Japan-Canada Joint Seminaron Advanced Electron Microscopy
and its Application『日本‐カナダ先端分析顕微鏡合同セミナー』

Third Japan-Canada
Microscopy Societies Joint Symposium 2022
第３回 日本‐カナダ
顕微鏡学会交流シンポジウム 2022

November 4 - 5, 2022
at
Kawasaki Sukenobu Memorial Lecture Hall
Kawasaki University of Medical Welfare, Kurashiki, Okayama, Japan

Japanese Society of Microscopy (JSM)
Microscopy Society of Canada (MSC)

This seminar is partially supported by JSPS Bilateral Program, Grant number [JP JSBP 220229906].

【Preface】
Dear Participants,
On behalf of the Organization Committee, we warmly welcome you toattend the third Japan-Canada Microscopy Societies Joint Symposium 2022,
supported financially by the Japan Society for the Promotion of Science(JSPS). Since 2020, the joint symposiums have been held with the aim of
deepening mutual exchanges between the Japanese Society of Microscopy(JSM) and the Microscopy Society of Canada (MSC). The 1st (held in Osakain May 2020) and the 2nd (held in November 2021 at the University ofAlberta) was held on-paper and on-line due to the COVID-19 pandemic. The
3rd Japan-Canada Joint Symposium on Advanced Analytical Microscopy will
be held in Kurashiki, Okayama in November 2022. The general purpose ofthis symposium is to provide a great opportunity for researchers, especially
young researchers, of JSM and MSC to communicate with world-leading
experts and discuss the next generation of microscopy.
In this symposium we will invite participants on a wide range of
topics, including not only electron microscopes but also ion microscopes, newequipment and technologies, and data processing methods and theirapplications that have made rapid progress in recent years. It also aims toadvance microscopy and further development of microscope technology
through direct exchanges between Japanese and Canadian researchers andthe two societies. Furthermore, in terms of mutual exchanges at theacademic society level, we will take advantage of the characteristics of bothsocieties to conduct exchanges not only in the fields of metals andsemiconductors, but also in the fields of medicine and biology.
Unfortunately, due to the travel limitation imposed by the COVID-19
pandemic, it is a pity that this year all the sessions in Canadian side will beheld in the online mode, although the committee did try our best fororganizing an on-site conference. We sincerely wish this symposium will beheld in-person next year!
Finally, we would like to thank all the researchers who participated1

in this symposium, Dr. Kazunori Toida, chairman of the 65th symposium of
JSM, and the members of the organizing committee of the 65th symposiumfor their support in planning this symposium, and for their cooperation inthis project. Also, we would like to thank all of you who attended in this
symposium. We hope that mutual exchanges between the Japanese andCanadian microscopy societies will continue in the future.
On behalf of all the organizers
Shigeo Mori (Osaka Metropolitan University)2

【Greetings】
Yuichi Ikuhara, (University of Tokyo)
President of The Japanese Society of Microscopy
This is the third symposium for bilateral exchange between theJapanese Society of Microscopy (JSM) and the Microscopy Society of Canada(MSC). This time, we planned to hold this symposium in conjunction withthe 65th symposium of the JSM, which will be held at Kawasaki Universityof Medical Welfare, Kurashiki city in Japan. This symposium is also in linewith the "Initiative toward Internationalization" that JSM is currently
actively promoting. In addition, the theme of the symposium “Microscopy
which is contributed to the state-of-the-art science” is very timely and usefulfor our field. When the first symposium was planned three years ago, withthe support of the Japan Society for the Promotion of Science (JSPS), it was
hoped that it would promote close exchanges and joint research between
microscopy societies between Japan and Canada. However, due to the spreadof the new coronavirus infection at the beginning, the first symposium washeld on paper. On the other hand, since there was a strong request from theparties concerned to keep the project of this important symposium, the 2nd
symposium was successfully held virtually. This is the 3rd time, following on
from that, and the organizers have set up lectures on cutting-edge science in
the Biological Science Session and the Materials Science Session in awell-balanced manner. It will be held in a hybrid system from Kawasaki
University of Medical Welfare. I would like to thank Prof. Shigeo Morichairman of the third Japan-Canada Microscopy Societies Symposium, allmembers of the organizing committee, Prof. Kazunori Toida, and Dr. Ken
Harada for their big effort and supporting this symposium. I also would liketo express my heartfelt gratitude to Prof. Kathryn Grandfield, President ofMicroscopy Society of Canada, and to the members of the organizing
committee, Prof. Marek Malac and Dr. Misa Hayashida, on the Canadianside. I really hope that the continuation of this symposium will further
develop exchanges between microscopy societies in two countries. I also hopethat the next symposium will be held face-to-face in Canada or Japan.

3

4

【Greetings】
Kathryn Grandfield, (McMaster University)
President of Microscopy Society Canada
On behalf of the Microscopy Society of Canada- Société duMicroscopie du Canada (MSC-SMC), I am honoured to welcome you to theThird Japan-Canada Microscopy Societies Joint Symposium held at the
Kawasaki University of Medical Welfare in Kurashiki, Okayama, Japan.
Together, our two societies have been pushing the frontiers of electron
microscopy with this exciting conference, now in its third year.
This year’s meeting focused on, “Advanced Electron Microscopy andits Application” will bring together the latest developments and eminent
speakers from across Japan and Canada presenting ground-breaking
research spanning techniques, such as correlative electron microscopy,
electron diffraction, analytical techniques, in situ electron microscopy,
ultrafast microscopy and electron tomography for broad applications
spanning both the Materials and Biological Sciences.
This annual meeting has proven to be a fruitful opportunity for
scientific collaboration and exchange between our two societies, and its
continued success is evidence of the advancements in microscopy oursocieties are making together.
I extend my deepest thanks to the sponsoring organizations andvolunteer organizers in Canada and Japan who have worked so tirelessly tobring you this exciting meeting. Thank you to my fellow society, the JSM, forextending the generous invitation to join you again in our third year.
We look forward to welcoming you to Canada next year for the 50th
Anniversary of the MSC-SMC and another stimulating Japan-Canada
meeting. I give my warmest welcome to all attendees, in-person or virtually,
and wish you a good scientific meeting.

5

【Scientific Program】
Day 1 (Tokyo time: Friday, November 4, 2022)
Opening

07:55 – 08:00

Biological Science Session
Bio-01 08:00 – 08:40
Michael J.Hendzel (University of Alberta)
The development of 3-channel imaging methods to discriminate biomolecules ininterphase nuclei
Bio-02 08:40 – 09:00
Hiroyuki Hioki, (Juntendo University)
A multiscale imaging from macroscopic to nanoscopic levels with a tissue
clearing method, ScaleSF
Bio-06 09:00 – 09:20
Haruyo Yamanishi (Shiseido Co., Ltd.)
Morphological changes of intracellular organelles during terminal differentiationof the epidermis
Bio-04 09:20 – 10:00
Kazunori Toida (Kawasaki Medical School)
Multimodal regulation of olfactory neural circuit, as revealed by correlated laser
and volume electron microscopy, ultra-high voltage electron microscopy, andelectron tomography
Short Break 10:00 – 10:10
Bio-05 10:10 – 10:30
Shuichi Hayashi (Kawasaki Medical School)
Developmental analysis of complex synaptic connections by correlative light andelectron microscopy
Bio-03 10:30 – 10:50
Joaquin Ortega (McGill University)
Getting ahead of the next pandemic: When antibiotics stop working
6

BINA-01 10:50 – 11:20
Claire M. Brown (McGill University)
National and international bioimaging networks

Materials Science Session
Mate-01 11:20 – 11:40
Ray F. Egerton (University of Alberta)
TEM of thick polymer or biological specimens
Mate-02 11:40 – 12:00
Tomohito Ishii (Osaka University)
Improving spatial resolution in rapid electron tomography for hundreds-nm-sized
3D materials

Lunch

12:00 – 13:00

Mate-03 13:00 – 13:20
Takahiro Shimojima (RIKEN)
Magnetic dynamics probed by ultrafast transmission electron microscopy
Mate-04 13:20 – 13:40
Hiroshi Nakajima (Osaka Metropolitan University)
Ferroelectric nanostructures observed by transmission electron microscopy
Mate-05 13:40 – 14:00
Licong Peng (RIKEN)
In-situ L-TEM observations of magnetic skyrmion and antiskyrmion dynamics
Mate-06 14:00 – 14:40
Hiroki Kurata (Kyoto University)
Electronic state analysis using monochromated STEM-EELS

7

Day 2(Tokyo time: Saturday, November 5, 2022)

Materials Science Session
Mate-07 08:00 – 08:40
Arthur M. Blackburn (University of Victoria)
Accessing sub-Ångström ptychographic information in a scanning electron
microscope below 30 kV
Mate-08 08:40 – 09:00
Jane Howe (University of Toront)
Automatic quantification of microplastic fibres in scanning electron micrographs
Mate-09 09:00 – 09:20
Daisuke Morikawa (Tohoku University)
Local structure analysis of interface and polar nano domains using
convergent-beam electron diffraction
Mate-10 09:20 – 09:40
Nadi Braidy (University of Sherbrooke)
Interpretation of 3D EDX maps: multivariate analysis and deep learning hybrid
approachMate-11 09:40 – 10:00
Naoya Shibata (University of Tokyo)
Atomic resolution magnetic field imaging by scanning transmission electron
microscopy
Closing

10:00 – 10:05

Continue to the 65th JSM Symposium
https://conference.wdc-jp-com/microscopy/sympo/65th/8

【Abstracts】
Day 1(Tokyo time: Friday, November 4, 2022)

Biological Science Session

9

Bio-01

The development of 3-channel imaging methods to discriminate
biomolecules in interphase nuclei

Hilmar Strickfaden, Natnael Abate, and Michael J. Hendzel
Departments of Cell Biology and Oncology
Faculty of Medicine and Dentist
3
University of Alberta, Edmonton, Canada T6G 1Z2*
mhendzel@ualberta.ca
1
2

The ability to image cell structures at molecular resolution makes transmission
electron microscopy the technique best-suited to study the molecular organization ofcells. Much of what we know about the organization of the cytoplasm is derived fromtransmission electron microscopy studies. Although fluorescence microscopy
progresses increasingly closer to transmission electron microscopy resolution, thetechnique is limited by the intrinsic inability to identify molecules without the use ofexogenous fluorescent labels. Thus, molecules and structures that are not labelledcannot be distinguished from a complete absence of molecules/structures in that space.
Transmission electron microscopy is mass sensitive and can contrast all biologicalstructure. However, the absence of colour has made it very difficult to compare
distributions and relationships of two or more types of biomolecules, at the same timethus limiting functional analysis. Discrimination of structures relies upon morphologicaldifferences rather than on contrast. This becomes problematic in the nucleoplasm where
fibrillar material could be chromatin, RNA, or protein. While specific stains exist to
discriminate amongst these molecules, they cannot be used in combination because
conventional transmission electron microscopy is a single-channel technology. A
potential path around this barrier is presented by electron spectroscopic imaging (ESI).
ESI can map distributions of chemical elements within an ultra-thin section. We havepreviously shown that the combination of nitrogen and phosphorus maps can be highly
informative on the interpretation of nuclear structures. A disadvantage of this approach
was that osmium tetroxide staining significantly impaired the acquisition of thephosphorus maps. Consequently, we were unable to preserve biological membranes. In
this study, we made preserved membranes with potassium permanganate and imaged
them with manganese, resulting in 3-color images. In separate 3-color experiments, weused potassium permanganate to contrast diaminobenzidine polymerized by10

photooxidation or using a singlet-oxygen generating enzyme to identify specificmolecules that we have incorporated enzymatic tags (APEX2) or fluorescently labelledmolecules to polymerize DAB at the location of the molecule of interest. Finally, we
have used RNA-specific staining to generate 3 color maps where RNA can be
distinguished from DNA. This enables the localization of RNA relative to theunderlying chromatin and will significantly advance our understanding of theorganization of RNA within the interphase nucleus.

11Bio-02

A Multiscale imaging from macroscopic to nanoscopic levels with atissue clearing method, ScaleSF.

Hiroyuki Hioki1,2,3,*, Kenta Yamauchi1,2, Takahiro Furuta4
Department of Neuroanatomy, Juntendo University Graduate School of Medicine
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of
Medicine3
Department of Multi-Scale Brain Structure Imaging, Juntendo University Graduate
School of Medicine
4
Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka
University
1
2*

h-hioki@juntendo.ac.jp

The mammalian brain contains a heterogeneous mixture of billions of neurons
with trillions of synapses. Connectomics, a description of wiring diagram of the nervous
system, is fundamental for understanding how a neural circuit processes information andgenerates behavior (Lichtman and Sanes, Curr Opin Neurobiol, 2008). While neurons
elaborate highly specialized processes that can span over a meter in length, synapses thatconnect neurons are several hundred nanometers in size. Thus, the imaging scale requiredfor deciphering brain-wide connectivity exceeds several orders of magnitude.
Our study overcomes the technical requirements required for whole-brain andnanoscale imaging by coupling a tissue clearing method with successive LM/EM imaging(multi-scale LM/EM neuronal imaging). We have established an imaging pipeline thatenables correlative light and electron microscopy (CLEM) in optically cleared tissues inthis study (Fig). Our multi-scale neuronal imaging makes it possible to describe synaptic
connectivity of brain-wide circuits by simultaneous interrogation of the neural circuit
structure mapped in optically cleared brain tissues, and synaptic connectivity imaged
with EM in a reasonable amount of time, without the need for specialized equipment. Our
study has expanded the applicability of tissue clearing techniques from LM to EM bydeveloping an ultrastructurally-preserved tissue clearing method, ScaleSF, andimplementing LM/EM dual labeling that remained stable in the clearing protocol. We
believe that our technique will contribute to our understanding of brain-wide connectivity
beyond the reach of current connectomic analyses with a single imaging modality.
12

Acknowledgements:
This study was supported by JSPS KAKENHI (JP21H02592 to H.H.; JP20K07231 to
K.Y.; JP21H03529 to T.F.) and Scientific Research on Innovative Area “Resonance Bio”
(JP18H04743 to H.H.). This study was also supported by the Japan Agency for Medical Researchand Development (AMED) (JP21dm0207112 to T.F. and H.H.), Moonshot R&D from the Japan
Science and Technology Agency (JST) (JPMJMS2024 to H.H.), Fusion Oriented Research for
disruptive Science and Technology (FOREST) from JST (JPMJFR204D to H.H.).
References:[1] Furuta T, Yamauchi K, Okamoto S, Takahashi M, Kakuta S, Ishida Y, Takenaka A, Yoshida A,
Uchiyama Y, Koike M, Isa K, Isa T, *Hioki H. (2022) Multi-scale light microscopy/electron
microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF. iScience.
25:103601.
[2] Yamauchi K, Furuta T, Okamoto S, Takahashi M, Koike M, *Hioki H. (2022) Protocol for
multi-scale light microscopy/electron microscopy neuronal imaging in mouse brain tissue. STAR
Protocols. 3:101508.
[3] Yamauchi K, Okamoto S, Takahashi M, Koike M, Furuta T, *Hioki H. (2022) A Tissue Clearing
Method for Neuronal Imaging from Mesoscopic to Microscopic Scales. J Vis Exp. 183:e63941.

Fig. Multi-scale LM/EM neuronal imaging pipeline. Multi-scale neuronal imaging
incorporates (1) macroscopic observation, (2) mesoscopic circuit mapping in cleared
brain slices, (3) microscopic subcellular imaging in re-sections, and (4) nanoscopic EM
imaging. Mouse and marmoset neurons are labeled with an AAV vector carrying afluorescent and electron-dense genetically encoded CLEM probe, EGFP-APEX2.

13

Bio-06

Morphological Changes of Intracellular Organelles
during Terminal Differentiation of the Epidermis

Haruyo Yamanishi（山西治代）1*, Akemi Ishida-Yamamoto (山本明美) 2
Shiseido Co., Ltd. MIRAI Technology Institute, 1-2-11, Takashima, Nishi-ku,
Yokohama, Kanagawa, 220-0011, Japan.
2
Department of Dermatology, Asahikawa Medical University, 1-1-1. Higashi-Ni-Jyo,
Midorigaoka, Asahikawa, Hokkaido,078-8510, Japan*
haruyo.yamanishi@shiseido.com
1

Skin is the largest tissue in our body and plays an important role in barrier
functions through the differentiation of keratinocytes. Intercellular lipids are released
from lamellar granules (LGs) and prevent water loss. LGs are small organelles found in
the cytoplasm of stratum granulosum (SG). LGs have been observed as oval-shaped
vesicles or vesicular tubular structures on electron micrograph, but thethree-dimensional (3D) structure of LG of the epidermis is unclear due to the technicallimitations of the previously applied microscopy techniques.
Recently, many 3D ultrastructural analyses by scanning electron microscopy(SEM), which generates high resolution and back scattered images resemblingtransmission electron micrographs, have been reported. This technology, called block
face imaging, is applied in several tissues including brain, kidney and testis, andexpected to be evolved rapidly.
In this study, we aimed to elucidate the 3D structures and spatial distributionsof LGs in normal human skin by focused ion beam-SEM (FIB-SEM) technique (Fig. 1).
The FIB-SEM images indicated that LGs fused the cellular membrane in the mostsuperficial layer of the SG. LGs in the second granular (SG2) layer was not only
localized in the cytoplasm but also secreted into the intercellular space [1]. In contrast,
LGs was maintained in the cytoplasm of the third granular layer. Trans-Golgi network(TGN) was visualized spreading into the cytoplasm with branched structures andconnected to the vesicles of LGs in the SG2 layer [2]. These results suggest that LGs
mature along with keratinocyte differentiation, and that the changes in LGs morphology
associated with the SG2 layer represent an important step preceding LGs fusion with thecell membrane. We consider that further research for the 3D structure of LGs and TGN
could elucidate pathological mechanisms of skin diseases which are associated withLGs.
14

Acknowledgements:
We appreciate Dr. Kazunori Toida (Kawasaki Medical School, Japan) for the contractive instruction
and Dr. Takuma Kanesaki (Cellink) for the technical advice. Special thanks go to the late Dr.
Toshihiko Hibino.
References:[1] Yamanishi H., Soma T., Ishida-Yamamoto A., Kenbikyo, (2019) Vol. 54, p.39-43[2] Yamanishi H., Soma T., Kishimoto J., Hibino T., Ishida-Yamamoto A., J Invest Dermatol. (2019)Vol. 139 (2) p.352-359.

Fig.1 The distribution of lamellar granules in human epidermis. Adapted andtranslated from “Three-dimensional ultrastructural analysis of lamellar granules andtrans-Golgi network in stratum granulosum by focused ion beam scanning electron
microscopy,” by Yamanishi H., et al., 2019, Kenbikyo, 54, p.39-43.

15

Bio-04

Multimodal regulation of olfactory neural circuit,as revealed by correlated laser and volume electron microscopy,
ultra-high voltage electron microscopy, and electron tomography

Kazunori Toida1,2,3*,
Sawa Horie1, Keita Satoh1, Nobuaki Matsuda3,4, Yukari Minami1,5, Satoshi Ichikawa2,
Shuichi Hayashi1, Emi Kiyokage4
1

Department of Anatomy, Kawasaki Medical School

2

Research Center for Ultra-high Voltage Electron Microscopy, Osaka University3

Bio-imaging Unit, Central Research Institute, Kawasaki Medical School

4

Department of Medical Technology, Kawasaki University for Medical Welfare

5

Department of Neurosurgery, Kawasaki Medical School*

toida@med.kawasaki-m.ac.jp

We have been examining the olfactory bulb (OB), the primary center of theolfactory system, as an attractive model for analyzing basic structure of the neuralcircuit in the brain. The OB is organized by laminar structure containing neuronalorganization with rich in chemical substances. These characteristic features areobserved beyond animal species and thus further enable us to do variable microscopic
experiments (Ref.1). At the outset, I would like to introduce just briefly the backgroundof our research (Ref.1), followed by integrative morphological research so far analyzed
by correlative laser and volume electron microscopy (serial-sectioning/reconstruction)
for synaptic organization of the OB (Ref.2-4). Urgent questions raised in these series ofour studies are how the OB is regulated by variable factors (Fig.1), and we need toestablish anatomical basis for multimodal regulation of the neural circuit of the OB. To
address, we have succeeded to label the OB neurons selectively by transgenic and/or
genetic induction to apply for microscopic analyses we have been consistently
performing (Ref.5-8, Fig.2).
Here, I would like to show our findings and let us consider all together
developing progress and possibility in recently advanced microscopy, such as
quantitative analyses by ultra-high voltage electron microcopy, electron tomography(Fig.3-5) and network tele-microscopy, to well understand the structure and function of
the brain.

16

Acknowledgements:

Grants supported by JSPS KAKENHI (JP24500418, JP15K06748, JP19K06932 to K.T.JP19K09922
to S.H.; JP23500419 to E.K.) Project Research in Kawasaki Medical School to K.T., S.H., K.S.,E.K.

References:[1] Toida K Anat Sci Int. 2008, 83:207-17[2] Kiyokage E, Kobayashi K, Toida K J Comp Neurol. 2017, 525:1059–1074, cover[3] Matsuno T, Kiyokage E, Toida K J Comp Neurol. 2017, 525:1633-1648.
[4] Notsu E and Toida K Microscopy 2019, 68:316-328.
[5] Suzuki Y, Kiyokage E, Sohn J, Hioki H, Toida K. J Comp Neurol. 2015, 523:262–280[6] Hamamoto M, Kiyokage E, Sohn J, Hioki H, Haratda T, Toida K
J Comp Neurol. 2016, 525:574–591[7] Satoh K., Kiyokage E., Ichikawa S., Toida K Neurosci. Lett. 2020, 738: 135386[8] Horie S, Kiyokage E, Hayashi S, Inoue K, Sohn J, Hioki H, Furuta T, Toida T
J Comp Neurol. 2021 529: 2189-2208, cover

Figures:
1

3

2

4

Fig.1: Para-sagittal plane of the mouse brain 5labeled by hoechst (blue), showing theolfactory bulb, which is regulated by5
multimodal, centripetal (red), centrifugal(green) and intrinsic (yellow) factors.
Fig.2: Centrifugal fibers of serotonin (5HT),
acetylcholine (Ach), and noradrenaline (NA)revealed by Cre-mice and AAV.
Fig.3: Diversity of GABA- expression
Fig.4:
Immunoreactivity
labeledbysilver-gold was analyzed by 3D-high-voltage
EM.
Fig.5: EM tomography indicated variation.
17

Bio-05

Developmental analysis of complex synaptic connections by correlative
light and electron microscopy

Shuichi Hayashi1*, Anna Hoerder-Suabedissen2, Nobuhiko Ohno3,4, Emi Kiyokage5,
Catherine Maclachlan6, Kazunori Toida1, Graham Knott6, Zoltán Molnár2
Department of Anatomy, Kawasaki Medical School, Okayama, Japan, 2Department of
Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK, 3Department of
Anatomy, Jichi Medical University, Tochigi, Japan, 4Division of Ultrastructural
Research, National Institute for Physiological Sciences, Aichi, Japan, 5Department ofMedical Technology, Kawasaki University of Medical Welfare, Okayama, Japan,
6
BioEM Facility, School of Life Sciences, EPFL, Lausanne, Switzerland*s.hayashi@med.kawasaki-m.ac.jp
1

It remains unclear how neuronal activity regulates synaptic development.
Axonal projections from layer 5 of the cerebral cortex form characteristic giant boutons
in the posterior nucleus of the thalamus (Po) and provide a powerful input to thalamic
neurons. To understand the 3D ultrastructure of the giant boutons and how neuronal
activity is involved in their synaptic morphogenesis, we first developed correlative lightand electron microscopy (CLEM) using confocal microscopy and serial block facescanning electron microscopy (SBF-SEM). Fluorescently labelled axons are correlated
in fluorescent and EM images without staining (Fig. 1) [1]. We found that multiple
excrescences from one Po dendrite connect with the same single layer 5 giant bouton thatcompletely encloses them (Fig. 2A,B). We next examined the layer 5 giant boutons in
Synaptosomal-associated protein 25 (Snap25) conditional knockout (cKO) mice, inwhich regulated vesicular release at synapses of layer 5 neurons was removed [2]. The
initial synapse formation in Po was not different between Snap25-cKO and control
boutons at postnatal day 8 (P8). However, the size of the Snap25-cKO boutons was
significantly smaller than that of control at P18. Although the Snap25-cKO boutons
retained single synapses with the shaft of Po dendrites, no dendritic excrescences
protruded into the boutons, and this caused a decrease in the total number of synapses oneach Snap25-cKO bouton (Fig. 2C,D) [3]. We also found that deletion of Snap25 in
granule cells in the dentate gyrus of the hippocampus leads to similar synaptic defects intheir axons called mossy fibers. These results suggest that regulated vesicular release isrequired for the maturation of the complex synaptic connections of corticothalamic
projections and hippocampal mossy fibers.
18

Acknowledgements
Funding: Medical Research Council (G00900901 to ZM’s laboratory). Daiichi Sankyo Foundation ofLife Science, The Uehara Memorial Foundation and KAKENHI (19K23786, 22K06242 to S.H.).
References[1] Maclachlan, C. et al., Frontiers in Neuroanatomy, 2018; 12:88[2] Hoerder-Suabedissen, A. et al., Cerebral Cortex 2019; 29(5):2148-2159[3] Hayashi, S. et al., Cerebral Cortex, 2021;31(5): 2625–2638

Fig. 1. Correlative light and electron microscopy of layer 5 corticothalamic axons.
(A-C) Fluorescent image (A) and EM micrographs (B, C) of labelled axons. Scalebars, 10 μm in (A) and (B), 2.5 μm in (C). Modified from Maclachlan et al., 2018.

A

C

B

D

Fig. 2. SBF-SEM of layer 5 corticothalamic axonal boutons. (A,B) Wild-type. (C,D)
Snap25-cKO. No dendritic excrescence is formed in Snap25-cKO brains. Scale bars,
1 μm. Modified from Hayashi et al., 2021.

19

Bio-03

Getting ahead of the next pandemic: When antibiotics stop working
Amal Seffouh1,2 and Joaquin Ortega1,2

Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A
0C7, Canada, and 2Centre for Structural Biology, McGill University, Montreal, Quebec
H3G 0B1, Canada,
1

Antimicrobial resistance is on track to become the next global pandemic. A recent
analysis of this emerging threat predicts grim scenarios and alarming economic estimatesthat are not difficult to imagine while still immersed in the Covid-19 pandemic. In the last
three decades, discovery efforts have been limited to generate improved versions oflegacy antibiotics that target conventional vital pathways in bacterial species causing
infectious diseases. Resistance to these modified drugs quickly emerges. Developing
novel antibiotics that target unexplored cellular processes not susceptible to existing
resistance mechanisms is urgent. Our research focuses on the assembly process of thebacterial ribosome, which is not currently targeted by any antibiotic. Ribosomes
synthesize all cellular proteins. If an organism cannot make ribosomes, it dies. Ribosomes
are complex molecular nanomachines made up of over 50 components. We found thatcritical steps within the ribosome assembly process have tremendous potential as targets
for developing novel antibiotics. Our recent work identified one of these critical steps(Seffouh et al., 2019; Seffouh et al., 2022). Three proteins, RbgA, YphC and YsxC, bindto the assembly intermediate that accumulates at this step. The three proteins are essential
for the 50S subunit to reach its mature state. We aim to understand how exactly RbgA,
YphC and YsxC act to complete the maturation of this critical assembly intermediate andtransform it into a functional 50S subunit. We use cryo-electron microscopy and single
particle analysis to directly image these proteins to gain insights into how they interact
with the assembling subunit and the maturation steps they catalyze. Uncovering these
critical steps in the assembly process will allow leveraging ribosome biogenesis factors as
a novel antimicrobial target and discovering new antibiotics that will help curve therapidly emerging threat of antimicrobial resistance.

20

Acknowledgements:
Funding: Canadian Institutes of Health Research [PJT-153044].
We thank K. Sears, K. Basu, M. Strauss and other staff members of the Facility for Electron
Microscopy Research (FEMR) at McGill University for help with microscope operation and datacollection. Titan Krios cryo-EM data were collected at FEMR (McGill).
References:[1] Seffouh, A., Jain, N., Jahagirdar, D., Basu, K., Razi, A., Ni, X., Guarne, A., Britton, R.A., andOrtega, J. (2019). Structural consequences of the interaction of RbgA with a 50S ribosomal subunit
assembly intermediate. Nucleic Acids Res 47, 10414-10425. 10.1093/nar/gkz770.
[2] Seffouh, A., Trahan, C., Wasi, T., Jain, N., Basu, K., Britton, R.A., Oeffinger, M., and Ortega, J.
(2022). RbgA ensures the correct timing in the maturation of the 50S subunits functional sites. Nucleic
Acids Res. 10.1093/nar/gkac059.

Fig. 1. Overview of the 50S subunit assembly process. Ribosomal
particles at early stages of assembly follow parallel assembly pathways
that converge into a critical maturation stage. The assembly intermediatethat accumulates is ‘locked’ and its maturation is paused. RbgA, YphC
and YsxC act on this intermediate to ‘unlock’ it and complete thematuration of the functional sites.

21

BINA-01

National and International Bioimaging Networks
Claire M. Brown1,2*

1 Associate Professor, McGill University, Montreal, Quebec, Canada

2 Advanced BioImaging Facility, McGill University, Montreal, Quebec, Canada*claire.brown@mcgill.ca

Over the past decade, a number of national and international networks have emerged
to support bioimaging scientists and their research. The focus of this presentation will beon two Canadian networks - the Canadian Network of Scientific Platforms its Canada
BioImaging technology node, BioImaging North America and the Global BioImaging
network. The Quality Assessment and Reproducibility for Instruments & Images in Light
Microscopy (QUAREP-LiMi) network has leveraged these communities to improvemicroscope quality management and will also be discussed.
Canada BioImaging (CBI) currently has 65 members from 7 provinces and 28
institutions within Canada, and falls under two umbrellas as a technology node of theCanadian Network of Scientific Platforms (CNSP) and a geographical node of
BioImaging North America (BINA). CBI and BINA share many of the same goals andobjectives, with a focus on providing a welcoming and supportive community forbioimaging scientists to grow and support each other, through training and professional
development activities. Both are light microscopy focused, advocating for bioimaging
platforms (a.k.a. core facilities) and imaging scientists working within the platforms whomake up a large proportion of their membership. The CNSP has a broader mandate andencompanses cutting edge scientific infrastructure and expertise within scientificplatforms across science and engineering. The CNSP provides professional development
opportunities, community engagements and advocates to funders and the Canadiangovernment. The CNSP represents 194 scientific platforms in 45 institutions and has
good representation from across Canada. Its efforts are aligned with those of CBI andtechnologies and expertise they need to do high-quality science. The CNSP does
focus more on aspects that apply to all technology areas and not those that apply
specifically to bioimaging. For example, there is a focus on the formation of a national
expertise database, development of Quality Management programs, and a general effort
to develop unique metrics for measuring the impact and showcasing the importance of
Scientific Platforms. BINA represents imaging scientists from the US, Canada and
Mexico and its membership is currently in excess of 700 people, (88% academic, 12%
corporate, 76% USA, 12% Canada, 3.2% Mexico, 9% international with an additional 16
countries represented).
Six BINA working groups, made up of volunteers, engage in activities focused onproviding opportunities for interaction, education, advocacy and standardization. The
BINA Quality Control and Data Management working group joined forces withQUAREP-LiMi and the broader global effort around improving quality assessment andquality control for light microscopy, spearheading the landmark FOCUS issue in Nature
Methods last December [1]. This issue featured 9 articles offering guidelines and tools for
improving the tracking, and reporting of microscopy metadata with an emphasis on
reproducibility and data re-use. This BINA working group also developed a metrology
suitcase program bringing the tools and protocols required for microscope QC to
individual platforms. The Training and Education working group has worked withinternational stakeholders to develop a centralized repository for microscopy resources(https://microscopydb.io/). This database system is intended to be a singular central
22

resource for the global community making it easier to submit, find and share resources(training resources, jobs. conferences, workshops). The database is searchable andpartners can access and display relevant content on their own websites.
Global BioImaging (GBI) represents a significant number of international partners
including CBI and BINA (Figure 1). GBI provides a united voice for all networkorganizations and has working groups focused on addressing the challenges of the global
community (Career Development, Demonstrating Impact). They have brought the global
community together to develop a number of International Recommendations [2] such as
the Impact of Imaging Facilities [3], the Added Value of Open Access Imaging Facilities[4] and Standards for Open Image Data Formats [5] to raise awareness and advocate for
imaging facilities and imaging scientists. They provide training opportunities andinternational job shadowing to support career growth and development. GBI hosts anannual Exchange of Experience meeting to bring global network community
representatives together to share ideas and strategize ways to build and strengthen thecommunity.
In summary, the support and advocacy provided by these national and international
bioimaging networks is democratizing access to advanced microscopes and expertisearound the world, working to sustain the critical research infrastructure and changing theculture by highlighting the importance of imaging facilities and imaging scientists toenable successful world class science.
Acknowledgements: CMB (CZI Imaging Scientist), BINA and GBI are supported by grants from the
Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation.
References: [1] Reporting and reproducibility in microscopy, Nature Methods 03 December 2021
https://www.nature.com/collections/djiciihhjh)[2]
Global
BioImaging
International
Recommendations https://globalbioimaging.org/documents [3] Added Value of Open Access Imaging
Facilities [4] Impact of Imaging Facilities [5] https://pubmed.ncbi.nlm.nih.gov/33948027/

Figure 1: The Global BioImaging Network includes Euro-BioImaging, Japan’s Advanced
BioImaging Support, Microscopy Australia, National Imaging Facility, Mexico’s National Laboratory
For Advanced Microscopy, South Africa BioImaging, India BioImaging Consortium, SingaScope,
Canada BioImaging, BioImaging North America and Latin America Bioimaging. Future members
include: Armenia, Chile, South Korea and the African BioImaging Consortium.

23

24

【Abstracts】
Day 1(Tokyo time: Friday,November 4, 2022)

Materials Science Session 1

25

Mate-01

TEM of Thick polymer or biological specimens

Ray Egerton1*, Misa Hayashida and Marek Malac2
Physics Department, University of Alberta, Edmonton, Canada
NRC-Nano, Edmonton, Canada*
regerton@ualberta.ca
1
2

The continued development of three-dimensional electron imaging (tomography)
has stimulated renewed interest in electron scattering in thick TEM specimens, and inprocedures for extracting structural information from this process. A judicious choice of
microscope parameters requires some knowledge of the angular distribution of electron
scattering as a function of specimen thickness and incident- electron energy.
Using a 300kV TEM, we have measured the bright-field collection efficiency F
and the mass-thickness contrast C (for a 10% thickness change) for carbon samples ofthickness up to 750 nm [1], equivalent to a 1.4 μm of polymer of biological tissue. Fromthese data values, the dose-limited resolution is calculated to be in the range 1 – 2 nm foran objective-aperture semi-angle of β ~ 2 mrad and a characteristic dose of 100 e/Å2,
typical of an organic material.
Of course, radiation damage provides only one limit to spatial resolution. In
fixed-beam TEM mode, chromatic aberration imposes a limit of typically 2 to 3 nm, as
deduced from the chromatic point-spread function [2]; see Fig. 1. In STEM mode, thecombined effect of probe diameter, beam divergence and beam broadening provide asimilar limit, with an optimum convergence semi-angle α = 1 – 2 mrad; see Fig.2. Such
resolution is an order of magnitude better than that offered by optical fluorescencemicroscopy and is substantially better than zone-plate STXM.
Acknowledgements:
NSERC Discovery Grant RGPIN-2018-03835 and NRC funding.
References:[1] M. Hayashida and M. Malac , Microscopy & Microanalysis, Vol. 28, (2022) 659-671..
[2] R.F. Egerton and M. Watanabe, Micron, Vol. 160, (2022) 103304.

26

Fig. 1. Blue: chromatic-aberration PSF for 300kV TEM of 500nm-thickcarbon with β = 2.4 mrad, Cc = 1.6 mm. Red: Fraction of electrons withina given radius, showing that half them lie within a diameter of 2.2 nm.

Fig. 2. 300kV STEM resolution for carbon of thickness 500 nm, plottedagainst probe semi-angle α, for the case of a large collection semi-angle
β (blue curve), and also for β = α (red curve) - demonstrating the effectof collection-aperture collimation on the beam-broadening component.

27

Mate-02

Improving spatial resolution in rapid electron tomography
for hundreds-nm-sized 3D materials

Tomohito Ishii（石井智仁）1, Jun Yamasaki（山﨑順）2, 3*
Department of Quantum Information Electronics, Graduate school of Engineering,Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan
2
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, 7-1,
Mihogaoka, Ibaraki, Osaka 567-0047, Japan
3
Institute of Materials and Systems for Sustainability, Nagoya University, Furo-cho,
Chikusa, Nagoya 464-860, Japan*yamasaki@uhvem.osaka-u.ac.jp
1

Electron tomography is a method for measuring the three-dimensional (3D)
structures and density distributions of materials smaller than 1 micron based onsequential tilt images (tilt series). For high-precision 3D reconstructions, acquisitions ofabout 100 micrographs at different angles are required, resulting in measurements
sometimes longer than 1 hour. In addition to accumulation of irradiation damages, such along measurement time limits the number of observations in a day’s experiment, that is,
the number of field of views and/or the number of sequential 3D measurements of time
evolutions of materials. Recently, rapid electron tomography (RET), in which a tilt series
is recorded as a movie for about 10 seconds, has been realized by a high-speed camera
developed for electron beam detections [1]. However, RET often suffers fromdeterioration of the resolution of the reconstructed 3D volume due to vibrations of thesample holder during the high-speed tilting. Hence the objective of this study is to solve
the problem to recover the original resolution in the 3D reconstruction.
In the present study, we used the high-voltage electron microscope (JEOL:JEM1000EES) of Osaka University, operated at the acceleration voltage of 1250 kV andequipped with a 1600 fps high-speed camera (Gatan: K2-IS). A tilt series of bright-field
TEM images was recorded for 14 seconds as approximately 20,000 frames, while
continuously tilting the sample holder from -70° to +70° at a constant rate (10°/sec).
Polystyrene latex spheres and a crystalline ZnO nanoparticle were employed as thestandard samples to examine the reconstruction quality.
As mentioned before, some frames in the tilt series are blurred due to the holder
vibration as shown in Figs. 1(a) and (d), while others are clear as shown in Fig. 1(b) and(e) although the SNR is poor due to the short exposure time of 1600 fps. We developed a28

method to quantify the image sharpness available even for flames with such poor SNRs.
Based on the method, the sharpest images in each group of 16 consecutive frames(consisting of a tilt range of 0.1 degree) were selected as the representative images ofeach tilt ranges. The 3D reconstruction using only them shows higher resolution thanthat using all the frames, as shown in Fig. 2. However, since the flames other than the
representatives are discarded, the SNR in the reconstructed volume becomes poor. To
enhance the SNR in the representative frames, we utilized robust principal component
analysis (RPCA), which is effective to separate the object image and the backgroundnoise [2], as shown in Figs. 1(c) and (f).
Figure 2 shows comparison of the 3D volumes of the latex spheres and ZnOnanoparticle reconstructed based on all the frames and on only the representative frames,
without and with the RPCA processing. As clearly shown in Fig. 2(i), the resolution
estimated from the 3D volumes has been improved by selecting the representative
frames in exchange for the decrease in the SNR, which has been recovered successfullyby the RPCA. Thus, the combined use of the sharp image selection and the RPCA iseffective to increase the quality of the reconstructed 3D volume in RET.
Acknowledgements:

This study was partly supported by MEXT KAKENHI (grant number 19H02600).
References:[1] V. Migunov, et al. Sci. Rep. 5 (2015) 14516.

[2] E. Candes, et al, J. ACM, 58 (2011) 11.

Fig. 1: Single frames sampled from RET tilt series consisting ofabout 20,000 frames for latex spheres(a-c) and ZnO (d-f).
(a)(d)Blurred frames. (b)(e)The sharpest frames selected as therepresentatives from a tilt range of 0.1 degree. (c)(f)The
representatives after denoised by RPCA. The values for image
sharpness (blue) and SNR (green) are shown in each image.

Fig. 2: Comparison of the reconstructed volumes of latex spheres(a-d) and ZnO(e-f). (a)(e) shows the
slice planes. The slice images reconstructed on (b)(f) all the frames and (c)(g) only the sharpest
frames, and (d)(h) after the RPCA processing. (i) Image resolution and SNR measured in thereconstructed 3D volume by FBP.

29

Mate-03

Magnetic dynamics probed by ultrafast
transmission electron microscopy

Takahiro Shimojima
RIKEN Center for Emergent Matter Science (CEMS), Wako 351-0198, Japan
It has been an important issue to track the magnetic dynamics in nanometerscale, since it provides a direct evidence for the manipulation of the self-organizedmagnetic nanostructures, such as skyrmions[1,2], under external stimuli. The
characteristic time scales for their motions are expected to be very fast which requires
better time resolution for the Lorentz transmission electron microscopy (LETM)
observation. A promising way to improve the time resolution of the electron microscopy
is the application of the ultrafast optical pump-probe technique[3]. Previous
pump-probe LTEM[4,5] studies reported the morphorogical changes of the magnetic
objects with the time resolusion being independent of the camera response time (ms).
Here, we report skyrmion dynamics in defect-introduced Co9Zn9Mn2 by using
pump-probe LTEM[6]. Following the nanosecond photothermal excitation, we resolve
160-nm skyrmion’s proliferation at <1 ns, contraction at 5 ns, drift from 10 ns to 4 µs,
and coalescence at ~5 µs. These motions relay the multiscale arrangement andrelaxation of skyrmion clusters in a repeatable cycle of 20 kHz. Such repeatable
dynamics of skyrmions, arising from the weakened but still persistent topologicalprotection around defects, enables us to visualize the whole life of the skyrmions anddemonstrates the possible high-frequency manipulations of topological charges broughtby skyrmions.
References:[1] S. Mühlbauer et al., Science 323, 915 (2009).
[2] X. Z. Yu et al., Nature 465, 901 (2010).
[3] B. Barwick, et al., Science 322, 1227 (2008).
[4] G. Berruto et al., Phys. Rev. Lett. 120, 117201 (2018).
[5] M. Möller et al., Communications Physics 3, 36 (2020).
[6] T. Shimojima et al., Science Advances 7, eabg1322 (2021).

30

31

Mate-04

Ferroelectric nanostructures observed by transmission electron
microscopy

Hiroshi Nakajima1* and Shigeo Mori1
1
Department of Materials Science, Osaka Metropolitan University, Sakai, Osaka
599-8531, Japan. *h-nakajima@omu.ac.jp
Ferroelectrics are known as important functional materials that play various
roles in electronics, various sensors, and energy harvesting applications. The functional
properties depend largely on the polarization reversal caused by the movement of theboundary between domains with different polarization directions within the solid.
Therefore, it is important to clarify how domain walls exist and how they move
microscopically in ferroelectrics [1]. In particular, charged domain walls of ferroelectrics,
in which ferroelectric polarizations face each other, have the property that the electricalconductivity changes significantly at the domain wall, even though the width is only a
few nanometers. Although such unique charged domain walls have been found in someferroelectrics, their structures are energetically unstable and thus it is an important issue
to elucidate the stabilization mechanism. In the layered perovskite ferroelectric oxide
Ca3-xSrxTi2O7 (x = 0.54), many charged domain walls have been observed in the crystal[2], but their microscopic structure has not been clarified. In this study, we visualized thecharged domain structure in Ca3-xSrxTi2O7 (x = 0.54) on an atomic scale using scanningtransmission electron microscopy (STEM) and clarified its structure [3].
Figure 1a shows a HAADF-STEM (High-angle annular dark-field STEM)
image of a charged domain wall at [] incidence. Noticeably, the crystalline domains

on both sides of the charged domain wall are translating. The domain wall structure isshown in Figure 1b. The translational distance of the crystal domain corresponds to0.1889 times the c-axis length, and such a boundary is called an out-of-phase boundary[4]. In addition, the atoms are brighter at the interface than in the interior of the crystal,suggesting elemental segregation at the interface.
Figure 2a shows a dark-field image of the observed region. In this region, linear
charged domain walls are formed, indicating the existence of domain walls on amacroscopic scale. The results of elemental analysis (Fig. 2b-f) show that the Sr and Ca
atomic rows face each other across the charged domain wall, which confirms the structure
shown in Figure 1b. The distribution of Ti also indicates that the crystal domain istranslationally shifted. Furthermore, corresponding to the strong intensity in the32

HAADF-STEM image, Sr elements are segregated at the interface as indicated by thewhite circles.
In summary, we observed the charged domain structure by a combination of
TEM dark-field method and high-resolution HAADF-STEM methods. The charged
domain wall is energetically unstable due to the divergence of the electric field caused by
the facing electric polarizations. As a stabilization mechanism, an out-of-phase boundary
is formed in the charged domain wall. In such a structure, the electric polarization of each
atom is partially parallel as indicated by the dotted lines (Fig. 1b). Therefore, such astructure is considered to make the charged domain wall more stable.
References:[1] G. Catalan, et al., Rev. Mod. Phys. Vol. 84, (2012) 119.
[2] Y.S. Oh, et al., Nat. Mater. Vol. 14, (2015) 407.
[3] H. Nakajima, et al., Commun. Mater. Vol. 2, (2021) 109.
[4] M.A. Zurbuchen, et al., J. Mater. Res. Vol. 22, (2007) 1439.

Fig. 1. Structure of a charged domain wall and its schematic. a. HAADF-STEM
image. b. Crystal structure. Ps is the direction of macroscopic electric
polarization. Arrows indicate the direction of displacement of each atom.

Fig. 2. Elemental analysis of charged domain wall. a. Dark-field image by 113
reflection. The yellow box indicates the area observed by STEM. b-f.
EDS-STEM images at various absorption edge energies.
33

Mate-05

In-situ L-TEM observations of magnetic skyrmion and antiskyrmion
dynamics
Licong Peng1*
1*

RIKEN Center for Emergent Matter Science (CEMS), Wako, Saitama 351-0198, Japanlicong.peng@riken.jp

Magnetic skyrmions (topological “particle”) and antiskyrmions (“antiparticles”)
hosting opposite topological numbers have attracted much interest in fields offundamental physics and spintronics. Driving and controlling their motions promises
skyrmion/antiskyrmion-based spintronic devices.
Here I will introduce the in-situ Lorentz TEM imaging of topological spin textures
and their dynamical behaviors. First, I will show the manipulation of a single skyrmion atroom temperature in the chiral-lattice magnet Co9Zn9Mn2-based microdevice using
nanosecond current pulses. We have directly observed the skyrmion translation andtransverse Hall motion and a dynamic transition from the static pinned state to the linear
flow motion via a creep event using Lorentz TEM [1]. In addition to skyrmions, I willthen present the real-space control of room-temperature antiskyrmions in magnets with
D2d [2] and S4 [3-4]symmetries. We have tuned the external magnetic field, temperature,
thickness, and sample geometries to induce the topological transformations among theantiskyrmions, elliptical skyrmions, and non-topological bubbles via the creation,
propagation, and annihilation of Bloch line pairs. We have also used the electric current
to drive the antiskyrmion movement efficiently.
Figure 1 shows the single-skyrmion motion stimulated by the pulsed current in the
Co9Zn9Mn2-based microdevice at room temperature [1]. Figure 2 shows the topologicaltransformation among antiskyrmions, skyrmions, and non-topological bubbles inmagnets with D2d [2] and S4 [3-5]symmetries.
In summary, we have utilized Lorentz TEM to tune the topological nature ofskyrmions/antiskyrmions, including the topological number, helicity, and lattice form,and directly observed the dynamical motions, paving a route to controlling topologicalspin textures with spintronic functions.

34

Acknowledgments:
These works were done in collaboration with Profs. Yoshinori Tokura, Naoto Nagaosa, Taka-hisa
Arima, Shinichiro Seki and Drs. Yasujiro Taguchi, Kosuke Karube, Rina Takagi, Wataru Koshibae,
Kiyou Shibata, Konstantin V. Iakoubovskii, Fumitaka Kagawa, Kiyomi Nakajima. This work waspartly supported by Grants-In-Aid for Scientific Research (A) (Grant No.19H00660) from JSPS, andJapan Science and Technology Agency CREST (Grant No. JPMJCR1874 and No. JPMJCR20T1)
from JST.
References:[1] L. C. Peng et al. Nat. Commun., 12 (2021), 6797.
[2] L. C. Peng et al. Nat. Nanotech. 15 (2020), 181-186.
[3] K. Karube#, L.C. Peng# et al., Nat. Mater. 20 (2021), 335–340.
[4] L. C. Peng et al. Adv. Sci. (2022), 2202950.

Fig. 1: (a) Crystal structure of Co9Zn9Mn2; (b) Schematic of Co9Zn9Mn2-based
microdevice. (c) Schematic of skyrmion motion with the translational and transverse
displacements induced by pulsed electric current j. (d) Over-focus L-TEM images
showing the single-skyrmion motion stimulated by j = −6.06 × 1010 A m−2 pulsed
current with a pulse duration of 150 ns [1].

Fig. 2. (a) Crystal structure of magnets with D2d [2] and S4 [3-4]symmetries. (b)Magnetic textures of antiskyrmion, non-topological bubble, and skyrmion.
35

Mate-06

Electronic state analysis using monochromated STEM-EELS

Hiroki Kurata1*, Takashi Nemoto1 and Mitsutaka Haruta1
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011,
Japan.
*
kurata@eels.kuicr.kyoto-u.ac.jp
1

Improving the energy resolution of electron energy-loss spectroscopy (EELS)
with a monochromator incorporated in a scanning transmission electron microscope(STEM) is driving new research in electronic state and vibrational analyses in local
regions with high spatial resolution. In order to maximize the features of high energy
resolution EELS, it is necessary to acquire high quality spectrum imaging (SI) data.
Recently we developed a new method to remove the dark reference of a charge coupleddevice (CCD) accurately [1], enabling the detection of a single signal count. Combining
high-precision dark reference subtraction with multi-frame acquisition technique is very
effective for measuring SI data consisting of monochromatic EELS with low signal
strength. In this contribution, we will present application results of electronic state
analysis using the energy-loss near-edge structure (ELNES) appeared in core-loss
spectra.
Figure 1 shows the hole mapping of high-temperature superconductor of
La2-xSrxCuO4±δ [2]. The holes are detected as the pre-peak shown by the vertical arrowin oxygen K-edge ELNES. Two-dimensional hole maps using the pre-peak intensity
visualize the spatial distribution of the doped holes. The hole is preferentially doped at
the oxygen site in the CuO plane in the under-doped sample of x=0.15, but the hole isalso distributed at the apical oxygen sites in the over-doped sample of x=0.3. Such thetwo-dimensional hole mapping has been firstly realized with atomic resolution. Figure 2
shows the temperature dependence of the Ti L2,3-edge ELNES of SrTiO3. The crystal
field splitting between t2g and the eg peaks narrows slightly with increasing temperature,
which is attributed to the elongation of bond length between Ti and oxygen ions. The
crystal field splitting decreases linearly with temperature with a slope of 0.2 meV/K as
shown in Fig. 2(c). Considering the lattice expansion rate of 4 fm/K, the change ofcrystal field splitting due to the bond length expansion appears in the spectrum as achange of 50 meV/pm, suggesting the possibility of the measurement of sub-pm bond36

length change by ELNES. We also discuss the spectrum simulation considering thethermal vibrations.
Acknowledgements:
This work was supported by the JSPS KAKENHI Grants (Nos. 17H02739, 19H02597 and
19K22106).
References:[1] M. Haruta, Y. Fujiyoshi, T. Nemoto, A. Ishizuka, K. Ishizuka and H. Kurata, Ultramicroscopy, 207,(2019), 112827.
[2] M. Haruta, Y. Fujiyoshi, T. Nemoto, A. Ishizuka, K. Ishizuka and H. Kurata, Phys. Rev. B 97,(2018), 205139.
[3] M. Haruta, T. Nemoto and H. Kurata, Appl. Phys. Lett., 119, (2021), 232901.

Figure 1 (a) Oxygen K-edge ELNES of La1-xSrxCuO1±δ. (b) HAADF image (c) Oxygen map (d)and (e) Hole mapping for x = 0.15 and 0.3, respectively.
(a)

L3-eg

L3-t2g

L2-eg
L2-t2g

(b)

eg

t2g

Figure 2 (a) Temperature dependence of Ti L2,3-edge ELNES of SrTiO3 (b) Enlarged L3 peakregion (c) Temperature dependence of the energy splitting between L3-t2g and L3-eg peaks.
37

38

【Abstracts】
Day 2(Tokyo time: Saturday, November 5, 2022)

Materials Science Session 2

39

Mate-07

Accessing Sub-Ångström Ptychographic Information in a ScanningElectron Microscope Below 30 kV

Arthur M. Blackburn1*, Cristina Cordoba1, Matthew Fitzpatrick1, Robert McLeod2
Department of Physics and Astronomy, University of Victoria, 3800 Finnerty Road,
Victoria, BC, V8W 2Y2, Canada.
2
Hitachi High-Tech Canada Inc., 89 Galaxy Blvd, Etobicoke ON, M9W 6A4, Canada.
*ablackbu@uvic.ca
1Advances in electron microscopy techniques and instrumentation aim to
maximize the useful information given from the sample under investigation. Ideally the
information is gained with minimal damage to the sample, and with minimal cost andtime. From these goals there has been a long-standing to break into the high-resolution(sub-Ångström, < 10-10 m) regime using general purpose, non-aberration corrected, lowenergy (< 30 keV) scanning electron microscopes. Experimentally we show that this goal
is now possible.
We achieve this using a relatively simple modifications to a conventionalscanning electron microscope combined with the latest generation high-speed, uncoatedhybrid direct-electron detectors and recent advances in multi-slice ptychographic
reconstruction algorithms. Specifically, we used a Hitachi SU9000 SEM/STEM with anadditional simple projector lens; an uncoated Dectris Quadro camera; and multi-slice
maximum likelihood solvers [1]. The solvers also used blurring convolution [2] on theforward projected model diffraction to help compensate for projective chromaticaberration effects.
Here, as an example, we used samples comprised of gold particles on
free-standing few-layer MoS2 and on thin amorphous carbon. Our experiments used a 20
keV beam energy and a defocused electron probe. The beam was placed at around 2000
points with a known random positional offset from a regular grid over an ~40 nm field ofview. Convergent beam diffraction patterns were collected at each point. Ptychographic
reconstruction, using this diffraction data, yielded a phase and amplitude transfer modelof the samples (Figures 1 and 2). Their fast Fourier transforms show the presence offeatures below 1 Å. These features can also be seen directly in the reconstructions. In thecase of the MoS2 sample we can make direct comparisons to near identical regions of the40

same sample observed at 60 kV in an aberration corrected TEM, where we see similar
high-resolution information is revealed on the material.
The ability to access sub-Ångström information with a 20 keV electron beam, ina non-aberration corrected microscope changes the landscape of options forhigh-resolution electron microscopy. This makes it possible for a wider community toaccess truly high-resolution information from thin materials samples at low energies.
Acknowledgements:
This work was funded by NSERC and Hitachi High-Tech Canada, within a Collaborative Researchand Development project. We thank Dectris AG (Switzerland) for their support and help on thisproject and acknowledge the computing resources provided from the Digital Alliance of Canada.
References:[1] Z. Chen, Y. Jiang, et al., Science, Vol. 372, (2021), 826.
[2] M. Odstrcil, J. Bussmann, et al., Optics Letters, Vol 40, (2015), 5574.

Fig. 1. Phase reconstruction of gold particle (left) and its fast Fouriertransform (FFT, right) formed from 20 keV diffraction data.

Fig. 2. Mixed phase and amplitude image of an Au island on MoS2 (left)
and its FFT (right) reconstructed from 20 keV diffraction data.

41

Mate-08

Automatic Quantification of Microplastic Fibres in ScanningElectron Micrographs

Jane Howe*, Bin Shi, Medhavi Patel, Dian Yu, and R. J. Dwayne Miller
University of Toronto, 184 College Street, Toronto, Ontario, Canada.
Jane.howe@utoronto.ca*

Microplastics are found everywhere, from samples in urban air to oceans of thearctic regions. It has increasingly become a problem to the environment and health ofhumans and other living beings. To evaluate the spread of microplastic pollution,
microplastics were often examined by visible light microscopy and quantified bylabor-intensive visual screening procedures. Among all shapes of microplastics,
microplastic fibres, the most abundant pollution in the marine environment, are difficult
to count due to their curvilinear and elongated shapes.
We first generated the scanning electron micrograph dataset of microplastic
fibres (MPF) collected from various resources, which includes the SEM images andground-truth manual segmentations [1]. We then applied two deep learning models, theclassical U-Net [2] and its viable successor MultiResUNet for the semantic segmentation.
The Semantic segmentation results are presented in Figure 1. Both models significantly
outmatched conventional computer vision techniques and achieved a high average
Jaccard index (Intersection-Over-Union, abbreviated to IoU) over 0.75.
The
pixel-embedding U-Net was implemented for instance segmentation on overlapping
fibres, shown in Figure 2. For shape classification, MPFs are classified using afine-tuned VGG16 neutral network with a high accuracy of 98%. With trained models,
it takes only seconds to segment and classify a new micrograph in high accuracy, which isremarkably cheaper and faster than manual identification.
In summary, the proposed deep learning approaches facilitated microplastics
quantification with high accuracy.
Acknowledgements:
The project is supported by the Water Seed Fund from Institute for Water Innovation, University ofToronto and by the Natural Sciences and Engineering Research Council of Canada (NSERC)'s
Discovery Grant. Electron microscopy was performed at the Open Centre for the Characterization ofAdvanced Materials (OCCAM), funded by the Canada Foundation for Innovation. Experiments were42

performed in part with the help of Dr. Jason Tam. This research was enabled in part by support
provided by Compute Canada.
References:[1] B. Shi, et al., Science of the Total Environment 825 (2022) 153903, and the references therein.
[2] O. Ronneberger, P. Fischer, T. Brox, International Conference on Medical image computing andcomputer-assisted intervention. (2015), 234–241.

Fig. 1. Semantic segmentation results. MultiResUnet/U-Net with # offilters (32, 64, 128, 256, 512) achieved high overall IoU in both cropped
clean/noisy (low SNRs) MPFs datasets with a 5-fold cross validation.

43

Fig. 2. Instance segmentation results. Note in (d) and (e), Mean-shift
and heuristic methods were used for post processing, respectively.
Different instances were labelled in different colours for counting.

44

45

Mate-09

Local structure analysis of interface and polar nano domains
using convergent-beam electron diffraction

Daisuke Morikawa（森川大輔）1*, Kenji Tsuda（津田健治）2
Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku
University, Katahira 2-1-1, Aoba-ku Sendai, 980-8577, Japan
2
Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University,
Aramaki aza Aoba 6-3, Aoba-ku, Sendai, 980-8578, Japan
1

Convergent-beam electron diffraction (CBED) is one of the powerful techniques
for nano-meter area structural analysis. From its specific features such as nano-meterprobe, dynamical scattering, and direct determination of electrostatic potential, CBED
can be applied for the analysis of interfaces and in-situ observation of polar nano-domains.
In this presentation, some examples for recent results obtained by CBED method aredescribed.
Fig. 1 shows CBED patterns obtained from an area of CaTiO3 containing a twinboundary. While the bulk sample of CaTiO3 has a centrosymmetric structure, the twinboundary is reported to have a polar structure. CBED patterns depending on the positionof the electron probe exhibit clear symmetry changes. Different types of twin boundaries
were found, even though the two boundaries show the same symmetry changes. A
quantitative analysis of the CBED pattern revealed a polar crystal structure at the twinboundary [1].
Fig. 2 shows a map of mirror symmetry breaking originates from the tetragonalphase of BaTiO3 under an electric field. Even when applying an electric field that is highenough for switching mesoscopic polar domains, inhomogeneous distributions ofrhombohedral nano domains were still observed. Three different types of nanodomain
configurations in the beam direction under electric field were proposed from comparing
simulated CBED patterns with inhomogeneous stacking models [2].
Acknowledgements:
The authors thank Professor Masami Terauchi for the fruitful discussions and Mr. M. Ageishi and Ms.
K. Sato of Tohoku University for their support during the experiments. This work was partially46

supported by JSPS KAKENHI Grant Nos. JP18H03674, JP18K18931, JP19K14623, and 20H05176,
the Research Program of “Dynamic Alliance for Open Innovation Bridging Human, Environment
and Materials” in “Network Joint Research Center for Materials and Devices.”
References:[1] D. Morikawa and K. Tsuda, Appl. Phys. Lett., 118, (2021), 092901.
[2] D. Morikawa and K. Tsuda, Appl. Phys. Lett., 119, (2021), 052904.

Fig. 1. (a) Reconstructed scanning transmission electron microscopy image of atwin boundary of CaTiO3 formed by summing the intensities of CBED patterns
at the position indicated by the red circle in (b) 1. The STEM image wasreconstructed from 40 × 3 CBED patterns. (b) CBED patterns obtained for the
positions shown in (a) 1–9. The letter “m” denotes mirror symmetry. Position 5
corresponds to the twin boundary. (c) CBED pattern obtained at a twinboundary using an exposure time of 10 sec. and the conventional CBED mode.
[1]

Fig. 2. (a) Symmetry-breaking index map of thetetragonal phase of BaTiO3 under an electricfield of 5.9 kV/cm. (b)–(d) Integrated CBED
pattern from 3 × 3 positions shown in (a). The
CBED pattern (b) shows clear mirror symmetry
corresponding to the tetragonal symmetry. The
pattern (c) shows a deviation from the mirror
symmetry and left-right reversal relation withthe pattern (d). The color arrowheads are
eye-guides for understanding the difference inintensity distributions and symmetries. [2]47Mate-10

Interpretation of 3D EDX maps: multivariate analysis and deeplearning hybrid approach

Frédéric Voisard1, Ryan Gosselin2, Jiří Dluhoš3, Hana Tesařová3, Vendulka Bertschová3,
Nadi Braidy1,2*1Institut Interdisciplinaire d’Innovation Technologique 3IT, Université de Sherbrooke,
3000 Boul. de l’Université Québec, J1K 0A5, Canada Department of Chemical and2
Biotechnological Engineering, Université de Sherbrooke, 2500 Boul. de l’Université,Quebec, J1K 2R1 Canada
3
Tescan Orsay Holding, a.s., 21 Libusina trida, Brno 62300, Czech Republic*nadi.braidy@usherbrooke.ca
Serial sectioning tomography (SST) in the focused ion beam-scanning electron
microscope (FIB-SEM)1 constitutes an extremely powerful tool to capture subsurface
microstructure in three dimensions. When coupled to energy-dispersive spectral
mapping (EDS), FIB-SEMs can generate analytical information of the sampled volume.
However, despite day-long acquisition times, low signal-to-noise ratio forces to choose
between spectral precision and spatial resolution. In addition, simple window integral
algorithms are inappropriate to appreciate the complex and rich data generated by
EDS-SST. On one hand, multivariate analyses can certainly tackle the data complexity
but not the load. On the other hand, the low signal-to-noise level severely impedes thegeneration of an acceptable training dataset for deep learning. There is therefore an
opportunity to leverage the capability of multivariate analyses algorithm to classify low
SNR data to produce the training dataset for the deep learning step. Here, we test thisidea using a multi-phasic tribological coating made of tungsten carbides in an ironaluminide matrix. The coating was deposited using high-velocity oxyfuel jet of aball-milled powder containing 30 % vol WC in Fe3Al powder.
The multi-modal 3D data set was collected on an area of 51.4× 51.4× Z µm3 with X-ray
maps collected using 1000 channels between 0-10 keV and 728× 817 pixel sampling and
186 slices using a TESCAN AMBER FIB-SEM equipped with a backscattered electron
detector (BSE) and an Oxford Ultim100 X-ray detector. Each EDS frame tookapproximately 6 minutes. The polyphasic and porous nature of the sample makes thesole BSE image segmentation difficult via simple thresholding methods. A typical X-ray
map (Fig. 2a) shows the presence of at least 5 phases, with phases sharing similarelements. In a first attempt to use deep learning, a training set was performed manuallyby thresholding the BSE signal and using the 4 EDS maps. 4 phases were used, believed48

to represent Al2O3, Fe3Al, W/WC as well as a “pore” phase. The 3D reconstruction of
the classified voxels was found rough, with many areas mislabeled by manual selection
error.
In a second attempt, the training dataset was produced using a hierarchal classification
scheme2 of the EDS dataset from 8 slices that greatly facilitated the identification of 8
endmembers. Using the endmembers, we could calculate abundance map for each sliceby loglikelihood maximization3 which was converted to an 8-phase mask for each BSE
slice. These segmented BSE slices were then used as the training dataset for a deeplearning step (3-level U-net) using Dragonfly. In this case, additional details are muchclearer and helps streamline the interpretation (Fig. 2c).
References:

1. Berger, S. D. et al. DualBeam metrology: a new technique for optimizing 0.13-um
photo processes. in (ed. Sullivan, N. T.) 423 (2001). doi:10.1117/12.436768.
2. Fauteux-Lefebvre, C. et al R. Anal Chem 90, 13118–13125 (2018).
3. Lavoie, F. B., et al. Chem. Int. Lab. Sys. C, 40–50 (2016).
a) Coregistered data
BSE
EDX

Hierarchal classification

Training

PCA

EDX

3 slices
MVA

12 slices
MVA

Slice selectionb)

3 slicesmanualEnd member
selection

U-shaped
Neural network

Log-likelihood optimization

Classification 3D rendering

MVA training
datasetFig. 1. (a) Algorithms for segmentation. (b) Details of the hierarchal
classification to generate multivariate analysis (MVA) training dataset

Fig. 2. (a) EDX map of a slice over BSE image (b) Machine learning
segmentation from manual selection (c) U-Net deep learning segmentation
using H-MCRLLM as training data49

Mate-11

Atomic Resolution Magnetic Field Imaging by Scanning Transmission
Electron Microscopy
Naoya Shibata1,2,3*

Institute of Engineering Innovation, The University of Tokyo, Tokyo 113-8656 Japan
Nanostructures Research Laboratory, Japan Fine Ceramic Center, Nagoya 456-8587,
Japan
3
Quantum-Phase Electronics Center (QPEC), The University of Tokyo, Tokyo 113-8656,
Japan.
*
shibata@sigma.t.u-tokyo.ac.jp
12

Aberration-corrected scanning transmission electron microscopy (STEM) is a
powerful technique for directly observing atomic-scale structures inside materials anddevices. However, in ordinary STEM, atomic-resolution observation of magnetic
materials is essentially very difficult, since high magnetic fields (>2T) are inevitably
exerted on samples inside the magnetic objective lens. We have succeeded in developing
a new magnetic objective lens system that realizes a magnetic field free environment at
the sample position. Using this new objective lens system combined with thestate-of-the-art higher order aberration corrector, sub-Å spatial resolution inmagnetic-field-free condition is realized by STEM [1]. This novel electron microscope(Magnetic-field-free Atomic Resolution STEM: MARS) is expected to be useful for
research and development of advanced magnetic materials and steels.
On the other hand, by using elaborate detectors such as segmented or pixelated
detectors in STEM, we can not only image atoms, but can also image electromagnetic
field distribution inside samples through differential phase contrast (DPC) imaging
techniques [2]. Applying DPC imaging in atomic resolution STEM, we can now directly
visualize the electric field distribution within single atoms [3], i.e. the electric field
between the positively charged atomic nucleus and the negatively charged electron cloud.
Therefore, it becomes tempting to directly observe magnetic fields at atomic resolution
by MARS. However, to visualize magnetic fields at atomic scale, we must solve
remaining major difficulties even after magnetic-field-free atomic-resolution STEM
imaging becomes possible. One major difficulty is that both the specimen’s electric andmagnetic fields inevitably contribute to the phase shift of the incident electron probe. To
visualize magnetic fields, we must differentiate between electric and magnetic phase shiftcomponents in the atomic-resolution DPC images. The other major difficulty is that the50phase shift induced by the atomic-scale magnetic field is predicted to be extremely small.
Thus, very high signal-to-noise ratio imaging conditions should be fulfilled. We havedemonstrated that the real-space visualization of intrinsic magnetic fields of an
antiferromagnetic material is possible by combining kernel filtering and unit-cellaveraging techniques to solve the difficulties of differentiating between electric andmagnetic phase shift components and extremely small magnetic phase shifts (Figure 1)[4]. The details of this magnetic-field-free STEM and the atomic-scale imaging ofmagnetic fields will be presented in the talk.
Acknowledgements:
The author thanks all the collaborators of this research, especially Y. Kohno (JEOL Ltd.), T. Seki, T.
Matsumoto, Y. Ikuhara (U. Tokyo), S.D. Findlay (Monash U.) for their contribution to the works
shown in this presentation. The author acknowledges support from the JST SENTAN Grant Number
JPMJSN14A and the JSPS KAKENHI Grant numbers 20H05659, 19H05788, 17H06094.
References:[1] N. Shibata et al., Nature Comm. 10, 2380 (2019).
[2] N. Shibata et al., Nature Phys., 8, 611-615 (2012).
[3] N. Shibata et al., Nature Comm. 8, 15631 (2017).
[4] Y. Kohno et al., Nature 602, 234-239 (2022).

_ _Fig. 1. (left) Unit cell averaged and tiled ADF image of α-Fe2O3 observed along the [1120] direction. (right) The corresponding projected magnetic field vector color map. The
antiparallel magnetic field component on the adjacent Fe-Fe double atomic layers isclearly observed, visualizing antiferromagnetic order in this crystal [4].

51

【Organizers】
Shigeo Mori (Osaka Metropolitan University)
森

茂生（大阪公立大学）

Kazunori Toida (Kawasaki Medical School, Osaka University)
樋田一徳（川崎医科大学、大阪大学）

Ken Harada (RIKEN)
原田

研（理化学研究所）

Marek Malac(National Research Council Canada, University of Alberta)
Misa Hayashida (林田美咲)(National Research Council Canada)

52

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【Venue Information】
Kawasaki Sukenobu Memorial Lecture Hall,
Located in Kawasaki University of Medical Welfare.
https://w.kawasaki-m.ac.jp/language/english/
Matsushima 288, Kurashiki, Okayama 701-0193, Japan
From Okayama station to Nakasho station,
JR Sanyo line: approx. 15 minutes.
JR Hakubi line: approx. 13 minutes.
From Kurashiki station to Nakasho station,
JR Sanyo line: approx. 6 minutes.
JR Hakubi line: approx. 6 minutes.

54

From Nakasho St. to Kawasaki University of Medical Welfare
Walk: approx. 10 minutes.

55

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