
documentation of microscopic specimens
becomes easier and more efficient.
Efficiency and Quality Are Key
Efficiency and high-quality microscopic
images are key in the lab. It takes time to
acquire detail-rich, true-color images of bio-
medical specimens. Many steps are required:
place the sample on the microscope stage,
focus the region of interest, switch to the
computer, adjust settings such as white bal-
ance, exposure time and gain, then acquire
an image, insert a scale bar, switch back to
the microscope ... and so on. That’s what a
typical documentation workflow looks like.
The same steps must be repeated again and
again and the settings of the microscope
and software must be adjusted. The constant
switch between microscope and PC is tiring
and time-consuming.
Acquiring Detail-Rich Images in
True Color at the Touch of a Button
To stay focused on the sample is simple with
Smart Microscopy. It is a new concept for
routine digital documentation of microscop-
ic samples. The combination of the different
microscopes with different cameras form a
smart microscopy system. In this system, the
routine microscope communicates directly
with the camera for digital documentation.
With PC, tablet or as a stand-alone solution,
the system automatically adjusts brightness
and white balance to keep digital documen-
tation easy. Simply place the sample on the
microscope stage, find the region of inter-
est, then press the ergonomic Snap button
on the stand to acquire the image. The sam-
ple can be constantly focused through the
eyepieces and eyes and hands never have to
be taken from the microscope. All elements
are within reach and can be operated with
one hand, including the stage drive, focus
knobs, light intensity control, and the Snap
button. The system documents the sample
reproducibly and precisely as seen through
the eyepieces – detail-rich and in true color
(fig.1). The correct scaling is always includ-
ed automatically.
Getting Multichannel
Fluorescence Images with one Click
In addition to the documentation of
stained samples in transmitted light, the
system also facilitates work on specimens
with multi-colored fluorescence markers.
Working in the lab with specific fluores-
cent labels, these labels need to be excited
by exactly the right wavelength. Combine
Axioscope 5 with the high-performance
LED light source Colibri 3 and the sensitive,
stand-alone microscope camera Axiocam
202 mono to have the perfect setup to ac-
quire brilliant fluorescence images with
ease. The LED light source delivers the right
wavelength and intensity to excite fluores-
cent dyes and proteins in a gentle way. The
switch between the channels for UV, blue,
green and red excitation is effortless. Just
select the relevant channels and press Snap.
The system takes over and automatically
adjusts the excitation intensity, exposure
time and gain, acquires the image, switches
the channel and starts again. The overlaid
multichannel fluorescence image including
scale bar can be obtained directly (fig.2.).

Clever Ergonomics for Relaxed Lab Work

Fig.1: Whether unstained cells, histologically stained sections, or other samples from the labora- With Axiolab 5 and Axioscope 5, the ergo-
tory – the microscopes come with all relevant contrasting techniques for detail rich images. Left: nomic details make it possible to work re-
Mouse kidney in fluorescence, cryosection, AF 488 - WGA, AF 568 Phalloidin, DAPI; right: laxed for several hours on the system. All
Corylus avellana, hazel, male flower, cross section in brightfield. operating elements can be reached with one

16 • Imaging & Microscopy 1/2020


Fig.2: Owl monkey kidney – overlayed multichannel fluorescence image and single channels for DAPI, Alexa Fluor 488 and Alexa Fluor 546 dyes.
hand: stage drive, focus knobs, light inten-
sity control and the image capture button
are all within reach. The specimen can be
constantly observed through the eyepieces
without the necessity to look for a spe-
cific control button. Both microscopes are
equipped with a light manager providing
uniform brightness at all magnifications,
eliminating manual lamp intensity adjust-
ments when changing objectives. This saves
time and prevents eye fatigue. Overall,
Smart Microscopy minimizes and eases out
manual steps, allowing to work more effi-
ciently and in greater comfort (fig.3).
More Economic and Reliable
The microscopes are efficient when it comes
to cost- and energy-saving. Activate Eco-
mode, for instance, and the microscopes
automatically go to standby after being
idle for 15 minutes. This saves energy and
extends illumination life time. Rely on effi-
cient LED illumination: In transmitted light,
the powerful white LED allows to visualize
the sample in natural colors. Even subtle
color differences can be clearly seen. For
fluorescence, the integrated LEDs in various
wavelengths are easier and safer to use than
e.g. classical mercury lamps. LEDs provide
a high long-term light intensity stability
and are particularly gentle on your sam-
ples. With LEDs, warm-up and cool-down
times can be avoided. Lamp replacements
and lamp adjustments are a thing of the
past. Due to their low energy consumption
and long lifetime, LEDs are also easy on the
budget.
Digital Documentation Made Easy
There is a choice of how to use Smart Mi-
croscopy:
▪ In the stand-alone version, only the mi-
croscopes Axioscope 5 or Axiolab 5 and
the cameras Axiocam 202 mono or Axio-
cam 208 color are needed. A PC with ad-
ditional software is not necessary. The on-
screen display of the camera can be used
and save the images directly to your USB
storage medium plugged into the camera.
▪ Use Smart Microscopy in conjunction
with Imaging App Labscope on an iPad
or PC. This solution is ideal for easy im-
age acquisition with annotation and
manual measurement possibilities, for
multi-channel fluorescence documenta-
tion, and networked laboratories.
▪ For further processing and analyzing the
images afterwards, Smart Microscopy
with the Zeiss Imaging Software ZEN can
be used.
Fig.3: All the main controls are accessible with
just one hand, including the Snap button,
stage drive, focus adjustment, and brightness
control. Even after long hours working with
the microscope you can work comfortably.
Cover Story
Camera settings such as white balance,
exposure time, and image enhancement
functions can always be done automatical-
ly. Just focus and snap an image - that’s
it. Simply enjoy and boost efficiency in the
lab (fig.4).
Conclusion
Smart Microscopy from Zeiss with Axiolab
5 and Axioscope 5 are made for routine mi-
croscopy work that goes on every day in the
lab. Compact and ergonomic microscope
designs save space and make for easy han-
dling. Take full advantage of the concept: a
completely new form of digital documen-
tation can be experienced. Just focus the
sample and press a single button directly
at the stand for crisp images in true color.
The digital image will look like it is seen
through the eyepieces, with all the details
and subtle color differences clearly visible.
Plus, the microscopes automatically add the
correct scaling information to the images.
All of this can be done even in a stand-
alone operation, without needing a PC or
any additional software. Save time, money
and valuable lab space with Smart Micros-
copy. Digital documentation has never been
easier.
Contact
Dr. Michael Gögler
Market Sector Manager for Routine Microscopy
Carl Zeiss Microscopy GmbH
Munich, Germany
michael.goegler@zeiss.com
Imaging & Microscopy 1/2020 • 17
Light Microscopy
Intracellular Journey of Cell Surface Receptors
Imaging Intracellular Trafficking Using Photoconvertible Fluorescent Proteins
Gregory Redpath1, Manuela Ecker2, Jérémie Rossy3
I
maging intracellular trafficking in live
cells is challenging. Mostly because la-
belling of cargo proteins with conven-
tional fluorescent proteins does not pro-
vide temporal or directional information
about the vesicles that transport these
cargoes. The use of photoswitchable or
photoactivatable fluorescent proteins
enable to “shine the light” on specific
membranes, vesicles or intracellular com-
partments at a given time and location.
The transport of cargoes can thereby be
visualized and quantified in order to ob-
tain key information about the mechanism
underpinning intracellular trafficking.
Introduction
Since photoactivatable GFP was engineered
in 2002 [1], the past seventeen years have
seen the development of a multitude of pho-
toswitchable (PS) or photoactivatable (PA)
fluorescent proteins of various spectra, in-
creasing photostability and quantum yields
[2-5]. They were first designed with live cell
imaging in mind, because of the obvious
edge they provide cell biologists by allow-
ing visualization and measure of protein
dynamics.
However, PA/PS proteins perfectly illus-
trate the gap that often stands between pow-
18 • Imaging & Microscopy 1/2020
erful tools developed by research groups that
specialize in method development and their
application to address biologically relevant
questions. The MEDLINE database references
almost every publication in life and biomedi-
cal sciences and can be publicly accessed with
the search engine PubMed. A quick search on
PubMed with the keywords “Photoconvert-
ible”, “Photoactivatable” or “Photoswitchable
fluorescent proteins” reveals that the number
of studies reporting development, characteri-
zation or methodologies related to PS/PA pro-
teins outweighs papers using them to gain
novel insights into biological questions by at
least forty to one. Biologists too often privi-
lege the use of a more widespread and charac-
terized approach, fluorescence recovery after
bleaching (FRAP). Partially because nowadays
most commercial fluorescence microscopes
include step-by-step wizards to generate and
analyze FRAP data. PS/PA fluorescent pro-
teins benefited from a renewed interest ten
years ago with the advent of photoactivatable
localization microscopy [6,7], which further
shifted the balance of publications towards
method papers [8]. Hence, these powerful
tools have been continuously developed and
improved, but too sparsely used to do what
they had been initially designed for.
Imaging intracellular trafficking in live
cells involves several challenges. The main
problem is that when cargo proteins are
labelled with conventional fluorescent pro-
teins such as GFP, the fluorescent signal will
not provide any information about the time
the cargo was packaged into a transport ves-
icle. It will not reveal either if this vesicle
is heading towards intracellular compart-
ments from the plasma membrane after hav-
ing been internalized, from one endosome to
another or being delivered from intracellular
stores to the plasma membrane. However, an
accurate quantification of these parameters
is required to understand the mechanisms
regulating how proteins are transported
through the cell. PS/PA fluorescent proteins
represent the ideal tool to access this infor-
mation. Cargo proteins fused to PS/PA fluo-
rescent proteins can be revealed at a specific
time and location within the cell. It is then
possible to visualize the cellular trafficking
of these proteins from the region where the
photoactivation/conversion has been per-
formed to a target destination of interest.
Accordingly, several studies have made
use of PS/PA fluorescent proteins to follow
proteins of interest while they are trans-
ported within various cell types [9-11]. Two
recent publications [12, 13] describe a global
microscopy approach based on the light-in-
duced activation of photoactivatable fluores-
cent proteins to visualize, and most impor-
tantly to quantify, the endocytic trafficking
of proteins associated with the plasma mem-

brane. This approach has allowed to extract
quantitative information about every step of
the endocytic recycling process: 1) internal-
ization of receptors from the plasma mem-
brane (endocytosis); 2) incorporation of
these receptors into intracellular compart-
ments; 3) return of the receptor from intra-
cellular compartment to the plasma mem-
brane (endocytic recycling).
Internalization
To investigate endocytosis, the cell surface
protein of interest is fused to PA-mCherry.
Defined regions of the cells are illuminated
for a few seconds with 405 nm laser us-
ing a confocal microscope. These regions
are selected on the edges of the cell, which
consist mostly of plasma membrane and
contain very little intracellular material, in
order to photoactivate only proteins with-
in the plasma membrane. The PA-mCherry
that is revealed at the plasma membrane
initially shows a globally uniform distribu-
tion before being eventually packaged into
vesicles, which appear as bright punctate
structures (fig. 1). These structures can eas-
ily be counted throughout a time series by
any analysis routine that identifies parti-
cles. The number of vesicles that form and
the speed at which they form provide direct
information about the processes that me-
diate the endocytosis of the protein fused
to PA-mCherry. This approach can also
be used to identify the cellular machin-
ery supporting endocytosis. Components
of this machinery are fused to EGFP and
a cross-channel nearest neighbor analysis
can be used to determine if the endocytosed
particles identified in the red channel (the
membrane protein fused to PA-mCherry)
and the particles in the green channel (an
endocytic protein fused to EGFP) are the
same vesicles or not.
Incorporation into
Intracellular Compartments
The target compartments – or endosomes – of
internalized cell surface proteins determine if
these proteins will be sent for degradation or
are reused, and thereby returned to the cell
surface. PA/PS fluorescent proteins also allow
to quantify how much of an internalized pro-
tein reaches a given endosome. As when in-
vestigating endocytosis, membrane proteins
Light Microscopy
fused to PA-mCherry are photoactivated at
the edge of the cell, but this time repetitively
(every 5-10 seconds), in order to reveal large
amounts of internalized proteins. Markers of
specific endosomes labelled with EGFP are
used to define a mask. The fluorescence in-
tensity of the PA-mCherry signal within the
EGFP-mask indicates if the internalized pro-
tein has reached the endosome, how quickly
it enters the compartment, as well as how
long it remains in this specific compartment
before continuing its endocytic journey.
Transport of Membrane Proteins from In-
tracellular Compartment to the Cell Sur-
face
While the mechanisms that regulate the
fusion of vesicles with the plasma mem-
brane have been extensively investigated,
much less is known regarding how cargoes
are loaded into transport vesicles that will
bring them to the cell surface for such fu-
sion. Two-photon illumination of fluorescent
proteins can answer this question, because
it allows the selective photoconversion of
membrane proteins strictly within intracellu-
lar compartment and nowhere else in the cell
Imaging & Microscopy 1/2020 • 19
Light Microscopy
Fig. 1: A. Examples of images and quantification of the internalization of a cell surface receptor (TCRζ)
fused to PA-mCherry after a single round of 405 nm illumination (upper row) or repetitively photoactivated
8 sec for 200 sec (lower row) with a region of interest at the edge of Jurkat T cells (white dashed line). B:
Number of TCRζ-positive after standard (blue) or repetitive (cyan) photoactivation. Scale bars = 5 µm.
(fig. 2). The membrane proteins
of interest are fused to PS-CFP2,
which in our hands proved to be
more resistant to photobleach-
ing than PA-mCherry and there-
fore is more suitable for this ap-
proach. Similar to the process for
visualizing the incorporation of
internalized proteins into endo-
somes, the cells express markers
of endosomes of interest labelled
with mCherry. The mCherry sig-
nal is used as an “aiming light”
to define which area inside the
cell will be illuminated with 800
nm light to visualize if the trans-
port to the plasma membrane
occurs from this compartment
of interest. The photoconverted
PS-CFP2 signal is measured by
confocal imaging at the plas-
ma membrane (using 488 nm
excitation). The intensity of
this signal is directly related to
transport of the PS-CFP2-fused
membrane protein from the
endosome where the photocon-
version has been performed to
the cell surface. Additionally,
vesicles positive for converted
PS-CFP2 can be imaged at a fo-
cal plan that is axially located
Fig. 2: A. Principle of two-photon specific photoconversion of cargo between the endosome of inter-
proteins of interest within intracellular compartments. B. Jurkat T cells est and the plasma membrane
transfected with TCRζ- mCherry (magenta) and TCRζ-PSCFP2 (cyan) to uncover the identity of the
were activated on glass coverslips and fixed 20 minutes post activation. vesicles transporting the cargo
Z-stacks of cells were imaged before (left) and after (right) photoactiva-
tion with one-photon 405 nm excitation (first row) or two-photon 800
nm excitation at the indicated z-depth. Dashed white line indicates pho-
toactivated region. Adapted with permissions from G. M. I. Redpath et al More on live-cell imaging:
et al. Flotillins promote T cell receptor sorting through a fast Rab5– http://bit.ly/WAS-live-cell
Rab11 endocytic recycling axis, Nat. Commun. 10 4392 (2019).
20 • Imaging & Microscopy 1/2020
protein. A cross-channel near-
est-neighbour analysis between
photoconverted vesicles and
vesicles positive for mCherry
can be used to determine if the
membrane protein of interest is
transported in the endosomal
population where PS-CFP2 was
photoconverted. Altogether, this
approach allows direct quantifi-
cation of the magnitude, kinetics
and molecular identity regulat-
ing the transport of membrane
proteins to the cell surface.
Conclusion
While PA/PS proteins tend to
be used more for single mole-
cule localization microscopy,
they are a very powerful tool to
investigate cellular trafficking.
They allow to follow and quan-
tify the entire endocytic journey
of a given membrane protein in
live cells. Hence, PA/PS proteins
can be used to obtain crucial
information about the cellu-
lar mechanisms regulating the
shuttling of membrane proteins
between the plasma membrane
and intracellular compartments
to fully understand how endo-
cytic trafficking regulates cell
function.
Affiliations
1 Department of Biochemis-
try, Otago School of Medical
Sciences, University of Otago,
Dunedin, New Zealand
2EMBL Australia Node in Sin-
gle Molecule Science, School of
Medical Sciences, University of
New South Wales, Sydney, Aus-
tralia
3Biotechnology Institute Thur-
gau at the University of Kon-
stanz, Kreuzlingen, Switzerland
Contact
Dr. Jérémie Rossy
Biotechnology Institute Thurgau 
University of Konstanz
Kreuzlingen, Switzerland
jeremie.rossy@bitg.ch
References: http://
bit.ly/IM-Rossy
 speed
sen
siti
vity

re
s
olu
tio
n
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MADE IN GERMANY
Light Microscopy

Biochemical Imaging by Stimulated

Raman Scattering Microscopy
Metabolic Imaging and Super-Multiplexed Vibrational Detection
Fanghao Hu1 and Wei Min1,2

S
timulated Raman scattering microsco-
py has emerged as a new frontier in
light microscopy with exciting devel-
opments. Recently, heavy water and deu-
terated glucose were applied for metabolic
imaging in vivo, which allowed visualization
of proteins, lipids, DNA, and glycogen turn-
over in animals. A Raman-tailored tissue
clearing method was developed to increase
the imaging depth by >10 folds in tumor
and mouse brain tissues. Two sets of Raman
dye palettes were developed for super-mul-
tiplexed optical imaging in cells and tissues,
which can visualize cell-type dependent
metabolic activities in neuronal culture and
subcellular organelles in living cells.

During the last decade, stimulated Raman

scattering (SRS) microscopy has become one
of the most promising techniques for chemi-
cal imaging in biomedicine [1,2]. SRS micros-
copy is a nonlinear optical imaging technique
based on Raman scattering of molecular vi-
brations, which can map the distribution of
chemical bonds in three-dimensional space
with high sensitivity and specificity [3]. Two
near-infrared lasers (pump and Stokes beams)
are applied in SRS to amplify the Raman
scattering process. When the frequency dif-
ference of two lasers matches with the vibra-
tional frequency of selected chemical bond,
the Raman scattering transition rate can be
enhanced by up to 108 folds, analogous to
that in the stimulated emission [4].
In SRS microscopy, both pump and Stokes
beams are spatially and temporally over-
lapped, and focused collinearly into a diffrac-
tion-limit spot inside the sample. A high-fre-
quency (MHz) modulation transfer scheme is
applied to detect SRS signal with near shot-
noise limit using a lock-in amplifier. By col-
lecting SRS signal at each pixel in either trans-
mitted or reflected light directions, three-di-
mensional images can be readily acquired
by raster-scanning the laser focus across the
samples. High-speed SRS imaging has been
achieved with ~100 frames/s [5,6], and in vivo
imaging has been demonstrated in living mice
and humans with high sensitivity [7].
Since its invention, SRS microscopy has
made significant progress in both optical
instruments and imaging probes, and has
Fig. 1: DO-SRS imaging of germline development in C. elegans (a) and volumetric SRS imaging of
mouse brain tissues with Raman-tailored clearing method (b). Images are adapted from ref. 13 and 16.
found broad applications in life sciences [2].
Label-free SRS imaging has been first applied
to many biomedical applications. By targeting
endogenous chemical bonds including O–H,
C–H, C=C, O–P–O, amide and ring-breathing
modes, many biomolecules have been success-
fully imaged in cells, tissues and organisms
by label-free SRS microscopy [1], including
total proteins, lipids, DNA, cholesterol and cell
walls. This article focuses on recent develop-
ments of metabolic imaging and super-multi-
plexed Raman detection.
Heavy Water and d7-glucose for
Imaging Metabolic Dynamics in Animals
Metabolic imaging is important to visualize
cellular states and their transformations in
disease conditions. To visualize cellular me-
tabolism, small vibrational tags have been
coupled with SRS microscopy for metabol-
ic imaging of proteins, lipids, and nucleic
acids with minimal perturbation [8,9]. Deu-
terium, as a minimal label, introduces little
change to the biochemical property of the
target molecule. The replacement of hydro-
gen with deuterium also doubles the mass
and shifts the vibrational frequency of as-
sociated chemical bonds for characteristic
detection in the cell Raman-silent window
(1800-2700 cm-1). Using deuterated amino
acids, our group has achieved SRS imaging
of newly synthesized proteins in living cells,
tissues and organisms [10-12].
More recently, to study cellular activity at
a global level, heavy water (D2O) has been
applied as a universal and inexpensive deu-
terium source to visualize in situ metabolism
of proteins, lipids and DNA in cells, tissues
and animals, by DO-SRS [13] (fig. 1). Addi-
tionally, using d7-glucose and spectral trac-
ing of deuterium isotopes (STRIDE), glucose
metabolism into biosynthesis can be traced
with high specificity [14]. With both DO-SRS
and STRIDE, multiplexed imaging of biomass
turnover can be monitored based on differ-
ent Raman spectra of C-D bonds in proteins,
lipids, DNA and glycogen.
DO-SRS and STRIDE have been applied
to study protein and lipid metabolisms in
developmental biology. D2O was used in
visualizing zebrafish embryogenesis and
germline development of C. elegans, which
showed active protein synthesis through-
out germline development and temporally
defined lipid incorporation into late-stage
oocytes (fig. 1a). By imaging protein and
lipid synthesis, DO-SRS can also visualize
brain tumor boundaries and heterogeneity

22 • Imaging & Microscopy 1/2020


in a mouse xenograft of colon
tumors, which was difficult to
distinguish using the label-free
approach. In addition, STRIDE-
SRS using d7-glucose was
applied to study the myelination
process of developing mouse
brain, which revealed strong
patterns of lipid turnover and
was correlated with the expres-
sion of myelin basic proteins. In
newborn mouse, fast and uni-
directional lipid absorption was
also captured in the intestine by
depth-resolved STRIDE imaging.
Moreover, SRS imaging
of heavy water has provided
insights into the metabolic het-
erogeneity and antibiotic tol-
erance of Pseudomonas aeru-
ginosa bacterial biofilms [15],
which are one of the major
causes of chronic lung infec-
tions. Through SRS imaging of
D2O incorporation, deep regions
of metabolic activity were
observed for the first time inside
biofilms grown on glucose. The
metabolism was found to link to
small metabolite phenazines and
Cco terminal oxidases, which
could promote biofilm protec-
tion against antibiotics.
Furthermore, to image tis-
sues with much greater depth,
a Raman-tailored optical clear-
ing technique [16] was devel-
oped by our group using urea
and low-concentration Triton
X-100. By reducing refractive
index mismatch and tissue scat-
tering, volumetric SRS imaging
of tumor and mouse brain tis-
sues have been achieved with
over 10-fold increase in the
imaging depth (~1 mm) (fig. 1b).
Super-Multiplexed Vibration-
al Imaging in Cells and Tissues
Simultaneously visualizing a
large number of species with
high spatiotemporal resolution
will allow systematic interro-
gation of the structure, activity,
and dynamics of complex sys-
tems. Fluorescence imaging is
highly limited in multiplexed
detection due to broad line-
width and large spectral cross-
talk, allowing no more than
5–6 targets. Raman imaging
can achieve much higher mul-
Fig.2: Super-multiplexed vibrational microscopy. (a) 20-color Carbow
palette; (b) 8-color MARS imaging in neuronal system; (c) 10-color or-
ganelle imaging in live cells. Images are adapted from ref. 17 and 18.
tiplexing capability, with about
50–100 times narrower peak
widths. Two sets of super-mul-
tiplexed Raman dyes have been
recently developed to achieve
direct imaging of tens of species
in cells and tissues (fig. 2).
Manhattan Raman scattering
(MARS) dyes was developed by
conjugating alkynes and nitriles
to near-infrared-absorbing xan-
thene [17]. 14 well-resolved
Raman peaks in the cell spec-
tral-silent region were obtained
through changing the central
atom (from O to C to Si) of xan-
thene, expanding the ring size,
and isotopic editing of alkynes
and nitriles. Another palette of
super-multiplexed Raman dyes
was developed based on the
polyyne scaffold, which is a lin-
ear chain of conjugated alkynes
[18]. Through the chain-length
modulation, bond-selective iso-
tope labeling and end-group sub-
stitutions, 20 distinct Raman col-
ors were obtained in the cell-si-
lent window, which were termed
the carbon rainbow (Carbow, fig.
2a).
With strong signal enhance-
ment under electronic pre-reso-
nance SRS excitation, MARS dyes
have superb sensitivity down to
sub-μM, which are well suited for
specific imaging of low-abundant
species such as protein receptors.
MARS probes were applied to
eight-color imaging of complex
metabolic activity in neuronal
co-culture (fig. 2b) and mouse
brain tissues [17]. Cell-type-de-
pendent metabolic heterogene-
ities were revealed with astro-
cytes forming more proteome
inclusions than neurons under
proteasome stress, suggesting an
active role by sequestering mis-
folded protein species.
With a neutral scaffold, Car-
bow probes are suitable for live-
cell imaging with high membrane
permeability and little non-spe-
cific background. Ten-color
imaging of subcellular structures
has been achieved with organ-
elle-targeted Carbow probes in
living cells (fig. 2c), including
mitochondria, lysosomes, lipid
droplets, endoplasmic reticulum,
plasma membrane, Golgi appa-
ratus, nucleus, microtubules, and
actins [18]. This can facilitate the
study of inter-organelle interac-
tions under both physiological
and disease conditions. Also, a
More on Raman imaging:
http://bit.ly/WAS-Raman2
Light Microscopy
ratiometric Raman sensor has
been reported based on Carbow
probes to detect H2S in the mito-
chondria of living cells by SRS
imaging [19].
Moreover, super-multiplexed
optical barcoding has been
demonstrated through combi-
natory spectral encoding of Car-
bow probes in μm-sized polysty-
rene beads, which can generate
up to 310~60,000 distinct spectral
barcodes with little crosstalk for
high-capacity cell labeling and
rapid identification by SRS [18].
With both MARS and Carbow
palettes, super-multiplexed SRS
microscopy has great potential
in high-dimensional cell imag-
ing and profiling to study com-
plex biological systems.
Summary
In summary, the use of deute-
rium labels in heavy water and
d7-glucose has allowed SRS im-
aging of metabolic dynamics in
animals with chemical speci-
ficity. With a Raman-tailored
tissue clearing technique using
urea and Triton X-100, a deep
imaging depth of ~1 mm can
be achieved by SRS microscopy
in tumor and brain tissues that
suffer from strong scattering
loss. Furthermore, the develop-
ment of super-multiplexed vi-
brational imaging with 20-color
Raman palette has open up new
possibilities to investigate the
organization, interaction and
dynamics of complex systems.
Affiliations
1Department of Chemistry, Co-
lumbia University, New York,
NY, USA
2Kavli Institute for Brain Sci-
ence, Columbia University, New
York, NY, USA
Contact
Dr. Wei Min
Department of Chemistry
Columbia University
New York, NY, USA
wm2256@columbia.edu
References:
http://bit.ly/IM-Min2
Imaging & Microscopy 1/2020 • 23
Light Microscopy

Single-Shot Volumetric Microscopy

Imaging Entire Optically Sectioned Volumes at an Instant
Florian Ströhl

A
new imaging concept that illumi-
nates the sample with two orthog-
onally polarized excitation lattices
is posed to allow single-shot optically sec-
tioned recordings of entire volumes. Its core
is a special illumination structure that was
originally developed for laser cooling appli-
cations. Combined with multi-focus optics
and a dedicated reconstruction algorithm,
the technique can retrieve conventional-
ly lost information about a sample’s axial
structure in next-generation microscopy
systems.
Introduction
Existing fluorescence microscopy methods
suffer from background haze produced by
emitters residing outside the image plane.
These out-of-plane emitters reduce contrast
and must be avoided to obtain a sharp, op-
tically sectioned image. Figure 1 shows the
point spread function (PSF) of a widefield
microscope. That means a single fluorophore,
much smaller than the resolution of the mi-
croscope, will look like the blob depicted in
panel a (in the x-z plane). You can see that
an imaged emitter has clearly visible ‘wings’
along the z-axis that contribute out-of-plane
signal. The effect of these wings can be even
better quantified in the transmitted spatial
frequency spectrum of the microscope, which
is known as its optical transfer function (OTF).
In widefield microscopy, the ‘missing cone’
(actually a double cone) of spatial frequen-
cies along the axial direction characterizes the
spectral support – filling that cone is equiva-
lent to optically section the image, boosting
contrast. The most common technique for
erasing the wings and hence filling the cone
is confocal microscopy, where unwanted light
from outside the focal plane is physically re-
jected by a pinhole. A different well-known
approach is light sheet microscopy where flu-
orophores are excited in a single plane only
such that no out-of-focus signal is generated
in the first place. The drawback of both ap-
proaches is, by design, that they require the
translation of the image point or plane along
at least one direction to create a fully sec-
tioned volume. This can reduce imaging speed
and lead to motion artefacts, a challenge in
live cell imaging.
Single-Shot Optical Sectioning
Recently, a half optical/half computational
concept termed single shot optical sectioning
(ssOS), was presented to circumvent the miss-
ing cone problem of widefield microscopy
[1,2]. In this new approach [1], special multi-
plane imaging elements [3] and a dedicated
illumination arrangement are used together
with a reconstruction algorithm [1] to allow
the recoding of optically sectioned volumes in
a single snap-shot. The basic layout of such a
system is shown in figure 2.
Linearly polarized laser light is split into
two beams by a grating and polarized into
counter-rotating circular polarization states.
This arrangement is known as sigma+/sigma-
configuration and is the circular-polarization
analogue of the lin-perp-lin configuration, an
important tool in laser cooling.
Relayed to the sample plane, the two beams
are brought together in order to interfere.
Remember that they are orthogonally polar-
ized, though! Thus, they do not produce an
intensity modulated illumination pattern, but
an alternating pattern in terms of electric field
directions. The intensity profile is flat.
Nevertheless, akin to structured illumina-
tion microscopy (SIM), the sample is illumi-
nated with light possessing spatial frequen-
cies of its own – i.e. two stripe patterns. These
stripes produce beat patterns with the sample
structure and, as known from SIM, the result-
ing Moiré-like patterns encode depth infor-
mation of the sample. In SIM it is necessary
to acquire many raw frames of the same view
under slightly different illumination patterns
to disentangle additional information. A clever
combination of just two independent record-
ings is enough though, if only axial informa-
tion content of the missing cone is sought
after (fig. 3). Intriguingly, these two inde-
pendent frames can be captured in a single
shot under orthogonally polarized excitation
lattices (as described above) when relatively
large fluorescent labels are used. Large in this
respect means that the fluorophores possess
a small rotational diffusion coefficient. Thus,
polarized excitation light retains some of its
directionality when being emitted as fluores-
cence after absorption by the fluorophore. This
phenomenon is known as fluorescence aniso­
tropy and can be made visible with polarizers
in the detection path. For ssOS, the important
takeaway is that multiplexed sample illumi-
nation via orthogonally polarized excitation
lattices allows en- and de-coding of two pat-
terns simultaneously.

Fig. 1: Loss and recovery of axial sample information in widefield and confocal microscopy. The 3D PSF of an incoherent widefield microscopy
system displays ‘wings’ that produce a background haze (a). They are blocked by the pinhole in confocal microscopy systems to enable optical sec-
tioning (b). The 3D Fourier transform of the PSF is the OTF, which displays a ‘missing cone’ of spatial frequencies in widefield microscopy (c). In
contrast, optically sectioned confocal microscopy fills the missing cone (and increases resolution as a side-effect) (d). Filling the missing cone in
widefield microscopy is already enough to enable optical sectioning similar to confocal microscopy.

24 • Imaging & Microscopy 1/2020

 Light Microscopy

Fig. 3: Illustration of the sectioning concept. Assume a hypothetical

Fig. 2: Realizing ssOS. A potential optical setup to realize orthogonally
polarized excitation lattices and successive polarization-based decod-
ing. Multi-plane prisms further allow recording of all axial planes si-
multaneously. Figure modified from F. Ströhl and C. F. Kaminski, Sci-
entific reports 9(1): 6425 (2019).
spectrum of an object with distinct features (green triangles) along the
spatial frequency z-axis, symbolizing the missing cone. Recording im-
ages and Fourier transforming them yields the spectra shown in (b) for
widefield (I0) and structured (I1) illumination. A deconvolution step
lays the non-attenuated spectra free (c), which can then be used to re-
trieve the missing cone components. Key element here is a complex
weighted subtraction (d) of several shifted widefield spectra S0 from the
spectrum S1. Superscripts denote shift directions with respect to the
original spectrum S0. A combination of the original widefield spectrum
with the averaged cone component (e) then yields the sectioned spec-
trum SOS (f). Figure modified from F. Ströhl and C. F. Kaminski, Scien-
tific reports 9(1): 6425 (2019).

The volumetric single shot aspect is real-
ized by recently introduced multi-plane split-
ter optics [2] that redirect light from multi-
ple image planes onto different locations on
the camera. They thus permit recording of
whole volumes in a single shot. On their own,
multi-plane splitter optics only capture vol-
umes without optical sectioning. The section-
ing comes into play via ssOS. The projected
lattices are translationally invariant along the
optical axis and are therefore visible in each
of the simultaneously recorded image planes.
Moiré patterns of each plane can thus be com-
bined via the ssOS algorithm for retrieval of
missing cone information and section the
whole volume.
quirement of scanning with a light sheet or
physical rejection of out-of-focus light to ob-
tain contrast. Maybe we are seeing a new type
of fast volumetric imaging systems emerge?
Acknowledgements
I would like to thank my PhD supervisor
Clemens Kaminski for his advice as well as
Thomas Huser and Kevin o’Holleran for fruit-
ful discussions on this topic. This article was
realized with support from the EU’s Hori-
zon2020 program (Marie Skłodowska Curie
Action #836355) and the research work was
funded by the European Molecular Biology
Organization (EMBO), the Engineering and
Physical Sciences Research Council (EPSRC)
(EP/H018301/1), Medical Research Coun-
cil (MRC) (MR/K015850/1, MR/K02292X/1),
Wellcome Trust (089703/Z/09/Z), and Infini-
tus China Ltd.
Contact
Dr. Florian Ströhl
Department of Physics and Technology
UiT The Arctic University of Norway
Tromsø, Norway
florian.strohl@uit.no
http://site.uit.no/nanoscopy

Summary
More about volumetric imaging: References:
In summary, ssOS makes use of all fluoro- http://bit.ly/WAS-VI http://bit.ly/IM-Stroehl2
phores in the sample volume, without the re-

See the essential.

High-performance optical filters Visit us at
for spectroscopy applications Hall B1
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Imaging & Microscopy 1/2020 • 25

Light Microscopy
Cool Lenses for Light Microscopy
Immersion Objectives and Media for Cryo-Fluorescence Imaging
M
icroscopy at cryogenic temperature
is highly attractive for correlating
light and electron microscopy in
cells that are preserved in a near native state
by flash freezing (vitrification). High-qual-
ity immersion objectives are important for
pushing the limits of resolution and contrast
in light microscopy, but such objectives have
long been lacking for temperatures below
the glass transition of water (-135°C). We
have overcome this challenge by a new class
of cryo-compatible immersion objectives
and by a new medium which matches the
refractive index of room temperature water
at cryogenic temperature.
Introduction
Cryofixation of cells and biomolecules opens
exciting opportunities in light and electron
26 • Imaging & Microscopy 1/2020
Thomas P. Burg
microscopy. Deep-frozen in a glassy – or
vitrified – state at liquid nitrogen tempera-
ture, the native structure of cells can be pre-
served without fixation artifacts and imaged
at high resolution with cryo-EM. However,
while the technology of cryo-EM is relative-
ly mature and well supported, correlating
light and electron microscopy in cryogeni-
cally frozen objects remains non-trivial. One
long-standing challenge in cryogenic light
microscopy is the lack of high numerical ap-
erture (NA) microscope objectives. The NA
of an objective is an important parameter
that ultimately limits the light-collection
efficiency and diffraction-limited resolution
of the entire optical system.
To avoid warming the sample and dam-
aging the optics, standard platforms for cor-
relative light and electron cryo-microscopy
exclusively employ air objectives. Air objec-
tives are fundamentally limited to NA val-
ues less than 1.0 and in practice to NA<0.95.
To surpass this limit, the space between the
object and the front lens needs to be filled
with an immersion liquid. At room tempera-
ture, this is routinely done, but for imaging
below the glass transition of water (-135 °C),
no suitable cryo compatible objectives and
matched immersion media exist.
To enable light microscopy with a numer-
ical aperture greater than 1.0 at cryogenic
temperature, we recently introducted a new
concept based on active thermal shielding
[1]. This principle guards the warm objective
body from the low temperature of the sample
and protects the sample from being thawed
by heat conducted through the immersion
medium. The approach is based on a strong
thermal isolation of the front lens by an
actively heated ceramic front lens mount.
Due to the high thermal resistance of the
ceramic, the temperature gradient between
the housing of the objective and the liq-
uid nitrogen-cooled sample remains con-
fined mainly within the lens mount. Front
lens and sample are nearly in thermal equi-
librium while the objective is maintained
far from thermal equilibrium. Power to the
electrical heating system near the connec-
tion between the ceramic front lens mount
and the housing of the objective is contin-
uously adjusted to maintain a stable steady
state (fig. 2). The front lens is able to tolerate
the deep temperature cycles by virtue of its
small size and good match between its own
thermal expansion coefficient and that of the
ceramic mount.
An important advantage of our approach
is that the sample resides in a thermally
shielded microenvironment that also con-
tains the immersion liquid and the front
lens. Refractive index gradients due to tem-
perature variations are therefore minimized
Fig. 1: Three-color widefield cryofluorescence
image of plunge frozen U2OS cells labeled
with Alexa Fluor 488 (vimentin cytoskeleton),
Alexa Fluor 594 (Tom20 mitochondrial pro-
tein), and DAPI (cell nuclei) [1].

Fig. 2: Immersion microscopy at cryogenic temperature is enabled by
an objective in which the front lens and the objective body are not in
thermal equilibrium. An electrical heater maintains a temperature gra-
dient across the ceramic front lens mount.
and associated wavefront distortions of the
wavefront are avoided.
Another important aspect besides the
mechanical design is the choice of the
immersion medium, the refractive index of
which should match the design value of the
lens system to at least ~10-3 refractive index
units. In addition, the immersion medium
needs to be optically clear, nonfluorescent,
and nontoxic, and have low vapor pressure
at the imaging temperature. Moreover, the
liquid range should extend above room tem-
perature for easy storage and handling.
Results
It has been discovered that the partially fluo-
rinated liquid ethoxynonafluorobutane (3M
HFE-7200), which has a low refractive index
of 1.28 at room temperature, is well suited
as a cryoimmersion medium for objectives
designed to be used with water immersion at
room temperature. The increase of refractive
index with decreasing temperature results in
a close match for the design of water ob-
jectives at -140°C [2]. HFE-7200, despite its
relatively high vapor pressure, does not boil
until ~70°C and solidifies just below -140°C.
The medium is also inexpensive, nontoxic,
and safe for the environment.
By comparing the point spread function
(PSF) of the objective with HFE-7200 immer-
sion to the PSF at room temperature, -140°C
has been identified as the ideal operating
point. At this temperature, the overall shape
and symmetry of the PSFs are very similar
to those observed at 23°C with the standard
medium (Zeiss W2010). These results were
consistent across the entire field of view.
One of the key advantages of immer-
sion objectives over air objectives is their
light collection efficiency (~NA2). This was
Fig. 3: Bleaching is significantly reduced at 140°C. This is shown here
using yeast cells expressing GFP as an example [1].
confirmed experimentally by measuring an
increase in brightness of 5.7 ± 0.6 times over
a 63/0.75 air objective, which is in good
agreement with the expected scale factor of
~NA4 for widefield fluorescence imaging.
Cryo-fluorescence imaging is attractive in
part due to the fact that many fluorophores
are significantly more stable at low tem-
perature than at room temperature. This is
exemplified by a comparison of the bleach-
ing rates of GFP in yeast during microscopy
at room temperature and at -140°C (fig. 3).
At cryogenic temperature, photobleach-
ing was suppressed by nearly two orders
of magnitude. The possibility of resolving
submicrometer cell structures has been also
elstabished by multicolor cryofluorescence
microscopy using the presented method. Fig-
ure 1 illustrates the attainable image qual-
ity in widefield fluorescence of immunos-
tained U2OS cells at -140°C. Networks of
mitochondria (Tom20 immunolabeled with
Alexa Fluor 594-decorated antibodies) and
vimentin filaments (Alexa Fluor 488) were
well resolved simultaneously.
Conclusions
In conclusion, here a concept has been
demonstrated to enable high-NA cryoflu-
orescence microscopy based on a modified
commercial water immersion objective. To
achieve this, a thermally shielded microen-
vironment has been created around the sam-
ple. A new immersion medium, HFE-7200,
More on lenses for microscopy:
http://bit.ly/WAS-lenses
Light Microscopy
provided accurate index matching at a sam-
ple temperature below the glass transition
of water. In the future, it will be likely that
this method will help to advance the com-
bination of advanced light microscopy with
electron cryomicroscopy to elucidate con-
nections between structure and function at
the subcellular and molecular scale.
Acknowledgements
This work was done in collaboration with
the group of Prof. Stefan Jakobs (MPI for
Biophysical Chemistry and University Med-
ical Center, Göttingen) and with Carl Zeiss
Microscopy (Göttingen). We espacially
thank the groups for optics design, optics
manufacturing, and application support
of Carl Zeiss Microscopy in Göttingen and
the chemistry laboratory of Carl Zeiss in
Oberkochen (Germany) for many invaluable
contributions. The research was carried out
at the Max Planck Institute for Biophysical
Chemistry (MPI-bpc). Funding was provided
by the Max Planck Society and the MPI-bpc.
Contact
Prof. Dr. Thomas P. Burg
TU Darmstadt
Department of Electrical Engineering and
Information Technology
Darmstadt, Germany
tburg@micronano.tu-darmstadt.de
www.etit.tu-darmstadt.de/micronano
References:
http://bit.ly/IM-Burg2
Imaging & Microscopy 1/2020 • 27
Light Microscopy
Fig. 1: Image of the adaptive lens
mounted on a microscope objective.
Smart Objective Compensate Aberrations
Free Form Programmable Lens for Any Type of Microscopes
M
icroscopes rely upon high quali-
ty optical systems to achieve the
optimum resolution. Nonetheless,
in life science microscopy, sample inomo-
geneities seriously degrade images quali-
ty and contrast. Spatial variations of the
refractive index in the sample generate
aberrations. Therefore, even with a per-
fect optical system, the microscope cannot
achieve the resolution given by the dif-
fraction limit. Free form adaptive lenses
placed on the microscope objective can
compensate for these aberrations.
Introduction
Adaptive optics has been widely studied for
improving the resolution and the contrast of
images of biological samples. In the last de-
28 • Imaging & Microscopy 1/2020
Stefano Bonora
cade several techniques have been developed
leading to significant improvements. In the
case of microscopy, with respect to classical
adaptive optics for astronomy, direct wave-
front measurement is not possible because of
the three dimensional nature of the samples
[1]. Therefore different techniques have been
developed. Indirect wavefront optimization is
based on algorithms that search the compen-
sating wavefront exploiting an orthogonal
base of modes. Pupil segmentation is another
interesting method for aberration correction.
Instead of considering the aberrated wave-
front as a series of modes, it cuts the pupil
into segments and the correction occurs on
one segment at a time [2].
Both methods demonstrated to be very
effective and they were implemented on
several imaging platforms [1,2]. Most of the
adaptive optics microscopes have inserted a
deformable mirror or a liquid crystal mod-
ulator as wavefront corrector. Both devices
work in reflection and therefore specific
optical paths have to be implemented to
insert them inside the microscope and to
block diffracted beams in the case of liquid
crystal modulators.
Correction systems using free form pro-
grammable lenses [3] lead to a significant
simplification of the optical setup. In its
most simple implementation the lens can be
mounted directly on the microscope objec-
tive to correct for the optical aberrations.
Material and Methods
The free form programmable lens (or Multi
Actuator adaptive Lens) is a refractive wave-
front modulator composed by two thin glass

Fig. 2: In vivo OCT images of mouse retina before and after aberration correction. Courtesy of
M.Sarunic: Scientific Reports 6, 27620 (2016) doi: org/10.1038/srep27620.
windows bonded to a series of piezoelectric
actuators placed on the periphery of the clear
aperture and filled with transparent liquid.
The actuators can be activated applying inde-
pendent voltages that bend the glass surface
inside the clear aperture. With 18 actuators it
is possible to generate free form aberrations
up to the 4th order of Zernike polynomials
with a response time of less than 1.5 ms.
Interesting results have been obtained
combining the adaptive lens with optimiza-
tion algorithms. As an example, Jian et al [4]
used the adaptive lens to correct for aberra-
tions of in-vivo mouse eye with Optical Coher-
ence Tomography. Images of retina without
the optimization and after the aberration cor-
rection are shown in figure 2. It is evident
that adaptive optics correction is necessary
to achieve the resolution necessary to image
cones photoreceptors. To carry out the correc-
tion, a sequence of aberrations was applied
by the lens while the system was acquir-
ing the retina’s images. The correspondence
of the lens aberrations with the images with
the higher merit values is used to calculate
the lens shape that compensate for the aber-
rations. This simple technique has also been
used for two-photon microscopy [5, 6].
An upgrade of this method consists in the
measurement of the aberration induced by the
sample rather than rely on an optimization
algorithm. To this purpose a movable iris is
faced to the free form programmable lens in
order to be able to select a small portion of the
clear aperture. Similarly to the pupil segmen-
tation, it is possible to sample the wavefront
gradient by moving the iris and measuring the
displacement of the images with respect to the
one acquired with the full aperture. In its more
compact implementation the movable iris is
a spinning disc that rotating moves a small
hole across the clear aperture of the lens. The
advantage of this method is that the correc-
tion is more accurate because the aberration is
Light Microscopy
4-Color Microscopy Laser
Cost-effective laser engine for advanced mi-
croscopy methods: The iChrome CLE integ-
rates four important microscopy colors in
one output fiber, all diode-based, combined with
turnkey operation and automatic alignment.
It provides plentiful output power to obtain
excellent microscopy results.
measured and the correction takes place with
a closed loop feedback control. In addition,
in this method it is not necessary to define a
merit function which is dependent on the type Four colors in one box
of microscope and sample. As an example the 405, 488, 561, 640 nm with > 20/50 mW each
implementation of this technique on a Light
Sheet Microscope is reported. The system [7] Complete-off and crosstalk-free
(fig. 3a) is composed by an illumination path,
where a cylindrical lens is used to create the COOLAC – hands free, self-aligning system
light sheet illumination, and from the imaging
path composed by an objective and the tube
lens. The adaptive lens with the spinning iris
is simply screwed up to the microscope objec- iChrome CLE
tive as shown in figure 3b. To evaluate the
Guaranteed output power (mW) after fiber
performances of the system fluorescent beads
50
suspended in agar solution were imaged. In
its rest position the adaptive lens is flat (the
40
rest position was preliminary calibrated in a
different setup).
Other wavelengths on request
30
Since both a wavefront measurement sys-
tem and a wavefront modulator are available 20
the same procedure used for a normal adaptive
optics system could be exploited. Initially the 10
system measuring the wavefront deformation
given by each single actuator (so called influ- 405 488 561 640
ence function matrix) was calibrated. Using Wavelength [nm]
a linear model to describe the adaptive lens
deformation it is assumed that its shape will
be the product of the influence matrix and the
voltage control given to each single lens actu- Visit us at analytica Munich 2020
ator. Therefore the commands to be sent to the A1 | 301
lens for the wavefront correction will be com-
puted by the inversion of the influence matrix
(so called reconstructor) and by its multiplica-
tion with the wavefront to be corrected.
In order to arrive to a wavefront error
smaller than 0.08 waves rms (Marechal crite-
rion for well corrected optical systems) usu-
ally 2 or 3 iterations are necessary.
Figure 4 shows an example of wavefront
correction. The aberrations in that case come
from some residual of spherical aberration
left from the correction collar and from the
sample holder. www.toptica.com/CLE
Light Microscopy

Fig. 3: a) Layout of the use of the adaptive lens with the spinning iris on a light sheet microscope. b) Picture of the system. It is possible to see the
adaptive lens next to the objective (courtesy of Prof. A.Bassi, Politecnico of Milan).

Conclusions

Here it has been demonstrated that an adap-

tive lens added to the microscope objective
can be used to compensate for aberrations
coming from the sample under examination.
The aberration correction can happen both
using an optimization algorithm or by the
measurement of the aberrations. In the lat-
ter case a spinning iris was inserted in the
adaptive lens box creating a full adaptive
optics system that can be implemented on
any type of microscope platform as simple
add-on to the objective.
This combination of adaptive lens and
measurement system creates a smart objec-
tive that can be used to image the samples at
the maximum possible resolution.

Acknowledgments

We thank our collaborators from CNR-IFN

Dr. Tommaso Furieri and Giulio Bursi. We
also thank Prof A. Bassi of Politecnico of
Milan where the ligh sheet experiment was
carried out.

Fig. 4: Top: Images of a fluorescente bead and its wavefront measurement (represented as an
interferogram at 633nm). Bottom: Image and interferogram after the aberration correction.
Contact
Dr. Stefano Bonora
CNR-Institute for Photonics and Nanotechnology
Padova, Italy More on spherical abberation cor- References:
stefano.bonora@pd.ifn.cnr.it rection: http://bit.ly/ibiology-SAC http://bit.ly/IM-Bonora2
www.pd.ifn.cnr.it

30 • Imaging & Microscopy 1/2020

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Image Processing

UCSF Chimera
My Favorite Image Analysis Tool, by Neubias Members
Lucas Schütz

I
n toto imaging became popular with
the development of light-sheet micros-
copy. While preparation of the sample
and mounting it are already difficult, the
main challenge comes after imaging: vi-
sualization. Using a stand-alone software
package UCSF Chimera you can look at
tiff stacks in 3D, or even its time series,
providing you with a good visual overview
of your 3D sample. It also offers several
opportunities for advanced visualization
and movie recording. UCSF Chimera is one
of the must have tools for visualization of
large volumetric microscopy data.
During embryonic development of an or-
ganism, shape changes with extensive re-
modeling processes take place [1]. For ex-
ample, in the process of gastrulation, the 3
germ layers ectoderm, mesoderm and endo-
derm are formed [1]. With the development
of light-sheet microscopy, scientists finally
acquired a tool in their hands to image the
whole organism in a sufficient temporal
and spatial resolution; a technique that be-
came known as in toto imaging [2-4]. How-
ever the biggest challenge comes usually
after imaging when terabytes of imaging
data from different viewing angles need
to be registered and fused into one sin-
gle image creating an isotropic 3d (+time)
volume5. Moreover, only having the fused
image does not help, it needs to be visual-
ized. The quick and dirty approach is to do
a maximum intensity projection over the
entire volume or half of it. This approach is
however very limited as one cannot change
the viewing angle. The better solution is
a 3D-viewer like UCSF Chimera [6] which
enables you to enjoy a full in toto view of
your data.
The Advantages of UCSF Chimera
While UCSF Chimera is well known among
scientists working in structural biology [6],
it seems rather to play the role of an under-
dog in the microscopy and image analysis
community. Considering its advantages as
a quite powerful 3D viewer, I would like to
recommend it for more use in the imaging
community.
Simple to Use
with a Good Rendering Quality
Its primary advantage is its simple usage.
You can just open any grayscale 8-bit or
16-bit tiff stack. Once the image is opened
you can adjust the visualization settings
in the Volume Viewer window that opens
with your 3d image. There, you can set the
threshold level for rendering and choose
from different visualization options like
surface, mesh or solid. The rendering qual-
ity is much better compared to other more
popular solutions like the Fiji [7] 3D Viewer
[8] or Vaa3D [9]. While high-quality ren-
dering usually requires decent hardware re-
sources, UCSF Chimera is quite humble and
runs without problems on notebooks with
onboard graphic processing units. Memory
usage is also on a good level. These features
might make it sound like just another 3D
viewer with a better quality, but there are
more.
3D Mouse Support
The main purpose of a 3D visualization is not
only for the use in a presentation to attract
the attention of readers and audiences. Es-
pecially when working with large volumet-
ric image data containing regions of interest
on every side of the volume, we sometimes
need a 3D viewer to visually inspect your
image data from multiple sides. To do this
inspection most 3D viewers utilize the clas-
sical controls using the mouse, sometimes
also the arrow keys and some digital tun-
able knobs. The user experience of adjusting
the 3D view with these apparatus is usually
rather painful. A more modern and better
solution is a small Joystick with 6 axes that
one can control individually, often referred
to as 3D mouse or SpaceMouse. Where-
as this device is very common in the field
of engineering for computer aided design
(CAD), it is almost unknown in biological
imaging domain, which might be due to the
fact that it is rarely supported. UCSF Chime-
ra does support this 3D mouse enabling you
to move your object in the 3-dimensional
space with a precision you probably never
experienced before. Even moving in and out
side of your object is possible.
Lucas Schütz
University of Heidelberg (B.Sc. and M.Sc.),
Centre for Organismal Studies, University
of Heidelberg (PhD), He now works as a
Postdoc in the Advanced Light Micros-
copy Facility (ALMF) of the European
Molecular Biology Laboratory (EMBL)
(Heidelberg), also associated with the
Translational Lung Research Center (TLRC)
at the University Hospital Heidelberg.
Time Series Support
There is more. Especially in developmental
biology like Drosophila development, we
need to inspect the development of the or-
ganism over time using our own eyes. The
ability to visualize 3D time-series data is
often absent in 3D viewers, either because
it is simply not implemented or because of
huge consumption of memory leading to
software crashes. In UCSF Chimera the Vol-
ume Series function provides the capability
to render a time series of 3D data. A great
advantage of this capability is that it allows
us to limit the rendering only to time points
we want, which can become quite handy on
notebooks when you want to save memo-
ry and therefore only open every second or
third image of a time series. Moreover, once
the time series is opened, we can set how
much memory is allocated to UCSF Chimera
and how many renderings it should store

32 • Imaging & Microscopy 1/2020


Fig. 1: View of a Drosophila embryo in Chimera.
Fig. 2: Red-cyan stereo view of a Drosophila embryo.
in that allocated memory. Once these set-
tings are configured, we can click on Play
and UCSF Chimera will start opening and
rendering the images, as well as caching
the renderings if there is sufficient space
in the allocated memory. When all render-
ings are cashed, the playback speed of the
time series can become very fast, making
the option Maximum playback speed very
handy. While the time series is playing, one
can interactively change the viewing angle
and look at the moving 3D object from var-
ious sides. Although UCSF Chimera is very
resource efficient, the visualization of a 3D
time series is definitely recommended to
be done on a workstation rather than on a
notebook, just like it is true for every 3D
viewer.
Now you might wonder, how large a data
set can be that one can actually visualize at
one time. From personal experience I can
say that once you load a 100 time points
with around 1 GB for each time point, so a
100 GB as time series, you are at the limit.
Even with a workstation powerful enough
for this task, the software will become quite
slow and the likelyhood of crashes increases.
Nevertheless, UCSF Chimera is the only open
source 3D viewer where I managed to visu-
alize this huge amount of data. As a general
advice, a file size of 200-400 MB per time-
point is usually recommendable.
Image Processing
THE FUTURE
DEPENDS ON
OPTICS
TECHSPEC® Ultra
Compact Objective
Assemblies
NEW
Optics and Brain
Diagnostics
Advancements
Millions of people are affected by
ailments of the brain, but advances
in optics and medical diagnostics are
3D Visualization Options enabling the non-invasive detection
and treatment of some of these con-
The images rendered in any 3D viewer in- ditions. Innovative technologies in-
cluding UCSF Chimera, are not really in 3D, cluding fluorescence microscopy and
but rather a 2D projection on the screen, CLARITY are dependent on optical
and various techniques are used to mimic a components such as infinity correct-
3-dimensional view. An option to get closer ed, apochromatic microscope objec-
to a real 3D image is to provide the left and tives and optical filters.
the right eye with different images, imitating
Find out more at
the real-world view. UCSF Chimera provides
several options to achieve this functionality www.edmundoptics.eu/
under Tools > Viewing Controls> Camera: the advanced-diagnostics
classical red-cyan stereo view; the wall-eye
stereo view, which can be used with virtual
reality goggles; the sequential stereo view,
which can be used with shutter goggles. The
use of these 3D viewing controls often occurs
at its very beginning for analysis and the vi-
sual inspection of samples in 3D helps us to
get a better understanding.
Recording and Animation Functions UK: +44 (0) 1904 788600
GERMANY: +49 (0) 6131 5700-0
While visualizing 3D data is nice and may- FRANCE: +33 (0) 820 207 555
be helpful to get a better impression and for sales@edmundoptics.eu
detailed inspection of large sample, it is also
important to be able to present these visual-
izations for talks and publications. For this
purpose, a decent 3D viewer needs also to
provide functionalities for recording a movie
including animations like rotations around
Imaging & Microscopy 1/2020 • 33
Image Processing
Fig. 3: Most important control panels.
the x.y or z axes. In UCSF Chimera, there
are the Movie recorder and the Animation
options. While the Movie recorder is rather
a simple utility for recording what the user
is currently doing, basically a screen re-
corder, the Animation option provides more
advanced functionalities. Basic animation
functionalities every 3D viewer should pro-
vide are of course there, such as rocking and
rolling of the object around a given axis.
However, it is not limited to such basic func-
tions, the user can define arbitrary frames,
including not only rotation and size changes,
but also cutting of the volume, arrange them
in a timeline and UCSF Chimera will render
the intermediate frames, a feature more com-
mon in 3D rendering software packages like
Blender. With a simple click on record UCSF
Chimera uses the frames defined by the user
and renders the movie adding also the inter-
mediate steps; afterwards the movie can be
stored in a common file format
Advanced Options:
Exporting Transformation Matrix
In light-sheet microscopy it is common to
acquire multiple views from different angles,
Bioimage Data Analysis:
http://bit.ly/BIDA-Miura
34 • Imaging & Microscopy 1/2020
which are then registered and fused into one
single volume image. During this process,
it sometimes happens that the registration,
which usually consists of 2 steps, a broad
and a fine alignment, fails. To rescue such
incomplete registration, one way is to feed
an appropriate transformation matrix for
correcting the alignment into the registration
software. Conveniently, this transformation
matrix can be generated in UCSF Chimera.
We can load multiple volumes at the same
time and align them manually by carefully
adjusting their orientation in the 3D space
(the Model Panel allows for individual con-
trol of all opened volumes). Once the volumes
are successfully aligned, the transformation
matrix for that aligning can be generated
with the function matrixget. This transforma-
tion matrix then can be fed into registration
software for the correct processing.
The Future: UCSF ChimeraX
While I would still consider UCSF Chime-
ra as one of the best available 3D viewers
even in comparison to available commercial
solutions, this software package is already
quite old. The good thing is that its succes-
Neubias:
http://bit.ly/Neubias
sor, called ChimeraX [10], is already under
development, and although with limited
features its first versions are already avail-
able for download. The rendering quality is
even better, and maybe improving with new
versions that are published regularly.
Conclusion
After multiple years of almost daily use of
UCSF Chimera, I consider it as one of the
best available 3D viewers. Being highly ver-
satile, resource efficient but also simple in
use it remains an unsolved question to me
why it is not well known among imaging
scientists. Its most unique feature is prob-
ably the support of a 3D mouse, enabling
a precise inspection of all sides of the data,
that is missing often even in commercial
software packages.
Contact
Dr. Lucas Schütz
European Molecular Biology Laboratory (EMBL)
Heidelberg, Germany
lucas.schuetz@membl.de
References:
http://bit.ly/IM-Schuetz
 Image Processing

Managing Big Imaging Data

Cell Imaging and Processing with Lattice Light-Sheet Microscopy
Nicholas D. Condon1,2, Adam A. Wall2, Jennifer L. Stow2

M
icroscopes such as the lattice
light-sheet microscope (LLS) can
now record high-resolution, 3D
live cell behaviors at high speed and over
long periods and has been used to study
ruffling in activated immune cells. While
the LLS provides unprecedented insights
into cell behavior, it can also create huge
image data files. New software and com-
puting resources are needed to process,
manage and analyze this big image data.
Here is described a set of scripts ‘LLS Big
Data Tools’ that facilitate and speed up big
data file transfers and processing, using
the open source platform Fiji.
Imaging Dynamic Cellular Behavior
Macrophages are immune cells that con-
stantly survey their tissue environments by
ruffling. Highly dynamic membrane ruffles
on the cell surface are in constant motion,
scooping up and ingesting fluid to screen
for any sign of infecting pathogens. Ruf-
fling and accompanying macropinocytosis
(for uptake or endocytosis) are linked with
recycling and exocytic processes to carry
out a complete turnover of the macrophage
cell surface every ~ 30 mins [1]. Membrane
ruffles and other cell surface projections are
erected on a scaffold of concentrated F-ac-
tin, which forms the basis for the bright la-
belling of cell surfaces.
The ability to image very rapid, struc-
turally complex events on cell surfaces has
recently advanced through the development
of new imaging modalities such as lattice
light-sheet microscopy (LLS) [2]. High-speed
multi-dimensional (3D time-lapse) imaging
by LLS allows for near real-time imaging of
biological processes, which are often over-
looked when imaging at slower speeds, or
only in 2-dimensions. Labelled ruffles can
now be optimally resolved and tracked over
time using LLS (fig. 1), which has revealed
novel mechanisms for ruffling and macropi-
nocytosis [3,4]. LLS has also been used to
image other cell processes such as, neuro-
nal activation [5], endosomal biogenesis [6],
mitochondrial membrane dynamics [7] and
T-Cell activation [2]. However, in the process
of observing such events in full 3D over long
duration time-lapse acquisitions, the image
data-sets become very large and processing
steps to analysis and visualization become
very much more complex.
LLS uses multiple, closely-spaced inter-
fering Bessel beams to create a thin laser
light-sheet which the sample is moved
through repetitively to record a high-res-
olution, 3D volume in less than a second
[2]. As with other Single Plane Illumination

Imaging & Microscopy 1/2020 • 35

Image Processing
Fig. 1: Example image of GFP-LifeAct labelled macrophages viewed by LLS. A) A single time-point 3D view of of 5 cells. B) Field of view from (A)
Z-projected using a Maximum Intensity Projection. Scale = 10 μm.
Microscopy (SPIM) techniques, only the
plane being imaged is illuminated, casting
a lower photon count over the entire sample
throughout image acquisition [8]. Thus, with
minimal photo-bleaching or photo-toxicity,
living cells can be imaged for many hours,
which creates the large data-sets (hundreds
of gigabytes and even terabytes per session)
characteristic of this microscopy. Image data
processing, visualizing and quantifying from
these images is time-consuming and also
requires considerable automation.
Live cell imaging generated by LLS and
other forms of microscopy now increasingly
operates in the realm of big data, requir-
ing high performance computing on a
whole new scale and new, customized soft-
ware and hardware to support data han-
dling, storage and analysis in microscopy
facilities and institutions around the world.
With files in the order of hundreds of giga-
bytes becoming the new norm, custom tools
and approaches are required to handle these
files. One example of such custom software
is LLSspy, a python library to facilitate file
handling, manipulation and processing for
lattice light sheet data (Talley Lambert, Har-
vard Medical School [https://github.com/
tlambert03/LLSpy]). While home-built LLS
microscopes run Lattice Scope (already pro-
viding multiple .tif stack outputs), commer-
cial systems (3i & Zeiss) provide files in their
proprietary containerized formats, which
36 • Imaging & Microscopy 1/2020
formed the impetus for this project. It is also
anticipated that these tools will be useful for
multiple microscopes where a single, large
containerized file is output, as long as it is
readable by Bio-Formats in Fiji [9,10]. Here
is reported a new ‘LLS Big Data tools’ col-
lection of Fiji-based scripts for batch pro-
cessing big containerised image data. The
first script (Directory_Deskew.ijm) separates
large data files into smaller ones, allow-
ing for faster processing of relevant data
on university scale GPU-based parallelized
high-performance compute (HPC) clusters,
greatly reducing the workload and avoiding
time-consuming manual intermediate steps
otherwise required to process LLS data files.
It also has been developed a ‘Live_Deskew.
ijm’ script which works interactively with
already open data-sets to export multiple
single image stacks. The second script (Direc-
tory_Z-projection.ijm) batch flattens multi-
ple image stacks by Z-projection and stitches
them together into a avi-file for quick data
surveying.
Creating and Processing
Big Microscopy Data
Standard Image Acquisition
Cells stably expressing GFP-LifeAct [4] (or
labelled with suitably bright fluorophores/
dyes) are seeded at low density and imaged
using a 3i Lattice Light-Sheet microscope
V2, (or Andor Dragonfly 500 Spinning Disk
Confocal or Zeiss LSM880 confocal) un-
der Nyquist sampling conditions running
3i Slidebook (or Andor Fusion/Zeiss Zen
Black). RAW data is output in propriety file
formats as a single containerized, Bio-For-
mats readable file. Large files are chopped
up and single frame Z-stacks are then pro-
cessed with Microvolution for both GPU-ac-
celerated deskewing and deconvolution us-
ing multiple cycles of iterative deconvolu-
tion on a GPU-HPC cluster (Deconvolution
could also be performed using alternative
software packages). Large directories of pro-
cessed image data are then batch Z-project-
ed and stitched into a avi-file for surveying.
Selected 3D frames are then recombined into
a 4D time-series in FIJI (or commercial soft-
ware) prior to analysis and visualization.
Automating Processing
for Large Data
With a standard 15 min, 4D acquisition re-
sulting in a single file in excess of 75 GB,
data migration, management and processing
steps must be designed to make optimal use
of computing resources. To realize the full
potential of multiple parallelized GPUs and
compute nodes of the HPC system, multiple
smaller files will process faster than a single,

Fig. 2: Image Destacking Script overview. A) Launch screen. B) Directory Warning. C) Example input directory. D) Script Parameters. E) Example
output directories including sub-directories with output images. F) Example log file.
larger file. Therefore, the first
step in the processing pipeline
is to reduce the dimensionality
of the data into multiple, single
time-point files consisting of
single 3D Z-stacks (using the
above example, this results in
500, 165MB ‘.tif’ stacks) each.
Two custom ImageJ macros
have been developed to either
process a directory of multiple
acquisition files, or to process a
4D image open in Fiji (fig. 2, Di-
rectory/Live_deskew.ijm).
Using a custom Microvolu-
tion execution script, and a HPC
scheduling script (e.g. SLURM),
directories of dimension-re-
duced (single Z-stacks) files
are both deskewed and decon-
volved on the university’s GPU
HPC cluster and exported out as
a subdirectory of deconvolved
single Z-stack files.
Prior to recombining these
individual processed files post
deskew and deconvolution
(~350MB each/~170GB total)
a prudent step is to survey the
data quickly by flattening indi-
vidual frames (by Z-projection)
and re-combining multiple time
points into a single file time-se-
Image Processing
ries. Using another ImageJ
macro, output directories can be
quickly flattened and combined
into a single file (fig. 3, Direc-
tory_Z-projection.ijm) for quick
pE-4000
data checking.
Using server grade hardware
Flexible
(Dell R740 server with 1TB of Microscopy
RAM and 3x Nvidia V100 Volta
GPUs) running bare metal Win-
Illumination
dows Server 2016 (a familiar
environment for researchers)
which can be accessed remotely,
larger re-combined, multi-di-
mensional data-sets can then
be analyzed using commercial
applications (Bitplane Imaris,
or Arivis Vision 4D) or open-
source software including Fiji,
for tracking, segmentation and
co-localization profiling.
Conclusions As a powerful, efficient and compact
Illumination System, the pE-4000 sets the
The development of novel im-
aging modalities including the standard for fluorescence.
LLS has allowed for the inves-
tigation and visualization of t: +44 (0)1264 323040 (Worldwide)
1-800-877-0128 (USA/Canada) Simply Better Control
novel cellular events in unpar- e: info@CoolLED.com
alleled 3D detail and resolution.
2002002
Such microscopy data comes at
FEB 20
www.CoolLED.com
a cost of massive data-sets and
Imaging & Microscopy 1/2020 • 37
Image Processing
Fig. 3: Batch Z-Projection and concatenation macro steps. A) Launch screen. B) Directory Warning. C) Example input directory. D) Script Parame-
ters. E) Avi parameter window (displayed if selected). F) Example log file. G) Example output directory including concatenated avi-file.
large files sizes, thrusting researchers into
the realms of big data. Custom tools and de-
velopment of robust pipelines such as the
LLS Big Data Tools are necessary to auto-
mate the processing and handling of these
big data-sets that greatly improves pro-
cessing time and access to GPU accelerated
computing.
Note scripts described here are available
from: https://github.com/NickCondon /LLS_
Big_Data_Tools
Acknowledgements
Microscopy was performed at the Institute
for Molecular Bioscience (IMB) Microscopy
Facility incorporating facilities funded by
the Australian Cancer Research Foundation
More on data analysis:
http://bit.ly/WAS-Data-Analysis
38 • Imaging & Microscopy 1/2020
(ACRF) with computing resources devel-
oped and supported by The University of
Queensland’s Research Computing Centre.
Read more about Fiji:
http://bit.ly/WAS-FIJI
Research grant funding NHMRC (1098710
and 1003021) and ARC (AAW LE170100206
and DP180101910 to J.L. Stow).
Affiliations
1Institute for Molecular Biosciences (IMB)
Microscopy, The University of Queensland,
Brisbane, Australia
2IMB Centre for Inflammation Disease Re-
search, The University of Queensland, Bris-
bane, Australia
Contact
Dr. Nicholas Condon
Institute for Molecular Biosciences (IMB) Microscopy
The University of Queensland
Brisbane, Australia 
n.condon@uq.edu.au
References:
http://bit.ly/IM-Condon2
 Scanning Probe Microscopy

Scanning-Probe-Assisted Nanowire Circuitry

A Novel Technique for the Fabrication of Metal Nanoelectrodes
Pablo Ares1, Miriam Moreno-Moreno2, Félix Zamora3,4, Julio Gómez-Herrero2,4

A
new procedure to fabricate highly
conducting nanocircuits based on
the manipulation and cold weld-
ing of gold nanowires using atomic force
microscopy has been introduced. The
technique allows the characterization of
electrical transport properties at the na-
noscale through clean and easily reconfig-
urable nanoelectrodes. It does not require
the use of polymers, chemicals, vacuum
or high temperatures, thus it can com-
plement and/or be an alternative to other
well-established techniques.
Introduction
The continuous miniaturization of devices
or the use of new nanomaterials are some
of the current challenges in the fabrica-
tion of electrical nanocircuits, which im-
ply the need for smaller and smaller metal
electrodes. There are several techniques to
fabricate micro- and nanoelectrodes, in-
cluding optical, X-ray or electron beam
lithographies [1], the latter being one of
the most commonly used. However, they
present some drawbacks, such as the need
of expensive equipment, contamination left
by the polymers needed in the fabrication
process or the need of vacuum and chem-
icals that might damage delicate samples.
Recently a new approach to fabricate metal
nanoelectrodes based on the adsorption and
subsequent manipulation of gold nanowires
with an atomic force microscopy (AFM) tip
has been presented. This novel technique,
named SPANC after Scanning-Probe-As-
sisted Nanowire Circuitry, allows the fabri-
cation of highly conducting, clean (it does
not require polymers, just a water based

Fig. 1: Artistic image as a result of the combination of optical dark field microscopy and AFM images. It shows gold nanowires randomly dis-
persed on a SiO2 substrate and a central nanoelectrode about 150 µm long assembled and imaged by AFM, connecting a gold microelectrode (bot-
tom left) to several few layer antimonene flakes (inside squares). The inset is an AFM image of a two-terminal device connecting a graphene na-
noribbon with nanoelectrodes fabricated using SPANC.

Imaging & Microscopy 1/2020 • 39

 Scanning Probe Microscopy
solution contaning gold nanowires), gen-
tle (no need of vacuum or chemicals) and
cost-effective (any conventional AFM with
standard tips can be used) nanoelectrodes.
Morevover, the circuits made with SPANC
are reconfigurable, allowing the electrical
characterization of multiple nanoobjects in
a given sample or different areas in a same
nanoobject [2].
Material & Methods
SPANC fabrication process comprises the
following steps, depicted in figure 2: a)
Deposition of the nanoobject(s) of interest
on an insulating substrate (typically SiO2
or mica). b) Fabrication of microelectrodes
by any conventional technique. These steps
are interchangeable, depending on the re-
sistance of the nanoobject(s) to the condi-
tions needed for these microelectrodes. c)
Gold nanowires adsorption. d) Connection
of the microelectrodes to the chip carrier. e)
Fabrication of the nanoelectrodes by AFM
nanomanipulation.
Both commercial (Nanopartz) and home-
synthesized gold nanowires obtained follow-
ing reported protocols have been used [3-4],
with typical diameter ~50 nm and length ~5
µm. Upon deposition by drop casting and
subsequent blow drying, it has been checked
with dark field optical microscopy the den-
sity of nanowires on the substrate, repeating
the deposition if necessary. For the nanow-
ires manipulation standard silicon cantile-
vers of ~75 kHz resonance frequency and 3
Nm-1 spring constant have been used. First
the area of interest in amplitude modulation
mode has been imaged, to avoid unwanted
nanowire manipulation. Then, the tip has
been brought into contact along predefined
trajectories, moving the gold nanowires
[5-6] to assemble the nanoelectrodes that
will connect the nanoobject(s) to the micro-
electrodes. After each of these nanomanip-
ulations, the area has been imaged again
in amplitude modulation mode to see the
new position of the nano­wires. The high sur-
face-area-to-volume ratio of the nanowires
originates a high surface energy resulting
in a natural tendency of the nanowires to
join forming a more energy favorable shape.
Thus, when two nanowires are brought into
contact, they spontaneously cold weld [7].
A nanoelectrode composed of almost one
hundred nanowires has been characterized,
finding that the nanoelectrodes obtained
with SPANC present near-bulk gold con-
ductivity, with just an extra resistance of
about 9 Ω in each of the junctions compat-
ible with a single grain boundary, excellent
for nanocircuitry fabrication [2].
40 • Imaging & Microscopy 1/2020
Fig. 2: SPANC procedure steps for a two-electrode configuration. a) Nanoobject preparation. b)
Microelectrode fabrication. c) Gold nanowires deposition. d) Connection to chip carrier. e) Gold
nanowires manipulation. Adapted with permission from M. Moreno-Moreno & P. Ares et al.
AFM Manipulation of Gold Nanowires To Build Electrical Circuits. Nano Lett. 19, 5459-5468
(2019). Copyright (2019) American Chemical Society.
Results trodes fabricated with SPANC, demonstrat-
ing the possibility of fabricating complex
The feasibility, robustness and versatility circuitry. This four-terminal setup is com-
of SPANC have been demonstrated by the monly used in transport measurements of
fabrication of devices in different configu- highly conducting objects as it allows the
rations for the electrical characterization of experimental measurement of their resis-
several nanoobjects [2]. These included: i) tance getting rid of the contact resistances.
Devices with one nanoelectrode fabricated This approach has been applied successful-
with SPANC and a conductive AFM tip as a ly to a few layer graphene flake. Figure 3a
second mobile electrode. This configuration shows an AFM image before nanoelecrode
has been used first as a proof of concept of fabrication, figure 3b after the four nano-
the technique, studying the transport prop- electrodes are connecting the graphene flake
erties of few layer graphene, obtaining a and figure 3c the current vs. voltage curves
sheet resistance of 670 ± 60 Ω/sq, in good acquired in both two and four-terminal con-
agreement with literature values reported figurations. A step further has been gone,
through different electrical contact schemes disconnecting one of the nanoelectrodes
[8-10]. This same scheme has been used to and using a conductive AFM tip as a fourth
probe the transport properties of thin few mobile electrode, thus obtaining a resistance
layer antimonene, finding a sheet resistance vs. distance plot in the absence of contact
of 1300 ± 400 Ω/sq. ii) Devices with two na- resistance (fig. 3d). iv) Finally, two devices
noelectrodes fabricated with SPANC. Under have been fabricated which go beyond stan-
this configuration the electrical transport dard spatial resolution and illustrate further
has been studied in a few layer graphene na- the possibilities of SPANC. To this end, on
noribbon and in a carbon nanotube. These the one hand it was possible to fabricate a
results gave evidence of the robustness of device connecting a ~10 nm gold nanopar-
the SPANC nanorcuits, allowing the electri- ticle between two SPANC nanoelectrodes.
cal characterization of these devices under On the other hand, a preliminary experi-
high vacuum (~10-6 mbar) and at variable ment introducing SPANC in the molecular
temperature (96-373 K), and its versatility, electronics realm has been presented. Two
probing the resistance vs. distance in the SPANC nanoelectrodes as close as possible
carbon nanotube by reconfiguring the po- have been fabricated, but with no electri-
sition of one of the nanoelectrodes relative cal connection. Then 1,4-benzenedithiol
to the other. iii) Devices with four nanoelec- (BDT) molecules have been deposited on the
 Scanning Probe Microscopy

Fig. 3: Few layer graphene four-terminal device fabrication and characterization. a) SiO2 substrate with the few layer graphene sample, microelec-
trodes and randomly deposited gold nanowires. b) Same as in a) after the manipulation of the gold nanowires to fabricate the four nanoelectrodes. c)
Current vs. voltage curves from the device in b) acquired in both two and four-terminal configurations. The inset is a zoom in of the same plot at a
low voltage. d) Resistance vs. distance plot obtained by replacing one of the inner electrodes by a metal-coated AFM tip used as a mobile electrode.
The inset is an AFM image indicating the nanowire terminals used for the measurement and the positions where IV curves were acquired. Adapted
with permission from M. Moreno-Moreno & P. Ares et al. AFM Manipulation of Gold Nanowires To Build Electrical Circuits. Nano Lett. 19, 5459-
5468 (2019). Copyright (2019) American Chemical Society.

device, so they filled the gap between the
nanoelectrodes. Electrical transport mea-
surements in this configuration confirmed
the molecular nature of the measured cur-
rent, with a conductance of about 0.06G0,
in good agreement with reported values [11-
13], and allowed stable BDT conductance
measurement at room temperature for more
than one hour. Typical molecular electronics
setups, such as scanning tunneling micros-
copy or break junctions, allow the molecules
the three spatial degrees of freedom, thus
measurements longer than 1 minute at room
temperature are a challenge. The inherent
two dimensional configuration of the device
made with SPANC gives the system the ob-
served outstanding time stability.
Conclusions
A new versatile and cost-effective tech-
nique for nanoscale electrical connectivity,
named Scanning-Probe-Assisted Nanowire
Circuitry, SPANC [2] has been developed.
It is based on the AFM manipulation and
cold welding of gold nanowires to fabri- Affiliations
1Department
cate near-bulk gold conductivity nano- of Physics and Astronomy &
electrodes. It does not require the use of National Graphene Institute, University of
polymers, chemicals, vacuum or high tem- Manchester, Manchester, UK
2Departamento de Física de la Materia Con-
peratures, allowing clean and reconfigu-
rable nanoelectrodes which are compatible densada, Universidad Autónoma de Madrid,
with delicate materials. It allows fabrication Madrid, Spain
3 Departamento de Química Inorgánica,
of circuits with different topologies which
are stable in vacuum, at variable tempera- Universidad Autónoma de Madrid, Madrid,
ture and on different substrates. It allows Spain
4Condensed Matter Physics Center (IFIMAC),
the fabrication of devices for the electrical
characterization of nanoobjects as small as Universidad Autónoma de Madrid, Madrid,
~10 nm, although future combination with Spain
shorter and thinner nanostructures such
as nanorods and nanoparticles would al- Contact
low going down to the nm regime. On top Dr. Pablo Ares
of that, it has shown a great potential for Department of Physics and Astronomy & National
molecular electronics devices. In summary, Graphene Institute
SPANC is now a reality allowing endless University of Manchester
possibilities when it comes to fabricating Manchester, UK
circuits at the nanoscale. pablo.ares@manchester.ac.uk
More on Material Sciences: References:
http://bit.ly/WAS-MS http://bit.ly/IM-Ares3

Imaging & Microscopy 1/2020 • 41

Electron Microscopy
TEM Training in Core Facilities
Effect of Duration and Number of Trainees on Improvement
S
taff responsible for TEM training at
core facilities were surveyed to iden-
tify critical factors relating to train-
ing effectiveness. Increasing the ratio of
trainees to trainers was associated with
shorter training durations but not with the
degree of improvement in trainees. Train-
ing duration varied between facilities, but
no association was found between training
duration and improvement.
Introduction
There is a trend towards complex technical
services being provided through core facil-
ities [1,2]. Expensive equipment can ser-
vice multiple different clients, sometimes
in entirely different fields of research in
a more cost-effective manner. If multiple
instruments are in a single multi-user fa-
cility, economies of scale are possible with
42 • Imaging & Microscopy 1/2020
James D. Riches
maintenance, support and training. User
training is a key activity in many elec-
tron microscopy core-facilities. Users are
trained by skilled staff in the operation of
the instruments so they can collect and in-
terpret their data by themselves.
User training in core-facilities should
ensure that users can operate the instru-
ments safely and competently, and col-
lect high-quality data that allows them to
answer their research questions. It is also
desirable that this training be carried out
in the minimum amount of time possible,
so that resources such as staff time and
instrument time are free for other activities.
In order to better understand how train-
ing in TEM is delivered at multi-user facil-
ities internationally, a survey was sent
to staff responsible for training in TEM.
This survey sought information about the
resources present at the core facilities (e.g.
number of staff/columns, etc), the manner
© galitskaya - Adobe-Stock.com
of training provided, and the outcomes of
the training (e.g. degree of improvement,
user satisfaction, etc). In this paper, atten-
tion is focused on the number of trainees
and trainers, the duration of training, and
the degree of improvement caused by the
training.
Three hypotheses were generated before
analyzing the survey data. The first (H1)
was that the average time required for
training before being allowed to use the
instrument independently would be lower
(negatively correlated) with higher ratios of
trainees to staff. The second (H2) was that
the degree of improvement in skill level of
trainees would be lower (negatively cor-
related) with higher ratios of trainees to
staff. The third hypothesis (H3) was that
the degree of improvement in skill level of
trainees would be higher (positively cor-
related) with longer average times required
for training.

Fig. 1: The average training time required for independent operation of routine TEM plotted against
the ratio of the number of students trained annually to the number of training staff at a facility.
Materials and Methods
Requests to participate were sent to the
staff member primarily responsible for
training at 329 facilities from a range of
different countries and 103 of these re-
sponded to the survey (31.3% response
rate). Not all participants answered every
question, so response rates between ques-
tions varied. This study was approved by
the QUT Human Research Ethics Commit-
tee (approval number 1800000423). The
results for the full survey can be obtained
from the author.
The skill level of the trainees was que-
ried with the following questions. “In your
assessment, what is the average skill level
of your clients immediately before they
start training (after they complete training)
in the operation of Routine TEMs at your
facility? In the scale below (1-7), a Novice
(1) is a person with no experience or knowl-
edge of TEM, and an Expert (7) is a person
with extensive practical experience and an
excellent knowledge of TEM”. The training
time required was queried with the follow-
ing question. “On average, how many hours
of training are normally required before your
clients are allowed to operate a Routine TEM
independently?”
Results
For each respondent, the ratio of the number
of trainees trained annually to the number
of staff available for training was deter-
mined. The degree of improvement in the
skill level of the trainees was determined by
subtracting the skill level before training,
from the skill level after training.
Electron Microscopy
Label-free
Microscopy
To test the first hypothesis, a scatter-
plot of the data was generated, as shown
in figure 1, and a Kendall’s tau-b correla-
tion was run to determine the relationship
between the average training time required
for independent operation and the ratio of
trainees to trainers, for the 54 participants.
In this case there was a moderate, negative Image courtesy of
correlation, with training duration required Park, Joo Hyun and
Lee, Sang-Wong
for independent operation decreasing with
increasing trainee to trainer ratios, and this
was found to be statistically significant (τb
= -0.346, p=0.0005).
The second hypothesis was tested in a
similar fashion (see fig. 2), with a Kend-
all’s tau-b correlation run on the level of
improvement following training and the
ratio of trainees to trainers, amongst the With tunable short-pulse
56 participants. There was a weak, negative laser sources for
association here, with skill levels decreasing
with increasing trainee to trainer ratios, but
this was not found to be statistically signif-
Three-photon imaging
icant (τb = -0.147, p=0.159). THG imaging
To test the third and final hypothesis (fig.
3), a Kendall’s tau-b correlation was run on CARS & SRS
the degree of improvement and the average
length of time required for training, amongst
the 57 participants. In this case essentially no
association was found (τb = -0.003, p=0.98).
Discussion
The significant (p<0.05) correlation found
between training time required and the
ratio of students to staff at a core facility
(H1) was expected. As the number of stu-
dents increases, the amount of staff time
that can be devoted to a single trainee will
tend to decrease and there can be pressure
to accelerate the training. The second and
www.ape-berlin.de
Electron Microscopy
Fig. 2: The degree of improvement plotted against the ratio of the number of students trained
annually to the number of training staff at a facility.
Fig. 3: The degree of improvement plotted against the average training time required for inde-
pendent operation of routine TEM.
third hypotheses, on the other hand, were
not supported by the data and this was quite
unexpected.
Learning theory from other fields [3,4]
consistently shows that trainees improve
with increasing training time, rapidly at
first and then with decreasing speed, even-
tually reaching a plateau. In this study, there
was essentially no increase in the degree of
improvement from quite short training times
(<5 hours) and those that were much longer
(>20 hours).
One explanation for this is that the skill
level of trainees may have already plateaued
after only a few hours, but this seems very
44 • Imaging & Microscopy 1/2020
unlikely given the complexity of TEM opera-
tion. Alternatively, it is possible that facilities
differ markedly in the efficiency with which
they provide training, and that some facili-
ties require far more time to train people to
the same standard. Finally, in the absence
of an objective test of the skill level of the
trainees, the opinion of the training staff has
More information:
http://bit.ly/WAS-TEM
been sought, and this will be, to some degree,
subjective. Research in other fields [5] has
found self-assessment can be prone to bias
which leads to compression of results. The
subjective nature of the rating system used
may be causing clustering of results for the
skill levels of trainees after training – with
those training for longer using a higher stan-
dard of ‘competence’.
Conclusion
This study has shown that high ratios of
trainees to trainers correlates with shorter
training times but does not significantly
correlate with lower degrees of improve-
ment. The finding that the length of the
training time also did not appear to cor-
relate with higher degrees of improvement,
is unexpected. The counter-intuitive nature
of this finding suggests caution in the in-
terpretation of the results, and that further
investigation is warranted. Definitions and
objective measures of competence should be
developed and used in any future investi-
gations. The findings also suggest that fa-
cilities mandating relatively long periods of
training in Routine TEM (i.e. greater than 20
hours) could look critically at the training
they provide and consider whether the du-
ration could be reduced without significant
reduction in outcomes.
Acknowledgement
The author would like to thank all of the
survey respondents that gave generously of
their time, and the QUT Office of Research
Ethics and Integrity for support.
Contact
Dr. James Riches 
Senior Research Officer (Microscopy)
Central Analytical Research Facility (CARF)
Brisbane, Australia
jamie.riches@qut.edu.au 
www.qut.edu.au/ife
References:
http://bit.ly/IM-Riches2
 X-Ray Analysis

Soft X-Ray Microscopy of Complex Samples

From Tissue Culture to Tissue Components
Casper Hempel1, Sergey Kapishnikov2, Matthias Fach1, Ana J. Pérez-Berná3, Andrea Sorrentino3, Stephan Werner4, Peter Guttmann4,Thomas Lars Andresen1

C
ryotransmission x-ray microscopy has
emerged as an imaging modality that
enables nanoscale resolution of cul-
tured cells in three dimensions. Previous
studies have so far mostly dealt with tissue
culture and unicellular organisms but here
we report the use of transmission x-ray
microscopy to study tissue components. By
gentle homogenization and isolation one
can in hydrated, native-state samples study
specific tissue components of interest.

Cryo soft x-ray tomography has emerged

as a powerful technique enabling nanoscale
resolution of native, hydrated cells with-
out the need for contrasting agents, dyes
or antibodies [1,2]. By using x-rays with
an energy range between the K-absorption
edges for oxygen and carbon intracellular
water molecules absorb almost no photons
while carbon-rich structures such as plasma
membranes and organelles absorb well re-
sulting in high contrast in images [3]. This
window of opportunity is termed the “water
window” [2]. In the water window one can
image unstained samples of up to approxi-
mately 10 µm in thickness [4].
Biological samples can be cryogenically
preserved in vitreous ice allowing for native-
state cells to be imaged at high resolution
resolving subcellular features with a reso-
lution around 25-30 nm [5,2,6]. This tech- Fig. 1: Cultured endothelial cells imaged with soft x-ray tomography resolves intracellular com-
nique has mostly been applied to study cells ponents like the nucleus (N), lipid droplets (black arrows) and mitochondria (white arrow). Mul-
grown in culture and monocellular organ- tiple endosomes are also clearly distinguished. Data acquired at the Mistral beamline, Alba light
isms though in one study tissue was sec- source. Scale bar: 500 nm.
tioned cryogenically and imaged [7,4,2,8,9].
We have inserted an example of how soft
x-ray tomography can visualize cultured (Taconic) were housed with access to ad as previously described [12] to enrich for
brain endothelial cells (fig. 1). libitum food and water. In deep anesthe- cerebral microvessels. In another set of
One downside of using tissue culture sia (Hypnorm/Midzolam) mice were tran- experiments we used cell strainers of dif-
models is the lack of translational value. One scardially perfused with isotonic saline ferent sizes (Corning) to enrich for cere-
example is the studies on endothelial gly- (15 mL). In some mice, gold nanoparticles bral blood vessels as described previously
cocalyx [10], and also in drug delivery the were injected via the tail vein and left to [13]. The tissue homogenate was diluted
translational value is not ideal [11]. With an circulate for 15 minutes prior to terminal 2-fold with cold isotonic saline and 2 µL
intention to study uptake of solid nanopar- anesthesia and perfusion. The transferrin of this mixture was applied to a Quanti-
ticles in tissues we investigated how trans- receptor-targeting gold particles (30 nm foil (R2/2) coated grid (mesh 200). The
mission x-ray tomography could assist in gold core) were in essence produced as grids were high-pressure frozen (HPM100,
this task. previously described in detail but with a Leica) without the use of ethanol as syn-
shortened production time [12]. The brain chronization fluid and were kept in liquid
was removed and homogenized using two nitrogen until imaging. All imaging was
Materials and Methods different approaches. In one set of exper- performed under cryogenic conditions at
iments the brain was homogenized using Alba (Mistral beamline [6]) and at HZB-
All animal studies were approved by the a Dounce homogenization pestle (loose) Bessy II (U41-TXM [2]). Pre-labeling blood
Danish Animal Inspectorate. Balb/c mice and mixed with Dextran (Sigma-Aldrich) vessels with fluorescent lectins allowed us

Imaging & Microscopy 1/2020 • 45

X-Ray Analysis
Fig. 2: Visualization of complex samples ex vivo allows for assessment of myelinated axons. A) Overview image showing myelinated axons (white
arrow) as well as multiple cellular components (likely a microglia, black arrow) and a nucleus (arrowhead) as well as multiple small vesicles. B)
Focused section of a myelinated axon. The individual myelin fibers are visible. Data acquired at the U41-TXM beamline, HZB-Bessy II. Scale bars:
A: 500 nm and B: 200 nm.
to localize blood vessels by using the vis-
ible light fluorescence microscopes build
into the x-ray microscopes at both beam-
lines. The following programs were used
for alignment and 3D reconstructions: Im-
ageJ (TomoJ [14]), Bsoft [15], IMOD [16],
Tomo3D [17].
Results
Homogenization of tissues disrupts parts
of the tissue organization while preserving
other parts. In our hands, homogenization
with cell strainers led to relatively impure
samples in terms of microvessels. The sam-
ples prepared this way however suffered
very little to beam damage and highly de-
tailed tomograms could be obtained.
As one appreciates from the micro-
graph, multiple sizes of vesicles, lipid bod-
ies and cellular structures are seen (fig. 2).
Blood vessels were regularly detected but
also other parts of the brain were detected.
In particular we found a high fraction of
myelinated axons (fig. 2A). Soft X-ray
tomography enabled visualization of
myelinated axons (fig. 2B). The high res-
olution of soft x-ray tomography enables
quantification of the thickness of myelin.
We measured a median thickness of 203
nm, which is approximately three times
thicker than what we reported in a previ-
ous publication using electron microscopy
[18]. This effect could be due to shrinkage
46 • Imaging & Microscopy 1/2020
as previously reported [19] or a large vari-
ation in myelin thickness, which has also
been reported previously [20]. The distance
between the myelin sheets was tempting
to measure but with a reported distance
of approximately two times less the limit
of resolution [21] this was not attempted.
Additionally, the relatively large volume
imaged additionally enabled visualization
of myelinated indentations along the length
of the myelinated axons. If using other
methods like volumetric electron micros-
copy this information can also be obtained
but such methods require that the samples
are dehydrated and contrasted heavily to
obtain contrast in electron microscopes [2].
One goal was to obtain information on
the trafficking of nanoparticles over the
blood-brain barrier (BBB). Drug delivery to
the brain is challenged by a tight BBB that
is almost impermeable to passive transport
of drugs into the brain parenchyma. Despite
the fact that only a small fraction of the
injected dose ends up in the brain paren-
chyma the use of targeted nanoparticles
has been suggested as one way to improve
brain drug delivery [22]. Temporal assess-
ment of uptake by using electron micros-
copy has shown that the majority of tail
vein-injected, gold nanoparticles target-
ing the transferrin receptor are transported
over the brain endothelium in about 0.5-2.5
hours after intra venous injection [22]. Cryo
soft x-ray tomography has previously been
used to study nanoparticle uptake in cells
cultured in vitro [23] but here we wanted to
extend these studies to ex vivo preparations.
In dextran isolated vessels we obtain a very
pure sample in terms of cerebral microves-
sels [12]. In cryo soft x-ray tomography we
observe the dextran beads being attached
to the isolated microvessels (fig. 3A). The
high amount of dextran present in the sam-
ples led to significant problems since we
observed that dextran particles were partic-
ularly subjective to beam damage (fig. 3B).
This hampered the collection of ideal tomo-
grams since we had to use low excitation
time (1 s) and a low number of tilt angles
(35 instead of 131). Samples with slightly
thicker ice seem to reduce radiation dam-
age to some extent. Obviously, these fac-
tors hamper the 3D reconstructions. Yet,
using soft x-ray tomography we were able
to visualize gold particles associated with
cerebral blood vessels (fig. 3C; D). Due to the
low quality of the reconstructions subcellu-
lar details are not resolved. Further proto-
col developments are needed but these data
suggest that imaging solid nanoparticles ex
vivo is possible.
Summary and Conclusions
Here we show the use of cryo soft x-ray to-
mography to study the binding and trans-
port of gold nanoparticles to the surface
of blood vessels isolated from brain tissue.
This allows for imaging and studying how
 X-Ray Analysis

Fig. 3: Microvessels isolated with dextran. A) Overview image showing part of an isolated blood vessel with dextran a bead attached to its side (ar-
row). B) Image taken from a tilt series of a blood vessel where beam damage was developing during acquisition (arrow). C-D) Gold nanoparticles
were localized in the endothelium from isolated blood vessels (arrows). Erythrocytes trapped in the vessel are marked with E. Data acquired at the
Mistral beamline, Alba light source. Scale bars: A: 1 µm, B: 500 nm and C, D: 200 nm.

nanoparticles interact with a complex bi-
ological system at nanoscale resolution.
The approach is not high-throughput
but allows for detailed analyses of solid
nanoparticle transport ex vivo in hydrat-
ed, native-state tissues. The approaches
described here for brain tissue could be
modified to other tissues and cell types of
interest. One important take home mes-
sage of this is the aspect of beam damage
that was experienced using dextran. Other
methods could also cause beam damage or
could result in unwanted high absorption
of photons leading to sub optimal imag-
ing of samples of interest. Optimizing on
these protocols would also avoid perform-
ing technically demanding cryo-sectioning
of thick sections that would be ideal for
three-dimensional studies of native-state
biological phenomena.
Acknowledgements
We acknowledge the superb support from
Eva Pereiro, responsible for the Mistral
beamline (Alba, Barcelona). We thank HZB
for the allocation of synchrotron radiation
beamtime. We acknowledge the financial
support from the Lundbeck Foundation as
part of the Research Initiative on Brain Bar-
riers and Drug Delivery.
Affiliations
1Dept Health Technology, Technical Univer-
sity of Denmark, Kgs Lyngby, Denmark
2Niels
Bohr Institute, University of Copenha-
gen, Copenhagen, Denmark
3ALBA Synchrotron Light Source, MISTRAL
Beamline-Experiments Division, Cerdanyola
del Valles, Barcelona, Spain
4Helmholtz Zentrum Berlin für Materialien
und Energie GmbH, Wilhelm-Conrad-Rönt-
gen Campus, Berlin, Germany
Contact
Dr. Casper Hempel
Department of Health Technology
Lyngby, Denmark
casperhempel@gmail.com

More on x-ray microscopy: Read more about soft matter: References:

http://bit.ly/WAS-xray http://bit.ly/WAS-soft-matter http://bit.ly/IM-Hempel

Imaging & Microscopy 1/2020 • 47

Products
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a Cryo-Correlative
Microscopy system
enabling the full work-
flow of Correlative Light
and Electron Microscopy
(CLEM). It is ideal for the correla-
tion of high resolution structural
information with biochemical
processes within cells. The latest
model can be integrated with a
wide range of research grade up-
right microscopes and offers en-
hanced sample stability for cryo
imaging, improved sample han-
dling and reduced sample contam-
ination. The system maintains the
vitrified state of the sample at a
Optical Frequency Measurement
Toptica’s DFC CORE+ frequency
comb has demonstrated record
stability transfer at a record level
of 10-21 at 105 s averaging time,
as reported in an article by scien-
tists of the Physikalisch-Technische
Bundesanstalt (PTB) Braunschweig,
Germany and the manufacturer.
Today’s best optical clocks achieve
an amazing accuracy and would
be off by less than one second over
the age of the universe. These clocks
rely on probing optical transitions
in cold atoms with very stable and
narrow linewidth laser light. Fre-
quency combs allow the combina-
tion of the most stable lasers with
the best reference atoms. This has
been achieved by suppressing the
influence of optical path-length
Terahertz Waves of 450 μm
Hamamatsu Photonics has suc-
ceeded in producing terahertz
waves at a wavelength of 450 mi-
crometers (μm: micro or μ is one
millionth), which is currently the
world’s longest wavelength avail-
able from a single semiconductor
laser operating at room tempera-
ture. To achieve this, the compa-
ny developed a long-wavelength
mid-infrared quantum cascade la-
ser, in which the laser structure was
based on research and the analysis
results of the terahertz wave gen-
eration principle. Results from this
research will be useful in applica-
constant -196 °C by means of liq-
uid nitrogen cooling and pro-
vides proven capabil-
ities to safely handle
and transfer cryo
samples and image
them with optical
microscopy. Samples are
kept free of contamination at all
times due to active self-cleaning
via the liquid nitrogen cold trap.
Sample cassettes are available for
different grid types, including FEI,
Planchette, Bessey, Polara and cus-
tom designs.
Linkam Scientific Instruments
www.linkam.co.uk
fluctuations with a combination of
active phase-stabilization and com-
mon-path propagation. This paves
the way for a future improvement
of some of the most sensitive in-
struments ever created: optical
clocks and gravitational wave detec-
tors. Both benefit from transferring
the ultimate stability to a specific
wavelength.
Toptica Photonics
www.toptica.com
tions such as quality testing and the
non-destructive inspection of drugs
and foods containing components
that absorb electromagnetic waves
in the sub-terahertz range, as well
as in submillimeter astronomy and
high-speed and high-capacity com-
munication over short distances.
Hamamatsu Photonics
www.hamamatsu.co.uk
 Products

Field Emission SEM with Desktop Scanning Electron Microscope

Automated Analytical Intelligence
Jeol has introduced a new Field
Emission Scanning Electron Micro-
scope with analytical intelligence
for optimizing electron beam setup
and tuning, embedded EDS with live
analysis for real time imaging and
elemental analysis, and Zeromag
navigation function, seamlessly
transitioning between optical im-
aging to nanoscale investigation
with the high-powered optics of
the device. Neoengine corrects elec-
tron trajectories and automatically
aligns the beam in real time, and
also automatically corrects focus,
brightness/contrast, and astigma-
tism. The SEM achieves single-digit
Angstrom resolution at low kV, ideal
for ultrahigh high-resolution im-
aging of nanostructures, specimen
surface details, biological speci-
mens, and magnetic samples as well
as elemental analysis of non-con-
ductive and beam sensitive samples.
An optional Soft X-ray Emission
Spectrometer provides efficient and
parallel collection of very low-en-
ergy X-rays while providing unprec-
edented chemical state analysis.
Jeol
www.jeolusa.com
Thermo Fisher Scientific has in-
troduced the Phenom Particle X,
a versatile and intuitive desktop
scanning electron microscope
(SEM) solution that is designed to
provide automotive suppliers and
additive manufacturing compa-
nies faster quality control analyses
of materials used in development
and production. The device in-
cludes a broad range of auto-
mated SEM analyses to identify
faults in materials or gauge the
impact development and produc-
tion changes have on a final prod-
uct. By keeping quality control
in-house, users can analyze ma-
terials up to ten times faster than
by outsourcing. Industrial man-
ufacturers can use it to confirm
whether components fulfill tech-
nical cleanliness specifications ac-
cording to the VDA19 or ISO16232
standards. It also provides detailed
feedback on the cleanliness or
purity of final product without
the manufacturer having to share
this confidential and valuable data
with third parties.
Thermo Fisher Scientific
www.thermofisher.com

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Products

Updated Laser Driver Gantry Motion System

Picoquant recently presented the
latest models and upgrades for
its high-end Taiko PDL M1 pico-
second pulsed laser driver, for the
high-throughput Multiharp 150
event timer and TCSPC unit as
well as for the Vis UV laser plat-
form. Thanks to a free firmware
update, the Taiko PDL M1 laser
driver is now capable of using the
full power mode of its diodes that
allows running all existing and new
laser heads at the maximum pulse
energy possible for any repetition
rate setting. The selection of laser
heads is expanded with modules
CMOS Cameras
emitting in the green (530, 560,
and 595 nm) as well as a new
generation of high powered multi-
mode diodes covering the visible to
near infrared range. The Multiharp
150 is now available as 4, 8 and 16
channel versions. The update en-
ables programmable dead-times up
to 160 ns for each individual chan-
nel. Extending the Multiharp 150’s
native dead-time of ca. 650 ps to
larger values helps eliminate arti-
facts due to detector after-pulses.
Picoquant
www.picoquant.com
The A-351 gantry motion system
from Physik Instrumente is related
to the A-341 hybrid air bearing
gantry. Driven by linear motors and
guided by precision mechanical
linear bearings, the system pro-
vides high speed and accuracy, at
a lower price compared to its air
bearings-based brother. Advanced
absolute linear encoders increase
accuracy and reduce start-up time
by rendering homing procedures
unnecessary. The integrated ironless
linear motors eliminate cogging,
providing smoother motion without
vibration and better velocity control.
The highest motion performance
and easy integration with factory
automation systems is provided by
an ACS-based motion controller
with Ethercat connectivity. For ap-
plications requiring a motorized ver-
tical axis, several Z-stage options are
available with either screw-driven
platforms or linear motors and a
pneumatic counterbalance. Other
customizations, such as mounting
platforms for vision systems or dis-
pensers are also offered.
Physik Instrumente
www.pi-usa.us

iocam 705 mono and 712 mono are

Zeiss has introduced four new
high-quality CMOS cameras for
digital imaging in light micros-
copy. These cameras complete the
Axiocam portfolio. The new 705
color and 712 color microscope
cameras deliver high image quality
for histology, pathology or material
research and analysis, thanks to ex-
cellent color rendition and greatly
improved dynamic range. Zeiss Ax-
Raman Imaging and Scanning
Electron (RISE) Microscopy
RISE Micro Witec’s solution for
correlative Raman-SEM imag-
ing is now available for the Zeiss
Sigma 300, a field emission scan-
ning electron microscope (FE-
SEM). With this jointly-developed
system, the companies provide a
ideal for demanding fluorescence
live-cell imaging with fast frame
rates and high dynamic range. Also,
their extended near-IR sensitiv-
ity allows for deeper insights into
sample structures. Small 3.45 µm
pixels and low noise levels in com-
bination with the fast USB 3.0 plat-
form enable researchers to carry
out extremely fast imaging exper-
iments while maintaining excellent
signal quality. Also, the new cam-
eras’ global shutter architecture
allows capturing dynamic samples
without creating motion artifacts.
Zeiss
www.zeiss.com
erations of experience in Raman
spectroscopic imaging and ad-
vanced structural analysis. The
seamless combination of the two
techniques offers a distinct advan-
tage when investigating samples,
improves ease-of-use and accel-
Broadband Multinuclear Benchtop NMR Spectrometer
Oxford Instruments has launched
X-Pulse, a high resolution 60MHz
benchtop NMR system. This offers
research chemists capabilities in
their laboratories that were pre-
viously available only on complex
and expensive high-field NMR
spectrometers in specialist fa-
cilities. The system can be easily technology delivers line shapes of
tuned to any nucleus from 29Si less than 0.35Hz/10Hz as standard,
to 31P, without having to change making it easier to separate over-
NMR probes. This means that us- lapping peaks and identify smaller
ers can select whichever nucleus concentrations of compounds. A
they want, on a single instrument. traditional magnet design with
A flow cell and variable tempera- a high thermal mass makes the
ture probe allows dynamic chem- device insensitive to sample tem-
ical reactions to be continuously perature variations, whether static
monitored, with a variable tem- or flowing, so that sample tem-
perature capability from 20°C to perature artefacts are eliminated.
70°C, helping users to understand
the processes and kinetics of their Oxford Instruments
reactions in detail. New shimming www.oxinst.com

fully-integrated instrument avail-
able as an OEM product through
Zeiss that features a standard,
unmodified vacuum chamber and
SEM column along with a com-
plete confocal Raman microscope
and spectrometer. It expands the
range of choices available to the
researcher and incorporates gen-
More Products:
erates experimental workflow. The
research-grade optical microscope
capability integral to every WITec
microscope also helps researchers
survey their sample and quickly lo- http://analyticalscience.wiley.com
cate areas of interest.
Witec
www.witec.de

50 • Imaging & Microscopy 1/2020

 Index / Imprint

AHF Analysentechnik 25 Hamamatsu Photonics  48 Royal Microscopical Society (RMS) 14

APE Angewandte Physik 43 io Light48 Technical University of Denmark 45

Carl Zeiss Microscopy  Cover, 16 Jeol 49 Technische Universität Darmstadt 26

Central Analytical Research Facility (CARF) 42 Linkam Scientific Instruments 48 Tescan  11

CNR-Institute for Photonics and Nanotechnology 28 NKT Photonics  Inside Front Cover The Arctic University of Norway (UiT) 24

Cobolt  7 Olympus  48 Thermo Fisher Scientific 49

Columbia University 22 Oxford Instruments  50 Toptica Photonics29, 48

Coolled 37 Park Systems Outside Back Cover, Insert University of Konstanz 18

Edmund Optics 33 PCO 48 University of Manchester 39

EMC 2020 12 Physik Instrumente 50 University of Queensland35

European Microscopy Society 15 Picoquant 50 Witec 21, 50

European Molecular Biology Laboratory (EMBL)  32 Prior Scientific 19 Zeiss  50

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Imaging & Microscopy 1/2020 • 51

 September 23 - 25, 2020 I Dublin, Ireland

REGISTER FOR NSFE 2020 TODAY

Keynote Speakers
• Prof. Franz J. Gießibl • Prof. Christine Kranz
University of Regensburg, Germany Ulm University, Germany
• Prof. Dr. Lukas Eng • Prof. Thorsten Hugel
Technical University Dresden, Germany University of Freiburg, Germany
• Ass. Prof. Kim McKelvey • Prof. Tom Hauffman
Trinity College Dublin, Ireland Vrije Universiteit Brussel, Belgium
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University College Dublin, Ireland Tokyo Institute of Technology, Japan
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correlative Microscopy, Forchheim, Germany • Prof. Pat Unwin
• Prof. Dennis Meier University of Warwick, United Kingdom
Norwegian University of Science and • Dr. Neus Domingo
Technology, Norway live.parksystems.com/nsfe2020
ICN2 Barcelona, Spain

Conference Topics Scope

Application NSFE series is an open European AFM User Forum focusing on sharing and
• Energy Storage exchanging the cutting-edge research for both materials and life science disciplines
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• Organic, Inorganic and Hybrid Semiconductors The 3rd NSFE 2020 will focus on nanoscale functional materials, such as organics,
• Nanomaterials and Biotechnology organic/inorganic hybrid semiconductors, nano- and biomaterials, as well as the
• Special Session: Electrochemical & Electromechanical development of novel nanometrology methods.
Properties in Functional Materials The conference program includes exciting nanoscientific lectures, instrument
workshops, poster session and networking events.
Method
• Nanomechanical and Electrical Characterization
• Characterization of soft materials in Liquid Environment Submit your Abstract at NSFE 2020
• Advanced Imaging ...and win up to EUR 500 AFM Scholarship.
Deadline: June 1, 2020

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