89 IUVSTA Workshop - NPL

89th IUVSTA Workshop

Biological and soft matter sample preparation for the high-resolution imaging by high-vacuum techniques

19th – 24th May 2019

Hotel Belvedere

Zakopane, Poland

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Contents

WELCOME ............................................................................................................................................... 5

CONFERENCE INFORMATION .................................................................................................................. 7

SPONSORS ............................................................................................................................................. 11

WORKSHOP SCHEDULE ......................................................................................................................... 13

PROGRAMME ........................................................................................................................................ 15

Day 1 - 20th May ................................................................................................................................ 15

NanoSIMS & SIMS ......................................................................................................................... 21

Day 2 – 21st May ................................................................................................................................ 29

NanoSIMS & SIMS ......................................................................................................................... 29

SIMS .............................................................................................................................................. 37

Poster Session ............................................................................................................................... 37

Day 3 - 22nd May ................................................................................................................................ 49

Atom Probe Tomography .............................................................................................................. 49

Day 4 - 23rd May ................................................................................................................................ 57

Spectroscopies .............................................................................................................................. 57

Day 4 - 23rd May ................................................................................................................................ 63

Applications, novel solutions and technologies ............................................................................ 63

Day 5 - 24th May ................................................................................................................................ 73

Applications, novel solutions and technologies ............................................................................ 73

NOTES.................................................................................................................................................... 80

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WELCOME

It is a great pleasure to welcome you to the 89th IUVSTA Workshop on the preparation of biological

and soft matter samples for high-resolution imaging by high-vacuum techniques.

This workshop is bringing together leading scientists, researchers and instrumentation developers in

electron microscopy, secondary ion mass spectrometry, atom probe tomography, cryo-spectroscopy

and related technologies to address important challenges in biological and soft matter sample

preparations. This is a forum where experts share their views and engage in in-depth discussions to

identify and solve current challenges and issues.

We are very grateful to our sponsors for supporting this workshop and to the scientific community for

your contributions making this workshop possible.

We hope you will enjoy your stay in Zakopane!

The Organising Committee:

Paulina Rakowska, National Physical Laboratory, UK

Kirsty MacLellan-Gibson, National Institute for Biological Standards and Control, UK

Anja Henß, Justus-Liebig University Gießen, Germany

Giacomo Ceccone, European Commission, Italy

Junting Zhang, National Physical Laboratory, UK

Stephan Gerstl, ETH Zurich, Switzerland

Senior Advisory Committee:

Ian Gilmore (National Physical Laboratory, UK)

Anouk Galtayries (Chimie ParisTech, France)

Miguel Manso (Universidad Autónomade Madrid, Spain)

Gregory L Fisher (Physical Electronics, USA)

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CONFERENCE INFORMATION

REGISTRATION, CHECK-IN AND CHECK-OUT

The registration to the conference will commence from 16:00 on Sunday 19th May at the hotel

reception desk.

Check-in to the hotel starts at 16:00 on the day of arrival. Check-out, on the day of departure, should

be done before 12:00.

WELCOME RECEPTION

The welcome reception will start at 19:00 on Sunday 19th May in Pilsudski conference room at level II.

SCIENTIFIC SESSIONS

The main conference presentations and discussions will take place in Pilsudski conference room at

level II. During the breaks, the coffee will be served in open foyer in front of the conference room.

WORKSHOP FORMAT

The workshop format has been developed to provide a forum for discussion and debate. There are

eight sessions, each with time allocated for discussions. The following provides information on the

way the sessions will operate. Very important aspects are the role of discussion leader, rapporteur

and, of course, contributions and discussion from the workshop participants:

• Each session will be chaired by discussion leaders.

• The discussion leaders are responsible for keeping sessions to time.

• The audience may ask questions after talks for technical clarification, but discussion questions

should be saved for the discussion period.

• The role of the discussion leader is to ensure that everyone gets an opportunity to contribute

and to stimulate discussion. Discussion leaders may come prepared with material to focus

discussion.

• We would encourage workshop participants to come prepared with material for the

discussion. The time allocated to participants is at the discretion of discussion leaders and, as

a guide, typically 2 slides taking no more than 5 minutes may be appropriate.

• With the aid of a rapporteur, discussion leaders will summarise the discussions.

• The final summary will be presented at the closing discussion on Friday where the discussion

leaders will form a panel and summarise the key items of the workshop, areas of

recommended future research, potential collaborations and outlook.

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POSTER SESSION

The poster session will take place in Pilsudski conference room at level II. The posters should be put

up during the Tuesday’s lunch break at the latest. They can stay up all week for discussion.

SLIDE PREVIEW

Speakers will be able to use their own laptops (with port HDMI or HDMI adapter) or use a laptop

provided by the venue. If you are planning to use the provided by the venue laptop, we would advise

you to check beforehand that your presentation works. The laptop will be available from Sunday

afternoon in the Pilsudski conference room.

WI-FI

Log in details and password can be obtained from the Reception. You may also connect without

password via social media.

EXCURSION – VISIT TO KOSCIELISKA VALLEY

This will be a guided tour to the Koscieliska Valley of the Tatra National Park. The bus will pick up

participants at 14:00 from outside the hotel.

The bus ride to the Valley entrance will take about 20 minutes. Then, walk into Valley will take 2 - 3

hours. The trip may be shortened depending on weather conditions and participants wishes.

It is advised to bring some comfortable shoes (trekking or sport shoes) and appropriate to the weather

outdoor clothing including, if necessary, a rain protection. There is a shelter on the end of the valley

but it's almost 2 hours walk one way.

BREAKFAST

Breakfast will be served in one of the restaurants on level I from 07:00 until 11:00

LUNCH

Lunch will be provided in Wieniawy Restaurant at level I of the Hotel

DRINK VOUCHERS

Participants will be given drink vouchers for the main events: welcome reception, poster session and

gala dinner. There will be a choice of alcoholic and soft drinks.

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BARS & RESTAURANTS

There are several places where additional drinks and food can be purchased:

• Lobby Bar at level 0, next to the Reception - open until 23:00

• Restaurant a'la carte 'Pod Aniołem' at level IV - open until 23:00

• Bar U-boot at level I - open until 24:00

There are also mini bars in the rooms, which are extra to pay and not included in the registration nor

the accommodation costs.

POOL, SAUNAS, GYM, JACUZZI

The use of pool, saunas, gym and jacuzzi is complementary. The facilities are open from 08:00 - 12:00

and 14:00 – 22:00. There is also a SPA but all the treatments need to be paid individually.

HELP

In case any assistance is needed, there is always 24/7 Reception staff ready to help. You can also speak

to a member of the organising committee.

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SPONSORS

This workshop has been made possible through generous sponsorship from the

International Union for Vacuum Science, Technique and Applications (IUVSTA)

The Organising Committee is also grateful for the additional generous sponsorship from:

Gold sponsor

Silver sponsors

Bronze sponsor

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WORKSHOP SCHEDULE

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PROGRAMME

Day 1 - 20th May

Session:

Electron Microscopy

Session co-chairs:

Kirsty Mac Lellan-Gibson & Paulina Rakowska

Electron Microscopy

Welcome: P. Rakowska 8:30 – 8:40

Introduction to IUVSTA: G. Ceccone 8:40 - 9:00

I: P. Hawes 9:00 - 9:30

C: K. MacLellan-Gibson 9:30 - 9:50

S: F. Leroux (Leica) 9:50 - 10:10

Coffee Break 10:10 - 10:30

I: Paul Verkade 10:30 - 11:00

C: L. M. Valencia 11:00 - 11:20

Discussion 11:20 - 13:00

Lunch 13:00 – 14:00

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Imaging the cell biology of virus-host interactions

Prof Pippa Hawes

Head of Bioimaging, The Pirbright Institute, Ash Road, Pirbright, Surrey, UK.

The inside of an electron microscope is not a natural environment for biological tissue, therefore a

significant amount of sample preparation has to occur before imaging can be attempted. Cells thrive

in aqueous surroundings and if dried, for example in a vacuum, many artefacts will be introduced

making identification of their ultrastructure impossible. Biological samples have been prepared by

chemical fixation for decades, however this process introduces an extensive set of artefacts which can

influence interpretation. Cryo methods of preparation preserve samples in a manner more

representative of the native state, but there are other technical difficulties to address. The best

preparation method depends on the type of sample and the information needed, and the key to

interpreting your data is understanding what happened to your sample during preparation.

In this presentation I will discuss some of the most common preparation methods in the study of cell

biology within the context of high consequence veterinary virus research.

When viruses infect cells they hijack cellular machinery in order to replicate. This can have dramatic

effects on the ultrastructure of the cell. These structural changes need to be correlated with changes

in the proteome of the cell as the virus induces production of new viral proteins. The function of these

proteins is key to understanding virus replication, and identifying the location of the proteins is an

important step in this process. Once the replication cycle for a virus has been described in detail it

may be possible to manipulate the system in order to produce a future vaccine or antiviral treatment.

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The challenges of adequate cryoprotection for UHVAC imaging techniques

Jean-Luc Vorng1, Paulina D Rakowska1, Robert J Francis2, Anwen Bullen2, Kirsty MacLellan-Gibson2*

1. Biological Imaging, National Institute for Biological Standards and Control, Blanche Ln., Potters Bar, EN6 3QG

2. NiCE-MSI, National Physical Laboratory, Hampton Road, Teddington, TW11 0LW.

* Corresponding Author

In an ideal world analysis of a biological specimen should be performed on a sample which is identical

to that found in nature; in practice this is rarely possible. Biological specimens need to be prepared

to ensure that analyses can be performed reproducibly under conditions not usually conducive to life,

such as under a vacuum. One approach is to immobilise the liquid water in the sample. This can be

done by cryogenic fixation, rendering the water solid and able to withstand analysis in a vacuum.

The biological material within a specimen is liable to damage by improper sample preparation, which

may result in the misinterpretation of data or even the generation of false results. Multiple techniques

have been developed within the microscopy world to try and reduce these artefacts. Many of these

methods rely on cryogenic fixation of the biological material, preserving the sample in a vitreous

(glass-like) state. Unfortunately freezing biological specimens often results in the formation of ice

crystals, which destroy the delicate ultrastructure of the cells. In order to reduce the incidence of ice

crystal formation a cryoprotectant strategy can be used. A good cryoprotectant strategy should direct

the sample towards vitrification, whilst leaving the sample unaltered.

One often overlooked area of biological sample preparation by freezing is the role that

cryoprotectants play. In this talk I will discuss the optimisation of cryoprotection for correlative

electron microscopy and nanoSIMS approaches. I will also describe the application of different

cryoprotection and sample preparation methods to the preparation of frozen hydrated material of

‘difficult’ samples including brain tissue, hard materials and plant tissue. Finally, I will discuss ways to

approach samples where composition cannot be modified, such as bulk vaccine preparations and

highly unstable biological constructs.

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Cryo-preparation workflows for high vacuum analysis

Frederic Leroux

Leica-Microsystems

Our understanding of how a biological system works is intimately tied to the capabilities of imaging

systems that allow us to resolve and analyze both their chemical and interacting constituents at high

spatial and temporal resolution. A variety of high vacuum techniques is available to study these

systems. These techniques provide valuable complementary information about the molecular

composition and morphology of biological samples. Cryo analysis is increasingly becoming a

mainstream technology and most of the high vacuum instruments can be operated under cryo

conditions. This lecture is intended to provide an overview on different strategies to prepare, process,

screen and transfer biological samples under cryo conditions for and to high vacuum instruments.

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Title Correlative Light Electron Microscopy (CLEM) strategies for life sciences

Paul Verkade

Professor of Bioimaging, School of Biochemistry, Biomedical Sciences Building, University Walk, University of Bristol, Bristol, UK

A wide variety of Microscopy techniques is underpinning key discoveries Biomedical research. For

instance, Live light microscopy can show us the dynamics of a system. On the other hand Electron

Microscopy (EM) gives us better resolution combined with a structural reference space.

Correlative Light Electron Microscopy combines the strengths of light and electron microscopy in one

experiment and the sum of such an experiment should provide more data / insight than each

technique alone (hence 1 + 1 = 3). There are many ways to perform a CLEM experiment and a variety

of microscopy modalities can be combined (see e.g. Brown et al., 2012, Olmos et al., 2015). The choice

of these instruments and the experimental approach should primarily depend on the scientific

question to be answered.

Any CLEM experiment can usually be divided in 3 parts; probes, processing, and analysis. I will discuss

3 processing techniques based on light microscopy in conjunction with Transmission Electron

Microscopy, each with its specific application and its advantages and challenges.

The application of CLEM technology is not limited to pure cellular based systems. One of the aims of

Synthetic Biology is to mimic and improve existing biological systems for instance for the delivery of

drugs. This can be achieved by vesicles that are formed of lipids but in our case we have been using

Self Assembling peptide caGEs (SAGEs, Fletcher et al., 2013). In order to analyse how such synthetic

systems are taken up, trafficked and processed in cells we can employ a large number of the

techniques we use for “standard” cell biological research. Sometimes a fairly simple approach suffices

to answer the question. In other cases however it requires the development of new tools and

instruments to adequately do so. In my presentation I will highlight this approach through a number

of examples.

References

Brown, E., J., Van Weering, T. Sharp, J. Mantell, and P. Verkade (2012). Capturing endocytic segregation events with HPF-CLEM. Methods in Cell Biology, Volume 111: Correlative Light and Electron Microscopy, 175-201.

Olmos, Y. L. Hodgson, J. Mantell, P. Verkade, and J.G. Carlton (2015). ESCRT-III controls nuclear envelope reformation. Nature, 522: 236–239.

Fletcher, J., R. Harniman, F. Barnes, A. Boyle, A. Collins, J. Mantell, T. Sharp, M. Antognozzi, P. Booth, N. Linden, M. Miles, R. Sessions, P. Verkade and D. Woolfson (2013). Self-assembling cages from coiled-coil peptide modules. Science, 340: 595-599.

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Polymer-based materials for Additive Manufacturing: specimen preparation methods for analysis by TEM

L. M. Valencia1, M. de la Mata1, M. Herrera1, J. Hernández2, S.I. Molina1

1. Departamento de Ciencia de los Materiales e Ingeniería Metalúrgica y Química Inorgánica, IMEYMAT, Facultad de Ciencias, Universidad de Cádiz, Campus Río San Pedro s/n, 11510 Puerto Real (Cádiz), Spain

2. Departamento de Ingeniería y Ciencia de los Materiales y del Transporte, Universidad de Sevilla, 41092 Sevilla, Spain

Nowadays, additive manufacturing (AM) is gaining interest and experiencing a significant evolution due to a number of advantages. This technology allows the fabrication of individual objects with complex geometries. Materials like polymers, metals and ceramics can be used to produce a large number of different products with countless applications (Gibson, Rosen, & Stucker, 2015). Due to the continuous growth in the use of these techniques, new materials with optimal properties for different applications and suitable to be implemented in AM are required. In particular, the use of polymer blends and polymer-based composites are under intense research due to their versatility.

Understanding the functional properties of these novel polymeric materials requires the analysis of their structural properties, where transmission electron microscopy (TEM) techniques may play an essential role. TEM can provide information about the composition of the material, the distribution of different phases (Uribe & Tarpani, 2017) or the distribution of nanoaditives (Du, Guo, & Cai, 2018), the orientation of the C chains, etc. However, the analysis of polymeric materials by TEM is challenging, as they are prone to radiation damage. Different solutions such as reducing the electron dose during the TEM analysis have been proposed in order to overcome these limitations. Additionally, specimen preparation for TEM in polymers is more complex than in other materials as they normally are rather soft. The preparation of clean and thin lamellae of polymeric materials is essential in order to avoid artifacts during the TEM analysis.

In this work, we compare the quality of different TEM specimens of polymer obtained using different preparation methods. In particular, the material of interest consists of a composite formed by a mixture of acrylates working as matrix and metal nanoparticles, such as Ag, as additive of the polymer. The preparation methods considered are the following: mechanical thinning followed by Precision Ion Polishing System (PIPs), ultramicrotomy using a diamond knife, ultramicrotomy with a glass knife and Focused Ion Beam (FIB) sample preparation. The quality of the specimens prepared is evaluated through their analysis by electron microscopy related techniques.

References

Du, L., Guo, A., & Cai, A. (2018). Polydopamine-functionalised graphene–Fe3O4–Ag magnetic composites with high catalytic activity and antibacterial capability. Micro & Nano Letters, 13, 518–523. http://doi.org/10.1049/mnl.2017.0593

Gibson, I., Rosen, D. W., & Stucker, B. (2015). Rapid Prototyping to Direct Digital Manufacturing. Additive Manufacturing Technologies. http://doi.org/10.1007/978-1-4419-1120-9

Uribe, B. E. B., & Tarpani, J. R. (2017). Interphase analysis of hierarchical composites via transmission electron microscopy. Composite Interfaces, 24(9), 849–859. http://doi.org/10.1080/09276440.2017.1299428

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Day 1 - 20th May

Session:

NanoSIMS & SIMS

Session co-chairs:

John Fletcher & Peter Sjövall

NanoSIMS & SIMS

I: K. Moore 14:00 - 14:30

C: L. H. Søgaard Jensen 14:30 - 14:50

I: M. L. Kraft 14:50 - 15:20

Coffee Break 15:20 - 15:40

I: S Reipert 15:40 - 16:10

C: N. Sakamoto 16:10 - 16:30

Discussion 16:30 -18:00

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Revealing trace element and stable isotope subcellular distributions in plants and bacteria with NanoSIMS

Katie L. Moore*, Mary Burkitt-Gray, Rebeca Lopez, Sadia Sheraz, Ryo Sekine

* School of Materials, University of Manchester, Manchester, M13 9PL, United Kingdom

In this presentation I will discuss how the NanoSIMS technique (high resolution secondary ion mass

spectrometry) can be applied to the analysis of plant and bacteria samples.

The analysis of plant material with NanoSIMS, which operates under ultra-high vacuum, is complicated

by the need to preserve not only the in vivo structure of the cellular components but also the

elemental distribution. There are two approaches for sample preparation, chemical and cryo fixation

with the choice dependant on the nature of the elements to be studied. In all cases the ideal sample

preparation methodology workflow is fixation, dehydration, resin embedding and microtomy to

create a sample that is flat and high-vacuum compatible. I will also discuss the use of stable isotope

labelling to help understand uptake and mobilisation mechanisms in plants and how samples can be

isotopically labelled.

The sample preparation for NanoSIMS will be discussed in the context of some of our results that have

been acquired from the NanoSIMS. The majority of this work has focused on the analysis of major

crops, rice and wheat, with a particular focus on the effect of micro- and macro-nutrient distributions,

notably iron and nitrogen, that affect human health. We have also investigated elements which have

a deleterious effect on human and plant heath such as arsenic and aluminium. Furthermore, we have

followed up our arsenic work in rice by investigating the mechanisms by which bacteria in the rice

paddy fields remobilise As and make it easily taken up by the rice plants.

Recent work has also investigated nanoparticle distributions in plants and algae. While the spatial

resolution and destructive nature of the NanoSIMS would suggest that experiments on nanoparticles

are not possible, we have some very interesting results that were either not achievable with other

more common techniques or the NanoSIMS data has proven complementary or key to validating data

from other techniques.

Throughout my presentation I will emphasise how we have used complementary and correlative

imaging to gain a deeper understanding of the samples than could be obtained from one technique

alone and the advantages of interdisciplinary research.

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Sample preparation for CryoNanoSIMS

Louise Helene Søgaard Jensen1, Tian Cheng2, Florent Olivier Vivien Plane1, Stephane Escrig1, Arnaud Comment2, Ben van den Brandt3, Anders Meibom1

1 Laboratory for Biological Geochemistry, École Polytechnique Fédérale de Lausanne, Switzerland

2 Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK

3 Laboratory for scientific development and novel materials, Paul Scherrer Institute, Villigen, Switzerland

The NanoSIMS instrument is a magnetic-sector, multi-collecting ion probe performing mass-spectrometry on secondary ions sputtered from a solid target by a primary beam of charged particles (either Cs+ or O-). The secondary ions sputtered from the sample are transferred with high transmission to a multi-collection mass-spectrometer, where they are counted in electron multiplier detectors, or as currents in Faraday cups. The NanoSIMS primary beam can be focused to a spot smaller than 100 nm in linear dimension, which allows us to clearly resolve structures larger than a few hundred nanometers.

The conventional NanoSIMS instrument operates at room temperature and ultra-high vacuum (10-9-10-10 Torr). Classical sample preparation procedures for biological samples developed for electron-beam imaging techniques (e.g. TEM and SEM) meet such constraints. However, these procedures involve steps such as fixation of the tissue, dehydration in ethanol series, and finally embedding into resin. The procedure effectively removes soluble compounds originally present in the samples. What remains in the samples are macromolecular structures, such as proteins, lipids, RNA, and DNA. These macromolecular structures can be isotopically imaged in great detail with a conventional NanoSIMS instrument. This has already created vigorous research programs and important biological insights have been gained across an impressive range of organisms; reviewed in (1, 2).

However, soluble compounds like ions, metabolites, drugs, etc. are lost or significantly displaced during classical sample preparation and thus cannot be analyzed by conventional NanoSIMS analysis. The only certain way to preserve and observe soluble molecular compounds and ions unperturbed in situ in a biological tissue is to create and maintain highly controlled cryo-conditions throughout the chain of preparative and observational procedures. Our method is based on state-of-the-art cryo-methods for sample preparation, starting with high-pressure freezing, followed by cryo-planing of the samples in a cryo-ultramicrotome using specialized holders and subsequent ultra-structural observations with cryo-scanning electron microscopy prior to transfer to the CryoNanoSIMS. The final step being to isotopically image the cryo-prepared samples with ultra-high spatial resolution, permitting precise correlation with the structural information provided by electron microscopy. From a technical development point of view, we have succeeded in this and we will present our workflow and developments to sample preparation for CryoNanoSIMS.

References

1 Hoppe P, Cohen S & Meibom A (2013). NanoSIMS: Technical Aspects and Applications in Cosmochemistry and Biological Geochemistry. Geostandards and Geoanalytical Research 37, 111-154, https://doi.org/10.1111/j.1751-908X.2013.00239.x

2 Nuñez J, Renslow R, Cliff III JB and Anderton CR (2018). NanoSIMS for biological applications: Current practices and analyses. Biointerphases 13, 03B301, https://doi.org/10.1116/1.4993628

Acknowledgements:

We would like to thank EPFL and the Swiss Science Foundation (R'Equipe program) for financial support and Dr. Bruno Humbel, Jean Daraspe, Dr. Celine Loussert, Antonio Mucciolo and Dr. Caroline Kizilyaprak for technical discussions during the early stages of this project.

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Imaging the distributions of cholesterol, distinct lipid species, and select proteins within the membranes of mammalian cells with high-resolution

secondary ion mass spectrometry

Mary L Kraft

Dept. of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA

A method that combines high-resolution secondary ion mass spectrometry (SIMS) and metabolic

isotope labeling has been developed for imaging the distributions of distinct lipid and protein species

in the membranes of mammalian cells with 100-nm-lateral resolution. High-resolution SIMS

performed with a Cameca NanoSIMS 50 enables mapping the elemental and isotopic distribution at

the surface of a sample with as good as 50 nm lateral resolution. To enable detecting specific lipid

species and imaging their distributions on the surfaces of cells using a NanoSIMS, metabolic labeling

is employed to selectively incorporate a rare stable isotope into each lipid species of interest in the

living cells. To permit visualizing these lipid in parallel with distinct membrane proteins, an antibody

functionalized with a distinct non-native element or rare stable isotope that can be detected with a

NanoSIMS are used to label the protein s so that they can be imaged in parallel with the lipid species

of interest. Then the cells are prepared for analysis under ultrahigh vacuum, the cells are chemically

fixed and dehydrated to prepare them for analysis under ultrahigh vacuum. Finally, the distributions

of the components of interest are imaged by mapping their component-specific isotope or elemental

enrichments on the surface of the cell. The application of this approach to imaging the distributions

of rare isotope-labeled cholesterol, sphingolipids, and the influenza virus envelope protein,

hemagglutinin, on the surfaces of mammalian cells will be presented.

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(Cryo)preparation of Cells and Tissues for TEM: Prerequisite for Combining NanoSIMS with Ultrastructural Studies

S Reipert1, AA Legin2, J-M Volland3, G Paredes4, H Goldammer1, M Eckhard1, M Wagner5 and ASchintlmeister5

1. Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, Austria

2. Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Austria

3. Dept. of Limnology and Bio-Oceanography, University of Vienna, Austria

4. Archaea Biology and Ecogenomics Division, University of Vienna, Austria

5. Large Instrument Facility for Advanced Isotope Research in Life Sciences, University of Vienna, Austria

For decades biological TEM has improved its means to preserve cells and tissues for ultrastructural

studies. In doing so it has addressed questions related to the immobilization and fixation of the living

state, the dehydration of fixed samples, and their embedding in resins for subsequent thin-and

ultrathin sectioning. Taken together the technical efforts have been applied for sample preparation as

close to the natural state as possible, under the premise of high-vacuum conditions in the columns of

TEMs.

Similar to TEM, nanometer-scale dynamic secondary ion mass spectrometry (NanoSIMS) also aims at

high resolution imaging under ultra-high vacuum conditions, but for the purpose of high-sensitivity

element specific, isotope-selective analysis. Notwithstanding its performance in analyte detection in

single cells and tissues, NanoSIMS analysts are confronted with limitations with respect to

unambiguous ultrastructure characterization. Since information on ultrastructure could be vital for

the interpretation of results, it makes sense to test the suitability of state-of-the-art sample

preparation for TEM also for NanoSIMS applications. Such studies should include the correlation of

data obtained by both techniques.

The proximity of the Electron Microscopy Facility of the Cell Imaging and Ultrastructure Research

(Faculty of Life Sciences University of Vienna) to the Large Instrument Facility for Advanced Isotope

Research in Life Sciences, with its centerpiece- the NanoSIMS 50L instrument (Cameca) - at our

University, inspired collaboration of both facilities. Our current approach builds up on experience that

sample embedding in epoxy resins for TEM (Agar100 or Low Viscosity; Agar Scientific Ltd., UK) also

suits applications of NanoSIMS to cells and tissues.

Initially, we applied routine chemical fixation and processing at room temperature to cell culture

monolayers exposed to anti-cancer drugs [1]. Consecutive sections were placed on EM-grids and Si-

wafers for TEM and NanoSIMS, respectively. Lysosomes were identified as cytoplasmic target for

accumulation of Pt-compounds, based on the fact that these organelles are large enough for

circumstantial semi-correlative re-localization within consecutive thin sections.

More recently, we started exploring the potential advantage of cryopreparation for correlative TEM /

NanoSIMS studies. In doing so, we have profited from experience in both freezing techniques, such as

plunge- and high-pressure freezing, and accelerated freeze substitution (FS), otherwise known as a

notoriously slow dehydration- and fixation process. For the latter we have realized sample agitation

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at low temperature within commercial automated freeze substitution systems (AFS) by insertion of

agitation modules in the cryochamber of AFS [2,3]. If compared with experimental setups on dry ice

[4], the agitation modules ensure rapid processing, reproducibly, and a high safety standard.

Moreover, we have demonstrated that agitation in AFS offers flexibility in the temperature/time

schedule for FS that is needed for certain samples endowed with strong cell walls, cuticles, or egg

shells [5]. While it appears to be an obvious advantage to apply FS to native samples that are rapidly

frozen, hybrid-processing of chemically fixed, subsequently frozen samples by FS under agitation has

been proven as useful for samples collected in field experiments [6].

Figure 1. Freeze substitution under agitation – an effective way for semi-correlative TEM-and NanoSIMS studies.

a) Patented agitation module for FS, manufactured by Cryomodultech, e. U. (holder: H. Goldammer) inserted in

an AFS2 [5] b) Localization of the stable isotope tracer 13C by NanoSIMS in a part of a microzooid of the ciliate

Zoothamnium niveum. c) The 13C signal correlates with the bacterial symbionts observed in the TEM image [4].

References:

[1] Legin AA et al. (2016). Chem Sci 7: 3052-3061.

[2] Goldammer H and Reipert S (2015). Patent No. AT515423 and German Utility Model DE 21 2015 000 100.

[3] Goldammer H, et al. (2016). Protist 167(4):369-376.

[4] McDonald KL and Webb RI (2011).Freeze substitution in 3 hours or less. J Microsc 243(3):227-33.

[5] Reipert S, et al. (2018). J Histochem Cytochem 66:903-921.

[6] Volland J-M, et al. (2018). ISME J 12:714-727.

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Cryogenic D-SIMS for Biology and Cosmochemistry

Naoya Sakamoto

Isotope Imaging Laboratory, Creative Research Institution, Hokkaido University, N21W10 Kita-ku, Sapporo, Hokkaido 001-0021 Japan;

Several cryogenic stage systems have been proposed for mass spectrometry [e.g. 1], but few systems

have mechanisms to introduce frozen sample to the cold stage. Secondary ion mass spectrometry

(SIMS) is widely used to know quantitative isotopic or elemental distributions of solid surface, but

cryogenic applications are still limited may due to lack of the mechanism. In biology, the ability to

analyze frozen sample allow us to avoid artifacts associated with sample preparation, such as

dehydration, resin substitution or a little melting to stick a frozen section on the substrate, and to

expose flat surface of the target region using independent sectioning instruments. Moreover, water-,

fat-soluble molecules can be analysis subjects for SIMS. In cosmochemistry, fluid inclusions trapped in

meteorites can be exposed on the flatten surface as ice to provide precise isotopic information of

hydrogen and oxygen for ancient water to understand the evolution of the Solar System [2,3].

Therefore, we developed a cryogenic SIMS system equipped with a frozen sample introduction

mechanism for CAMECA ims family.

The cryogenic SIMS system is composed of a cryo-sample-stage (Techno I.S. Corp.), cryo-holder, cryo-

transfer-vessel and cryo-loadlock equipped with a CAMECA ims-1270 instrument at Hokkaido

University. The cryo-sample-stage was cooled down to about -190 C using liquid nitrogen with roots

vacuum pump to suppress vibration caused by drawing liquid nitrogen to the stage. Sample

preparation techniques of cryo-EM to make flat surface, such as cryo-ultramicrotome, cryo-FIB or

freeze fracture method are available with a universal holder (Leica Microsystems) integrated into a

cryo-holder. Large frozen tissues up to 1-inch size flatten by cryostat can be mounted in shaving-out

type cryo-holders. The frozen sample coated with Au thin film was mounted in the cryo-holder and

stored into a cryo-transfer-vessel filled with liquid nitrogen (Fig.1a). A cryo-loadlock can be attached

to the cryo-transfer-vessel using a slot compatible to the airlock of SIMS instrument (Fig.1b). A valve

of the cryo-loadlock was closed to keep the stored cryo-holder in nitrogen gas to prevent frost

formation. The cryo-holder was transferred to the cryo-sample-stage from the cryo-load lock (Fig.1c).

Fig.1d and 1e show a 16O- image and mass spectrums of frozen H2O ice using a Cs+ beam of 15keV with

a normal incident electron gun. The intensity of 16OH- was similar to the 16O- peak of H2O ice formed

from saturated saline solution. Tails of several tens ion counts were observed both sides of 16O- and 16OH- peaks due to the high intensity whereas the 18O- peak had no tail in this intensity range. The

interference of tail from 16OH- to 17O- was estimated to be about 10‰. Oxygen isotope analysis using 16OH- and 17,18OH- signal would be effective for further high precision oxygen isotope analysis for H2O

ice as well as the tail correction method used in [3].

28

Fig.1. (a,b,c) Procedure to transfer a cryo-holder onto a cryo-sample-stage of SIMS instrument, (d) isotope image

(squared) and (e) mass spectrum of H2O ice in halite crystal.

References

[1] Nambu et al., Mining Geology (Japan), 27, 41 (1977).

[2] Sakamoto et al., Science, 317, 231-233 (2007).

[3] Yurimoto et al., Geochem. J., 48, 549-560 (2014).

29

Day 2 – 21st May

Session:

NanoSIMS & SIMS

Session co-chairs:

Junting Zhang & Ian Gilmore

NanoSIMS & SIMS

I: A. Ewing 9:00 - 9:30

C: J. LovricSTA: TBD 9:30 - 9:50

C: A. Henβ 9:50 - 10:10

Coffee Break 10:10 - 10:30

I: P. Sjövall, 10:30 - 11:00

C: J. Fletcher 11:00 - 11:20

I: S. Sheraz 11:20 - 11:50

Discussion 11:50 - 13:00

Lunch 13:00 - 14:00

30

Combination of NanoSIMS with other methods to understand the chemical structure of neurotransmitter vesicles and neurotransmission

A. Ewing

Department of Chemistry and Molec Biol, University of Gothenburg, 41296 Gothenburg, Sweden

Mass spectrometry imaging combined with other methods is a powerful approach. We have

developed a method, intracellular vesicle impact electrochemical cytometry (IVIEC), where a nanotip

electrode is placed into a cell with the vesicles opening on the electrode tip for quantification of vesicle

content in live cells. Comparing the content of large dense core vesicles (approx. 200 nm) and small

synaptic vesicles (50-60 nm) shows the fraction released in each event ranges from 5-20 %.

We have used NanoSIMS imaging to spatially resolve the content across nanometer neuroendocrine

vesicles in nerve-like cells to show the distribution profile of newly synthesized dopamine across

individual vesicles. Furthermore, intracellular electrochemical cytometry at nanotip electrodes has

been used to count the number of molecules in individual vesicles and to compare to the amount

imaged in vesicles. This allows us to add a novel quantitative aspect to the mass spectrometry imaging

experiment. These nanoanalytical tools quantitatively reveal that dopamine loading/unloading

between vesicular compartments, dense core and halo solution, is a kinetically limited process, and

we have been able to quantitatively image in 3D across the cell and vesicles.

The combination of these analytical approaches and their application is leading to a unified model of

vesicle structure, exocytosis, and the beginning stages of plasticity, memory, at the cellular level.

31

In-Situ Correlative Helium Ion Microscopy and Secondary Ion Mass Spectrometry for High-Resolution Nano-Analytics in Life Sciences

Jelena Lovric, Jean-Nicolas Audinot and Tom Wirtz

Advanced Instrumentation for Ion Nano-Analytics, Department of Materials Research and Technology, Luxembourg Institute of Science and Technology, Belvaux, Luxembourg

In nearly all research areas development of innovative characterization techniques is of great

importance to advance the frontiers of science. The limitations imposed by individual imaging

techniques can be overcome with in-situ correlative microscopy approaches. Helium Ion Microscopy

(HIM) offers an excellent spatial resolution, but until recently there was no possibility to perform

chemical analyses by HIM. On the other hand, Secondary Ion Mass Spectrometry (SIMS) is a powerful

technique for chemical analyses of surfaces owing to its excellent sensitivity, high dynamic range

(a same signal can be followed over several orders of magnitude), high lateral and mass resolution.

In order to combine the high-spatial resolution performance of HIM with analytical sensitivity of SIMS,

we developed an integrated HIM-SIMS instrument [1-3]. One of the main advantages of the HIM-SIMS

technique with its high-spatial resolution capability (sub-nm secondary electron combined with sub-

20 nm SIMS imaging) is a possibility to achieve in-situ correlative imaging. This approach allows SE

images of exactly the same zone analyzed with SIMS to be acquired easily and rapidly, followed by an

image fusion between the SE and SIMS data sets [4]. The integrated approach allows fast and multiple

interlacing between the different imaging and analysis modes reducing significantly the analysis time.

Furthermore, we are developing the npSCOPE instrument, a new integrated instrument optimised for

physico-chemical characterisation of primarily nanoparticles in biological samples. Beside the

possibility to perform SE and SIMS imaging, this instrument will allow obtaining structural data with

an in-situ Scanning Transmission Ion Microscopy (STIM). Moreover, the instrument will operate in a

cryo-mode allowing analysis of biological samples in frozen-hydrated state and thus avoiding artefacts

from a classical sample preparation used for UHV imaging at room temperature [5].

In this work, we focus on the application of the HIM-SIMS technology in different life science research

fields. As a first step, we investigated the methodology to analyse a typical biological sample consisting

of a chemically fixed and resin embedded bacteria exposed to nanoparticles (NPs). The standard HIM

imaging by detecting secondary electrons of thin flat sections showed difficulties to reveal sample

features. However, when using the SIMS system to detect all negatively charged particles (electrons

and negative ions) emitted from the sample, more structural details were obtained. Secondary ion

maps of endogenous CN- ion cluster and ion signal originating from NPs were acquired allowing to

assess the interaction between bacterial cells and NPs. Furthermore, we optimised the sample

preparation and imaging methodology in order to obtain high-resolution secondary electron images

and correlate these with high-spatial resolution SIMS imaging of specific cell features highlighted using

labelling.

We show that the distinctive capability of HIM-SIMS in terms of correlative microscopy combining

both high-spatial resolution structural information and high sensitivity chemical information allows

32

research in various areas of life sciences such as nanotoxicology, geomicrobiology, virology and lipid

research, sub-cellular and sub-organelle analyses, and many others.

Acknowledgements:

We kindly acknowledge C. Cartier and M. Mercier-Bonin, Toxalim, France, I. Fourquaux, CMEAB, France, S. Winter and P. Chlanda, University Hospital Heidelberg, Germany and S. Jiang and X. Rovira Clave, Stanford University, USA for providing biological samples.

References:

[1] T. Wirtz et al., Nanotechnology 26 (2015) 434001

[2] T. Wirtz et al., Helium Ion Microscopy, ed. G. Hlawacek, A. Gölzhäuser, Springer, 2017

[3] D. Dowsett, T. Wirtz, Anal. Chem. 89 (2017) 8957-8965

[4] F. Vollnhals, J.-N. Audinot, T. Wirtz, M. Mercier-Bonin, I. Fourquaux, B. Schroeppel, U. Kraushaar, V. Lev-Ram, M. H. Ellisman, S. Eswara, Anal. Chem. 89 (2017) 10702-10710

[5] www.npscope.eu

33

Effects of sample preparation and measurement conditions on the mass spectrometric analysis of bone

Anja Henß1, Kaija Schäpe1, Anne Hild2, Marcus Rohnke1

1) Institute for Physical Chemistry, Justus-Liebig University of Giessen, Germany; Center for Materials Research, Justus-Liebig University Giessen, Germany

2) Institute for Veterinary Anatomy, Histology & Embryology, Clinic for Small Animals, Giessen, Germany

Mass spectrometry imaging (MSI) is increasingly applied for analytical tasks in the field of biomedical

research. Therefore, proper sample preparation plays an important role and is a prerequisite for high-

quality analysis and indispensable for reliability and reproducibility.(1) Herein we focus on sample

preparation of bone tissue and its impact on mass spectrometric analysis. The analysis of bone is

crucial for the understanding of bone diseases, developing new drugs and innovative replacement

materials. For bone analyses, embedding is typically employed to stabilize the tissue in a solid resin

block that aligns and supports the tissue and prevents it from distortions during sectioning. For

histological and histochemical examinations, the samples are usually embedded in resins or in

paraffin, the latter after decalcification. For mass spectrometric studies, however, the embedding

process must ensure that interesting components are not rinsed out, remain at their original location

and that no damage of the sample occurs due to chemical reactions. It must be the major aim to find

the optimal sample preparation route depending on the analytical question thus combining the

benefits of MSI with histological and histochemical studies. Therefore, we focus on the analysis of

bone samples from human femoral heads that have been embedded in typical resins. Cut and ground

sections of bone as well as native samples without embedding for comparison were analysed by time

of flight secondary ion mass spectrometry (ToF-SIMS). It is shown that epoxy resin as well as

methacrylate based plastics (Epon and Technovit) do not infiltrate the mineralized tissue and that cut

sections are better suited than ground sections for the analysis of the mineralized bone.(2) To analyse

fatty acids and lipids in bone and bone marrow, the classical preparation route with an ascending

alcohol series is not suitable. The bone must be prepared so that the native state is preserved.

Therefore, a cryo-embedding route has been established and ToF-SIMS and matrix-assisted laser

desorption ionization (MALDI) have been used to measure the lipid composition of bone and bone

marrow.(3)

References:

1. Goodwin RJA. Sample preparation for mass spectrometry imaging: Small mistakes can lead to big consequences. Journal of Proteomics. 2012;75(16):4893-911.

2. Henss A, Hild A, Rohnke M, Wenisch S, Janek J. Time of flight secondary ion mass spectrometry of bone-Impact of sample preparation and measurement conditions. Biointerphases. 2015;11(2)

3. Schaepe K, Bhandari DR, Werner J, Henss A, Pirkl A, Kleine-Boymann M, et al. Imaging of lipids in native human bone sections using ToF-SIMS, AP-SMALDI Orbitrap MS and Orbitrap-SIMS. Anal Chem. 2018.

34

TOF-SIMS imaging of biological samples: cryo-SIMS and sample preparation techniques

Peter Sjövall

RISE Research Institutes of Sweden, Borås, Sweden

TOF-SIMS is a powerful method for characterizing the spatial distribution of molecular species in

biological samples. Label-free, parallel imaging of specific molecules can be obtained at lateral

resolutions down to the submicrometer range and 3D mapping is obtained by repeated cycles of

surface etching and 2D imaging analysis. The molecular weight range (up to 1500-2000 Da) covers

most lipids and many drug molecules and metabolites, thus filling an analytical gap not easily accessed

by immuno-based imaging techniques. However, TOF-SIMS analysis of biological samples also brings

challenges, many of which are related to the sample preparation procedures enforced by the vacuum

requirement of the analysis.

The basic options available for TOF-SIMS analysis of biological samples are to analyse the sample in

the frozen hydrated state or to remove the water prior to analysis (usually by freeze drying). Both

options need careful considerations as to how well they retain the spatial distributions and molecular

integrity of the analytes. An important aspect is that many of the sample preparation strategies

developed for electron microscopy and fluorescence microscopy of biological samples may not be

suitable for TOF-SIMS, due to the added molecular complexity they may introduce (certain embedding

and fixation schemes) or the modifying effect they may have on the analyte distribution (for example,

procedures including ethanol or other solvents will remove, or seriously alter the distributions of all

lipids). In this presentation, different strategies for sample preparation and analysis of biological

samples by TOF-SIMS will be reviewed, separately for different types of samples, including tissues,

cells, and lipid membrane systems. The strategies will be discussed in relation to the overall purpose

of the analysis, including the specific information requested and possible complementary analyses of

the same sample with other techniques.

35

Sample preparation of cells and tissue for ToF/Hybrid-SIMS.

John Fletcher

University of Gothenburg

Sample preparation is a crucial step in the analysis of many samples particularly biological specimen

that contain many potentially mobile chemical species and water and hence exposure to the vacuum

environment of a mass spectrometer can result in considerable perturbation of the chemical

distribution from the life like state. Increasingly additional techniques such as fluorescent or electron

microscopy are used to validate the SIMS sample preparation and aid in the interpretation of the MS

data.

Frozen hydrated analysis of single cell samples has been shown to improve lateral and vertical

chemical distribution however the fine detail of the sample preparation can vary depending on

whether the sample is an adherent cell line (e.g. HeLa) attached to a suitable substrate or is in

suspension (e.g. a free swimming single celled organism such as tetrahymena pyriformis). [1,2]

The analysis of tissue samples often presents additional challenges as the analyst often has less control

over the entire sample preparation chain – the SIMS expert does not normally dissect the animal or

perform the surgery on a human patient. As with single cell cell analysis frozen hydrated analysis of

the tissue sample has been shown to preserve the integrity of the tissue and minimise lateral and

vertical displacement of different chemical species in mouse brain and heart.[3,4] If this is not possible,

methods to remove artefacts such as cholesterol migration in dry mouse/rat brain have been

demonstrated.[3,5]

Additional sample treatments to enhance particular classes of molecules, such as the derivatisation of

catecholamines present opportunities for improving detection of low abundance, low ionising species

[6] but present additional challenges in order to be applicable to sub-cellular scale imaging.

References:

1. Fletcher et al. Rapid Commun. Mass Spectrom., 2011

2. Angerer & Fletcher, Surf. Interface Anal., 2014

3. Angerer, Mohammadi & Fletcher, Biointerphases, 2016

4. Sämfors et al, Int. J. Mass Spectrom. 2017

5. Kaya et al, Anal. Chem. 2018

36

Biological sample preparation, a critical step for the success of secondary ion mass spectrometry imaging

Hua Tian1

Presented by Sadia Sheraz (née Rabbani)2

1 The department of Chemistry, Pennsylvania State University, US

2 School of Materials, Photon Science Institute, University of Manchester, U.K

With unprecedented spatial resolution coincident with rich chemical information, Time-of-Flight

secondary ion mass spectrometry (ToF-SIMS) imaging has gained popularity in biological studies. Due

to biological incompatibility with ultra-high vacuum, sample preparation is critical for imaging of the

unperturbed chemical distribution of targeted/untargeted molecules. At least two requirements must

be fulfilled for sample preparation, preserving the analyte of interest and eliminating surface

contamination. Combining extensive experience on biological sample handling in our lab, we review

the current methodologies for tissue and cell preparation, and their influence on sample morphology

and chemical information (e.g., lipids and metabolites). The success of SIMS imaging also depends

upon a suitable primary ion source. Currently, we focus on gas cluster ion beams (e.g., CO2 and H2O

cluster ion beam) for enhanced ionization and 1 µm spatial resolution. A mathematical approach is

also considered to distinguish the exogenous materials introduced through sample preparation.

37

Day 2 - 21th May

Session:

SIMS &

Poster Session

Session co-chairs:

Gregory Fisher & Andrew Ewing

SIMS

C: M. Rohnke 14:00 - 14:20

C: J. Zhang 14:20 - 14:40

S: A. Bellew (Ionoptika) 14:40 - 15:00

Discussion 15:00 - 16:00

Coffee Break 16:00 - 16:20

Poster Presentations

S. Jung,

W. Akwani

C. Newell,

Se-Ho Kim,

M. Białas,

R. Podlipec,

C. Stoffels,

16:20 - 17:00

Discussion @ posters and drinks 17:00- 19:00

38

Sample preparation of bone cells for 2D and 3D mass spectrometric imaging

Marcus Rohnke, Kaija Schäpe, Julia Kokesch-Himmelreich, Anja Henß

Institute for Physical Chemistry, Justus-Liebig-University of Giessen, Germany

Secondary ion mass spectrometry (SIMS) is the only mass spectrometric imaging (MSI) technique, which offers a sufficient lateral resolution for the investigation of subcellular structures. As a vacuum technique, SIMS poses particular challenges to sample preparation and makes direct analysis of the living and native system impossible. Cells are particularly delicate samples that tend to burst or shrink when exposed to hypotonic or hypertonic solutions and during the process of drying in vacuum as illustrated in Figure 1. A suitable vacuum sample preparation should provide vacuum compatibility and be adapted to the analytes of interest, while it preserves the sample in terms of morphology and analyte distribution and composition.

Here we compare and discuss different sample preparation routines for cell fixation with the aim of lipid analysis of the cell membrane as well as finding the best suitable storage conditions for fixed cells. For this purpose, human mesenchymal stem cells were analyzed by ToF-SIMS: a) as chemically fixed, b) freeze-dried, and c) frozen hydrated. Subsequent data evaluation of SIMS surface spectra in both, positive and negative, ion mode was performed by principal component analysis by use of peak-sets representative for lipids.1

Since ToF-SIMS analysis of many single cells is a time-consuming process, we examine in the following storing conditions of cells. Best results were obtained for chemically fixed cells, which were stored for 4 weeks in cell culture media or water. Here no degradation effects were observed.2

The monitoring of lipid composition during differentiation of human bone-derived mesenchymal stromal cells is presented as a practical example for successful cell preparation.3

Finally, we evaluate different sample preparation routines for high-resolution imaging of myeloma cell sections. The multiple myeloma is a serious type of bone cancer. Here the aim is to localize the drug Bortezomib inside the cells by mass spectrometric imaging. Results of ToF-SIMS and nano-SIMS measurements will be presented and compared.

References:

1. Schaepe, K.; Kokesch-Himmelreich, J.; Rohnke, M.; Wagner, A. S.; Schaaf, T.; Wenisch, S.; Janek, J., Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS. Biointerphases 2015, 10 (1). 2. Schaepe, K.; Kokesch-Himmelreich, J.; Rohnke, M.; Wagner, A. S.; Schaaf, T.; Henss, A.; Wenisch, S.; Janek, J., Storage of cell samples for ToF-SIMS experiments-How to maintain sample integrity. Biointerphases 2016, 11(2). 3. Schaepe, K.; Werner, J.; Glenske, K.; Bartges, T.; Henss, A.; Rohnke, M.; Wenisch, S.; Janek, J., ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis. Anal Bioanal Chem 2017, 409 (18), 4425-4435.

Figure 1: Throughout dehydration, the integrity of cell samples is compromised as a consequence of water evaporation, which increases pressure on the cell caused by surface tension and leads to it structural collapse.

39

Cryo-3D-OrbiSIMS – metrology of biological sample preparation methods for studies of frozen-hydrated bacterial biofilm

Junting Zhang1, James Brown2, David Scur4, Anwen Bullen3, Kirsty MacLellan-Gibson3, Kim Hardie2, Morgan R Alexander4, Paul Williams2, Ian S Gilmore1,4 and Paulina D Rakowska1

1 National Physical Laboratory, National Centre of Excellence in Mass Spectrometry Imaging, Teddington, UK

2 University of Nottingham, School of Life Sciences, Nottingham, UK

3 National Institute for Biological Standards and Control, Analytical Sciences, Potters Bar, UK

4 University of Nottingham, School of Pharmacy, Nottingham, UK

Secondary ion mass spectrometry imaging is one of the most powerful techniques for high-resolution

label-free imaging of biological samples. However, one of the challenges is that bio-samples must be

analysed in ultra high vacuum, which is quite different from their hydrated in vivo situation. The

established vacuum preparation pre-treatment processes such as freeze-drying, resin-embedding or

histological fixation are known to influence cell morphology, and lead to re-localization of chemical

composition or drug compounds within cells or tissues, along with other artefacts. Thus, a careful

consideration of sample preparation is required to achieve an analysis of bio-samples most closely

approximating their native state.

A biofilm is a structured consortium of bacteria distributed in a highly hydrated extracellular matrix,

which shows increased tolerance to antibiotics and can cause chronic infections. Visualization of

biofilm structure and localisation of antibiotics in a biofilm is very important to understand the

development and antibiotic resistance. However, the high water content of a biofilm makes it difficult

to image by SIMS without loss of their native architecture. Therefore, advanced cryo-preparation

methods are necessary for immobilisation of water in biofilm to prevent the ultrastructural

reorganisation and the loss or translocation of water-soluble molecules, to circumvent the use of

chemical fixation and to enable their analysis in the vacuum during SIMS analysis.

This presentation will show our developments of the cryo-SIMS methodologies mainly focused on

application to 3D-image of frozen-hydrated bacterial biofilm. Combining high pressure freezing

technique, cryo transfer system and the advanced cryo 3D-orbiSIMS instrument, the architecture of a

Pseudomonas aeruginosa biofilm in ‘hydrated’ state will be visualized. The protocol of cryo-SIMS

methods will be presented and the difference between frozen-hydrated and freeze-dried samples will

be discussed.

Acknowledgements:

This work has been done under the 15HLT01 MetVBadBugs project funded by the European Metrology Programme for Innovation and Research

40

High Resolution Bio-imaging with a 70kV Water Cluster Ion Beam

Allen T. Bellew

Technical Sales Manager, Ionoptika Limited, Unit B6, Millbrook close, SO53 4BZ, UK

.

In recent years, GCIB TOF SIMS has emerged as a powerful tool for investigating biological systems at

spatial resolutions below 5 microns. Large gas cluster ions significantly reduce the fragmentation that

is normally typical of SIMS, increasing sensitivity to large molecules by several orders of magnitude. In

addition, as surface damage is minimised, consecutive layers can be analysed without loss of signal,

making GCIB TOF SIMS a powerful technique to study samples in 3D with extremely high depth

resolution. To date the technique has been used extensively to study phospholipid, cardiolipin, and

ganglioside distribution in a variety of animal tissues, most recently planarian flatworms. [1]

In order to achieve sub-cellular resolutions, spot sizes of less than 1 micron are required. However, as

the limits of spatial resolution are pushed and volumes of analyte reduced, new strategies are required

to improve ion yield and increase sensitivity. While there have been several possible solutions

proposed to achieve this, most involve additional, complex sample preparation steps, or compromise

performance, e.g. spatial resolution.

Here we explore a simple strategy to achieve almost orders of magnitude increase in sensitivity, with

no additional sample preparation steps, and no loss of performance. We present the very latest in

cluster beam technology for biological imaging SIMS, Ionoptika’s new high-energy water cluster ion

beam, which is enabling 3D imaging SIMS of drugs, lipids, and other important molecules at sub-

cellular resolutions and with unprecedented sensitivity

References:

[1] T. B. Angerer, N. Chakravarty, M. J. Taylor, C. D. Nicora, D. J. Graham, C. R. Anderton, E. H. Chudler and L. J.

Gamble, “Insights into the histology of planarian flatworm Phagocata gracilis based on location specific, intact

lipid information provided by GCIB-ToF-SIMS imaging,” Biochimica et Biophysica Acta (BBA) - Molecular and

Cell Biology of Lipids, pp. 733-743, 2019.

41

Poster

Cryo-sample preparation of fresh leaf material TOF-SIMS analysis

Stefanie Junga, Anja Henssb, Jürgen Janekb and Volker Wissemanna

a Institute of Botany, Justus Liebig University Giessen, Germany

b Institute of Physical Chemistry, Justus Liebig University Giessen, Germany

Invasions of alien species into new habitats are a major threat to biodiversity due to the competition

for sunlight, space and nutrients with native species. Originating in eastern Asia and being cultivated

for coastal conservation purpose, the japanese rose Rosa rugosa Thunb. became an invasive alien

species at German coasts and islands displacing native coastal dune vegetation like the scots rose

Rosa spinosissima L.

Vegetation near the european north sea is constantly exposed to salt containing aerosols. A possible

reason for the invasive potential of R. rugosa might be an increased tolerance to salty air compared

to native species. Previous studies revealed that R. rugosa developed efficient mechanisms against

the impact of salt leading to a reduced sensitivity to saline soils than native species. However, the

underlying pathways that lead to increased salt tolerance are poorly understood.

The aim of this study is to use Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to reveal

differences in chloride ion accumulation and/or exclusion of leaves between the invasive R. rugosa

and the native R. spinosissima. The plants shall be stressed with aerosols containing a concentration

of 28 % synthetic sea water for at least two weeks. Subsequently, 2D and 3D ToF-SIMS analyses of the

leaves of both species, R. rugosa and R. spinosissima, are planned to localize the salt ions. In addition,

depth profiles will reveal a possible ion accumulation in deeper layers of the leaves. Focused Ion Beam

Scanning Electron Microscopy (FIB-SEM) will be used to obtain morphological information and to

prepare FIB-cross sections of an entire leaf for subsequent SIMS imaging.

Since ToF-SIMS and FIB-SEM are high vacuum techniques, the high water content and the hydrophobic

properties of the epicuticular waxes of leafs make the sample preparation quite challenging.

Moreover, the very mobile saline ions have to be kept and fixed at their original localization. In order

to keep the leaves in an almost native state, we employ cryo-sample preparation to freeze the leaves

in dry-ice cooled hexane or liquid nitrogen. Several workflows were tested using different embedding

materials such as TissueTek (Sakura Finetek, USA), Kawamoto gel or gelatin and different cutting

procedures at various temperatures (- 40 °C, under liquid nitrogen). After cryo-fixation the samples

are stored at -80°C or in liquid nitrogen, transferred and analyzed under cryo-conditions with SIMS

and FIB-SEM.

42

Poster

Use of Nano- SIMS at the single cell-level to evaluate drug penetration into mycobacterial biofilms

Winifred Akwani1,2, Paulina Rakowska2, Mark Chambers1, Greg McMahon2 and Suzie Hingley-Wilson1

1. Department of Microbial Sciences, University of Surrey, Guildford, UK

2. Mass Spectrometry Imaging, National Physical Laboratory, Teddington, UK

Non-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause

high mortality and morbidity amongst patients who are immunocompromised or have chronic lung

diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients

following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated

from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate

and are associated with the development of increased antimicrobial resistance (AMR). Treatment

for M. abscessus and chimaera infections often requires 2-3 antibiotics over 2 years.

The question we want to address is whether the increased AMR and treatment time in NTM biofilm

formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative

levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-

SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic

bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms

(M. abscessus and M. chimaera). Nano-SIMS maps the relative abundance of different ions down to

the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum

inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to

determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the

AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and

morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce

treatment duration and AMR development.

43

Poster

Imaging of Drosophila skin lipids using 3D OrbiSIMS

Clare Newell1,2, Jean-Luc Vorng2, Andrew Bailey1, Ian Gilmore2, Alex Gould1

1. The Francis Crick Institute, 1 Midland Road, London, NW1 1AT

2. NiCE-MSI, National Physical Laboratory, Hampton Road, Teddington, TW11 0LW

Secondary ion mass spectrometry imaging (SIMS) is a high vacuum technique that is recently gaining

traction for biological applications. However, the surface topology and charging effects of biological

samples present a significant challenge for SIMS analysis. The 3D OrbiSIMS is a new SIMS instrument

optimised for the detection of intact biomolecules with high spatial resolution and accurate mass

determination. Here, we apply 3D OrbiSIMS to image the lipidome on the surface of the cuticle (skin)

of the insect Drosophila. Using this model system as a proof-of-principle, we develop technical

improvements that help to overcome problems arising from uneven surface topology and sample

charging. We also develop cryo-OrbiSIMS, a new methodology for analysing volatile lipids such as

Drosophila hydrocarbons.

44

Poster

Detecting organometallic surfactant micelle using atom probe tomography (APT)

Se-Ho Kim†, Andrew Breen†, Kevin Schweinar, Torsten Schwarz, Priyanshu Bajaj, Dierk Raabe, Leigh Stephenson, Baptiste Gault*

Max-Planck-Institut für Eisenforschung, Düsseldorf, Germany

Atom probe tomography (APT) is a high-resolution characterization technique for mapping the elemental distribution in nanostructured materials. APT has a unique combination of three-dimensional imaging with sub-nanometer spatial resolution and a detection sensitivity in the range of tens of ppm for all elements. Because of challenges for preparation of the needle-shaped specimens necessary for APT from liquid phases, only a limited number of analyses have been reported for liquids1–3. Two major issues must be solved to turn a frozen liquid into a specimen. First, the solution must have a high electrical conductivity to avoid charging during electron beam imaging and enable field evaporation during APT. Second, the sublimation of the frozen liquid should be low under ultra-high vacuum conditions (~10-9mbar) so that the frozen sample remains stable during fabrication, cryogenic transfer and analysis. In order to acquire meaningful data for as-synthesized nanoparticle-surfactant complex solutions, we propose a new approach for APT tip sampling from a Au nanoparticle solution using a cryo-plasma focused ion beam and a specimen transferring system under ultra-high vacuum and cryogenic conditions.

Fig. (a) colour differences of nanoparticle precursor solution with different micelle structures. (b) Ice-quenching

process of the precursor solution for cryo-sampling for atom probe tomography measurement.

References:

1. Gerstl, S. S. A. & Wepf, R. Methods in Creating, Transferring, & Measuring Cryogenic Samples for APT. Microsc. Microanal. 21, 517–518 (2015).

2. Stephenson, L. T. et al. The Laplace Project: An integrated suite for preparing and transferring atom probe samples under cryogenic and UHV conditions. PLoS One 13, 1–13 (2018).

3. Schreiber, D. K., Perea, D. E., Ryan, J. V, Evans, J. E. & Vienna, J. D. A method for site-specific and cryogenic specimen fabrication of liquid/solid interfaces for atom probe tomography. Ultramicroscopy 194, 89–99 (2018).

45

Poster

Steering of electron beam in MEMS electron microscope

M. Białas, M. Krysztof, P. Szyszka, A. Górecka-Drzazga

Wroclaw University of Science and Technology, Faculty of Microsystem Electronics and Photonics

A concept of MEMS transmission electron microscope fully integrated on one chip was published in 2014 [1]. It assumes fabrication of all parts of the electron microscope using the MEMS technology. The device consists of an electron gun with an extraction electrode, an Einzel lens and anode with a very thin Si3N4 membrane, which is transparent for the electron beam (Fig. 1a). Those electrodes are made of high conductive silicon substrates and are isolated from each other by borosilicate glass separators. Electron gun is integrated with an ion-sorption micropump which generates high vacuum up to 10–6 mbar inside the MEMS microscope [2]. The observed sample can be located in air, directly onto the Si3N4 membrane or introduced using a microfluidic chip. A detector, e.g. image sensor is mounted on a top of MEMS electron microscope.

The advantages of MEMS microscope are small dimensions, low power consumption and the ability to test biological (wet) samples. However, the construction of such a miniature device requires the development of new methods of signal detection and sample imaging, as well as conducting many experiments to make the obtained image satisfactory.

Currently, research is going in two directions. One is the development of a transmission microscope, in which an electron beam illuminates the entire surface of the membrane and, as a consequence, a sample. The second is the sample's lighting through the membrane by a focused beam whose motion is controlled by a multi-element electrode (“octupole”, Fig. 1b) powered by a special electronic system.

In this paper the first results of study on construction, technology of octopole electrode for electron beam deflection will be presented. The first images obtained in the test structure of the MEMS microscope will be discussed.

a) b)

Fig. 1. Schematic view of MEMS microscope integrated with vacuum micropump (a) and octupole electrode with conductive paths made of copper tape (b)

The work was financed by National Science Centre Poland, project number UMO-2016/21/B/ST7/02216.

References: [1] M. Krysztof et al., A concept of fully integrated MEMS-type electron microscope, Technical Digest of 27th International Vacuum Nanoelectronics Conference, Engelberg, Switzerland, 6-10 July, 2014, pp. 77-78 [2] T. Grzebyk, A. Górecka-Drzazga, J. A. Dziuban, Glow-discharge ion-sorption micropump for vacuum MEMS, Sensors and Actuators A 208 (2014) 113-119

46

Poster

Towards correlative live-cell imaging using super-resolution optical and ion/electron based techniques and development of special holders

Rok Podlipec1, Aleksandar Sebastijanovic1, Katarina Vogel Mikus2, Primoz Pelicon1, Janez Strancar1

1 J. Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia,

2 Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia

Clear understanding of molecular phenomena of cells/tissue response after interacting with

dangerous metal nanoparticles is still lacking. In our research we thus focus on highly relevant

scientific/toxicology problem, which is understanding of the mechanisms of molecular initiative events

between nanoparticles (typically smaller than 100 nm) and lung tissue cells during inhalation [1]. It is

strongly believed that nanoparticles can trigger toxic effects with increasing risk for further

cardiovascular diseases [2]. However, limited spatial resolution of optical microscopy, even in super-

resolution mode, prevents visualization of interaction phenomena at smaller, nanometer scales which

could reveal some of still unclear molecular events. In order to get better insight on the phenomena

at such small scales one needs to introduce proper correlative microscopy techniques which in our

case are electron (eSEM) and ion based (PIXE, HIM). Since these techniques are commonly employed

in high vacuum and detection principles differ reasonably in comparison to optical imaging techniques,

special sample holders have to be developed in order to perform efficient correlative microscopy on

different submicron scales.

In this pilot study we first tested the performance of correlative microscopy on epithelial cells

nebulized with TiO2 nanoparticles using optical, eSEM and RISE techniques, while in the second pilot

study we developed the concept for robust special holder preparation in order to enable super-

resolution optical imaging (STED) as well as ion based imaging on the same sample site. Despite

integrating few to few tens micron sized layers consisting of Mylar foil, water and glass which

introduce refractive index mismatches, we were able to improve confocal resolution by at least factor

2-3 using STED imaging. This was a proof-of-concept that we can perform high resolution optical

imaging on these holders which can successfully be implemented as well in correlative electron/ion

based imaging, thus giving the opportunity to gain better insight of the measured complex samples

from nano to micro scale.

References:

[1] Iztok Urbančič et al., “Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer,” Nano Letters 18, no. 8 (August 8, 2018): 5294–5305.

[2] Robert D. Brook et al., “Air Pollution and Cardiovascular Disease: A Statement for Healthcare Professionals From the Expert Panel on Population and Prevention Science of the American Heart Association,” Circulation 109, no. 21 (June 1, 2004): 2655–71.

47

Poster

Employing high-resolution correlative electron microscopy and secondary ion mass spectrometry imaging to study toxicology in biological samples

Charlotte Stoffels, Jelena Lovric, Jean-Nicolas Audinot and Tom Wirtz

Advanced Instrumentation for Ion Nano-Analytics, Department of Materials Research and Technology, Luxembourg Institute of Science and Technology (LIST), Belvaux, Luxembourg

This project aims to employ a state-of-the-art correlative imaging approach in order to explore toxic effects of emerging pollutants such as nanoparticles (Au, Ag, ZnO) and perfluorinated organic compounds that are omnipresent in our environment. Perfluorinated carboxylic acid has been widely used for many industrial purposes and consumer-related applications and therefore it is ubiquitous in our environment. The gastrointestinal tract is the first physical barrier against these toxicants, and meanwhile their first target. Surprisingly, their effect on the intestinal wall is largely unknown.

Imaging methods at cellular and subcellular levels are increasingly used in life sciences such as environmental toxicology and food toxicology. Specifically, in-situ multimodal imaging approaches provide an impact in this research field due to the possibility to obtain multiple characterisation approaches in the same time. Hence, we employ a HIM-SIMS instrument that allows correlative secondary electron and secondary ion mass spectrometry imaging to reveal the toxic effects of nanoparticles and perfluorinated organic compounds. Imaging with helium ions leads to resolutions of 0.5 nm for secondary electron (SE) based imaging [1]. However, the analysis capability of the instrument is currently limited. In order to add nano-analytical capabilities to the HIM, we have developed a Secondary Ion Mass Spectrometry (SIMS) system specifically designed for the Zeiss ORION NanoFab HIM [2-4]. In SIMS, the typical interaction volume between the impinging ion beam and the sample is around 10 nm in the lateral direction. As the probe size in the HIM is substantially smaller (both for He and Ne ions), the lateral resolution on the integrated HIM-SIMS is limited only by fundamental considerations and not, as is currently the case on commercial SIMS instruments, the probe size. We have demonstrated that our instrument is capable of producing elemental SIMS maps with lateral resolutions down to sub-20 nm. Furthermore, HIM-SIMS opens the way for in-situ correlative imaging combining high-resolution SE images with elemental maps from SIMS. This approach allows SE images of exactly the same zone analysed with SIMS to be acquired easily and rapidly, followed by a fusion between the SE and SIMS data sets.

We show that high-resolution imaging capability of HIM-SIMS instrument that combines structural information with high-sensitivity chemical information allows research in a toxicology field and provides insightful information to better understand negative impact of nanoparticles and perfluorinated organic compounds onto gastrointestinal tract.

Acknowledgements:

We kindly acknowledge C. Cartier and M. Mercier-Bonin, Toxalim, France, I. Fourquaux, CMEAB, France for providing biological samples.

References:

[1] B. W. Ward, J. A. Notte, and N. P. Economou, Journal of Vacuum Science & Technology B 24 (2006) 2871

[2] T. Wirtz, P. Philipp, J.-N. Audinot, D. Dowsett, S. Eswara, Nanotechnology 26 (2015) 434001

[3] T. Wirtz, D. Dowsett, P. Philipp, Helium Ion Microscopy, edited by G. Hlawacek, A. Gölzhäuser, Springer, 2017

[4] D. Dowsett, T. Wirtz, Anal. Chem. 89 (2017) 8957-8965

48

49

Day 3 - 22nd May

Session:

Atom Probe Tomography

Session chair:

Katherine Rice

Atom Probe Tomography

I: D. E. Perea 9:00 - 9:30

C: I. McCarroll 9:30 - 9:50

I: G. Sundell 9:50 - 10:20

Coffee Break 10:20 - 10:40

C: C. Macauley 10:40 - 11:00

C: K. Rice 11:00 - 11:20

I: K. Vogel-Mikus 11:20 - 11:50

Discussion 11:50 - 13:00

Lunch 13:00 - 14:00

50

FIB-based Preparation of Cryogenic Specimens for APT analysis

Daniel E. Perea,* Daniel K. Schreiber, James E. Evans

Pacific Northwest National Laboratory, Richland, WA USA

The utilization of the focused ion beam scanning electron microscope (FIB-SEM) opened up the

application of Atom Probe Tomography (APT) to a continually-growing variety of materials, including

soft biological materials. However, the regular application of APT to hydrated materials, such as

biological and environmental materials, is lacking due to challenges in specimen preparation using a

FIB-SEM under high and ultra-high vacuum conditions and cryogenic temperatures. Although a FIB-

SEM based cryogenic specimen preparation approach offers a rational route to the preparation of

such specimens for TEM analysis, there are still many unaddressed aspects such as welding and

limitations in the cryo stage manipulation which makes it challenging for FIB-based cryo specimen

preparation of APT specimens. Our recent efforts in the development of a FIB-based site-specific

liftout and attachment scheme of cryogenically cooled specimens involving a combination of

redeposition and overcoating of organic and organometallic molecules will be described. Using a

modified shuttle device and an environmental transfer hub at PNNL is used to facilitate

environmentally-protected specimen transfer between the cryo FIB and the APT tool, allowing for the

first time, APT analysis of a water/solid interface in 3D to reveal the complex nanoscale water-filled

porous network of corroded glass. Application of this unique specimen preparation approach to

biological specimens will also be discussed, as well as the application of a unique cryo

nanomaniupulator with integrated gas injection system to enable controlled FIB-based welding under

cryogenic temperatures.

51

UHV-Cryo transfer suite for preparation and transfer of biological and soft matter between glovebox, SEM-FIB and atom probe tomography

Ingrid McCarroll1, Naveena Gokoolparsadh1, Alexander Rosenthal2 and Julie M. Cairney1

1. Australian Centre for Microscopy and Microanalysis, The University of Sydney, Camperdown 2006, NSW, Australia

2. Microscopy Solutions Pty. Ltd., 9/57 Orrong Cres., Caulfield North 3161, Vic., Australia

Fifty years ago atom probe tomography (APT) enabled the first 3D tomographic atom-by-atom

visualisation of conductive materials. Within the past 20 years the capacity of APT, through the

implementation of a pulsed laser system, has expanded into the study of non-conductive materials,

such as oxides and geological specimens. Today, the capabilities of APT are developing such that is

possible to incorporate the study of biological and soft matter specimens. Key to enabling this capacity

within APT has been developments in sample preparation and transfer systems.

The University of Sydney is looking to facilitate these developments with its (ultra) high vacuum (U)HV

and cryogenic interconnected suite of equipment, including: a purpose built glovebox, a scanning

electron microscope-focused ion beam (SEM-FIB), and a laser-assisted local electrode atom probe,

developed in collaboration with Microscopy Solutions, Ferrovac GmBH, and CAMECA. The new

transfer suite enables production of the nanoscale geometries required for APT under controlled

cryogenic temperatures. In this talk, we will provide an overview of this custom-designed system, and

provide initial results that validate the capabilities of the integrated cryogenic and (U)HV transfer

system.

52

Atom probe tomography of silica-encapsulated biomolecules

Gustav Sundell, Mats Hulander, Astrid Pihl, Simon Isaksson, Martin Andersson

Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden

Proteins are the building blocks of life and participate in virtually all processes within living organisms. A critical aspect of the field of structural biology is to characterize the structure of proteins in three dimensions. Knowledge of the shape of a e.g. a transmembrane protein can allow for design of new pharmaceutical substances that inhibit virus infections1. Traditionally, the functional structure of proteins has been determined using X-ray crystallography or nuclear magnetic resonance (NMR). More recently, development of direct electron detectors has sparked a revolution in cryo electron microscopy for structural biology applications.

Here, we show that atom probe tomography may constitute a complementary method for proteomics, providing both chemical information as well as with 3D structure. We have encapsulated the human antibody Immunoglobulin G (IgG) in a solid silica glass matrix to provide the requisite mechanical stability to allow for atom probe analysis.

(a) TEM micrograph of a silica-embedded IgG, (b) and (c 3-4) atom probe reconstructions of IgG, (c 1-2) reference pdb structure of IgG as retrieved from the protein databank.

We present atom probe reconstructions of the antibodies, both in an aggregated and monomer state. The resulting tomograms are compared with known structures retrieved from the protein databank2.

Encapsulation in silica may also be a feasible platform for studying membrane proteins with APT, without need for detergents. We present a route for depositing a lipid bilayer containing aquaporins on a mesoporous silica substrate3, followed by an embedding step to produce a solid structure suitable for APT.

The challenges and opportunities that are associated with these two approaches are discussed.

References:

1. Kielian, M. & Rey, F. A. Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Micro 4, 67–76 (2006).

2. Berman, H. M. et al. The protein data bank. Nucleic Acids Res. 28, 235–242 (2000).

3. Isaksson, S. et al. Protein-Containing Lipid Bilayers Intercalated with Size-Matched Mesoporous Silica Thin Films. Nano Lett. 17, 476–485 (2016).

53

Developing a custom cryogenic-transfer system to the atom probe

Chandra Macauley*, Martina Heller, Peter Felfer

Department of Materials Science and Engineering, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany

To study materials that are sensitive to air or thermal exposure, environmentally-controlled transfer

of samples between instruments is essential. Atom probe tomography is a powerful technique to

obtain 3D chemical and structural information, however the ‘conventional’ transfer of samples into

the atom probe occurs at ambient conditions. In this study, we describe a versatile transfer system

that enables cryogenic- or room-temperature-transfer of specimens in vacuum or atmospheric

conditions between sample preparation stations, the focused ion beam (FIB) and the atom probe. The

transfer system includes a custom-built transfer arm that maintains high vacuum conditions and

actively cools samples during transport. This transfer arm can transport samples from the FIB or from

sample preparation stations such as a custom cryogenic-electropolishing set-up. The transfer arm also

interfaces directly with a modified CAMECA LEAP 4000X HR atom probe to facilitate transfer into the

analysis chamber in less than 5 minutes. The utility of this transfer system is demonstrated by the

acquisition of previously unattainable chemical information from the grain boundaries of aluminum

alloys prepared at cryogenic temperatures with a Ga ion FIB. Cryogenic temperatures (down to -140°C)

prevent the segregation of Ga to aluminum grain boundaries, enabling the true chemistry and

structure of the grain boundaries to be characterized. Precisely controlled transfers enabled by the

system open up a multitude of scientific possibilities in interdisciplinary fields beyond metals, such as

liquid-containing materials.

54

Cryogenic UHV Specimen Preparation for Atom Probe Tomography

K.P Rice1, R.M. Ulfig1, U. Maier2, R.G. Passey3

1. CAMECA Instruments Inc., 5470 Nobel Drive, Madison, WI 53711 USA.

2. Ferrovac GmbH, Thurgauerstrasse 72, Zurich CH-8050 Switzerland.

3. Thermo Fisher Scientific, 5350 NE Dawson Creek Drive, Hillsboro, OR 97124 USA.

As the application space for atom probe tomography (APT) expands, new hardware innovations are required to prepare and transfer specimens under cryogenic and/or ultra-high vacuum (UHV) conditions. For example, the analysis of biological materials, hydrogen containing materials [1], or surfaces prone to rapid oxidation, require that the specimen be maintained in a controlled environment from focused-ion-beam (FIB) manufacture through APT analysis. Designing hardware to successfully meet these application demands typically requires extensive experience in multiple areas of expertise, which may be beyond the typical laboratory [2,3]. Here we present a commercial cryo-UHV solution for FIB-APT that can be implemented to meet the transfer process requirements between FIB and APT for non-experts.

The CAMECA® vacuum cryo-transfer-module (VCTM) is a portable, cryogenically cooled, UHV module that enables transfer and short-term storage of prepared specimens from DualBeam™ FIB-SEM systems to the atom probe [4]. Ferrovac, working in collaboration with Thermo Fisher Scientific and CAMECA, has engineered a Quick-Loader™-based system that attaches to a DualBeam chamber and enables transfer into the VCTM for transport to a LEAP® 5000. The module or “suitcase” features a portable, battery-operated ion pump and a non-evaporable getter (NEG) to maintain UHV conditions, and a cold stage to maintain low-temperature conditions. The temperature and vacuum control prevent the formation of crystalline ice on the prepared samples, which would inhibit the subsequent APT analysis.

A primary consideration for transfer between FIB and APT is attachment of the VCTM to the FIB without compromising the FIB imaging resolution, due to vibrations from the attached suitcase while maintaining UHV conditions within the VCTM. Ferrovac’s novel docking station was engineered meet all requirements for the transfer process. The docking system is installed using the standard mechanical and vacuum interface provided on ThermoFisher Scientific instruments. The small loading-docking chamber is equipped with an O-ring sealed quick-connector that allows for a fast and simple attachment of the VCTM while maintaining the functionality of the standard sample loader on the DualBeam instrument. This is achieved by providing the ability to switch back and forth, between VCTM and a standard sample-loader transfer arm.

The loading-docking chamber is pumped either via the existing roughing pump of the FIB or a separate scroll pump. The loading sequence of the FIB’s software control can be applied without any software modification. Experiments performed with a VCTM have shown that UHV conditions in the VCTM are maintained after exposure to a vacuum level present in a standard FIB.

References:

[1] Y.-S. Chen et al., Science 355 (2017), pp. 1196–1199.

[2] S.S.A. Gerstl et al., Microscopy and Microanalysis 23 (2017), pp. 612–613.

[3] D.K. Schreiber et al., Ultramicroscopy 194 (2018), pp. 89-99.

[4] L.T. Stephenson et al., PLoS ONE 13 (2018), pp. 1-13.

55

Cryo-preparation of plant tissues for imaging of element and biomolecular distribution by X-ray and MS based focused beam techniques

Katarina Vogel-Mikuš1,2, Paula Pongrac2, Anja Kavčič1

1 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

2 Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

Development of high-resolution X-ray (µ-PIXE, SR-µ-XRF) and MS (LA-ICPMS, SIMS) based imaging

techniques has opened new possibilities to study spatially resolved metabolic processes in plants.

Ability to focus the photon or particle beam to micron or even below micron size has placed stringent

demands on sample preparation, since any measurement can be meaningless and misleading, unless

changes in tissue morphology/ anatomy and chemical redistribution have been limited to dimensions

that are smaller than the lateral resolution of the particular technique.

To preserve plant tissue, cellular and sub-cellular structures and biochemical composition to resemble

as much as possible the "in vivo" state we have focused on development of cryo-based sample

preparation techniques. In this talk, the cryo workflows developed and optimized in our laboratory

during the last 15 years will be presented.

56

57

Day 4 - 23rd May

Session:

Spectroscopies

Session chair:

Giacomo Ceccone & Hiram Castillo-Michel

Spectroscopies

I: H. Hertz 9:00 - 9:30

C: A. SorrentinoIUVSTA: TBD 9:30 - 9:50

I: H. Castillo-Michel 9:50 - 10:20

C: G. Ceccone 9:50 - 10:10

Coffee Break 10:10 - 10:40

Discussion 10:40 - 12:30

Lunch 12:30 - 13:40

58

Laboratory cryo x-ray tomography

Hans M Hertz

Biomedical and X-Ray Physics, Dept of Applied Physics, KTH/Albanova, Stockholm, Sweden

X-ray microscopy allows high-spatial-resolution 3D imaging of intact cells with natural contrast. We

developed the first high-resolution laboratory x-ray microscope operating in the water-window

(E=284-540 eV).1 The present version of the microscope relies on a liquid-nitrogen-jet-target laser-

plasma x-ray source, multilayer condenser optics, zone-plate imaging optics and cryogenic sample

handling.2 This microscope design enabled proof-of-principle biological imaging in 2D and 3D with

synchrotron-like image quality.3

Recent improvements have focused on shorter exposure times and higher reliability to allow routine

tomographic cryo imaging in cell biology. Key improvements include higher source power, higher

multilayer mirror reflectivity,4 and reliable cryogenic sample preparation and sample handling. The

present microscope provides 2D images in 10-20 seconds and allows repeated tomographic imaging

with up to hundred projections during typically 1 hour total data acquisition time. Demonstrated

applications include imaging autophagy-relevant behavior of starving HEK cells and interaction

between NK-cells and HEK target cells,4 as well as assessment of virus infection dynamics in amoebas.5

References:

1. Berglund et al, J. Microscopy 197, 268 (2000)

2. Takman et al, J. Microsc. 226, 175 (2007); Bertilson et al, Opt. Lett. 36, 2728 (2011).

3. Hertz et al, J. Struct. Biol. 177, 267 (2012).

4. Fogelqvist et al, Sci. Rep. 7, 13433 (2017).

5. Kördel et al, ms in preparation

59

Characterizing calcium storage organelles in biological cells using Cryo-Soft X-Ray transmission microscopy at MISTRAL

A. Sorrentino1, A. Gal2, A. Procopio4, K. Kahil2, E. Malucelli4, A.J. Perez-Berna1, J.J. Conesa1, S. Weiner2, L. Addadi2, S. Iotti4 A. Scheffel3 and E. Pereiro1

1 ALBA Synchrotron Light Source, MISTRAL Beamline−Experiments Division, Barcelona, Spain

2 Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel

3 Max-Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany

4 Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

Mistral is the soft X-ray full field transmission microscopy beamline at the ALBA light source [1]. It is

devoted to cryo-tomography in the water window energy range, allowing for the 3D morphological

characterization of whole cells in their native state with 50 nm spatial resolution. In addition, cryo-

spectromicroscopy, i.e. energy resolved microscopy, can be performed. It allows the chemical

characterization of objects inside the cells, providing pixel-by-pixel absorption spectrum. In this work

we will firstly introduce the basics working principles of the X-ray transmission microscope installed

at Mistral and the method of application of the technique to frozen hydrated samples. Then, recent

results about the characterization of Ca storage organelles in different biological eukaryotic cells

obtained combining cryo-tomography and cryo-spectromicroscopy will be presented [2-5].

References

1. Sorrentino A. et al. J. Synchrotron Rad. (2015), 22, 1112-1117. 2. Sviben, S. et al. Nat. Commun. 7:11228 (2016). 3. Gal A. et al. PNAS October 23, 2018 vol. 115 no. 43 11005. 4. Kahil, K. et al. “Understanding Calcium transport pathways in Skeleton Forming Cells of Sea Urchin Larvae”,

in preparation. 5. Procopio, A. et al. “Early steps of bone mineralization mechanisms investigated combining cryo-soft X-ray

tomography with XANES at the Ca L2,3 edges”, in preparation.

Figure 1. Analysis of the single-celled green alga Chlamydomonas reinhardtii by cryo-tomography and cryo-spectromicroscopy. (A) A slice in the 3D reconstructed data. Color-coded arrowheads indicate the chloroplast (green) and the nucleus and nucleolus (pink and purple, respectively). (Inset) A light micrograph of a live cell. (B) A 3D volume rendering of an entire cell. The nucleus (pink) and X-ray dense bodies (red) are surrounded by the cup-shaped chloroplast (green). (C) Spectromicroscopy at the calcium L-edge revealed the X-ray dense bodies to be Ca-rich and not lipid bodies. (D) A 3D representation of the cell in C showing only the Ca-rich bodies (red) and the enclosing membranes (beige). (E) High magnification showing the organization of the Ca-storage organelles, which are called acidocalcisomes, in C. reinhardtii. Ca-rich body (red arrowhead), compartment membrane (beige arrows). (F) Ca L-edge XANES spectra extracted from different regions of image stacks traversing the energy range around the Ca L-edge. From Gal A. et al. PNAS October 23, 2018 vol. 115 no. 43 11005 [3].

60

The ID21 beamline at ESRF: sub-micron spectroscopy under cryogenic conditions for life and environmental sciences

Hiram Castillo-Michela, M. Cottea, M. Salomea, D. Bugnazeta, W. De Nolfa, A.E Pradas del Reala, C. Guillouda, and E. Gagliardinia

a European Synchrotron Radiation Facility, France

Localization and speciation of trace elements at ID21 is done using micro-X-ray fluorescence (µXRF)

and micro X-ray absorption spectroscopy (µXANES) in the tender X-ray domain (2-9.1 keV) [3]. The

X-ray beam is focused using KB optics to a sub-micron spot (~500 nm), which then allows localization

of trace elements at subcellular level. The beamline is equipped with a passively cooled cryogenic

stage that allows the study of frozen hydrated specimens (cells and cryo-sectioned tissues) preventing

elemental redistribution and minimizing radiation damage.

Sample preparation is a critical step for imaging techniques such as µXRF, and even more critical for

techniques focusing on elemental speciation such µXANES. For this purpose, a cryo-microtome Leica

RM2265 is available at the beamline. For storage and transport of samples a 70 L LN2 cryodewar, and

5 L voyager dry cryo-dewar approved for airplane trips are also available to our users. Here, we will

discuss the tools and followed protocols to prepare samples for micro-analysis under cryogenic

conditions at ID21.

61

Interaction of nanoparticles and cells studied by different spectroscopy and microscopy techniques

G. Ceccone1, J. Ponti1, A. Bogni1, A. Gianoncelli, G. Kourousias 2, L. Pascolo3, M. Salomé4

1 European Commission, Joint Centre, Ispra (VA), 21027, Italy

2 ELETTRA, Sincrotrone Treste, Italy

3 Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy

4 European Synchrotron Radiation Facility (ESRF), 38000 Grenoble, France

The use of nanomaterials and nanoparticles (NPs) in many applications ranging from electronics to

food and biology has strongly increased in the last years bringing to potential human and

environmental exposure.1,2 Even if many studies on nanomaterials' biocompatibility have been

completed, there is still the need in understanding the mechanisms of nanomaterials' cell interaction.

Thus, in the recent years different methods to investigate cellular internalisation and distribution have

been developed.3,4,5,

In this work we report our investigations using EM and synchrotron-based spectro-microscopy

techniques on the interactions of nanoparticles (i.e. SiO2, CoFe2O4 and Fe2O3) with different cell lines,

namely Balb/3T3 mouse fibroblasts, UMG87 glioblastoma-astrocytoma and A549 human alveolar

epithelial type‐II‐like.6-9

In particular, the behaviour of the magnetic nanoparticles was studied on Balb/3T3 and UMG87, whilst

the A549 cell line and human monocytes were used to investigate the effects of SiO2 nanoparticles

both materials considered of biomedical and therapeutic interest.

The results show that uptake of SiO2 NPs is more pronounced in the A549 that in human peripheral

blood monocytes. Furthermore TEM-EDS data indicate the intracellular localization of NPs

demonstrating their accumulation in vacuoles such as endosomes/lysosomes.

As far as the magnetic NPs, complementary synchrotron-based X-ray imaging and SRXRF microscopy

experiments and STEM analysis have shown that NPs are confined in the perinuclear region of the cell.

References:

1. C. Power, Nanotechnol Rev (2018), 7(1): 19–41

2. Buzea, I. Pacheco, K. Robbie, Biointerphases (2007), 2, MR17.

3. V. Mailander and K. Landfeste, Biomacromolecules (2009), 10, 2379–2400

4. Fadeel, Nat Nanotechnol. (2018), 13, 538-43

5. S. Bohic, et al., Journal of Structural Biology(2012), 177, 248–258

6. P. Marmorato et al., Toxicology Letters (2011), 2017, 128– 136

7. Gianoncelli, X-Ray Spectrom. (2013), 42, 316–320

8. G. Kourousias, et al., X-Ray Spectrom. (2015), 44, 163–168

9. M. Rio‐Echevarria, et al., X‐Ray Spectrometry. (2019);1–8.

62

63

Day 4 - 23rd May

Session:

Applications, novel solutions and technologies

Session chair:

Anja Henss & Junting Zhang

Applications, novel solutions and technologies

C: D. Breitenstein 13:40 - 14:00

S: J. Javůrek (Tescan) 14:00 - 14:20

C: C. Loussert-Fonta 14:20 - 14:40

C: D. Petrovykh 14:40 - 15:00

Coffee Break 15:00 - 15:20

S: F. Kollmer (IONTOF) 15:20 - 15:40

C: L. Bürgy 15:40 - 16:00

C: M. Heller 16:00 - 16:20

Discussion 16:20 - 18:00

64

Analysis of Biological Tissue Samples by ToF-SIMS and Orbitrap-SIMS

Daniel Breitenstein1, Karsten Lamann1, Elke Tallarek1, Alexander Pirkl2, Ewald Niehuis2, Birgit Hagenhoff1

1 Tascon GmbH, Mendelstr. 17, 48149 Muenster

2 IONTOF GmbH, Heisenbergstr. 15, 48149 Muenster

Over the last decades Secondary Ion Mass Spectrometry (SIMS) has developed into a valuable tool for

the analysis of biological samples. One advantage of SIMS is that organic as well as inorganic sample

components can be simultaneously detected. Ideally, the lateral or even three-dimensional

distribution of these analytes within the specimen can be probed.

In the last years one research focus of our group has been the comparison of SIMS results with the

results of other techniques. The focus of these studies was on both, cross-validation as well as

benchmarking.

At the last IUVSTA workshop respective approaches performed on inorganic tissue sample

components (mostly nanoparticles) were shown. In these set-ups Time-of-Flight-SIMS (ToF-SIMS)

experiments were performed and compared to the results obtained by techniques such as Ion Beam

Microscopy1, Micro X-ray Fluorescence Spectroscopy (μXRF)2, Laserablation-Inductively Coupled

Plasma Mass Spectrometry (LA-ICP-MS)3 and different microscopic approaches4,5.

Currently, our research focus is set to the detection and identification of organic sample components.

For this purpose beside ToF-SIMS also Orbitrap-SIMS is applied. The latter technology is comparatively

new as it was introduced in 20175. It should offer a superior mass resolving power, compared to state-

of-the-art ToF-SIMS, which should be beneficial with view to the identification of organic analytes.

This study gives an overview on our current research projects.

References:

[1] Veith L., et al.: (2018): Nanomaterials, 8(1), 44 ff. doi: 10.3390/nano8010044.

[2] Veith L., et al. (2018). J. Anal. At. Spectrom.., 33(3), 491-501. doi: 10.1039/C7JA00325K.

[3] Veith L., et al. (2018). J. Anal. At. Spectrom., in preparation.

[4] Veith L., et al. (2017). Analyst. 142(14), 2631-2639. doi: 10.1039/c7an00399d.

[5] Veith L., et al.: (2018): Nanomaterials, 8(8), 571 ff, 10.3390/nano8080571.

[6] Passerelli M.K., et.al. (2017). Nature Methods 14, 1175–1183; doi: 10.1038/nmeth.4504

65

TESCAN Cryo-FIB-SEM: The key for successful inspection of sensitive samples

Jakub Javůrek1, Samuel Záchej2, Tomáš Nováček2, Kristýna Rosíková1 and Jana Havránková1

1. TESCAN ORSAY HOLDING, a.s., Brno, Czech Republic.

2. TESCAN Brno, Brno, Czech Republic.

Current trends in electron microscopy are focused on analysis of beam sensitive samples. Studying

beam sensitive materials containing water represents one of the most demanding but the most useful

approaches. Low temperature scanning electron microscopy (Cryo-SEM) has become an established

technique for capturing and observing such samples close to their natural state. It is a method of

choice, where the traditional biological sample preparation (e.g. critical point drying) causes unwanted

changes in the sample structure. A Cryo-SEM workflow involves sample fixation using fast freezing to

temperatures of liquid nitrogen. The frozen samples are then transferred under vacuum to an SEM

chamber equipped with a cryo-stage and observed in high vacuum environment.

A combination of SEM with nanomachining capabilities of Focused Ion Beam (FIB) opens wide range

of possibilities. FIB-SEM systems are widely used for their capability of precise cross-sectioning, 3D

volume imaging, as well as for routine preparation of ultra-thin TEM specimens. Moreover, FIB can be

used for sample ionization, production of secondary ions, which can be analyzed by mass

spectrometer. TESCAN integrates Time-of-Flight Secondary Ion Mass Spectrometer (TOF-SIMS) to a

FIB-SEM, and thus introduces unique combination of simple navigation over the whole sample with

advanced imaging capabilities and acquisition of elemental information from the observed material.

This integration considerably extends the possible applications of FIB-SEM workstation. While typical

TOF-SIMS analyses requires sample to be introduced into the (ultra) high vacuum, use of cryo stage

enables the cryo-immobilized samples to be analyzed, which is of great importance for e.g. chemical

mapping of dissolved materials.

The aim of this presentation is to demonstrate the exceptional range of capabilities within single

versatile workstation.

66

The colder the better. A correlative workflow combining cryo-preparation, fluorescence/electron microscopy, and NanoSIMS imaging.

Celine Loussert-Fonta*, Gaelle Toullec, Thomas Krueger and Anders Meibom

EPFL ENAC IIE LGB, GR C2 524 (Bat GR), station 2, CH-1015 Lausanne, Switzerland.

Climate change threatens populations and ecosystems all over our planet. A direct consequence of the warming of the oceans is coral bleaching. Coral reefs support the most biologically diverse marine ecosystems and are extremely sensitive to changes to their physical environment (Baker et al., 2008). Mass bleaching events have been accelerating dramatically over the last decade and they manifest through a loss of the symbiotic photosynthesizing algae as a result of the symbiotic breakdown. These pigmented dinoflagellates algae translocate photosynthates to their coral host, and thus provide an essential component of the corals’ nutritional requirements. This symbiosis thus keeps the corals alive in the typically highly oligotrophic (nutrient-poor) tropical ocean conditions (Kopp et al., 2015; Krueger et al., 2018). Prolonged bleaching leads to the death of the coral host, essentially from starvation (Hoegh-Guldberg, 1999), and has dramatic consequences for the full reef ecosystem that individual corals support. Entry and establishment of an endosymbiotic relationship involves a sequence of highly controlled stages that are linked to the exchange of nutrients and molecules that permit a) the symbionts to survive inside the host and b) the modulation of the host metabolism.

In order to study the fundamental process of endosymbiosis and the associated metabolic impacts, we extend a correlative workflow based on cryo-preparations previously published (Loussert Fonta et al., 2015) to spectrometric imaging technique such as NanoSIMS. This approach allows the direct correlation of samples prepared according the hybrid method, combining chemical fixation with cryo-preparation (Tokuyasu, 1973) and immunolabelling (fluorescence or TEM). Moreover, by avoiding classical protocol steps, such as ethanol dehydration and resin embedding, this technique reduces loss of cellular content and and minimizes dilution of isotopic enrichments, in particular 13C, thereby enhancing the quality of NanoSIMS images.

During the lecture, we will present the newly obtained data on the symbiotic coral Stylophora pistillata and its symbiont algae Symbiodinium by this cryo-based workflow. Results between classical protocols (resin embedding) and cryo-preparation method will be compared and discussed.

References:

Baker, A.C., Glynn, P.W., and Riegl, B. (2008). Climate change and coral reef bleaching: An ecological assessment of long-term impacts, recovery trends and future outlook. Estuar. Coast. Shelf Sci. 80, 435–471.

Hoegh-Guldberg, O. (1999). Climate change, coral bleaching and the future of the world’s coral reefs. Mar. Freshw. Res. 50, 839.

Kopp, C., Domart-Coulon, I., Escrig, S., Humbel, B.M., Hignette, M., and Meibom, A. (2015). Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis. MBio 6.

Krueger, T., Bodin, J., Horwitz, N., Loussert-Fonta, C., Sakr, A., Escrig, S., Fine, M., and Meibom, A. (2018). Tissue- and cellular-level allocation of autotrophic and heterotrophic nutrients in the coral symbiosis — A NanoSIMS study.

Loussert Fonta, C., Leis, A., Mathisen, C., Bouvier, D.S., Blanchard, W., Volterra, A., Lich, B., and Humbel, B.M. (2015). Analysis of acute brain slices by electron microscopy: A correlative light–electron microscopy workflow based on Tokuyasu cryo-sectioning. J. Struct. Biol. 189, 53–61.

Tokuyasu, K.T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551–565.

67

Preparation of Bacterial Samples for Nanocharacterization

C. Sousa, I. M. Pinto, D. Y. Petrovykh*

International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal

The unique physicochemical properties of nanoparticles (NPs) are the basis for their potential

biomedical applications, whereby NPs interacting with biological particles provide a means for

detecting, monitoring, or controlling biological functions via engineered non-biological (magnetic,

electronic, optical, mechanical) properties of NPs. Characterization of samples that include nanoscale

interactions between non-biological NPs and biological particles (Figure 1) requires considering both

biological and physicochemical properties of the system [1,2].

Bacterial cells are an example of biological particles that are practically important in biomedical and

food sectors. Having typical features with sizes in the sub-micron and nanoscale range also makes

bacterial cells a convenient model for extension and validation of nanocharacterization methods from

solid inorganic NPs to soft biological particles [1]. While some biological methods are available for

characterizing bacterial cells in suspension, reliable measurements of their physicochemical

properties remain challenging, even for basic parameters, such as concentration (Figure 2) or size

distribution.

As a model system for quantitative characterization of biological particles (Figures 1–2), we are using

Staphylococcus aureus (S. aureus) bacteria, which offer the advantages of nearly spherical shape and

of robust viability under a wide range of experimental conditions [1]. The ca. 1 µm diameter of live S.

aureus cells also makes them representative of the challenges encountered in the characterization of

bacterial cells. In microscopy, for example, the apparent size of individual S. aureus bacteria changes

dramatically as they are prepared for measurements with increased spatial resolution: from confocal

optical microscopy, to environmental scanning electron microscopy (ESEM), to SEM in vacuum.

As an example of using S. aureus bacteria as a model system, we describe an analytical protocol [1]

that starts from NP–cell interactions in solution and preserves their evidence in dried samples to be

subsequently revealed in high-resolution SEM images (Figure 1). The validation of this protocol also

provides insight into a quantitative comparison of a physicochemical parameter (colloidal stability) of

NPs and bacteria based on combining systematic physicochemical (sedimentation), biological (colony

forming units, CFU), and optical (optical density, OD) measurements.

68

Figure 1: SEM images of the pellet (left) and supernatant Figure 2: Cell concentration measurements

(right) after differential sedimentation of an NP–cell mixture. For S. aureus dilution series with 20%

increme increments.

References:

1. Analytical Protocols for Separation and Electron Microscopy of Nanoparticles Interacting with Bacterial Cells, C. Sousa, D. Sequeira, Y. V. Kolen'ko, I. M. Pinto, D. Y. Petrovykh, Anal. Chem. 87, 4641 (2015). DOI: 10.1021/ac503835a

2. Characterization of Bio-nanosystems, C. Sousa, D. Y. Petrovykh. In Advances in Processing Technologies for Bio-based Nanosystems in Food; O. L. Ramos, R. N. Pereira, M. Cerqueria, J. A. Teixeira, A. Vicente, Eds.; CRC Press 2019 (in press)

CF

Us

0.08 0.1 0.120.08 0.1 0.12

Relative Concentration

OD

64

0

0

0.05

0.1

0.15(a)

0

25

50

75

100

(b)125

69

TOF-SIMS Analysis of Complex Samples with High Lateral and High Mass Resolution in Parallel

Felix Kollmer1, Anja Henß2, Alexander Pirkl1, Wolfgang Paul1, Andreas Dütting1, Rudolf Möllers1, Ewald Niehuis1

1. IONTOF GmbH, Germany

2. Justus-Liebig University Giessen, Germany

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a very sensitive surface analytical

technique. It provides detailed elemental and molecular information about surfaces, thin layers,

interfaces, and full three-dimensional analysis of the sample. A general strength of the applied time-

of-flight mass analyzer is the very high transmission which is due to the fact that the entire mass range

is analyzed in parallel. Any selection of peaks prior to the analysis is not required.

However, the time-of-flight analyzer suffers from a trade-off between mass resolution, lateral

resolution, and signal intensity. Even under low intensity conditions highest mass resolution and

highest lateral resolution are not obtained at the same time. In our contribution, we will discuss an

improved analyzer setup that allows us to overcome this limitation.

A promising approach in order to overcome this fundamental trade-off is to pulse the analyzer and to

extract the secondary ions just after the primary beam hits the surface (delayed extraction). Under

delayed extraction conditions, the width of the primary ion pulse no longer influences the uncertainty

of the start signal of the time-of-flight analysis. High lateral resolution and high mass resolution are

obtained in parallel with long ion pulses at higher, and thus more useful, ion currents. Moreover, the

delayed extraction mode improves the analysis of topographic surfaces in terms of transmission and

mass resolution. We have optimized this mode and we will show that the achieved improvements are

very useful for the analysis of most kinds of samples and analytical tasks so that this mode presently

is becoming a standard mode for state-of-the-art TOF-SIMS instrumentation.

Especially complex real-world systems like biological samples often require the combination of high

lateral and high mass resolution in order to separate interfering signals. Moreover, these systems are

even more demanding since often additional sample cooling is required to preserve the distribution

of volatile and mobile elements and molecules. The new design of the cooling system, presented in

the contribution, overcomes several limitations. It allows to analyse samples up to 2.5 cm in diameter,

which can be easily rotated, tilted and laterally moved by several centimeters in cooled state.

Furthermore, circulation pumps are used to pump liquid nitrogen through the system. Thus, higher

cooling rates and easy on/off-switching of cooling are achieved. Also, large dewars can be used for

>24 hours stable operation. The setup is fully compatible with Leica‘s VCT 500 for combining advanced

cryo sample preparation techniques with SIMS analysis.

70

Multidimensional imaging reveals fusion and anisotropic growth of semi-crystalline starch granules.

Leo Bürgy1, Simona Eicke1, Stéphane Escrig2, Anders Meibom2, Samuel Zeeman1

1 Group of Plant Biochemistry, ETH Zürich, Universitätstrasse 2, CH-8092 Zürich

2 Laboratory for Biological Geochemistry, EPFL, CH-1015 Lausanne

Starch is a plant storage carbohydrate that plays a central role in buffering the diel patterns of

photosynthetic carbon assimilation. Although composed of simple glucose polymers, starch forms as

discrete, semi-crystalline granules, with defined locations, numbers and shapes within the chloroplast.

Understanding starch biosynthesis requires knowledge of both its biochemistry and its cell biology.

While much biochemical progress has been made, there is little cell biological knowledge. To address

this we combined several complementary imaging techniques. Serial block-face scanning electron

microscopy revealed either single or clustered granule initials form in stromal pockets between the

thylakoid membranes. By combining 13CO2 stable isotope labelling with nanometer-scale secondary

ion mass spectrometry (nanoSIMS) we demonstrate coalescence of starch granule initials and

subsequent anisotropic granule growth, resulting in their characteristic lenticular shape. These

insights provide a new conceptual framework for understanding the synthesis of this vital plant

product.

71

Developing a cryo-workflow for the FIB-SEM characterization of fuel cell membranes

Martina Heller1, Chandra Macauley1, Gabriel Sievi2, PeterWasserscheid2, Peter Felfer1

1 Department Werkstoffwissenschaften, Lehrstuhl WW1: Allgemeine Werkstoffeigenschaften, Martensstraße 5, 91058 Erlangen

2 Helmholtz-Institut Erlangen-Nürnberg, Forschungszentrum Jülich GmbH

Liquid-containing materials have interesting applications, especially in the fuel cell field. An

investigation of these materials with a FIB/SEM dual beam microscope could offer structural

information to improve the overall efficiency. Due to the low pressure in a SEM, the investigation of

liquid-containing samples is not possible [1]. A FIB/SEM equipped with a cryo-stage offers the

possibility to investigate liquid-containing materials in a frozen state. With a custom designed plunge

freezer and transfer arm, it is possible to cryo-fixate the sample and transfer it directly into the SEM

on the cryo-stage enabling the investigation. During the transfer, only a slight frost formation occurs.

Despite this frost formation it is a fast and easy process that decreases the time needed to perform

cryo-characterization while also preventing chamber contamination. The developed cryo-workflow

offers an efficient way to investigate liquid-containing materials under more relevant conditions

including near operando states. An example of this would be a membrane electrode assembly (MEA)

from a direct methanol fuel cell. Directly after operation, a small part of the MEA filled with reactants

(methanol and water) was plunge frozen, transferred into the SEM and investigated at cryo-

temperatures (-140°C) to get valuable insights into device performance.

References:

[1] H. Mulders, The use of a SEM/FIB dual beam applied to biological samples, G. I. T. Imaging Microsc. 2 (2003) 8-10

72

73

Day 5 - 24th May

Session:

Applications, novel solutions and technologies

Session chair:

Giacomo Ceccone & Paulina Rakowska

Applications, novel solutions and technologies

C: P. Benettoni 9:00 - 9:20

C: M. Krysztof 9:20 - 9:40

S: F. Horreard (Cameca) 9:40 - 10:00

C: J. Watts 10:00 - 10:20

Coffee Break 10:20 - 10:40

Discussion 10:40 - 13:00

Lunch 13:00 - 14:00

74

Versatile sample carriers for complementary high-resolution microscopy

Pietro Benettoni,a Jia-Yu Ye,a,b Hryhoriy Stryhanyuk,a Stephan Wagner,c Mashaalah Zarejousheghani,d Thorsten Reemtsma,c,e Hans-Hermann Richnowa

a Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstraße 15, 04318 Leipzig, Germany.

b Institute of Macromolecular Chemistry, Albert-Ludwigs-University of Freiburg, Germany.

c Department of Analytical Chemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstraße 15, 04318 Leipzig, Germany.

d Department of Monitoring and Exploration Technologies, Helmholtz Centre for Environmental Research - UFZ, Permoserstraße 15, 04318 Leipzig, Germany.

e Institute of Analytical Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany.

High-resolution microscopy techniques have advantages and limitations defining their requirements

for sample preparation and applicability. Thus, often different techniques are applied sequentially in

order to complement each other and facilitate a comprehensive understanding of physical, chemical,

and biological phenomena. A commonly employed complementary approach combines Electron

Microscopy (EM) with Secondary Ion Mass Spectrometry (SIMS) thus providing high resolution EM

imaging together with high-sensitivity molecular-specific SIMS analysis. To further increase the lateral

resolution and obtain more surface-detailed images, helium ion microscopy (HIM) may also be applied

in combination with EM and SIMS.

However, electron- and ion-probe experiments require an Ultra-High Vacuum (UHV) sample

environment, demand different sample properties (e.g. conductivity, roughness, thickness etc.) and

therefore samples have to be prepared according to instrument requirements. In particular, for

nanoSIMS experiments (especially in positive extraction mode), a conductive sample surface with low

roughness is required; thus, nonconductive species are chemically/cryo-fixed, embedded, thin-

sectioned and coated with a thin conductive layer prior to nanoSIMS analysis. Even though EM

instruments with Variable Pressure (VP-option) allow for charge compensation in cost of resolution,

Transmission-EM (TEM) requires the sample to be conductive, thin and stained with heavy metals to

achieve good imaging contrast and elemental resolution. On the other hand, HIM technique provides

flexible charge compensation, sub-nanometer lateral resolution, extreme surface sensitivity and deep

focus allowing for high-resolution imaging of pristine architecture. However, to preserve and maintain

sensitive features, HIM requires prior sample preparation with a solvent series followed by CPD or cry-

fixation which limits its applicability to certain samples and carriers. At the same time, different sample

carriers (e.g. wafers, filters, etc.) might be necessary to accommodate different samples (e.g. NPs,

bacteria, biofilm, viruses, tissue sections, etc.). Not all samples and/or sample carriers can successfully

fit all preparation steps to meet instrument requirements.

For the implementation of complementary microscopy analysis, a common sample carrier has to meet

the requirements of different microscopy techniques. Here, we suggest a new type of sample carrier

compatible with different methods of sample preparation and microscopy techniques. This type of

newly developed carrier comprises a polished silicon substrate treated with a UV-Ozone cleaner and

75

coated with a hydrophilic polymer. The polymer properties can be tuned according to the analyte to

be hosted and the analysis technique to be applied. Additionally, the polymer coating was crosslinked

and cured to increase its robustness towards solvents used during dehydration, CPD and improve its

adhesion on the substrate. The hydrophilicity of the polymer coating is a key feature that controls the

transfer of solutes from the top to the bottom of the polymer layer, thus limiting the crystallization of

possible salt residuals on the surface as well as the formation of the “coffee ring” effect. The suggested

sample carrier can accommodate a broad variety of sample types (e.g. NP suspensions, cells, biofilms,

tissue sections, viruses, etc.), undergo different preparation steps, and therefore allows the

implementation of complementary microscopy studies.

76

Imaging of samples through the thin silicon nitride membrane using low energy electron beam

M. Krysztof, M. Białas, A. Górecka-Drzazga

Wroclaw University of Science and Technology, Faculty of Microsystem Electronics and Photonics

The use of thin membranes in electron microscopy is a known issue. There can be found multiple

literature on the subject [1–6]. Usually the membrane works as a sample grid in TEM. In more

sophisticated solutions two membranes are glued together to form a capsule inside of which there is

a biological sample [1]. The newest trend is to make a microfluidic device which can introduce sample

to the measurement chamber and get the sample out for further investigation [2–6]. But what all of

the mentioned solutions have in common is that their design and application are limited to use with

TEM and STEM microscopes, with very high energy electron beam. For such imaging conditions the

thin membranes, usually <100 nm thick, are practically invisible for the electron beam.

At the Faculty of Microsystem Electronics and Photonics of Wroclaw University of Science and

Technology, a research on using thin silicon nitride membranes with low energy electron beam is being

done. This research is done for two main reasons. Firstly, to develop a miniature MEMS electron

microscope [7] which can be integrated with a microfluidic device, which introduce a biological sample

to the electron beam axis. Such miniature microscope will allow to generate an electron beam with

energies lower than 10 keV, that is why it is important to investigate the parameters of thin

membranes and how they influence the imaging process. Second, the research team is working on the

microfluidic device which can be mounted inside a classical SEM or LV TEM (Low Voltage TEM) to

enable imaging of aqueous samples.

In the first stage of the research parameters of the membranes such as endurance and electron

transmissivity were checked [8]. Second the imaging of several samples through the thin membranes

were done inside JEOL JSM IT100 SEM to establish the best imaging parameters (Fig. 1). In the paper

the concept of MEMS electron microscope, technology and measurements of thin membranes and

imaging of the samples will be described.

Fig. 1. Images of salt crystals, obtained through the 50 nm thick silicon nitride membrane, with

different electron beam energies: a) E = 2 keV; b) E = 7 keV.

The work was financed by National Science Centre Poland, project number UOM-2016/21/B/ST7/02216.

a) b)

77

References:

[1] K-L. Liu, et al., Lab Chip 8, pp. 1915-1921, (2008). [2] N. de Jonge, F. M. Ross, Nature Nanotechnology, vol. 6, 695-704, (2011). [3] J. F. Creemer, et al., Journal of Microelectromechanical Systems 19, pp. 254-264, (2010). [4] D. B. Peckys, G. M. Veith, D. C. Joy, N. de Jonge, PLoS ONE 4, e8214, (2009). [5] J. M. Grogan, L. Rotkina, H.H. Bau, Physical Review E 83, 061405, (2011). [6] J. M. Grogan, N. M. Schneider, F. M. Ross, H.H. Bau, Journal of the Indian Institute of Science 92, pp. 295-308, (2012). [7] M. Krysztof, T. Grzebyk, A. Górecka-Drzazga, J. Dziuban, Technical Digest, 27th IVNC, 6-10 July, Engelberg, Switzerland, pp. 77-78, (2014). [8] M. Krysztof, T. Grzebyk, P. Szyszka, K. Laszczyk, A. Górecka-Drzazga, J. Dziuban, Journal of Vacuum Science and Technology B, 36, 02C107 (2018).

78

CAMECA microanalysis solutions for soft matter analysis

François Horréard

CAMECA 29 Quai des Grésillons, 92630 Gennevilliers France

CAMECA is providing micro- and nano-analysis instruments based on three physical techniques: electron probe microanalysis (EPMA), secondary ion mass spectrometry (SIMS) and atom probe tomography (APT).

In the SIMS range the NanoSIMS is the CAMECA instrument applied the most to life sciences. It offers 50nm lateral resolution together with high sensitivity for trace elements and isotopic ratio mapping. Presently working under ultra-high vacuum environment, the method requires the sample to be dehydrated for analysis.

The atom probe, elemental mass spectrometry technique with 3D reconstruction and atomic scale resolution, has been historically applied to metals. The use of UV laser to desorb ions then enabled the analysis of materials such as semiconductors, oxides, minerals and biominerals. The sample, prepared as a sharp tip is analyzed at extremely low temperature (down to 20K).

NanoSIMS distribution maps are most of the time coupled with a labelling strategy: “feeding” cells, plants or animals with nutrients, drugs or any molecules labelled with one or several stable isotopes (2H, 13C, 15N, 18O…) or/and rare chemical elements. The fate of the labelled molecules is imaged and measured through the local isotopic or elemental enrichment from small regions of interest. The time scale can be obtained through the analysis of different samples extracted at various times of the experiment before/during/after labelling.

In cell biology sample preparations are often similar to the one used for TEM and SEM microscopies: cryo or chemical fixation, freeze drying and/or resin-embedding, sectioning to thickness at ~micron thickness. NanoSIMS maps are often used correlatively with TEM or SEM giving resolution at the nm level. In contrast with the NanoSIMS, EM can require chemicals for contrast enhancement. Working on serial microtome sections is one solution.

In microbiology preparation of samples filtered on a grid can be limited to dehydration in an oven and metal coating for charge evacuation. But in most cases NanoSIMS images are correlated with fluorescence in-situ hybridization microscopy (FISH) giving the identity of the individual cells. This imposes fixation, permeabilization of the membrane and hybridization, plus adding reference marks around cells with laser dissection.

We will review some existing methods and some prospects for enlarging the applications:

In APT the larger access to cryo-FIB (focused ion beam) and the new cryo-UHV transfer vessel for the LEAP 5000 atom probe enable the analysis of frozen hydrated samples. The workflow available for cryo TEM can now be applied to APT, cryo method by definition.

Applying NanoSIMS analysis to frozen hydrated samples is a challenging technical request but not out of reach. There will be some effort to be produced to ensure reliable analysis of ice. But surely it would be useful for example to analyze subcellular distribution of small, labile pharmaceutical drugs, susceptible to be redistributed during resin substitution.

79

Impact of sample preparation on hydrogel characterisation

Julie A. Watts, Nicola J. Starr, Louisa Perez-Garcia, David J. Scurr

Advanced Materials and Healthcare Technologies, School of Pharmacy, University of Nottingham, NG7 2RD, UK

Supramolecular hydrogel formulations have potential for topical applications, affording several

advantages over existing commercial products, such as increased drug retention in the skin with rapid

drug release [1]. Facile formation of such hydrogel formulations from the novel use of cationic

surfactant bis-imidazolium salts, in combination with neutral or anionic drugs, has been achieved at

room temperature with simple conditions [2]. Furthermore, the use of these hydrogels has been

shown, by chemical imaging using time of flight secondary ion mass spectrometry (ToF-SIMS), to

improve skin permeation of the anti-oxidant ascorbic acid and enable the and delivery of ascorbic acid

into the epidermal layer via the breakdown of the precursor ascorbyl glucoside [3].

The morphology and chemical microstructure of water-based hydrogels may be determined by

scanning electron microscopy (SEM) and ToF-SIMS respectively [4]. This work considers the effect of

sample preparation method upon the structure and chemical composition of supramolecular

hydrogels, in a dehydrated, slush nitrogen and high pressure frozen hydrated state. This work

demonstrates the importance of using the appropriate sample preparation process to gain insight into

hydrogel structure and chemistry.

References:

[1] D. Limón, C. Jiménez-Newman, M. Rodrigues, A. González-Campo, D. B. Amabilino, A. C. Calpena, L. Pérez-García, ChemistryOpen (2017), 6, 585.

[2] D. Limón, E. Amirthalingam, M. Rodrigues, L. Halbaut, B. Andrade, M. L. Garduño-Ramírez, D. B. Amabilino, L. Pérez-García, A. C. Calpena, European Journal of Pharmaceutics and Biopharmaceutics, (2015), 96, 421-436.

[3] N. J. Starr, K. Abdul Hamid, J. Wibawa, I. Marlow, M. Bell, L. Pérez-García, D. A. Barrett, D. J. Scurr, Journal of Pharmaceutics, (2019), 10.1016/j.ijpharm.2019.03.028.

[4] J. Boekhoven, J. M. Poolman, C. Maity, F, Li, L, van der Mee, C. B. Minkenberg, E. Mendes, J. H. van Esch, R. Eelkema, Nature Chemistry, (2013), 5, 433.

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