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VOLUME 26
MARCH
2024

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for sustainability:

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anytime, anywhere with our
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We are delighted to offer you technical articles, news,
application reports, interviews, and profiles on microscopy
topics in a digital format. The choice is yours.
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Editorial

Dear Readers,
It is with great pleasure that we welcome
you to EMC24, Sunday, August 25 - Friday,
August 30, 2024.
EMC2024 is the 17th of its kind and willbring the world of scientific imaging together
in one of Europe’s most beautiful cities Copenhagen, Denmark.
Incorporating a balanced conference program of light and electron microscopy inboth physical and life sciences - microscopists, manufacturers, and suppliers will cometogether to share new and exciting techniques, applications, and technology.
The EMC24 will take place at Bella Center,
Center Boulevard 5, 2300 Copenhagen, Denmark, and will on Sunday, August 25 feature pre-congress workshops and an opening reception.
Monday the 26th, Eva Olsson IFSM President, will give the opening address followed
by Prof. Carolyn Larabell as the first plenary speaker. The following days will feature Prof. Claus Ropers, Prof. Emma Lundberg, Prof. Vincenzo Grillo, and Prof. Moritz
Helmstaedter as morning plenary speakers.
The conference days will continue with six
parallel sessions and each day will close withdrinks in the Poster and Exhibition area. The
last day of the Congress will conclude witha farewell reception.
The main symposia feature Life Science(Chair, Eija Jokitalo), Physical Science (Chair,

Jakob Wagner), and Instrumentation andMethodology (Chairs, Julia Fernandez-Rodriguez and Randi Holmestad) – all session
topics are described on the conference homepage. Abstract submission will start in early
January 2024, but registration for the conference is already open – early bird registration is open until July 6, 2024.
Ongoing fundraising will attempt to fundas many travel bursaries (€ 400) as possible
for young students and technicians.
The exhibition is one the largest of its
kind featuring more than 100 exhibitors
and a Nordic Corner for smaller enterprises,
start-ups, and organizations, and 50% of
the available exhibition space has alreadybeen reserved. Day passes will be available
for a visit to the exhibition only. On January 17 the EMC2024 Corporate Advisory
Board (Chair, Kornelia Weidemann, Thermo
Scientific) will meet for an introduction of
EMC2024 scientific content and not least an
inspection of the venue.
The congress will among other addressa range of microscopy-related subjects,
ranging from dynamic interactions in cells,organoids, tissue, and entire organisms,
pathology, immunocytochemistry, biomolecular labeling, volume Electron Microscopy inLife Sciences, semiconductors, heterostructures, and devices, geological materials, andbio-mineral systems, quantum materials,

advances in 3-dimensional image reconstruction, new Instrumentation, and dynamic
studies using micro-nano labs.
For information on the program, exhibition, sponsorship opportunities, and conference venue, please visit the conference
website.
We welcome you to Copenhagen for the
EMC2024 Congress and Exhibition.
On behalf of the EMC2024 Local Organizers:

Prof. Klaus Qvortrup
Conference Chair

More information:www.emc2024.eu

Imaging & Microscopy 1/2024 • 3

© Creative Commons Attribution- ShareAlike 4.0 Int. license

Contents
EDITORIAL

3

NEWSTICKER

6

ANNOUNCEMENTS
RMS Events: Action-Packed Spring and Summer Ahead

8

NEW BOOKSuper Resolution Optical Imaging and Microscopy:
Methods, Algorithms, and Applications

8

RMS IN FOCUS© Dr. Thomas R. Neu

Register Now for elmi2024!

10

NEWS FROM EMS
EMS Newsletter #84, March 2024

11

COVER STORY© Dr. Gillian Kiliani, Universität Konstanz

Easy Access to Molecular Dynamics in Living Samples

12

Investigation of Molecular Behavior in Combination with LSM Imaging Experiments

A. Bergter and H. Gut

LIGHT MICROSCOPY
MyScope: Free Online Microscopy Training 

14

A Comprehensive, Open-Access, Training Resource

J. Whiting et al.

A Global Reproducibility Initiative in Light Microscopy

18

Community Standards for Instruments, Metadata, and Publications

R. Nitschke and H. Hartmann© Nordenfelt

Sign up for our newsletter andreceive issue alerts!

4 • Imaging & Microscopy 1/2024© Farknot Architect - stock.adobe.com

Read Imaging & Microscopy
anytime, anywhere with our
digital edition.

Out of this World –Hidden Biofilms Under the Microscope

22

The Matrix of Cave Snottites Studied by Lectins and Confocal Microscopy
T. R. Neu

26

High-Speed Molecular ImagingLine-Illumination Raman Microscopy with Slit Array Detection
K. Fujita et al

ELECTRON AND ION MICROSCOPY
Attosecond Electron Microscopy 

30

Movies of Light in Space and Time
P. Baum et al.

SCANNING PROBE MICROSCOPY
Next-Generation Time-Resolved Scanning Probe Microscopy 34
Ushering in a New Era of Nanoscale Exploration with Enhanced Usability
H. Shigekawa et al.

COVER STORY
IMAGE PROCESSING
Data-Driven Microscopy Improves Image Acquisition

37

Finding the Right Cells to Image
O. André and P. Nordenfelt

Investigation of Molecular Behavior
in Combination with LSM Imaging
Experiments Going Confocal

ADVERTORIAL
Olympus Scientific Solutions Is Now Evident

40

New Name, Same Commitment—Visit us at analytica 2024!

PRODUCTS41
INDEX / IMPRESSUM

Easy Access to Molecular
Dynamics in Living Samples

INSIDE BACK COVER

Understanding the dynamics of life is one ofthe driving forces in microscopy technology
development. Resolving ever-so-small structures
while reaching the needed temporal resolution allows
to investigate subcellular and sub-organelle behavior
in living samples in more detail and complexity thanever before [1]. However, molecular behavior in living
samples is often overlooked, since image-based
microscopy cannot always provide sufficient answers.
But microscopy-based technology and techniquessuch as Fluorescence Correlation Spectroscopy(FCS) do allow deeper insights into molecularcharacteristics in vivo [2].

12

1

69721

VOLUME 26
MARCH
2024

Official Partner of the EMS

Welcome to the knowledge age. Wiley builds on its 200-year
heritage by partnering with universities, businesses, research
institutions, societies and individuals to develop digital content,
learning, assessment and certification tools. Wiley continues toshare and deliver the answers to the world’s challenges
helping you to further your mission.

Read oureMagazine

Newsticker
Electron Microscopy in Neuroscience Research© Koppensteiner et al./PNAS

Analyzing Brain Region Using Light Flashes, High-Pressure Freezing and Fracturing

Fear and addiction exert significant influence within society.
Managing them is often challenging, as they are driven by intricate
neuronal circuits in our brains.
Understanding the underlying
molecular mechanisms is crucial
to intervene when these processes
malfunction. Pioneered by scientists at the Institute of Scienceand Technology Austria (ISTA),
the novel “Flash and Freeze-fracture” technique provides a uniqueglimpse into the respective brain
region. While looking for food,a bird encounters a fox. It getsaway just in time, but the sightand the sound of the predator lingers. The negative experience will
form a memory in its brain and

Beamline Analysis

Researchers employed an electrolyte additive to improve the functionality of energy-dense lithium
metal batteries. By adding cesium
nitrate to the electrolyte that separates the battery’s anode andcathode, the charging rate of lithium metal batteries significantly
improved while maintaining a long
cycle life. To better understandhow the cesium nitrate additive
influenced the electrolyte com-

tional processing. The scientists
investigated this particular partof the brain to understand how its
neurons communicate with eachother.
Original publication:
doi: 10.1073/pnas.2301449121
More information:
https://bit.ly/IM-012024-d

Expansion Microscopy

A Dual-Functional Additive for Fast Charging andLong Cycle Life of Lithium Metal Batteries© Jessica Rotkiewicz/BNL

will be associated with fear andstress from now on. Whenever itmeets a fox again, the fear memory is revived. The bird’s attention
spikes, its heart rate goes up, andit changes its behavior to reduce
the risk of predation. Such memory is mediated by a specific brain
region called the medial habenula,
one of the epicenters for emo-

Human Brain Tissue in Greater Detail than Ever Before

position and battery performance,
the scientists used four different
beamlines at National SynchrotronLight Source II (NSLS-II), a DOE
Office of Science user facility at
Brookhaven Lab. The results using
an X-ray Powder Diffraction (XPD)
beamline showed that the cesium
nitrate additive increased thepresence of components known
to make the interphase more protective. Remarkably, in addition to
the typical crystalline components,a compound called cesium bis(fluorosulfonyl)imide was also identified.
Original publication:
doi: 10.1038/s41467-023-44282-z
More information:
https://bit.ly/IM-012024-b

Using a novel microscopy technique, researchers have imaged
human brain tissue in greater detail than ever before, revealing cells
and structures that were not previously visible. The researchers hope
this technique could eventually be
deployed to diagnose tumors, generate more accurate prognoses, andhelp doctors choose treatments.
The new imaging method is basedon expansion microscopy, developed in Boyden’s lab in 2015. Bylabeling the proteins with fluorescent antibodies before expansion,
their location and identity could
be visualized after the expansion.
However, the antibodies commonly
employed for this type of labelingface difficulty penetrating densely
packed tissue before expansion. In
the study the team created a dif-

© Valdes, P.A., et al./MIT

ferent tissue-softening protocolthat breaks up the tissue but preserves proteins in a sample. Oncethe tissue is expanded, commercially available fluorescent antibodies are used to label the proteins.
Original publication:
doi: 10.1126/scitranslmed.abo0049
More information:
https://bit.ly/IM-012024-c

Optical Filters
For imaging & microscopy
Expert support · Customized designs · OEM

6 • Imaging & Microscopy 1/2024

www.ahf.de

Newsticker

Open-Source Compression Software© Anika Grüneboom/ISAS

EfficientBioAI Allows to Run Existing Bioimaging AI Models Fasterwith Significantly Lower Energy Consumption

The open-source, user-friendly EfficientBioAI can dramatically increase
the efficiency of neural networks
in bioimaging without limiting theaccuracy of the models. Artificial intelligence (AI) has become an indispensable component in the analysisof microscopic data. However, as AI
models become more complex, therequired latency (processing time)
for images significantly increases,
as does the associated energy consumption. To overcome the energy
issue, sophisticated algorithms areused to compress the AI models andavoid high latency in image analysis,

especially on devices with restrictedcomputing power; i.e., the computational extent is reduced in thesemodels while retaining comparable
prediction accuracy. Several strategies are combined to reduce memory consumption, speed up model
inference, and the ‘thought process’of the model. Pruning, for example,
can be used to remove excess nodes
from the neural network.
Original publication:
doi: 10.1038/s41592-024-02167-z
More information:
https://bit.ly/IM-012024-e

SuperSTEM: In Situ Electron Microscopy

Insights into the Origin of Life Inside a Rare Carbonaceous Meteorite

Infrared Photothermal Microscopy

Monitoring Lipid Metabolism in Living Cells
Researchers have successfully developed a novel technology to investigate neutral lipids within lipid
droplets of living cells. Two-Color
Infrared Photothermal Microscopy(2C-IPM) can be used with isotope
labeling, which allows for the detailed monitoring of neutral lipid
synthesis within individual lipid
droplets. Unlike previous fluorescence microscopy-based methods,
the 2C-IPM technology relies on
infrared spectroscopic (IR) and does
not require the use of fluorescentdyes. The new approach directly
monitors neutral lipids within LDs
by detecting changes in IR absorbance. Remarkably, it allows researchers to monitor the synthesisof neutral lipids within individual
LDs in living cells over a long period.
The team investigated the synthesisof neutral lipids in cells exposed toexcess fatty acids. They could distinguish freshly synthesized neutral© Institute for Basic Sciencelipids from pre-existing neutral lipids within cells by subjecting deuterium-labeled fatty acids, whichhave distinct spectroscopic properties from non-deuterated forms.
The results demonstrated that excess fatty acids led to lipid toxicity,and cells responded by increasing
the synthesis of neutral lipids.
Original publication:
doi: 10.1039/D3SC04705A
More information:
https://bit.ly/IM-012024-f

Explore The Nanoworld
With ModuleSci© D.M. Kepaptsoglou, SuperSTEM

An international research team reported the first in-depth analysis oforganic matter within the Winchcombe meteorite at the nanoscale.
The study shows that the meteorite
still contains pristine extraterrestrial organic molecules that might
have been crucial to the advent oflife on early Earth. They were able touniquely correlate synchrotron-radiation data with complementary
ultra-high resolution spectroscopic
information about the nature of

the functional chemical groups
present in the organic matter, using
one of the most powerful electron
microscopes in the world at the SuperSTEM Laboratory—this allowedin situ detection of nitrogen-bearing biorelevant molecules, including amino acids and nucleobases.
Original publication:
doi: 10.1038/s41467-024-45064-x
More information:
https://bit.ly/IM-012024-a

The PV-100 tabletop SEM from ModuleSci offers
exceptional resolution in a compact package. Featuring
a 5-axis stage and PICOSMART software, the PV series
is the ideal solution when lab space is at a premium.
info@modulesci.com
Modulesci.com

Imaging & Microscopy 1/2024 • 7

Announcement

RMS Events:
Action-Packed Spring and Summer Ahead
A wide range of fantastic RMS meetings, conferences, and courses are lined up for the microscopy, imaging, and flow cytometry community in the coming months.
Our Spring offerings get underway with AFM& SPM Meeting 2024 (25 – 28 March, Durham,
UK). This international conference on Scanning
Probe Microscopies is perfect for graduate students and early career scientists, as well as renowned experts in the field of SPM.
Another essential Spring event, EBSD 2024,
takes place on 29 – 30 April, in Glasgow, UK.
This annual gathering draws together the whole
Electron Backscatter Diffraction (EBSD) community, including leading international researchscientists and engineers, early career researchers, and students.
Fast forward to June, and the RMS will be
proudly hosting elmi2024 in Liverpool, UK. Thisis an essential event for the Light Microscopy▪ Light Microscopy Summer School 2024
▪ Getting the most from your Confocal Course
2024▪ Electron Microscopy Summer School 2024
▪ Strathclyde Optical Microscopy Course 2024
(RMS-sponsored event)▪ Flow Cytometry Course 2024
© Stefan Diller

community – bringing together both researchscientists and the manufacturers of the equipment. Find out more and book your ticket online
at the elmi2024 website:
Throughout the summer, the RMS is also
hosting a series of courses and summer schools
covering a host of different branches of microscopy, techniques, and applications.
Find out more about the following RMS
courses, and book your place:

All upcoming RMS events:
www.rms.org.uk/rms-eventcalendar.html

Super Resolution Optical Imaging and Microscopy:
Methods, Algorithms, and Applications
This book covers both the basic principles andspecific technical details of super-resolution
microscopy techniques. It covers the criteria
to choose different fluorophores for various
SRM methods and critically assesses the nitty-gritty of associated problems that are often encountered in practical applications. A
progressive guide to designing the next generation of advanced fluorophores to meet thegoal of advanced SR imaging studies is also
put forward.
Written by two well-qualified authors, thebook contains exclusive content to enhancereaders’ understanding on innovation of newer
SRM technologies. Sample topics covered in thebook include:▪ Optical techniques, fluorescent probe design,
and algorithm development▪ Recent highlight and breakthroughs in biology
using SRM methods

8 • Imaging & Microscopy 1/2024▪ The overall success of SRM in biologicalinventions▪ The future direction and scope of the field
This book is an invaluable resource for chemists
and researchers/scientists involved in designingnewer fluorescent materials for SRM studies. It
can also assist biologists engaged in advanced
biological studies using SRM by guiding themthrough sample preparation, image processing,and precautions to be taken in practical imaging
studies.

Editor(s): Junle Qu, Zhigang YangFirst published:10 January 2024
Print ISBN:9783527349869
Online ISBN:9783527835539
DOI:10.1002/9783527835539© 2024 Wiley‐VCH GmbH© Ant Clausen

RMS in Focus

ACC Convention Centre and Exhibition Centre.

Register Now for elmi2024!
The RMS Is Very Proud to Be Hosting elmi2024, Taking Place in Liverpool, UK, from 4 - 7 June

With a blockbuster scientific programme, worldclass exhibition, ‘hands-on’ workshops andmore, the annual meeting has become regarded
as an essential event for the Light Microscopy
community.
‘Early bird’ booking rates are available until 9
April – providing a 23% discount on the cost ofa standard ticket, and 30% off for students. Findout more about registration rates and book now
on the event page (link in crossmedia bar).

Unique Communication Network
The European Light Microscopy Initiative (elmi)
was created in 2001 to establish a unique communication network between European scientists working in the field of light microscopy andthe manufacturers of their equipment. Its aim is
to promote the quickly developing field of lightmicroscopy as a fundamental research tool forthe life sciences and to strengthen the channelsof communication between researchers, core facilities and industry.
The event has been running for two decades
at various venues across Europe, and has an excellent reputation within the microscopy community, making it a key fixture in the calendar of
hundreds of scientists and developers.
The strength of this meeting lies in the mixture of scientific lectures on state-of-the-art,

10 • Imaging & Microscopy 1/2024

high-end microscopy, combined with ‘hands-on’
workshops and an exhibition of the latest technology, organised by the leading companies in
the field.

Great Venue in the Heart of Liverpool
elmi2024 is being held at ACC Liverpool, a purpose-built arena and convention centre in theheart of the iconic city, on the King’s Dock. The
venue has great transport links as well as being
within walking distance from a large number ofhotels, restaurants, shops and bars. Liverpool is
one of the UK’s most iconic cities with a rich,cultural heritage, a wide range of attractions
and buzzing nightlife. With the conference
venue just a short walk from the bustling waterfront and commercial centre, there is no shortage of things to see and do during your stay.

What you Can Expect at elmi2024:▪ A blockbuster meeting programme covering
all the latest techniques, applications andtechnology. Topics include: New Technologies,
Imaging Across Scales, Super-resolution andNanoscale Imaging, The AI Revolution, The
Science of Tomorrow Today, and Multimodal
Imaging.

▪ A wide range of companies showcasing
their latest technology and running workshops timetabled outside of the main meeting
programme.
▪ An accompanying exhibition in the purpose-built hall alongside posters, food anddrink;▪ A community workshop space at the heartof the exhibition, with many groups hostingmeetings and running workshops.
▪ An event dinner with networking at the Rum
Warehouse.

Contact
Royal Microscopy Society
Oxford, UK
info@rms.org.uk

More information and registration:www.elmi2024.org

News from EMS

EMS Newsletter #84
March 2024

José Maria Valpuesta,
EMS President

Virginie Serin,
EMS Secretary

Dear EMS members,
Let me first wish you a happy 2024. Last year our society celebrated its
25th anniversary. To commemorate this milestone, a booklet containing
articles written by leading scientists of our community will be distributedat the upcoming European Microscopy Congress (EMC2024), to be held in
Copenhagen from August 25th to August 30th, 2024.

In addition, EMS will offer scholarships (link in cross-media bar) to young
researchers participating at EMC2024. The application deadline is April 17,2024. Visit our webpage for more information. We look forward to receiving your applications.
Hoping we can meet next August at EMC2024!

The new EMS year has started with the call for nominations for the EMS
Outstanding Paper Awards 2023 and for the EMS awards, which will bepresented at EMC2024.
As usual, the EMS is committed to supporting the activities of our members, so please visit our webpage for the various meetings and workshopsthat will take place over the next year, some of which are going to befinancially supported by EMS:

Contact
Prof. Dr. Virginie Serin
EMS Secretary
Toulouse, France
sec@eurmicsoc.org▪ Molecular Diagnostics Training School (MDTS) & Digital
Pathology and Image Analysis Training School (DP & IATS)
25 to 28 February 2024, Vienna – Austria▪ BIST Symposium on Microscopy,
Nanoscopy, and Imaging Science 2024,
10 March 2024, ICFO - Auditorium – Barcelona

EMS scholarships:www.eurmicsoc.org/en/funding/scholarships/

Prof. Dr. José Maria Valpuesta
EMS President
Madrid, Spain
jmv@cnb.csic.es▪ Winter School 2024 –Practical course in advanced microscopy​
22 to 26 January 2024, University of Zurich Center for
Microscopy and Image Analysis – Zurich – Switzerland▪ Electron Microscopy Spring School 2024
03 to 06 March 2024, Berlin – Germany

All EMS events:
https://bit.ly/EMS-spons-Events

EMS web page:https://bit.ly/EuroMicr

Imaging & Microscopy 1/2024 • 11

CoverStory
Cover Story

Easy Access to
Molecular Dynamics in Living Samples
Investigation of Molecular Behavior in Combination with LSM Imaging Experiments
Annette Bergter1 and Hanna Gut1

Understanding the dynamics of life is one ofthe driving forces in microscopy technology development. Resolving small structures
while reaching the needed temporal resolution allows investigation of subcellular andsub-organelle behavior in living samples inmore detail and complexity than ever before [1]. However, molecular behavior inliving samples is often overlooked, since
image-based microscopy cannot always
provide sufficient answers. But microscopy-based technology and techniques suchas Fluorescence Correlation Spectroscopy(FCS) do allow deeper insights into molecular characteristics in vivo [2].
Laser Scanning Microscopes (LSM) are
high-end microscopy systems that provide
great flexibility in imaging experiments fora wide variety of samples [3,4]. The single
excitation volume of a laser beam, which isconventionally used in the process to generate instant high-resolution optical sections of one or several fluorescent labels, isthe perfect tool for FCS measurements. The
excitation volume is kept stationary andpositioned at a point of interest within thesample (Fig. 1). Fluorescently labeled molecules move through the excitation volume
and the constantly changing emission intensity is recorded. With a matching analysis,
the pattern of this intensity trace can reveal

molecular characteristics, such as concentration or velocity.
Until recently, FCS was often disconnectedfrom imaging experiments routinely conducted with an LSM, since FCS only worked
with extremely low fluorescent expression
levels in the sample, needed specialized hardware, and required specific training.
Zeiss Dynamics Profiler uniquely overcomes this limitation and gives easy accessto molecular dynamics with a wizard-based
workflow. It utilizes the Zeiss Airyscan detector to collect 32 individual FCS intensity
traces per measurement for enhanced molecular information, even in bright samples.
Adding spatial information to FCS measurements with the Airyscan area detector[5] expands the opportunities from straightforward mobility and concentration measurements. Using multiple Airyscan detector
elements allows investigation of asymmetricdiffusion as in biological condensates anddetermines flow speed and direction in bloodvessels or microfluidic systems (Fig. 2) [6].

Remarkably Easy Access to
Molecular Dynamics
To ensure reliable and reproducible data, Dynamics Profiler guides the user by a 3-step

Fig. 1: A stationary excitation volume, created by an objective lens focused laser beam (top) is positioned in a sample. Fluorescence intensity
fluctuations are recorded for this spot over several seconds, resulting in
an intensity trace measurement (bottom).

12 • Imaging & Microscopy 1/2024

wizard. It ensures correct hardware selection
and detector alignment and supports steps
to improve data quality. A reference image
is taken in which the positions to be analyzed can be chosen. Each of the measurement spots is color-coded and its displayed
dimensions precisely depict the investigatedarea in the sample. After data acquisition,
the results are provided as a combination ofimages, graphs, and tables. All data can beexported to be used for further investigation,
documentation, or publication.
Thus, information about the concentration and the dynamic behavior of a fluorescently labeled protein can be easily added toa live cell or live organism experiment, as in
the example depicted in Figure 3. mCherry isexpressed under the control of the enhancer
for the hand gene in the heart cells of the
Drosophila melanogaster embryo. The AI
Sample Finder was used to create an overview image and identify an embryo of thecorrect age and orientation (Fig. 3, top left).
A z-stack was taken with the Airyscan detector, providing super-resolution structural
information while keeping light exposure
to a minimum (Fig. 3, bottom left). Several
cells of different intensities were chosen for
Dynamics Profiler measurements, recording their precise concentration levels of theexpressed protein (Fig. 3, right).

Fig. 2: Applications enabled by Dynamics Profiler.

Cover Story

Access to New Molecular Discoveries
Utilizing the Airyscan area detector, theDynamics Profiler adds spatial information
to every FCS measurement, providing theunique opportunity to measure liquid flow
at a precise position within a volume. The
flow direction and speed (µm/s) of fluorescent proteins or dyes that are actively transported in a liquid can be determined in single measurement spots.
The behavior of blood flow in vessels canbe investigated, resulting in exact speed values for individual positions within the bloodstream. The conditions for cells, organoids,or tissue cultures in microfluidic systems canbe documented and included in the experiment (Fig. 4).

New Insights from Living Samples

Fig. 3: Live imaging experiment of Drosophila melanogaster embryos. Top left: Overview scan.
Bottom left: Z-Stack acquisition with Airyscan, Ortho Maximum Intensity Projection. Right: Dynamics Profiler experiment with 3 spots, showing the individual and color-coded intensity traces(graph) and analysis results (table). Sample courtesy of Prof. Dr. Achim Paululat and Dr. Christian Meyer, Osnabrück University, Department of Zoology and Developmental Biology, Germany.

Due to the dramatic increase in brightness
during the process of protein accumulation
into condensates, it is impossible to study
these high concentrations of molecules
using conventional FCS methods. Dynamics Profiler can measure even these brightcondensates. Furthermore, by utilizing element pairs of the Airyscan detector, differences caused by cellular compartments,
barriers, or molecular binding processes
can now be assessed (Fig. 5). The changing behavior of proteins inside and outside
cellular condensates formed by liquid-liquid phase separation can be studied to analyze their changing dynamic behavior.

Summary
Zeiss Dynamics Profiler provides a new
method to investigate molecular behavior
in combination with LSM imaging experiments. In addition to the integrated analysis tools, raw data of 32 individual detector elements is always saved, allowing forseemingly endless possibilities to investigate
molecular movement.

Fig. 4: Analysis of flow characteristics in a microfluidics channel. The Automated Sequential
Injection System Aria by Fluigent was used to pump a rhodamine 110 solution through a microfluidic flow cell (1000 µm channel width).

Affiliation
1Zeiss Research Microscopy Solutions,
Jena, Germany
Contact
Dr. Annette Bergter
ZEISS Research Microscopy Solutions
Jena, Germany
annette.bergter@zeiss.com

Online version and references:https://bit.ly/IM-Zeiss-0124

Fig. 5: Live U-2 OS cells transiently co-expressing tetrameric EGFP and the nucleolar protein
Fibrillarin tagged with monomeric Red Fluorescent Protein. Measurements at the border of nucleoli (left) reveal an asymmetric diffusion behavior (heatmaps, right). Sample kindly provided by P.
Hemmerich and T. Ulbricht, Core Facility Imaging, Leibniz Institute on Aging, Jena, Germany.

Imaging & Microscopy 1/2024 • 13

© Creative Commons Attribution-ShareAlike 4.0 International license

Light Microscopy

MyScope: Free Online Microscopy Training
A Comprehensive, Open-Access, Training Resource
Jenny Whiting1, Peta Clode2, Lisa Yen1, Nikki Stanford3

M

icroscopy training is a time-consuming and labor-intensive process and tools that support thistraining can aid efficiencies for staff inmicroscopy core facilities. MyScope is
one such resource that has been developed by Microscopy Australia, a providerof open-access research infrastructure formicroscopy and microanalysis in Australia.
The content is completely free, openly accessible, and doesn’t require any registration. This comprehensive online tool covers
ten different broad microscopy techniquessuch as SEM, TEM, Light, and Fluorescence
Microscopy as well as basic concepts, research data management, and work healthand safety. MyScope has been developedby experienced trainers and is structured
into separate modules that can be tailored further if required. The modules
are structured to contain well-illustrated
theory, instrument simulators, and assessments that can generate a certificate if
a high-level pass is achieved. The use of,and rationale for, MyScope development
are discussed.

14 • Imaging & Microscopy 1/2024

Introduction
Microscopy Australia developed the free,open-access online microscopy training
website MyScope in 2011. It was developed
to be part of a blended learning approachto microscopy training, providing a preliminary online component for new microscopists, thereby saving time during subsequent
hands-on training. Although developed
to support Microscopy Australia users, theplatform is freely available to everyone andhas emerged as a world-leading resource forthe international microscopy community.

Rationale for MyScope
Microscopy Australia is Australia’s national research infrastructure for microscopy
and microanalysis and is composed of anAustralia-wide network of university-based
microscopy facilities. This organization
provides open access to microscopy instruments and expertise to researchers acrossAustralia. A major part of the facility staff’s
time is involved with training new users andsupporting more experienced users to get
the most out of the instruments. To increase

the efficiency of this new user training,
MyScope was developed and is used across
Microscopy Australia. It has been shown to
reduce hands-on training time by 11–25%(depending on training regimes at different
facilities) [1] by bringing inexperienced newusers up to speed on the fundamentals of
specific microscopy techniques and, throughsimulators, giving them a feel for using areal instrument.
MyScope contains ten technique-specificmodules, plus others on Microscopy Concepts, Research Data Management, and Work
Health and Safety. Initially, it focused on sixof the major microscopy techniques offeredto our users. As the microscopy landscapehas diversified, the platform has been developed to where it is now (Fig. 1).
The approach to developing content isto provide a range of complementary pedagogical approaches: clear theory and diagrams, real images and data, animations,flowcharts, interactive instrument simulators, assessments, glossary, and links to moreresources, including videos. This approachremains front of mind when extending
MyScope.
Instrument simulators are at the core ofmany of the modules and are designed to© Creative Commons Attribution-ShareAlike 4.0 International license

Light Microscopyteach the concepts and processes behind
instrument operation rather than to mimic
the interface on any specific instrument.
Through these simulations, the user experiences the components, sequences, and setting adjustments encountered when operating a real instrument, allowing users to ‘learn
through doing’ before encountering a real,and often very expensive, instrument (Fig. 2).
Two of the more recently added modules, however, do not have simulators. The
Cryo-EM module, for instance, covers cryoTEM, cryo-SEM, and cryo-FIB, all techniques
where basic instrument operation is coveredby simulators in other modules. The focus of
the Cryo-EM module is on specimen preparation and the variety of differing approaches tocapturing and processing data.
The SIMS module focuses on the theoretical basis of the different types of SIMS
instruments, how they work, what is achievable, and why. Although our network has
several SIMS instruments, the majority are
too advanced and complex for users to learn
in a time- and cost-effective way. The staff
runs these instruments for the researchers
so that optimal outcomes are obtained. This
module gives users an understanding andappreciation of the technique, as well as

Fig. 1: The 13 MyScope training modules.

provides an opportunity for the global SIMS
community of facility staff and researchers to learn more about the fundamentals
of SIMS.
In both these cases, the developers havechosen to include several interactive components to enhance learner engagement

and reinforce critical concepts addressedin the text. Figure 3 shows examples ofthese. In cryo-EM, single particle analysis
depends heavily on 2D projections of 3D
molecules, and this concept is illustrated in
the Cryo-EM module by an illuminated 3D
shape that can be rotated so that the chang-

Imaging & Microscopy 1/2024 • 15© Creative Commons Attribution-ShareAlike 4.0 International license

Light Microscopy

Fig. 2: Selected screen grabs from MyScope and some of its simulators.

ing projection can be observed. Anotherrequires the user to sort 2D projections intoclasses corresponding to different protein
orientations (Fig. 3).
Assessment is an essential component of
MyScope. It allows users to monitor theirlearning and demonstrates to facilities that
relevant knowledge has been acquired.
When a user achieves an 80% pass on theassessment quizzes, a certificate is earned.
Achievement of this high level was determined to be necessary if significant timesavings in hands-on training were to result.
This certificate can be presented to facility staff as evidence of completion of theonline training component and hands-on
training can then be booked. Each assessment session draws ten questions randomly
from a bank of multiple-choice questions.
All answers, whether right or wrong, have
instructive feedback to help the learner understand the correct answer as part of thelearning process.

16 • Imaging & Microscopy 1/2024

Underpinned by Expertise
The content for MyScope has been developed by Microscopy Australia expert staff
largely through in-kind contributions fromour network of facilities. Highly skilled in
microscopy and user training, the staff
maintain their expertise through a range ofnational and international collaborations
and exchanges through which they share
best practices and innovative approaches.
Central coordination of the platform content ensures a level of consistency acrossthe resource.

Uptake and Usage of MyScope
MyScope has developed a global reputationfor excellence in training and has thousandsof users each year from around 100 countries with the majority from Australia, theUS, China, the UK, and Mexico.

One of the powerful aspects of the platform
is that it accommodates the diverse backgrounds and levels of experience of thelearners who come to use it. It is self-paced
and freely available, making it a valuable
complementary resource for use before andduring the early stages of hands-on training.
Another helpful feature is that modules
can be tailored to just those componentsthat are relevant to the students in question.
Teachers and trainers who include microscopy in their courses provide us with very
positive feedback.

Challenges and Outlook
Tracking the use of MyScope consistently overtime is currently challenging due to the change
of Google Analytics to GA4. However, many
thousands of users from nearly 100 countries
are engaging with all the platform modules
with extended periods of use per session. A

© Creative Commons Attribution-ShareAlike 4.0 International license

Light Microscopy

A)

B)

C)

D)

Fig. 3: Examples of interactive components in the Cryo-EM (A & B) and SIMS (C & D) modules.
A. Projection of a rotatable 3D object (image courtesy: Martyn Cook). B. Class sorting activity.
C. Activity where the user adjusts and selects the optimal slit size for an electrostatic analyzer.
D. Activity where the user selects the optimal slit size for a magnetic sector analyzer.

clearer pattern of use will emerge as GA4 analytics are collected going forward. Also, withconstraints on staff availability and funding,
it is increasingly challenging for us to continue developing MyScope, with impacts felt incontent preparation and online development.
However, we are committed to improving andmaintaining the platform, including more integration of Microscopy Australia videos andlinks to external resources available throughreputable international sources.
To finish, this comment from an independent external review of Microscopy Australia summarises the impact of MyScope:“The MyScope microscope simulators andonline training modules are one of Microscopy Australia’s most internationally visible
and cost-effective investments ... There is no

MyScope Training:https://myscope.training

comparable resource to MyScope anywhere
else in the world.”
Affiliation
1 Microscopy Australia, University of Sydney,
Sydney, Australia
2 Centre for Microscopy, Characterisation
and Analysis, University of Western Australia,
Perth, Australia
3 Future Industries Institute, University ofSouth Australia, Adelaide, Australia
Contact
Jenny Whiting, Ph.D.
Microscopy Australia
University of Sydney
jenny.whiting@sydney.edu.au
ORCID: 0009-0006-4377-0859

More information:
https://micro.org.au

References:https://bit.ly/
IM-Whiting© World map by www.freeworldmaps.net; edited by R. NitschkeLight Microscopy

A Global Reproducibility Initiativein Light Microscopy
Community Standards for Instruments, Metadata, and Publications
Roland Nitschke1 and Hella Hartmann2

R

eproducing published research results can be challenging. In the field of light microscopy, there is a global open community bridging the gap between academia
and industry to improve the reproducibility of light microscopy experiments in thelife and materials sciences. The community is working towards establishing standards for
quality assessment and quality control (QA & QC) for imaging instrumentation, improving
reporting of imaging experiments, and data quality regarding metadata contained within.
The development of the initiative QUality Assessment and REProducibility for instruments
and images in Light Microscopy (QUAREP-LiMi) is presented, and the current topics andachievements of the 15 working groups are summarized. Several accessible online resources utilized by QUAREP-LiMi are introduced here, as well as the future goals, followed byan invitation to join and contribute to the success story of QUAREP-LiMi.

Fig. 1: Dynamic development of the QUAREP-LiMi memberships from the foundation in April
2020 until the end of 2023.

18 • Imaging & Microscopy 1/2024

Introduction
The reproducibility crisis of research results has emerged as a major challenge inscience [1], resulting in increased resource
consumption due to additional experiments
required. A lack of awareness, knowledge,
and agreement on common standards andguidelines for quality assessment and reproducibility in the field of light microscopy and image data analysis is the main
reason for the under-reporting of imaging
methods. The community-driven initiative“Quality Assessment and Reproducibility
for Instruments & Images in Light Microscopy (QUAREP-LiMi)” addresses this crisisby (1) defining robust and easy-to-implement Standard Operating Procedures (SOPs)
characterizing relevant instrument parameters for data acquisition, (2) establishing acommon metadata model for image data, including quality control data and associatedanalysis, (3) developing community agreed
guidelines for publication of image dataand its analysis, ensuring reproducibility
and AI-compatible data mining. Ultimately,
these efforts will lead to more robust results

Light Microscopy

Fig. 2: Overview of the members from companies, standardization organizations,academia networks, and research groups working together in QUAREP-LiMi(due to space restrictions, not all member logos were included).

and a significant reduction in the use of resources. This will be achieved by providing
better conditions for new research projects
based on previous, more profound findings.

Groundwork and Organization
Initially, QUAREP-LiMi started as a grassroots collective within the German BioImaging Society and informal meetings of small
groups of scientists at ELMI and other conferences. After an initial international online
meeting with about 50 participants during
the early Covid pandemic, it quickly gained
traction and, since then, has continuously
grown. Figure 1 summarizes the tremendous
development of the QUAREP-LiMi membership numbers since 2020. Today, 590 people
from 41 countries have joined QUAREPLiMi, and 30% of the members are females.
The majority of the 413 academic members
work in light microscopy core facilities or
research labs and have a strong focus onlight microscopy techniques. The 156 members from equipment and software manufacturers are designated specialists in their fieldof expertise like optics engineers, specificproduct experts, and hardware or softwaredevelopers. From those, 21 QUAREP-LiMi
members are involved in national and international standardization organizations suchas DIN or ISO or work as scientific editors
for journals. Figure 2 provides an overview
of the various organizations, networks,
and industry partners working together in
QUAREP-LiMi. All members participate voluntarily and without any financial contribu-

tion or benefit, ensuring complete independence for the best possible progress.
QUAREP-LiMi is organized in working
groups (WGs) focusing on specific topics andtasks. Interested individuals can subscribe to
specific QUAREP-LiMi WGs via online forms.
About 250 members are actively contributing
to 15 WGs, as listed in Table 1 with the main
topics they are focusing on. WGs 1 to 6 arededicated to technical parameters described in
the ISO norm 21073:2019 - Optical data of fluorescence confocal microscopes for biological imaging. WG 7 - Microscopy data provenance and QC metadata and WG 10 - Image
quality interact with most of the other WGs,
as their topics are overarching. WG 8 - Education, training, and outreach, WG 14 -Environment, and WG 15 - FLIM have recently
been established. WG 11 - Microscopy publication standards and WG 12 - Image visualization and analysis concentrate on establishing agreed-upon guidelines for extensive
description and visualization of light microscopy data. WG 13 - Phototoxicity is raisingawareness of the phototoxicity induced by
microscopy and evaluating the best methods
for assessing, minimizing, and reporting phototoxicity.
The work within each WG is coordinatedand guided by at least two elected co-chairs incharge of all WG-related organizational tasks.
Most WGs come together online monthly,and dates are published in an online calendar. All meetings are recorded, and meeting
agendas and minutes, shared working documents, literature, recorded image data sets,
and measurements are collected on a Nextcloud server that is openly accessible to all

QUAREP-LiMi members. QUAREP-LiMi is
active on social media channels like LinkedIn and X, with about 800 followers. Further
details and information are summarized on
the initiative’s extensive webpage.

Achievements and Work in Progress
The QUAREP-LiMi WGs 1-6 are working onor have already published detailed protocols
for the assessment, control, and analysis ofimportant imaging system parameters andproperties:▪ Illumination power WG 1
▪ Detection system performance WG2▪ Uniformity of illumination field-flatness
WG 3▪ System chromatic aberration and co-registration WG 4
▪ Lateral and axial resolution WG 5
▪ Stage and focus precision WG 6QUAREP-LiMi uses the platform protocols.
io for the joint creation, management, andpublication of the protocols. PDF versions of
the protocols are also available for downloadfrom the QUAREP-LiMi protocol website.
Moreover, WGs have developed macros formany protocols to automate image acquisition and instrument control such as microscopes and their devices or power meters. In
addition, WGs created various data and image
analysis program routines together with further tools for visualization of the measured
data over time and their storage. All information is publicly available on the QUAREPLiMi Github account. Working groups 1, 4,

Imaging & Microscopy 1/2024 • 19

Light Microscopy

and 5 also make use of Airtable,
an online platform for gatheringand presenting data. WG 1 collected hundreds of laser power measurements on confocal
microscope systems, which are
publicly available, and new measurements can be conveniently
uploaded via a form or as bulk
data. QUAREP-LiMi operates aYouTube channel that currently
holds 20 videos. Each WG has
published short videos summarizing their work and achievements, and additional videos
explain protocols and measurement routines developed in different WGs. Steered by the newly founded WG 8 - Education,
training, and outreach, moreteaching and training videos
will be put together in the future. In general, QUAREP-LiMi
is putting a lot of effort into
spreading our message within
the light microscopy community by organizing sessions during
the last three ELMI Core Facility
days. Moreover, an ELMI community room has been established that serves as an informal
meeting and discussion point
and playground to see demos ofprotocols in use, and newly developed tools and devices.
As a work in progress, a
QUAREP-LiMi OMERO image
database server has been set up.
All image data currently stored
on the Nextcloud will be transferred to OMERO and will beannotated according to FAIR
data standards. In the future,
the QUAREP-LiMi OMERO database will be open to the generalimaging community and canserve as a repository for standard image data sets of QA &QC measurements.
The QUAREP-LiMi community
initiative has accomplished an
impressive number of 12 high-impact publications since 2020. The
overall aims and the organizationof QUAREP-LiMi were describedin two whitepapers in the Journal of Microscopy [2] and Nature
Methods [3] and are setting theframe for future QUAREP-LiMi
developments. Most remarkably,
Nature Methods highlighted in the
December 2021 issue the importance of improving rigor andreproducibility in microscopy aswell as proper reporting of meta-

20 • Imaging & Microscopy 1/2024

Table 1: QUAREP-LiMi members work together in fifteen working groups(WGs), each dedicated to specific topics and tasks. Meetings of the WGs
are usually every month with about 10-15% of the total WG members attending each time. Members are often part of more than one WG.

data in a collection of nine articles with significant contributions from QUAREP-LiMi members. Four publications describedthe efforts of finding and setting
standards for open image data
formats [4], recommended metadata standards for light microscopy [5,6], and the developmentof a next-generation file format
to expand bioimage data-accessstrategies [7]. Another set of publications focused on tools for collecting, annotating, and publication of image data including
rich metadata as specified by thecommunity and that comply with
FAIR image data principles [8–10].
That impressively demonstrates
that the initiative evolved into
a productive consortium uniting
researchers, facility managers, aswell as companies, standardization organization representatives,and publishers. In July 2022, two
editorials in Nature Methods[11,12] highlighted the joint work
of QUAREP-LiMi on developing
the revision of the 4DN-BINAOME-QUAREP (NBO-Q) Microscopy Metadata specifications
data model related to cameras.
This major task was achieved in
more than 10 online meetings ofcamera specialists from academia
and industry and shall be finalized by the end of 2024. Recently,
the QUAREP-LiMi WG 12 - Image
Visualization and Analysis published “Community-developed
checklists for publishing images
and image analyses” in Nature

Methods [13]. It was further highlighted in the Journal of Cell Sciences [14], resulting in several
publishers expressing their strong
interest in implementing the suggested guidelines. The Journal of
Histochemistry and Cell Biology
already implemented the guidelines [15].

Conclusions
The QUAREP-LiMi initiative has
gained tremendous momentum
in the light microscopy community in a relatively short period
to advance the QA & QC issues
that have been difficult to address for the last 20 years. The
breakthrough seems to have beenachieved by the very open andproductive collaborative approachtaken by all people involved, including academia, industry, standards organizations, and scientificpublishers. The goal of improving
the reproducibility and qualityof research results based on imaging data by monitoring anddocumenting instrument quality
parameters over time with scientifically accepted, harmonized
SOPs, and thoroughly tested tools
is coming closer.
QUAREP-LiMi aims to become
a visible and essential actor in

More information:
https://quarep.org

the scientific landscape. It stands
for sustainable use of imaging
infrastructure, robust and reproducible results, and transparent
reporting. High-quality FAIR
image data, rich in metadata, will
facilitate data reuse. It will also
be a prerequisite for innovative,
next-generation AI-supported
data analysis methods.
Would you like to be part ofthis amazing new community?
Participating in QUAREP-LiMi iseasy and free – please complete
the membership form. Contributing actively can be quite somework, but you will meet an open
community and amazing colleagues along the way. Everyexpertise counts, and you could
be part of the next success story

Acknowledgment
We want to thank all the
QUAREP-LiMi members who
dedicate a part of their free timeto this initiative. R.N. would liketo thank the team at the Life Imaging Center for their support
of QUAREP-LiMi (in particular,
Arne Fallisch) and their comments on this manuscript. R.N. is
supported by grant NI 451/10-1
from the German Research Foundation and grant 03TN0047B‘FluMiKal’ from the German Federal Ministry for Economic Affairs and Climate Action.
Affiliation
1 Life Imaging Center and
Signalling Research Centres
CIBSS and BIOSS, University ofFreiburg, Germany
2 Light Microscopy Facility,
Technology Platform, Center for
Molecular and Cellular Bioengineering (CMCB), University ofTechnology Dresden, Germany
Contact
Dr. Roland NitschkeLife Imaging Center and SignallingResearch Centres CIBSS and BIOSS
University of Freiburg, Germany
Roland.Nitschke@biologie.unifreiburg.de
ORCID: 0000-0002-9397-8475

References:https://bit.ly/IM-Nitschkespeed

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y

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3D Raman image of a pharmaceutical ointment.

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www.witec.de© Dr. Thomas R. Neu

Light Microscopy

Three-dimensional semi-transparent and isosurface view after dual lectin staining in combination with nucleic acid staining. Take notice ofthe two lectin signals showing volumetric versus filamentous features and the large nucleic acid stained bacterial cells partly inside the green
lectin signal. Color allocation: lectin AAL-Alexa488 (green, isosurface semi-transparent), lectin PNA-TRITC (red, isosurface), Syto60 (blue,
isosurface). Grid size: 20 µm.

Out of this World –Hidden Biofilms Under the Microscope
The Matrix of Cave Snottites Studied by Lectins and Confocal Microscopy
Thomas R. Neu

M

icrobial biofilms are present in numerous locations including medical, technical, and natural habitats. A specific form of biofilms found ina semi-artificial cave is the subject of thepresent study. The biofilm formed pendulous
snottites at the ceiling of the cave. Confocal
laser scanning microscopy represented theideal technique for investigating the snottite architecture and assessing their hydrated structure. Fluorescently labeled lectins
proved to be a very useful probe for examining the extracellular matrix of microbial
snottites. The lectin approach, comprisingfluorescence lectin bar-coding (FLBC) andsubsequent fluorescence lectin binding
analysis (FLBA), identified the glycoconjugates that represented the main volumeof the snotittes. In combination with otherfluorochromes, the composition and structure of these snotittes were characterized
for the first time.
Introduction
Biofilms are defined as microbial communities associated with an interface (solid, liquid, gas) together with their self-produced
extracellular polymeric substances (EPS).
Microbial biofilms come in many different

22 • Imaging & Microscopy 1/2024

shapes, textures, colors, and dimensions.
They range from bacterial cell monolayers
via micro-colonies to biofilms (immobile)and bio-aggregates (mobile). They may
reach macroscopic dimensions in the formof massive biofouling layers (industrial) or
microbial mats (natural). Biofilms are highly relevant in the environment, technicalsystems (positive and negative), and the
medical field (mostly negative). Initially, themicroscopic analysis of biofilms was doneon dehydrated samples using scanning electron microscopy. However, the invention ofconfocal laser scanning microscopy (CLSM)
revolutionized the view of this universally present microbial growth mode. Using
CLSM, the fully hydrated biofilm system
could be examined by employing several
different fluorescent probes. Thereby, notonly the bacterial cell distribution could beassessed but also the so-called “dark matter” of biofilms, the extracellular polymericmatrix [1,2].
Microbial biofilms in the environment arepresent in all kinds of habitats, including
aquatic, sediment, soil, and rocks, and they
may be associated with plants, fungi, andanimals. A specific type of biofilm can befound in caves where they grow on humid
rock surfaces, in flowing water, and in theform of snottites. The latter develop at theceiling of caves and are characterized bysoft, gel-like, pendulous structures up to asize of 10 cm and longer. The snottites investigated in the present study were collected
in a semi-artificial, methane-driven cave,which was used as a former medicinal springrich in iodate [3].

Methods
In the case of snottites, the finger-size gelatinous material was sectioned with a scalpel
and mounted in a cover well chamber withthe appropriate spacer. Samples were examined using 25x NA 0.95 or 63x NA 1.2 water
immersion objective lenses.
The methodology of lectin staining has
been published in detail [4,5]. It’s a straightforward and easy approach, including staining and washing the sample. In short, thelabeled lectin is applied to the sample at aconcentration of 0.1 mg/mL. Usually, thebiofilm sample is covered with a few droplets of the working solution. A time series
revealed that an incubation in the dark for
20 min is appropriate. Then, the samples arecarefully washed 3-4 times to remove theunbound lectin. Usually, a nucleic acid-specific fluorochrome is applied as a counterstain.

Light Microscopy

The examination of biofilms by CLSM is ideally performed with a common instrument
having an upright microscope. This setup ismost flexible for different types of biofilms.
It will allow the use of 1) slides with coverslip and spacer, 2) cover well chambers withspacers of different dimensions, and 3) imaging the sample in a small Petri dish using
water-immersible lenses.

Lectins
Latin nameCommon name

3-letter code

Inhibiting carbohydrate

Aleuria aurantia

fungusAAL

α-Fuc

Musa paradisiaca

bananaBan

Glc, Man

Lycopersicon esculentum

tomatoLEAβ-GlcNAc

Arachis hypogaea

peanutPNA

β-Gal

Table 1: Characteristics of the four lectins identified as useful for fluorescent lectin binding
analysis (FLBA) after fluorescence lectin bar-coding (FLBC) screening.

Main Developments
bacterial cell surface, bacterial footprints,
extended capsules of bacteria, microenvironment of micro-colonies, or matrix of
biofilms and bio-aggregates. As lectins are
specific for carbohydrate residues, they do
not always show the overall biofilm matrix.
Despite this issue, they often result in a reasonably good signal showing glycoconjugate
features of interest in microbial communities. Hence, lectins are not a general probe
for all glycoconjugates present in a biofilm
sample; however, they are currently the bestcompromise for glycoconjugate imaging via
fluorescence in combination with CLSM.
To examine the snottite samples, we initially tried a range of different fluorochromes
to get a first idea of their basic structure, e.g.,with nucleic acid and protein-specific stains.
As an example, SyproOrange showed different types of bacterial shapes as well as
filamentous features (Fig. 1). Then we performed the screening via FLBC to identify
the most valuable lectins. As a result, wefound four lectins that were employed on
various snottites as well as in different areasof the snottite. The lectins AAL, PNA, LEA,
and Ban (Table 1) showed amazing voluminous structural features. Figures 2, 3, and 4
show lectin signals in the form of capsulesand globules, tubular structures, and filamentous features together with bacterial cell
distribution. The fluorochrome “stains all”
allowed the separation of bacterial cells from
globular signals in two emission channels(Fig. 5). The lead image shows a triple combination of fluorescent stains showing tubelike features, filaments, and large bacterial
cells. The specificity of the four lectins suggested α-Fuc, β-Gal, β-GlcNAc, and α-Manas major glycoconjugates. Throughout thedominating lectin signal, bacteria of different sizes, shapes, and lengths were found. In
fact, some of the filamentous bacteria formed
a dense network and might be responsible
for the additional strength of the snottites.
Experiments with fluorescent beads showed
an outer layer that was not penetrated by
the beads (data not shown). This indicateda surface layer around the snottites, which
could not be characterized in further detail.
For more details on snottite architecture, thereader is referred to the original article [9].
Additional approaches might be suggested to reveal more detailed information.
For example, non-commercial lectins fromresearch labs could be tested. Another option
is carbohydrate-binding modules (CBMs) as
a probe. The next logical strategy would© Dr. Thomas R. Neu

Microbial biofilms and bio-aggregates were
examined by CLSM in numerous studies
with a focus on bacterial cell distribution.
For this purpose, nucleic acid-specific fluorochromes were usually employed. Morerecently, molecular techniques have taken
advantage of fluorescent proteins such as
GFP, YFP, RFP, or others for labeling bacteria in biofilms. Nevertheless, the extracellular matrix proved to be an intractablefeature of biofilms [6]. The main reason isthe complexity of the matrix consisting ofpolysaccharides, extracellular nucleic acids, various types of proteins (e.g., enzymes,
structural proteins, amyloids), lipophilic
constituents, as well as bacterial-derived
refractory substances. For all these matrix
compounds, there is no single fluorescent
stain available. Even more, there is no universal fluorochrome on hand for one of the
major compounds, polysaccharides. One fluorochrome, somehow specific for polysaccharides, is calcofluor white, but it will bindto β 1-3 or β 1-4 polysaccharides only. This
linkage type is typically found in chitin andcellulose.
As a consequence, there is an urgent needto have a fluorescent probe for polysaccharides or, more specifically, for glycoconjugates. Such a probe is available in the formof fluorescently labeled lectins. Lectins havebeen used as a probe for several years, often
as a single probe only. During our confocal
work and the investigation of biofilm systems, we used more and more different typesof lectins. Finally, we collected all commercially available lectins (about 80) to assess
the biofilm glycoconjugate matrix. Thus,
when we got a new type of biofilm sample, we tested each of the lectins individually on 80 pieces of biofilm. This screening, we named fluorescence lectin bar-coding (FLBC). Based on FLBC, we identified
some useful lectins binding to the structureof interest. In a second step, we employed
this panel of lectins for the actual study. This
subsequent assessment of the samples from adefined experiment we named fluorescencelectin binding analysis (FLBA) [7,8].
Due to their specificity, lectins will binddifferentially at various locations within
microbial biofilms. They may stain theFig. 1: Transparent 3D view of a snottite stained with SyproOrange showing bacteria of different
sizes and shapes together with filamentous structures. The reflection signal inside bacteria indicates solid inclusions. Color allocation: SyproOrange (red), reflection (grey). Grid size: 20 µm.

Imaging & Microscopy 1/2024 • 23

© Dr. Thomas R. Neu© Dr. Thomas R. Neu

Light Microscopy© Dr. Thomas R. Neu

Fig. 3: Transparent 3D view of a snottite stained with the same lectin
but with another label and a different nucleic acid-specific stain. The
bacteria are embedded in extended glycoconjugates in the form of globules and tube-like shapes. Color allocation: AAL-Alexa488 (green),
Syto60 (blue). Grid size: 20 µm.

© Dr. Thomas R. Neu

Fig. 2: Transparent 3D view of a snottite stained with a lectin and anucleic acid specific counterstain showing bacteria with and without
capsules as well as large globular lectin signals. Color allocation:
AAL-Alexa568 (red), SybrGreen (green). Grid size: 20 µm.

Fig.4: Transparent 3D view of a snottite showing a group of bacteria
embedded in globules with filamentous structures and mineral particles. Color allocation: reflection (grey), AAL-Alexa568 (red), SybrGreen(green). Grid size: 20 µm.

include gene probes for the different bacteria
already identified by molecular analysis. This
fluorescence in situ hybridization (FISH) or
CARD-FISH approach can be combined withthe lectin approach [10]. Thereby, the identityof the bacteria can be linked to the identityof the glycoconjugates. Lately, new probes
for matrix constituents using oligothiophenes
were suggested for so-called optotracing.
Finally, other fluorescent techniques such as
FRAP and FCS, may help measure cave snottites’ diffusion characteristics.

Conclusions
Confocal laser scanning microscopy is an
ideal technique for studying hydrated, gelatinous snottites. Fluorescence lectin bar-cod-24 • Imaging & Microscopy 1/2024

Fig. 5: Transparent 3D view showing the edge of a snottite treatedwith “stains all” and recorded in two emission channels. Take notice ofthe differentiation between bacterial cells and globular signals. Color
allocation: emission 570-620 nm (red), emission 650-720 nm (blue).
Grid size: 50 µm.

ing (FLBC) with subsequent fluorescencelectin binding analysis (FLBA) revealed thearchitecture of snottites. The lectins showedvoluminous, globular, and tube-like structures interwoven with bacteria of different
sizes and lengths. Of the 78 lectins tested,
the lectins AAL, PNA, LEA, and Ban indicated α-Fuc, β-Gal, β-GlcNAc, and α-Man as
major glycoconjugates. Methane and iodate,
together with the microbial metabolism of
methano- and methylotrophic bacteria, may
be the primary reason for the snottite architecture.

tite project is highly appreciated. I am very
thankful to Ute Kuhlicke, who skillfully
worked with me at the laser microscope forover 25 years.

Contact
Dr. Thomas R. Neu
Department of River ResearchHelmholtz Centre for Environmental Research – UFZ
Magdeburg, Germany
ORCID: 0000-0001-6990-8787
thomas.neu@ufz.deAcknowledgments
The cooperation with Tillmann Lüders andClemens Karwautz concerning the snot-

References:https://bit.ly/IM-Neu1

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Light Microscopy

High-Speed Molecular Imaging
Line-Illumination Raman Microscopy with Slit Array Detection
Tomoaki Okumura1,2, Yasuaki Kumamoto1,3, Katsumasa Fujita1,2,3

Raman microscopy is an emerging molecular imaging technique that does
not require the labeling of targetedmolecules in specimens. A key to the emergence of Raman microscopy is the development of high-speed imaging techniquesthat enable the analysis of biological specimens susceptible to temporal changes oralterations. Here, we introduce a highspeed Raman hyperspectral imaging technique developed based on line-illumination
Raman microscopy. The technique, namely
multiline-illumination Raman microscopy,
equips periodic arrays of line-shaped laser
focus and confocal slits, enabling simultaneous spatial sampling of 10,000-20,000
Raman spectra and spectral sampling of
100 wavenumbers per exposure, which
performs high-throughput Raman hyperspectral image acquisition of (105 spectra
× 102 wavenumbers)/min. We anticipate
that our technique will enhance the utilityof Raman microscopy in practical applications in biology and medicine.

26 • Imaging & Microscopy 1/2024

Introduction

Strategy for Accelerating Raman Imaging

Raman microscopy has emerged in the last
decade as a molecular imaging techniqueenabling label-free molecular analysis [1].
The capability of Raman microscopy has
been studied for a variety of biomedical applications, i.e., observing drug response inliving cells at the molecular level [2], cellsorting [3], intraoperative rapid diagnosis[4], and identification of cell differentiation[5]. However, the slow imaging speed of Raman microscopy hampers the examinationof practical applications of Raman microscopy in the life sciences, mainly due to thesmall Raman scattering cross-section andpoint-by-point scanning scheme for image
construction. Although multiplexing spatialsampling, e.g., by line-illumination [6] andmultifocal illumination [7], has been applied
to increase the speed by 2-3 orders of magnitude, Raman hyperspectral imaging often
takes a few tens of minutes or even longer
than an hour [6, 8].

In conventional confocal Raman microscopy, a laser beam in the form of a spot illuminates the sample, and a single Raman
spectrum is recorded using sensors only inseveral lines of a two-dimensional sensor
array (Fig. 1A). In multiplex spatial sampling technique using line-shaped illumination, the spatial distribution of the spectra in the sample is recorded on the sensor
array by expanding the spatial information along y-axis, which is orthogonal to
the wavelength dispersion axis (x-axis), as
shown in Figure 1B. The number of spectra
simultaneously acquired here is determinedby the number of sensors in the y-axis used
for recording, i.e., typically 100-1,000, resulting in the imaging speed faster by twoor three orders of magnitude compared toconfocal Raman microscopy [9]. To further
improve the imaging speed by multiplex
spatial sampling, we increased the numberof illumination lines in the x-axis to acquireLight Microscopy

more spatial information simultaneously ina trade-off with reducing the wavenumber
information (Fig. 1C).

Multiline-illumination Raman Microscopy
with Slit Array Detection
We have developed a multiline-illumination
Raman microscope equipped with a confocal slit array (Fig. 2) [10]. A cylindricallens array formed a high-power laser beamwith a wavelength of 532 nm into multiple
line-shaped laser beams. The multiple lineshaped beams were relayed to the sample
plane and excited Raman scattering in asample. The Raman scattering was collected with an objective lens, separated fromthe excitation beam path by using an edge
filter, and refocused at the entrance of thespectrophotometer, where a periodic slit array was located instead of an ordinary entrance single slit. Each slit was conjugatedto one of the irradiated regions at the sample and worked as a confocal slit. To avoid
unwanted spectral mixtures among different
sample regions in the sensors of the spectrophotometer, the wavelength range of Raman
scattering entering the spectrophotometer
was limited by a bandpass filter. A comblike Raman hyperspectral image excited bymultiple illumination lines is acquired by asingle sensor exposure, where each Raman

Fig. 1: Schematic illustration of laser-scanning and spectral detection in three different types oflaser-scanning Raman microscopy, i.e., (A) confocal, (B) line-illumination, and (C) multiline-illumination Raman microscopy. In line-illumination Raman microscopy, spatial information isrecorded along y direction on the spectrophotometer sensor array. In multiline-illumination Raman microscopy, spatial information is expanded in the x direction as well for further multiplexing spatial sampling.

spectrum includes specific Raman peaks
from target molecules in the sample.
Raman hyperspectral image from polystyrene (PS) and polymethylmethacrylate(PMMA) beads dispersed on a quartz coverslip is shown in Figure 2. In the spectra
corresponding to the CH vibration region(2,850–3,100 cm-1), the characteristic Ramanpeaks of PMMA (green) and PS (magenta)
are found at 2,953 and 3,047 cm-1, respectively. To acquire a two-dimensional Raman
hyperspectral image (Raman spectra distributed in two-dimensional space), the sample
was scanned with the slit array and multiple
illumination lines by steering a galvanometer mirror. A shutter in the beam path opened

Fig. 2: A schematic diagram of the multiline-illumination Raman microscope equipped with a confocal slit array. Each Raman band in the Raman
hyperspectral image corresponds to the CH region, where characteristic Raman peaks from polystyrene (magenta) and polymethylmethacrylate(green) beads are found at 2,953 and 3,047 cm-1, respectively.

Imaging & Microscopy 1/2024 • 27

Light Microscopy

Fig. 3: (A) Raman spectra and image of living cultured HeLa cells. The image shows the distributions of the Raman signal at 2,854 cm-1 (magenta)
and 2,935 cm-1 (green). (B) Raman spectra and images of human dermal tissue. The Raman image was reconstructed using the Raman intensities of
2,850 cm-1 (blue) and 2,945 cm-1 (yellow). All the protocols for treating the clinical specimen were performed by the declaration of Helsinki andwere approved by the Institutional Review Board in Osaka University Hospital (20108). The written consent form was obtained from the participant
who underwent skin transplant surgery, and a surgical discard was used.

and closed synchronously with the start andend of a sensor frame exposure, respectively,
to prevent the excitation laser beams fromexcessively irradiating the sample.

High-throughput Raman Hyperspectral
Imaging of Cell and Tissue
Here, we present Raman images of biological
specimens to show the utility of the multiline-illumination Raman microscope for molecular imaging of biological samples. Figure
3A displays the Raman spectra and image forliving cultured HeLa cells. In the spectra, theband at 2,854 cm-1 can be assigned to theCH2 asymmetric stretching vibrational mode,
while the band at 2,935 cm-1 can be assigned
to the CH3 symmetric stretching vibrationalmode. The Raman images reconstructed using
the intensities at 2,854 and 2,935 cm-1 canillustrate the distributions of lipid and protein, respectively, in the cells [11]. Small dotsof lipid, i.e., lipid droplets, are imaged without significant distortion, indicating that themultiline-illumination Raman microscope has
sufficiently high spatial resolution to observe
organelles in living cells.
A measurement result for clinical specimensof frozen dermal tissue is shown in Fig. 3B. In
the spectra, two characteristic Raman bands
were identified at 2,850 and 2,945 cm-1. The
Raman image reconstructed with the intensity
at 2,945 cm-1 shows collagenous fibrous structures that occupy most of the part in the dermal tissue [12], while the Raman image recon-

28 • Imaging & Microscopy 1/2024

structed at 2,850 cm-1, exhibiting a warped
shape, presumably shows elastin fibers thatconnect collagen structures which distributeamong them [13]. These results indicate thatthe multiline-illumination Raman microscopevisualizes tissue structures that can be usefulfor studies of tissue chemistry and morphology in a label-free manner [14,15].
Each Raman hyperspectral image in Figure 3
is composed of 920 × 1,300 spectra. The dataacquisition time was 8.0 min (5.0-s exposureand 0.2-s readout per single frame acquisition of the sensors) for each dataset, corresponding to a throughput of 2 × 105 spectra/
min. This throughput is an order of magnitude higher than that in line-illumination
Raman microscopy with single-slit detection, which can realize the ultrafast Raman
imaging of biological specimens in practicalconditions.

Conclusion
We have developed a high-speed line-illumination Raman microscope equipped witha confocal slit array, where the image acquisition speed was improved by increasing thenumber of spectra that can be acquired witha single exposure. With the developed technique, we achieved a throughput with theorder of 105 spectra/min in cell and tissue
imaging, marking a 10-fold improvement inspeed compared to the single-line-illumination technique. Currently, to accelerate theimage acquisition further, we are developing

a technique capable of achieving speeds on
the order of 106 spectra/min by efficiently
detecting Raman scattering in a shorter exposure time. We believe that the presented
high-speed molecular imaging techniquewill further expand the utility of Raman microscopy in the fields of biology and medicine, not only for pursuing basic sciencebut also for developing new applications inclinics and related industries.
Affiliation
1Department of Applied Physics, Graduate

School of Engineering, Osaka University,
Osaka, Japan
2AIST-Osaka University Advanced Photonics and Biosensing Open Innovation laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Osaka,Japan
3Institute for Open and Transdisciplinary
Research Initiatives (OTRI), Osaka University, Osaka, Japan
Contact
Prof. Dr. Katsumasa Fujita
Department of Applied Physics
Graduate School of Engineering
Osaka University, Japan
ORCID: 0000-0002-2284-375X
fujita@ap.eng.osaka-u.ac.jp

References:https://bit.ly/IM-Fujita

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Electron & Ion Microscopy

Attosecond Electron Microscopy
Movies of Light in Space and TimeDavid Nabben1, Joel Kuttruff1, Peter Baum1

T

he fundamental process behind almost any light-matter interactionis the motion of electrons in theextremely fast oscillations of the electromagnetic wave. By advancing electron
microscopy to attosecond time resolution[1], we can now measure light waves andtheir interactions with complex materials
as a movie in space and time. We simultaneously achieve a spatial resolution of
nanometers and a time resolution in theattosecond range, smaller and faster thanthe oscillations of light. Selected resultsin nanophotonics and metamaterials show
the potential implications of our technique for future research.
Introduction

Almost all matter around us consists ofatomic nuclei surrounded by a shell of electrons. The arrangement of these constituents
into molecules, solids, or nanostructures
determines almost all of the macroscopic
functionality. When a reaction occurs, ora material interacts with light, the atoms
and electrons must shift from initial to final
configurations. For example, the outcome
and properties of phase transitions, chemical reactions, or optical phenomena are alldetermined by microscopic movements andrearrangements of the atoms and electrons
within.

30 • Imaging & Microscopy 1/2024

Knowing as much as possible about suchprocesses on an atomic scale is therefore
crucial for modern research, not only to provide a fundamental perspective on complex
material dynamics but also for developing
innovative electronic and photonic devices
for novel application regimes. However, thedirect spatial and temporal observation ofelectronic motion in atoms, molecules, solids, or nanostructures is still an unreachedmilestone in modern physics.
The difficulty lies primarily in theextremely small spatial and temporal scales
on which all these elementary processes aretaking place. An atom is approximately 10-10
m in size, ten thousand times smaller than
the wavelength of light. The time scales on
which electronic processes take place areattoseconds, that is 10-18 s, which is ten to
hundred times faster than one oscillation
period of light. Experimental access to these
unimaginably fast domains of time poses
an enormous challenge, in particular when
the spatial resolution of the atoms has tobe maintained. Figure 1 shows some fundamental physical processes in their relevant
dimensions with the limits of current experimental measurement techniques.

Attosecond Electron Microscopy
Recently we have succeeded in finding anovel approach to tackle this challenge [1].

We combine a transmission electron microscope which is capable of atomic spatial resolution with a continuous-wave laser that
advances the time resolution into the attosecond domain. With this new technology,
we can now create movies of light-matterinteractions in nanophotonic objects andmetamaterials with attosecond and nanometer resolutions in space and time [1]. Thisnew methodology can help researchers observe and understand fundamental atomic
and electronic processes in complex materials and then utilize these results for advanced material design.
Figure 2 shows the principles of our attosecond electron microscopy. It is based ona stroboscopic measurement principle, similar to a classical pump-probe experiment,
only that the optical oscillation periods ofcontinuous laser light rather than ultrashortlaser pulses are used for the excitation of thespecimen. We drive the process under investigation repetitively with the optical cyclesof laser light and then interrogate the system
response with high-energy electron pulsesthat have short enough de Broglie wavelength to resolve the atomic details of thespecimen. To achieve the necessary time resolution as well, the probe pulses in the electron microscope must be shorter than half
an optical cycle of the excitation light, that
is, attoseconds.
To generate our attosecond electron
pulses, we cross the continuous electron

Electron & Ion Microscopy

Fig. 1: Fundamental physical processes and their dimensions in space and time. Color shades
denote the available measurement methods, that is, laser spectroscopy (red), X-ray diffraction(blue), and electron microscopy (green). Most processes above the dashed line at one femtosecond cannot be addressed as a movie in space and time with existing technologies because either
the temporal or the spatial resolution is too low. However, our recently developed attosecond
electron microscopy (green arrow) can now provide access.

beam of the transmission electron microscope with the electromagnetic fields of acontinuous-wave laser. The extremely fast
oscillations of the laser wave then periodically modulate the energy and thus the speedof the electrons. Later/faster electrons eventually catch up with earlier/slower electrons[2], and the electron beam transforms into a
rapid sequence of attosecond electron pulses
which we then use to interrogate the ultrafast processes and material reactions at the
sample site [1].
In more detail (Fig. 2), a continuous-wave
laser (red) is split into two paths that are
directed into the transmission electron
microscope (gray) at two different points. The
first part of the laser modulates the electron
beam (blue) into a sequence of attosecond
electron pulses with the help of an ultra-thindielectric membrane (green) that is needed
to provide energy and momentum conservation [3-4]. The second part of the laser,
phase-locked in time, triggers the dynamics
to be investigated in the specimen (yellow).
The enlarged section in Figure 2 shows theultrafast excitation of electromagnetic nearfields (orange and turquoise) in the optical
cycles of the laser light (red) in the example of a nanometer-sized needle resonator(black). Depending on their arrival time, theattosecond electrons (blue) are either accelerated or decelerated in the temporally frozen near-fields, and the energy ∆E(r, ∆t) of
the electrons changes as a function of space
and time (r and ∆t).
A magnetic spectrometer in the electron microscope (the 90° curve) then spectrally separates the electrons in the image

and selects only those electrons for imaging that have gained energy at the specimen.
The magnified image then shows the spatial distribution of the time-frozen electric
near-fields in and around the sample at the
selected time ∆t. By varying ∆t with a micromechanical mirror (gray arrows), we obtain amovie of the dynamics at the nanostructurewith attosecond time resolution. The head
figure on the first page shows a photographof the experiment.
Extremely sharp metal needles with a
tip radius of only a few nanometers havea wide range of applications in classicaland ultrafast electron microscopy, but also
in nanoplasmonics, where they are used innear-field scanning electron microscopes for
optical surface analysis. The curved surfaceof our tungsten needle and its small radiusof 150 nm enables the formation of surface
waves in the form of plasmon-polaritons,
whose propagation along the needle tip we
can film in space and time (Fig. 3). Complete slow-motion movies of the measured
dynamics can be found in the supplementarymaterial of our original report [1].

Diagnostics of Metamaterials
By cleverly arranging many such resonators
on nanometer dimensions in the form of socalled metamaterials, researchers can create
macroscopic optical responses that cannot
be achieved with any natural materials. Examples include flat lenses, cloaking layers,
sensors for chiral molecules, condensers forsolar cells, or quantum optical circuitry. In

Fig. 2: Concept of our attosecond electron microscope. A continuous wave laser (red) modulates the continuous electron beam (blue) ofa transmission electron microscope (gray) at
an ultra-thin dielectric membrane (green) into
a sequence of attosecond pulses. A second
part of the laser beam excites the object to be
examined (yellow) and generates location-dependent and time-dependent energy changes∆E(r, ∆t). An energy-filtered image and variation of the time delay ∆t between the two laser paths can then be used to measure theelectromagnetic response of the sample as amovie in space and time.

realistic metamaterials, however, only theoverall optical response can be measured,
while the functionality of the individual
resonators remains unclear. For the effective
development of such structures, however,the field and emission dynamics of the individual elementary building blocks, the socalled meta-atoms, must be understood andthe time delays and interferences between
several meta-atoms must be determined andtaken into account.
Figure 4 displays a series of time-frozen snapshots from our attosecond electron
microscope. The specimen is an arrangement of four dielectric slot resonators in atilted, zig-zag geometry which has potential
applications in the field of meta-lenses. In
the attosecond movies, we see that localizednear-field resonances appear within all of
the slits, like in radio antennas, but smaller
than the wavelength of light. We also observe
how a complex, dynamic interference pattern of the radiated fields arises aroundthe nano-antennas which determines anddefines the macroscopic optical response.
In particular, we see a temporal symmetry
break between the upper and lower halves
that is probably generated by non-planarcomponents in the material. We conclude
that our attosecond electron microscopy can

Imaging & Microscopy 1/2024 • 31

Electron & Ion Microscopy

Fig. 3: Movies of light-matter interactions with the attosecond electron microscope. Plasmon-polaritons are excited with a laser on a tungsten needle
and propagate downwards along the surface. Attosecond electron microscopy provides a movie of the electric fields (blue, red) in space and time.

not only measure the functionality of individual antennas but also trace the collective
optical responses of nanophotonic superstructures back to their elementary components. Our measurement principle cantherefore contribute to the development ofnovel and better nanophotonic materials by
understanding their functionality on fundamental dimensions in space and time. Moreadvanced results, in particular on the chirality of nanometer light, can be looked up in
our original report [1].

Fig. 4: Optical field-cycle dynamics in a dielectric metamaterial. The functionality of the individual
nanoresonators and their collective response in the far-field is measured with our attosecond electron microscope as a movie in space and time.

Conclusions and Outlook
So far, the reported results [1] are only a first
demonstration of the new capabilities of ourattosecond electron microscope on selectedmaterials from optics and nanophotonics.
However, we believe that attosecond electron microscopy and the insight it delivers
into light-matter interaction can open up
many more application fields.
For example, a long-standing goal inchemistry is to increase the speed of chemical reactions through the use of catalysts.
However, most industrial catalysis requires
high temperatures and/or pressures that
often limit the reactivity, selectivity, stability, or throughput rate. An emerging areaof research in catalysis is therefore the utilization of plasma resonances in metallic
nanoresonators in combination with lightand aims to use hot charge carriers or tailored electromagnetic near-fields in nanomaterials to specifically control chemical
reactions. The distribution and dynamics
of the laser-induced optical near-fields inand around the resonators are crucial here.
As nanotechnology approaches the atomic
frontier and plasmonic decay processes
become as fast as one oscillation periodof the exciting light, the problem becomes
highly involved. Here, our attosecond electron microscopy could make a decisive contribution, because nanometer structures andtheir attosecond dynamics can be measured
explicitly in space and time, to understandthe influence of local optical fields on chem-

32 • Imaging & Microscopy 1/2024

ical processes and in turn design more effective catalysts.
Another area of research where we foresee significant practical benefits is computerscience. Our desire for ever smaller and everfaster electronic circuitry, operating ultimately at the frequency of light, creates afundamental problem: How can one measure a world-fastest prototype when electronic methods themselves are obviously
too slow? We believe that ultrafast electron
microscopy can become a crucial tool here,
because electron-optical measurement methods are already standard in chip manufacturing and quality control, and nothing fundamental stands in the way of adding time
resolution here.
In addition to the use of continuous laser
light for attosecond pulse formation in ourelectron microscope, pulsed lasers can also
be applied and will provide stronger specimen excitations at the expense of the average signal strength. Forthcoming publications from other research groups [5,6]already show some of these emerging possibilities, for example, in ultrafast scanningelectron microscopy [5] or in attosecond
electron interferometry [6]. In our laboratories, the next steps for further optimization of the measurement procedures will
include the improvement of the signal-tonoise behavior using improved modulation
membranes, the use of femtosecond sample
excitation for investigating nonlinear opticalresponses as well as measurements of fully
three-dimensional electric and magnetic
fields by attosecond electron tomography.
In all these activities, it seems important
to us to recall that the electron microscope is
one of the most versatile measuring instruments of our time, as is the laser. We therefore expect a very broad application rangeof our new attosecond imaging approach to
many open questions of modern research,by providing space-time access to opticalcomponents, lasers, metamaterials, chemical reactions, solar cells, or photocatalysis
on an attosecond and atomic scale.
Parts of this text and of the figures are
adapted from the PhD thesis of David Nabben, University of Konstanz, 2023.
Affiliation
1
Universität Konstanz, Germany

Contact
Prof. Dr. Peter Baum
Universität Konstanz
Konstanz, Germany
peter.baum@uni-konstanz.de

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Scanning Probe Microscopy

Next-Generation Time-Resolved
Scanning Probe Microscopy
Ushering in a New Era of Nanoscale Exploration with Enhanced Usability
Katsuya Iwaya1, Hiroyuki Mogi2, Shoji Yoshida2, Yusuke Arashida2, Osamu Takeuchi2, Hidemi Shigekawa2

Understanding the nanoscale carrier
dynamics induced by light excitationis the key to unlocking futuristic
devices and innovative functionalities inadvanced materials. Optical pump-probe
scanning tunneling microscopy (OPP-STM)
has opened a window to these phenomena.
However, mastering the combination ofultrafast pulsed lasers with STM requires
high expertise and effort. We have shattered this barrier and developed a compact OPP-STM system accessible to all.
This system precisely controls laser pulse
timing electrically and enables stable laser
irradiation on sample surfaces. Furthermore, by applying this technique to atomic force microscopy (AFM), we have captured time-resolved force signals with an
exceptionally high signal-to-noise ratio.
Originating from the dipole-dipole interactions, these signals provide insights into
the carrier dynamics on sample surfaces,
which are activated by photo-illumination.
These technologies are promising as powerful tools for exploring a wide range ofphotoinduced phenomena in conductive
and insulating materials.

34 • Imaging & Microscopy 1/2024

Introduction
The need for exceptional spatial and temporal resolutions is at the heart of deciphering
the intricate carrier dynamics in nanoscalematerials. Conventional scanning tunneling microscopy (STM), although providing
unparalleled spatial and energy resolutions, hits a temporal resolution ceiling at
sub-millisecond levels owing to preamplifier
bandwidth constraints. With the integrationof transformative OPP techniques into STM,
such barriers have been overcome, realizing
higher temporal resolutions. This fusion ispivotal in probing materials‘ complex nonequilibrium carrier and spin dynamics [1,2].
Another technique, the use of a subcycle
electric field as bias voltage, has been incorporated as an electric-field-driven STM.
This technique provides temporal resolutions of less than 1 ps and 30 fs, maintaining the spatial resolution of STM using terahertz (THz) and mid-infrared pulses [3-6].
This leap in technology widens the horizonof time-resolved STM; however, it still requires high expertise and simplification of
the system to make it accessible for a broad

spectrum of research endeavors. In addition,
since the application of STM is limited to
conducting materials, extending this OPP
technique to atomic force microscopy (AFM)would further expand the capabilities of thistime-resolved measurement technique.

Optical Pump-Probe Scanning TunnelingMicroscopy (OPP-STM)
In the conventional OPP method, pump lightand probe light, which are delayed in time,
are irradiated onto a sample, as shown in
Figure 1a. When carriers such as electrons
and holes excited by the pump light remain
in an excited state, the excitation by theprobe light is suppressed (absorption bleaching). Therefore, by measuring the reflectivityof the probe light as a function of the delaytime, one can investigate the dynamics ofthe states excited by the pump light with atime resolution corresponding to the pulse
width of the excitation light. However, thespatial resolution is limited to the diffraction and light-spot size (around micrometerorder).

Scanning Probe Microscopy

In OPP-STM, the sample surface under the
STM tip is first excited by a pump pulse andsubsequently by a probe pulse with a delay
time td, and the tunneling current is detectedusing a conventional preamplifier (Fig. 1b)[1]. When td is sufficiently long ((1) in Fig. 1c),most of the photocarriers excited by the pumppulse relax to the ground state before thesubsequent probe pulse illumination so thata similar number of carriers would be excitedby the probe pulse as with the pump pulse,
resulting in a large transient current I*probe. In
contrast, when td is short, the excited statesremain occupied with the photocarriers excited by the pump pulse when the probe pulse
illuminates the sample so that the optical absorption saturates, resulting in a small I*probe((3) in Fig. 1c). By illuminating a pair of pumpand probe pulses sequentially and by varying
td, one can detect a time-averaged tunnelingcurrent <I> as a function of td (Fig. 1d). By
fitting the time-resolved tunneling current
with an exponential function, we can obtaina decay time at the tip location, as examples
are shown in Figure 1e [1].
In the macroscopic OPP technique, themodulation of optical intensity is conventionally utilized to detect a weak OPP-induced signal. However, the optical intensity
modulation causes severe problems, such as
the thermal expansion of the STM tip. Sincechanges in tip-sample distance are exponentially multiplied in the tunneling current,
the optical intensity modulation cannot bedirectly applied to STM. To suppress the thermal expansion effect, we have developed an
excellent delay-time modulation technique[1]. In this technique, we use two delay times(td and tmax in Fig. 1b). The longer delay time
tmax is generally set to one-half of the laser
pulse interval (for example, 0.5 μs for 1 MHz
repetition rate), corresponding to the longest delay time available for the selected
repetition rate. We modulate the delay time

Fig. 1: (a) Conventional OPP configuration. (b) Schematic of OPP-STM with the delay-time
modulation method. (c) Transient tunneling current I* induced by the pump light and probe lightat the representative delay time td. Time-averaged tunneling current <I(td)> is shown for eachcase (green rectangular). (d) <I> as a function of td. The time-averaged <I> corresponding toeach case in (c) is plotted. (e) Time-resolved signals obtained for several samples. [1]between td and tmax at, for example, 1 kHz
and detect the resultant tunneling current
ΔI(td) = <I(td)> − <I(tmax)> using the lock-in
amplifier (Fig. 1b). This modulation technique enables us to keep the thermal load at
the tunnel junction constant, substantiallysuppressing the thermal expansion effect.

Compact and Stable OPP-STM
The delay time modulation technique has enabled reliable OPP-STM measurements. Since
then, many studies such as the atomic scale
carrier dynamics around a single impurity on

a GaAs surface [7,8] and the visualization of
the ultrafast carrier dynamics in a GaAs-PIN
junction [9] have been reported. However, theoptical system has been generally complex
and large in scale, hindering the widespreaduse of this technique. To overcome this difficulty, the OPP-STM system, whose laser-pulse
timing is electrically controlled by external
triggers, has significantly improved the easeof use, but its temporal resolution has been
limited to the nanosecond range [10]. In addition, fluctuations in optical intensity cause
unexpected issues, such as the thermal expansion effect, making the accurate observationof physical phenomena challenging.

Fig. 2: (a) Photograph of the compact OPP-STM system. (b) Three-dimensional illustration of the OPP-STM unit. (c) Optical image of the tip andits mirror image on GaAs(110) surface with a laser spot illuminated at the tunneling junction before (top) and after 16 h (bottom), showing thestability of the laser spot. [11]Imaging & Microscopy 1/2024 • 35

Scanning Probe Microscopy

Fig. 3: (a) Grid-point time-resolved tunneling current measurement on GaAs(110) cleaved surface [11]. Characteristic features along the step edge and the perimeter of the nanoscale bump
structure are identified in the time-resolved tunneling current map (right). By fitting a time-resolved tunneling current curve at each grid point, we can obtain the nanoscale relaxation timemap (top left). (b) Snapshots of ultrafast motion of photoinjected electrons in a C60 multilayer/
Au sample obtained by THz-STM. [5]electrically controlled delay-time modulation with tuning-fork-type frequency modulation (FM) AFM (Fig. 4b) [14]. By focusing
optical pulse pairs onto the apex of the AFM
probe, we were able to detect the amplitudeof the frequency shift Δf as a time-resolved
signal. The markedly stable time-resolved
measurements on bulk WSe2, a layered semiconductor, have unveiled dynamics withtwo decay components through an ultrafast
photoinduced force (Fig. 4c). These signals,
decoded as the surface recombination anddiffusion of photocarriers through tunnelingcurrent and force spectroscopy, mark a significant stride in our understanding of material properties. This innovative approach not
only resolves the limitations encountered intime-resolved STM due to tunneling current
but also paves the way for its application
across a diverse array of materials, setting a
new standard in nanoscale imaging.

Conclusion

Fig. 4: (a) Dipole-dipole interaction mechanism. αt, αs, and E0 are the complex effective polarizabilities of the tip and sample, the incident light electric field vector, respectively. (b) Schematicof tuning-fork-type time-resolved FM AFM setup. (c) Time-resolved signal obtained for a multilayer WSe2 sample and fitting results. [14]To improve the temporal resolution and thestability of laser illumination based on acompact electrically controlled laser system(Fig. 2a), the OPP-STM system with a temporal resolution of tens of picoseconds has
recently been developed [11]. The long-term
stability of laser illumination was realizedalso by placing the focus lens on the STM
stage (Fig. 2b) and confirmed by monitoring
the tip and its mirror image on the sample
surface together with a laser spot focused on
the tip-sample junction (Fig. 2c).
To demonstrate the performance of thesystem, we conducted a grid-point time-resolved tunneling current measurement on
GaAs(110) surfaces at T = 6 K (Fig. 3a). By
measuring the time-resolved tunneling current at each grid point, we can compile thetime-resolved tunneling current maps at eachdelay time and determine characteristic features along the nanostructure (Fig. 3a, right).
Furthermore, it is possible to map a relaxation
time by fitting each curve with an exponential function. The relaxation time map (Fig.
3a, top left) demonstrates that the relaxation
time inside the nanoscale bump structure

36 • Imaging & Microscopy 1/2024

is substantially shorter than that of its surroundings. This mapping technique is powerful in visualizing carrier dynamics associated with nanoscale structures. Figure 3b
shows an example of a time-resolved signal
map obtained by THz-STM. The improvementin the usability of the optical system can beextended to electric-field-driven STM, which
is currently our ongoing investigation.

OPP Atomic Force Microscopy
The optical system we have developed is also
applicable to AFM, expanding its potential
beyond conductive materials such as semiconductors and metals, traditionally targetedin STM. Integrating AFM into our repertoire
significantly broadens the horizons of nanoscale time-resolved microscopic techniques. Previous studies have clarified thephotoexcited dynamics of forces originatingfrom surface photovoltage and dipole-dipole
interactions (Fig. 4a) based on optical intensity modulation (Fig. 1a) [12,13]. We haveadvanced these achievements by combining

Our streamlined optical system markedly eases researchers‘ path to harness the transformative power of the OPP-STM technique. With
the multiprobe (MP) system, features such as
small islands on an insulating substrate canbe observed by using one tip as an electrodewhile conducting STM measurements withthe other tip. The OPP-MP measurements
have already been demonstrated on monolayer transition metal dichalcogenides to investigate nanoscale exciton dynamics [15,16].
Regarding the limitations of wavelength andtemporal resolutions, cutting-edge laser technology, which has been rapidly developing,may overcome these limitations, thereby realizing a higher-performance optical systemenabling a wavelength-variable, externallycontrollable laser system with a smaller pulse
width in the future. The development of easyto-use optical systems and their applications
to various scanning probe techniques will expand the capability of OPP-SPM techniquesand contribute to a deeper understanding ofvarious photo-induced phenomena.
Affiliation
1UNISOKU Co., Ltd., Hirakata, Osaka, Japan
2University of Tsukuba, Tennnodai, Tsukuba,
Japan
Contact
Prof. Dr. Hidemi Shigekawa
University of Tsukuba
Tsukuba, Ibaraki, Japanhidemi@ims.tsukuba.ac.jp

References:https://bit.ly/IM-Shigekawa

Image Processing

Data-Driven Microscopy
Improves Image Acquisition
Finding the Right Cells to Image
Oscar André1 and Pontus Nordenfelt1

D© Nordenfelt

eciding where to take images in lightmicroscopy is commonly realized bymanual decision-making or complete
scanning. By using information about theobjects in the sample, a data-driven approach, it is possible to steer a motorized microscope to the best spots at the right time.
This provides better data at a much fasterrate and can be applied to most microscopy
scenarios.

Imaging & Microscopy 1/2024 • 37

Image Processing

Introduction
Today, researchers can quickly amass vast
amounts of cellular process data throughhigh-speed image collection. However, current methods for microscope image acquisition rely on manual decisions or brute-force
techniques like slide scanning. High content screening (HCS), when combined with
automated image analysis, helps extract
valuable information from overwhelming
datasets [1–9]. HCS is the primary approach
for analyzing large single-cell datasets ina population-wide context [10–14]. Yet, in
high-resolution microscopy, images are often captured from specific points, lacking
population context and risking bias due tohuman selection. This challenge intensifies
during live imaging, like tracking cell migration or host-pathogen interactions.
Integrating image analysis, including
machine learning, into data selection has
enhanced efficiency and reduced bias, termed
event-driven feedback microscopy or intelligent microscopy [15, 16]. Various proprietary(Nikon, Zeiss, Leica, Olympus) and opensource solutions control microscope hardware [17–19] and enable feedback microscopy [16, 20–22], allowing real-time response
to image content. However, these decisions
usually rely on predetermined image characteristics and do not adapt to sample population data distribution. Notably, no solutions
currently focus on targeted image acquisition
based on analyzing population characteristics to identify relevant and representative
objects or events.

Methodology
We have recently introduced a general microscopy framework, data-driven microscopy(DDM) (Fig.1), which uses population-wide
data to improve and control microscopy andenables cross-experiment image data validation [23]. A data-independent acquisition
phase performs high-throughput imaging
and generates a population-wide phenotype assessment. This data includes the relative coordinates of each data point for thesystem, which feeds into the automated data-dependent acquisition of selected phenotypes. Combining the two data sets yields
population-wide data with high-fidelity object characterization data. DDM also inherently includes what constitutes an objective
representation of the sample population. The
general DDM framework applies to any motorized microscope, and all steps can be fully
automated, including phenotype targeting.
Placing the combined data in a population
context yields a more robust, reproducible,
and efficient method for selecting and acquiring data in microscopy.

38 • Imaging & Microscopy 1/2024

Fig. 1: Principle of data-driven microscopy. Data-independent acquisition (DIA) aims to characterize the full sample population in real-time. Generated single-cell data is continuously stored
in a database, allowing for data filtering and single-cell targeting. Predefined or generated targeting criteria are fed back to the microscopy for data-dependent acquisition (DDA). The
high-fidelity data is stored in a database interconnected to the DIA database. The interconnection of DIA and DDA databases allows for high-fidelity data to be placed in the sample-wide
context, allowing for potential new insights that motivate future experiments.

Targeting and Capturing Infection Events
In live imaging of host-pathogen interactions, predicting rare events is a challenge.
To validate DDM’s capabilities, we used
HeLa cells expressing mScarlet-Lifeact andYersinia pseudotuberculosis expressing
GFP. Interactions were categorized basedon distance. DIA monitored HeLa cells (N= 16,988) and Y. pseudotuberculosis (N =990), achieving a 100% hit rate in identifying interactions [23]. Traditional manual
monitoring would only yield a 1.4% hit rate,
demonstrating DDM’s adaptability in acquiring population-wide data and targetingrare events for live-cell imaging.

Targeting and Capturing
Migratory Phenotypes
To assess DDM’s capacity in live feedback
microscopy, we studied cancer cell migration at high spatiotemporal resolution using
an adaptive feedback microscopy modulewith DDM. We imaged H1299 mKate-paxillin cells (N = 24,940) through DIA every 10 minutes for 6 hours, characterizing
single-cell migration and collapsing theparameter space using UMAP analysis tocategorize migration modes. Cells exhibited diverse migratory behaviors, categorizedbased on mean migration speed and meandering index (Fig. 2A and 2B). In low-reso-

lution DIA data, a wide heterogeneity in cell
migratory behavior was observed.
To gain deeper insights, we conductedhigh-magnification DIA on select cells, tracking them every 10 minutes for 100 minutes(Fig. 2C). Based on mean speed, we targeted
the fastest cells for DDA (N = 100) and tracked
their migration at higher temporal resolution (Fig. 2D). Continuous tracking between
DIA and DDA was seamless (Fig. 2E), and we
expanded the dataset by tracking neighboring cells, resulting in a total of 1,336 observations. (Fig. 2F). DDA provided increased resolution and variance in cell tracking (Fig. 2G),
showing that fast-migrating cells maintained
their speed profile over time, while DIA data
indicated an overall regression to the population mean without altering speed group
allocation. In summary, DDM enhances cell
monitoring on a population scale with targeted high-spatiotemporal acquisition, ensuring data fidelity and integrity.

Conclusion
Light microscopy, a potent single-cell
method, provides subcellular resolution for
quantitative spatial data. However, unlike
flow cytometry and single-cell sequencing
techniques, it faces challenges in achieving
high-quality population-wide sample characterization while preserving resolution. We
introduce a versatile approach called da-Image Processing

Fig. 2: Targeting and capturing migratory phenotypes. Cells were imaged every 10 min and analyzed (total N = 24 940) in multiple migration
properties and categorized into slow and fast (<10th and >90th quantile, respectively) in terms of mean speed. a, b UMAP space colored using
mean-speed (a) and meandering index (b) revealed distinct migratory phenotypes. c,d Example image of cells migrating at different speeds in DIA(c) and a targeted cell in high-magnification DDA (d) migrating in the top percentile with overlayed coordinates from the DIA and DDA (large andsmall markers, respectively). Highlighted are fast (group 10; top left, top right, and middle rectangle) and slow (group 1; bottom left rectangle)
cells in terms of mean speed. e. Cell migration was continuously tracked between DIA and DDA. f. From the DDA datasets, neighboring cells (blue)
to the targeted cells (yellow) significantly increased the overall cell count. A small portion of the cells (gray) were excluded during analysis due toautofocus or tracking error. g. Speed variation over time during DIA vs. DDA for the two speed groups.

ta-driven microscopy (DDM). DDM leverages
real-time population-wide object analysis tofacilitate data-driven, high-fidelity imaging
of relevant phenotypes based on population
context. This method synergistically combines data-independent and data-dependent
steps, enhancing data collected through various imaging modalities.
As a proof of concept, we have developed and applied DDM along with plugins
to enhance high-content screening andenable live adaptive microscopy for cell
migration and infection studies. Thisapproach allows for the precise capture ofboth rare and common events of interest
with high precision and resolution. DDM

has the potential to mitigate human bias,
boost reproducibility, and provide single-cell characteristics within the context
of the sample population during microscopy data interpretation. Consequently, it
can significantly enhance overall microscopy data fidelity.

Acknowledgment
This project has received funding from theSwedish Research Council (Vetenskapsrådet), Cancer Foundation (Cancerfonden),and Strategic Research Foundation (Stiftelsen för Strategisk Forskning).

Affiliation
1 Department of Clinical Sciences, Faculty of

Medicine, Lund University, Lund, Sweden.
Contact
Dr. Pontus Nordenfelt
Biomedical Center
Medical Faculty
Lund University
Lund, Sweden
pontus.nordenfelt@med.lu.de
www.nordenfeltlab.com
References:https://bit.ly/IM-Nordenfelt

Imaging & Microscopy 1/2024 • 39

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40 • Imaging & Microscopy 1/2024

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CellVoyager High-Content Analysis System
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Camera-Based System for Fluorescence Lifetime Imaging Microscopy
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Products

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42 • Imaging & Microscopy 1/2024

A AI pixel classifier is an integral part of its big-data-capablesurface segmentation model. Using the classifier simplifies andimproves the segmentation andobject detection in challenging
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for scientific cameras and ensure
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Index / ImprintAHF Analysentechnik 

6Carl Zeiss Microscopy

Cover, 5, 12

Craic Technologies

42

EMC24

3

European Microscopy Society

11

Evident Europe 

40Media Cibernetics
ModuleSci
NKT Photonics

7, 42
Outside Back CoverOsaka University

26

Oxford Instruments - Andor

42

Royal Microscopical Society (RMS)

Helmholtz Centre for Environmental Research – UFZ 22

42

8, 10

Shimadzu

41

Teledyne Photometrics

41

Lambert Instruments

41

Lund University

37

Unisoku34

Mad City Labs

9

Universität Konstanz

30

Imprint
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Prof. A. Diaspro, Italian Institute of Technology
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Dr. M. Dürrenberger, Univ. of Basel, Switzerland
Dr. R. Fleck, King’s Collage London, UK
Prof. M. Gu, Swinburne Univ., Australia
Prof. B. Hecht, Univ. of Wuerzburg, Germany
Prof. M. Hegner, Trinity College Dublin, Ireland
Prof. F.-J. Kao, National Yang-Ming University, Taipei, Taiwan
Prof. N. Kruse, Washington State University, WA, USA
Prof. D. Nicastro, Thermo Fisher, The Netherlands
Dr. J. Rietdorf, Centre for Technological Development
in Healthcare (CDTS), Brasil
Dr. D. Spitzer, ISL, France
Prof. G. A. Stanciu, Univ. of Bucharest, Romania
Prof. G. Valdré, Univ. of Bologna, Italy
Dr. Roger Wepf, EMBL, Heidelberg, Germany
Dr. T. Zimmermann, ORG, Barcelona, Spain

University of Freiburg

18

University of South Australia

14

University of Sydney

14

University of Technology Dresden

18

University of Tsukuba

34

University of Western Australia

14

Wiley Science Solutions

25

WITec

21, 42

Yogokawa

41

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