NATIONAL UNIVERSITY OF IRELAND, GALWAYsymposia.microscopy.ie/MSI2014/Doc/Programme_MSI2013.pdf · 2016-04-11 · MSI2013 | National University of Ireland, Galway, 21st- 23rd August

37TH ANNUAL SYMPOSIUM

21ST – 23RD AUGUST 2013

NATIONAL UNIVERSITY OF IRELAND, GALWAY

This meeting has been sponsored by the following companies whose support is greatly appreciated.

Gold Sponsors

Bronze Sponsors

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___________________________________________________________________________ MSI2013 | National University of Ireland, Galway, 21st- 23rd August 2013 2

Microscopical Society of Ireland Committee

President: Prof. Fiona Lyng, FOCAS Institute, Dublin Institute of Technology, Kevin St., Dublin.

Secretary: Dr. Kerry Thompson,

Centre for Microscopy and Imaging, Anatomy, NUI Galway, Ireland.

Treasurer: Ms. Tiina O'Neill, Electron Microscopy Laboratory, Agriculture & Food Science Centre, UCD.

Outreach Officer: Dr. Dimitri Scholz, Director of Biological Imaging, Conway Institute, UCD.

Public Relations Officer: Mr. Danny Fox, CRANN and School of Physics, TCD. Public Relations

Officer.

Committee Members: Prof. Dmitri Papkovsky, Department of Biochemistry, University College Cork.

Dr. Hongzhou Zhang, CRANN and School of Physics, Trinity College Dublin. Dr. George Burke, School of Engineering, University of Ulster.

Dr. Thomas Flanagan, School of Medicine & Medical Science , UCD. Dr. Gerard Brennan, School of Biology, Queen's University Belfast.

Mr. Alexander Black, Anatomy, NUI-Galway. Dr. Tatiana Perova, Department of Electronic Engineering, TCD.

Prof. Martin Steer, Retired from School of Biology & Environmental Science, UCD. Dr. David Cottell, Retired from Electron Microscopy Laboratory, Agriculture & Food Science

Centre, UCD. Dr. Peter Owens, Centre for Microscopy and Imaging, NUI Galway.

Dr. Yina Guo, Materials & Surface Science Institute, University of Limerick.

Local Organising Committee:

Prof. Peter Dockery, Centre for Microscopy and Imaging, Anatomy, NUI Galway, Ireland. Mr. Alexander Black, Anatomy, NUI Galway, Ireland.

Dr. Kerry Thompson, Centre for Microscopy and Imaging, Anatomy, NUI Galway, Ireland. Dr. Peter Owens, Centre for Microscopy and Imaging, NUI Galway, Ireland.

Ms. Alanna Stanley, Anatomy, NUI Galway, Ireland. Dr. Jennifer Connolly, Nanobiophotonics and Imaging, Physics, NUI Galway, Ireland.

Webpage: http://www.microscopy.ie/

http://www.nuigalway.ie/msi/


General Information Posters: Poster sessions will be held during the symposium in the foyer of the Arts Millennium building. It is requested that all who are presenting posters be in attendance at the times detailed in the symposium schedule. Prizes: Monetary prizes will be awarded at the conference for the following:

• Most technologically innovative poster/talk • Best student oral presentation • Best student poster presentation

Winners will be announced at the conference dinner.

Guest wireless connectivity: This service will be free to all delegates of the conference. The username and password are as follows: Username: 9876259T Password: gvaxp2694

Symposium Location: All lectures are being held in the Arts Millennium Building on the 1st floor. Trade exhibition and posters are downstairs in the foyer. Conference Dinner: The conference dinner will be held in the College Bar on Thursday the 22nd August



Programme

Wednesday 21st August: 12:00 – 14:30 Arrival and Registration - Ground floor, Arts Millennium

Building Poster Setup

Session 1: 14:30 – 14:40 Opening address

14:40 – 15:10 Invited speaker: Hugh J. Byrne Spectroscopic Microscopy - disease diagnostics and beyond

15:10 – 15:20 Trade speaker: James Tumelty (Andor) Spinning disk confocal versus laser scanning confocal microscopy for live cell imaging

15:20 – 15:35 Valerie Barron Imaging cell distribution throughout a 3D scaffold using carbon nanotube nanoprobes in combination with photo-thermal optical coherence tomography

15:35 – 16:05 16:05 – 16:20 16:20 – 16:30

Invited speaker: Dimitiris Zuegolis Engineering functional in-vitro microenvironments Yvonne Lang Thiol-functionalization of the living diatom – microscopy approaches to monitor modification of the diatom Trade speaker: Michael Dixon (Hitachi) Novel strategies for non-destructive sample cleaning in TEM and SEM

16:30 – 17:00 COFFEE BREAK / REGISTRATION

Session 2: Sponsored by 17:00 – 17:15 Danny Fox

Controllable modification vs. non-destructive imaging: Helium ion microscopy for grapheme


17:15 – 17:25 Trade Speaker: Dan Metcalfe (Unitech) Development of fluorescence microscopy techniques and their application to basic and applied cell biology research

17:25 – 17:40 Alicja Antonczak

Does the structure of the pericentriolar material control checkpoint responses to DNA damage?

17:40 – 17:55 17:55 – 18:05

James Brown Mesenchymal stem cell cilia: Characterising novel pathways and components Trade Speaker: Natalie Hackett (ISS Group) Fera 3 - the world's first fully integrated plasma FIB

18:05 – 20:30 POSTERS, EXHIBITION Reception in Arts Millennium Foyer

20:30 – Late TABLE QUIZ IN MASSIMO’S Thursday 22nd August: Session 3: 09:00 – 9.30 Invited speaker: Martin Leahy

Multiscale label-free imaging of structure and function in living organisms

9:30 – 9:45 Manus Biggs Probing the limits of rigidity sensing with nanoscale patterning of substrate elastic modulus

9:45 – 10:00 Yina Guo Spatial variation of microtexture in linear friction welded Ti-6A1-4V

10:00 – 10:15 10:15 – 10:25

Abhigyan Satyam Micro-environment interface: Macromolecular crowding meets cell-sheet engineering Trade Speaker: Ellen Barker (Hamamatsu)


Lightsheet Mode™ : A novel firmware modification to achieve higher resolution in lightsheet microscopy

10:25 – 11:00 COFFEE BREAK Session 4: 11:00 – 11:30 Invited Speaker: Alan Ryder

Time-resolved fluorescence microscopy and spectroscopy for hydrocarbon fluid inclusion analysis

11:30 – 11:45 Yangbo Zhou Secondary electron contrast mechanism and layer determination for graphene

11:45 – 12.00 12:00 – 12:10

Zsolt Fábián TTPCK targets elements of mitotic spindle and induces cell cycle arrest in prometaphase

Trade Speaker: Joe Casey (Sarstedt) Lumox Film - Based Cell Culture Systems

12:10 – 12:25 Sivaramakrishnan Ramadurai MAPK activation-reduced nuclear diffusion of HIF-1α in hypoxic U-2 osteosarcoma cells

12:25 – 12:40 12:40 – 12:50

Luis Felipe CS Carvalho Raman micro-spectroscopy for rapid screening of oral squamous cell carcinoma

Trade Speaker: Keith Dicks (Oxford Instruments) An Introduction to Electron Backscattered Diffraction (EBSD) and it's applications

12:50 – 14:00 LUNCH & TRADE STANDS Session 5: Sponsored by 14:00 – 14:45 Keynote Speaker: Richard Horobin

Biomedical imaging using dyes and fluorochromes - 150 years and counting

14:45 – 15:00 Daniel Mortell In-situ micromechanical testing of composite materials under


scanning electron microscope

15:00 – 15:15

15:15 – 15:25

Benoit Houeix Using lectin and glycan microarrays to investigate the ‘cross-talk’ between host and commensal bacteria

Trade Speaker: Steve Cham (Elektron Technologies) Graphene oxide - a new super thin TEM support film

15:25 – 15:40 Kellie Adamson Peptide modified nanostructured surfaces for single platelet capture

15:40 – 15:55

15:55 – 16:05

Bogumila Reidy Optimisation of high content screening platform for label-free quantification of nanoparticle uptake and colocalization studies

Trade Speaker: Jasmin Moss (Invitrogen/Biosciences) Evos Cell Imaging Systems

16:05 - 16:30 COFFEE BREAK & TRADE STANDS

Session 6: 16:30 – 17:00 Invited Speaker: Sergey Alexandrov

Spectral encoding of spatial frequency approach to probe 3D structures.

17:00 – 17:15 Laurena Holleran Investigating crossing fibers in white matter using ex vivo human diffusion MRI and polarized light microscopy

17:15 – 17:30 Trade Speaker: Mhairi Crawford (Gatan) Low Energy Ion Beam Polishing for Low kV SEM

17:30 – 18:00 Annual General Meeting of the MSI

18:00 – 19:30 Posters, Exhibition, Reception

19:30 – Late Conference Dinner/BBQ with Live Music in the College Bar

Presentation of Awards


Friday 23nd August: Session 7: Sponsored by 09:40 – 10:00 COFFEE & TRADE STANDS

10:00 – 10:30 Invited Speaker: Antony Wheatley

Blood flow and other events in the microcirculation – what intravital fluorescence microscopy reveals

10:30 – 11:00 Invited Speaker: Laoise McNamara Microscopy investigations to find bone cell mechanosensors – TEM and confocal imaging of integrins, primary cilia and adhesion junctions on bone cells in vivo and in vitro

11:00 – 11:45 Keynote Speaker: Ian Dobbie Super Resolution: What's it good for? Comparison of SIM, STED and localisation techniques

11:45 – 12:00 CLOSING REMARKS CLOSE OF MEETING


Keynote Speaker Biographical Information Peter Evennett: Who Unfortunately Could Not Attend

Peter Evennett took his BSc in Zoology at Liverpool, then moved to St Andrews in Scotland for his PhD. From 1963 he lectured in the Zoology Department at the University of Leeds, in animal physiology, histology and cell biology. During this time he became increasingly interested in microscopy and in the educational work of the Royal Microscopical Society. Having taken early retirement from the University (many years ago) he now teaches microscopy courses around Europe. His special interests are in encouraging an understanding of basic principles of microscopy, and in the work of Ernst Abbe. He is particularly interested in helping amateur microscopists, and is President of the Leeds Microscopical Society. Peter is an Honorary Fellow of the Royal Microscopical Society.

Richard Horobin

Rather a long time ago I did a PhD in a chemistry department. Then I was resocialised scientifically, and subsequently have worked as a biologist, mostly at the small end of the size range. Logically, at the beginning of this second “phase” I became a member of the Royal Microscopical Society. I have, though, maintained a lifelong interest in the chemistry of dyes, at least when they are used as reagents to investigate biological structure and function. Currently I have particular interest in understanding fluorochromes which act as markers and reagents within living cells and organisms. Recent collaborations have ranged from labelling lobster larvae, to quantitative modelling of dye-DNA interactions, to investigating artefacts arising with calcium probes and with the MTT viability stain. And

in parallel, when applying dyes to cells, I always want to know if these reagents are pure or not … which concern with dye purity and standardisation led to my becoming a member of the Biological Stain Commission. Oh, and as cells are (usually) colour blind, I have sometimes been involved with studies of intracellular localisation of colourless xenobiotics, such as how to target drugs to mitochondria, or of why certain (prescribed) psychoactive drugs are so toxic.


Ian Dobbie

Ian has over 15 years’ experience in biological imaging gained in a range of leading academic institutes. He gained a degree in physics and a masters in computer modelling before moving on to do a PhD in muscle mechanics at the Randall Division of Cell and Molecular Biophysics at Kings College London. Since then he has been working in imaging with a range of biological systems at number of world class research centres including, Cancer Research UK, NUI Galway, Kings College London and The University of Oxford. Over the last 15 years he has specialised in advanced fluorescence microscopy. During the past 5 years Ian has setup up the Micron Oxford

Advanced Bioimaging Unit (www.micron.ox.ac.uk) within the Department of Biochemistry. This unit has a wide range of facilities but is specialising in Super-Resolution fluorescence imaging, especially 3D-Structured Illumination Microscopy (3D-SIM).


Invited Speaker biographical information Hugh J. Byrne

Professor Hugh J. Byrne is the Head of the Focas Research Institute, DIT. He has over 25 years’ experience in research science and has published over 250 peer reviewed journal and conference papers. He has been responsible for over €20 million in funded projects, including PRTLI Cycles 1,4 and 5 aswell as SFI, EI and EU projects and has over 15 years of experience in the management and development of research infrastructure. His principle research interests are in applications of spectroscopy and the study of molecular and nano-materials. Recent activities have extended to biospectroscopy for diagnostics and biochemical analysis and nano-bio interactions.

Martin J. Leahy

Martin Leahy is scientific director of the National Biophotonics and Imaging Platform Ireland. He completed a DPhil in BioPhotonics at the University of Oxford and he and a colleague established Oxford Optronix Ltd., where he was Director of R&D. From 1995 he had various research and teaching posts at the University of Oxford. He then joined the Physics Department at the University of Limerick where his group specialised in tissue optics and microcirculation imaging, a number of projects in the area of biomedical instrumentation and lecturing in physics. He has secured more than €7M in external R&D funding since 2007. In

addition to his Chair of Applied Physics at NUI Galway, he is adjunct professor at the Royal College of Surgeons, Fellow of the Institute of Physics, Fellow of the Royal Academy of Medicine in Ireland and Fellow of SPIE. For 2010 he was programme committee member, panel moderator for SPIE Photonics West BiOS in San Francisco and host of the BioPIC European Bioimaging conference in Dublin. From 2011 he is Editorial Board member of the Journal of Biomedical Optics, a conference co-chair and panel moderator at SPIE Photonics West and serves as chair or programme committee member of several other conferences in the US, EU and Russia. Prof. Leahy has held senior management roles in industry (e.g. Founding Director of R&D at Oxford Optronix Ltd.) and academia (e.g. Founding Director of CPI www.cpi.ul.ie and First Scientific Director of the National Biophotonics and Imaging Platform www.nbipireland.ie). He was founding MD of Limerick West Windfarms Ltd. and Millstream Energy and PI on the research projects that spun out Biomass Heating Solutions Ltd. and Wheelsbridge AB. Since his return to full-time academia, he has played a leading role in graduate biophotonics education through www.nbipireland.ie/moodle and www.biophotonics.ul.ie/resources.html and the Biophotonics and Imaging Graduate Summer School www.nbipireland.ie/events/bigss. In each of 2010 and 2011 he delivered a dozen international invited/keynote lectures and published more than a dozen ISI journal


articles, chapters and books. Martin Leahy succeeded Chris Dainty as Chair of Applied Physics at NUI Galway in 2011, where he aims to establish the best equipped microcirculation imaging facility in the world. Dimitrios Zuegolis

Dimitrios Zeugolis completed his PhD at the University of Northampton in UK in 2005. Following that, Dimitrios spent 2.5 years at the National University of Singapore, as a Post-Doctoral Research Fellow. In 2007, Dimitrios joined NUI Galway as a Government of Ireland Research Fellow. Since 2008, Dimitrios is a lecturer in Biomedical Engineering (Biomaterials) and Principal Investigator at the Network of Excellence for Functional Biomaterials (NFB) at NUI Galway. His research group (a) uses bottom up approaches to build nano-textured constructs that

closely imitate the properties of native extracellular matrix assemblies; (b) utilises recent advancements in scaffold functionalisation, through incorporation of biophysical cues and biochemical signals, to provide therapeutic interventions for the treatment of soft tissue injuries; and (c) investigates how modulation of the in vitro microenvironment will facilitate cell phenotype and functionality preservation and enable wide acceptance and clinical translation of cell-based therapies. Dimitrios has authored 24 peer-reviewed articles; over 70 peer-reviewed conference publications; 1 peer-reviewed book; 6 peer-reviewed book chapters; and 2 patents. He leads a team of 8 PhD candidates; 2 research assistants; and 4 post-doctoral scientists. He has been awarded over €7 million as Principal Investigator (26 awards); and is co-founder of Vornia Biomaterials. Laoise McNamara

Dr. Laoise McNamara is a Science Foundation Ireland Stokes Lecturer in Biomedical Engineering at the National University of Ireland Galway. She completed a PhD in Biomedical Engineering at Trinity College Dublin, a postdoctoral fellowship at the Department of Orthopaedic Research at Mount Sinai School of Medicine, New York and was a lecturer in Mechanobiology and Musculoskeletal Biomechanics at the University of Southampton in the United Kingdom. Her research studies in bone mechanobiology and osteoporosis, encompassing in vitro and in vivo experimental studies and computational modelling, have been recognised

through awards from leading international bodies (American Society for Bone and Mineral Research, American Society for Mechanical Engineering, European Society of Biomechanics, Orthopaedic Research Society), publications in major international multidisciplinary peer-reviewed journals, and > 60 presentations at international conferences and schools, and she was awarded a European Research Council Starting Independent Research Grant in 2011.


Antony M Wheatley Professor Antony M (Tony) Wheatley is Professor of Physiology at NUIG. Tony is a graduate of Trinity College Dublin (PhD, 1981) and previously held appointments in South Africa (University of Cape Town & University of the Witwatersrand), Switzerland (University of Berne) and New Zealand (University of Otago). His main research interests have been in microcirculation research particularly in the liver (liver transplantation, liver regeneration, I/R injury), GI tract, and kidney. He has over 80 publications in peer-reviewed journals. Sergey Alexandrov Sergey Alexandrov started this research at the University of Western Australia as a Research Assistant Professor and continued at the University of Pittsburgh, Pennsylvania, USA. From May 2012 he works at the National University of Ireland in Galway, with Tissue Optics and Microcirculation Imaging (TOMI) group. His research interest is the development of new optical methods and tools, such as microscopic imaging and measuring techniques (holographic, interference, polarization, spectral, photoacoustic, etc.) for biomedical and other applications. A significant contribution has resulted in the creation of new optical microscopy methods and techniques (combination of digital holographic microscopy and light scattering spectroscopy, synthetic aperture microscopy, spectral encoding of spatial frequency approach, ultra-high sensitive optical coherence tomography…) for imaging and characterization of different samples, including biomedical samples and human beings in vivo. Alan Ryder Dr. Alan G. Ryder is a Senior lecturer in the School of Chemistry at the National University of Ireland, Galway (NUIG). He obtained a B.Sc.(1.1) in Chemistry (1989) and Ph.D. in Inorganic chemistry (1994) from NUIG. After a stint as a postdoctoral researcher in UCC, he rejoined NUIG in 1997 to work on developing quantitative Raman spectroscopy based methods for measuring illicit narcotics. In 1998, awarded a Forbairt post-doctoral fellowship, began work on fluorescence methods for petroleum analysis. In 2003 he formed the Nanoscale Biophotonics Laboratory (NBL) with a grant from Science Foundation Ireland. The group is focussed on the use of photonics and chemometrics technologies for life and physical science applications. In 2006 he was appointed as a senior lecturers in the School of Chemistry at NUIG where he lectures mainly in spectroscopy. Dr. Ryder has two core research areas: Analytical Sciences and Photonics which cover a wide range of applications. In the Analytical Sciences domain the main research area is the development of rapid, quantitative analytical methods for the analysis of complex materials with particular focus on biopharmaceutical manufacturing. Key methods employed include Raman and Fluorescence spectroscopies and chemometrics. In the bioanalytical space he has collaborated with a range of industry partners including Bristol-Myers Squibb, Janssen-Biologics, Merck, Agilent, and Kaiser Optical Systems. In the photonics domain, specific projects involve the use of advanced fluorescence spectroscopy and microscopy for the analysis of protein adsorption on surfaces, petroleum fluids, biomedical polymers, instrument development, and novel fluorophore photophysics. He has authored in excess of 70 publications, generated 3 patents, and graduated 9 PhD students.


Oral Presentation Abstracts

Imaging cell distribution throughout a 3D scaffold using carbon nanotube nanoprobes in combination with photo-thermal optical coherence tomography

Emma Connolly1, Hrebesh M. Subhash2, Martin Leahy2, Niall Rooney3, Frank Barry1, Mary Murphy1 and Valerie Barron1

1Regenerative Medicine Institute, National University of Ireland, Galway/IE,

2Tissue optics and microcirculation imaging Facility, National Biophotonics and Imaging Platform, National University of Ireland, Galway/IE,

3Proxy Biomedical Ltd., Galway/IE

Regenerative medicine offers great promise for the repair of a range of damaged tissues and organs. In particular, mesenchymal stem cells (MSC) are an attractive cell therapy for cartilage repair. However, retaining sufficient numbers of functional MSCs at the site of injury still remains a challenge. One method of overcoming this limitation is to employ a scaffold. Nonetheless, once implanted, little is known about the fate of the cells in terms of distribution, migration and tracking. Despite the fact, that a range of clinically viable imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), photo emission tomography (PET), ultrasound and bioluminescence imaging are being optimised to track cells in vivo, many of these techniques are subject to limitations such as the levels of contrast agent required, toxic affects of radiotracers, photo attenuation of tissue and backscatter. With the advent of nanotechnology, nanoprobes are leading the charge to overcome these limitations. In particular, single wall nanotubes (SWNT) have been shown to be taken up by cells and as such are effective nanoprobes for cell imaging1. Consequently, the main aim of this research is to employ MSC containing SWNT nanoprobes to image cell distribution in a 3D scaffold. To this end, MSC were cultured in the presence of 32µg/ml SWNT in cell culture medium (αMEM, 10% FBS 1% penicillin/streptomycin) for 24h as described previously2. Upon confirmation of cell viability, the MSC containing SWNT were encapsulated in hyaluronic acid gels and loaded on the scaffolds. After 28 days in complete chondrogenic medium, with medium changes every 2 days, chondrogenesis was confirmed by the presence of glycosaminoglycan (GAG) using biochemical assays and immunostaining with toluidine blue. In parallel, the distribution of the cells in the 3D scaffold was successfully imaged using photothermal optical coherence tomography (PT-OCT). As shown in Figure 1, the cells can be seen to be distributed through the scaffold with a resolution better than that previously described for cell-seeded scaffolds3,4. In summary, these data reveal that MSC containing SWNT nanoprobes in combination with PT-OCT imaging modality offer an exciting opportunity for stem cell tracking in vivo.

References: 1. Welsher, K.; et al Nat Nanotechnol 2009, 4, 773–780. 2. Mooney, E.; et al Nano Lett. 2008, 8, 2137–2143. 3. Veksler , B. Proc. of SPIE 2008 Vol. 7139, 71390C 4. Holmes C, J Tissue Eng Regen Med. 2013 Feb 12 doi: 10.1002/term.1687


Thiol-functionalization of the living diatom – microscopy approaches to monitor modification of the diatom

Yvonne Lang1,2, Liam Collins3, Brian Rodriguez3, Kerry Thompson4, Peter Dockery4, David P. Finn2, Francisco del Monte5, Abhay Pandit1

1Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland

2Pharmacology and Therapeutics, School of Medicine, and Centre for Pain Research, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

3Conway Institute of Biomolecular and Biomedical Research and School of Physics,University College Dublin, Belfield, Dublin 4, Ireland

4Centre for Microscopy and Imaging, Anatomy, School of Medicine, National University of Ireland, Galway, Ireland 5Instituto de Ciencia de Materiales de Madrid, Consejo Superior de Investigaciones Científicas, Campus de

Cantoblanco, Madrid, Spain

Biomineralization processes identified within diatoms have inspired the design of nanoscale synthetic silica structures in vitro using silane precursors

[1-3]. Silanes have also

been employed to functionalize the cleaned non-living diatom by introducing amino[4-7]

, mercapto

[8, 9] and vinyl

[9] groups onto the surface. However, modification of the living diatom via metabolic insertion of silanes and organosilanes remains under explored. Herein, we investigate the use of the machinery within the living diatom to fabricate organo-silica constructs using a combination of silane and organosilane precursors. It was hypothesised that a solution of tetramethylorthosilicate (TMOS) and 3-mercaptopropyltrimethoxysilane (MPTMS) will provide an alternative source of silica for the growth of the diatom T. weissflogii enabling metabolic insertion of an organosilane into the diatom frustule. Formation of valves within the parent diatom was monitored using fluorescence microscopy. The chemical composition of the diatom was determined using

29Si-NMR and

energy dispersive X-ray analysis (EDX). The architecture of the diatom was characterised and quantified using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). Frustule synthesis, monitored using the fluorescent probe PDMPO, was observed in the cultures incubated with TMOS/MPTMS. EDX analysis confirmed the presence of sulphur in the TMOS/MPTMS modified diatom.

29Si-NMR analysis confirmed the presence of an organic

moiety extending from the Si backbone of the TMOS/MPTMS modified diatom skeleton. The gross morphology of the TMOS/MPTMS modified diatom is unaltered. AFM analysis revealed that the distance between ribs in both a radial and a rotational direction is decreased in the TMOS/MPTMS modified diatom compared to the native diatom. There is significant reduction in the pore dimensions of width, length, perimeter and area in TMOS/MPTMS modified diatoms compared to the native diatom. This is the first study to demonstrate that organosilanes can be used as a source of silica for the growth of the diatom T. weissflogii. The possibility to use the living diatom following modification of the chemistry of the frustule is an exciting area of research and requires further investigation.


References: 1. Pohnert, Angew. Chem. Int. Ed. 2002, 41, 3167 2. Patwardhan et. al, Chem. Commun. (Camb.) 2003, 21, 1122 3. Wieneke et. al, Org. Biomol. Chem. 2011, 9, 5482 4. Rosi et. al, Angew. Chem. Int. Ed. 2004, 43, 5500 5. De Stefano et. al, Acta Biomater. 2008, 4, 126 6. Townley et. al, Nanotech. 2007, 18, 295101 7. Gale et. al, Adv. Funct. Mater. 2009, 19, 926 8. Yang et. al, Sci. Technol. Adv. Mater. 2012, 13, 015008 9. Fowler et. al, Appl. Surf. Sci. 2007, 253, 5485

Keywords: diatom, organosilane, sol gel, modification Acknowledgements: This material is based upon works supported by the Science Foundation Ireland under Grant No. (07/SRC/B1163), Science Foundation Ireland/Enterprise Ireland TIDA (11/TIDA/B2026), and MICINN (MAT2009-10214 and PET2008-0168-01).


Controllable modification vs. non-destructive imaging: Helium ion microscopy for grapheme

D. Fox1, Y. Zhou1, A. ONeill1, S. Kumar2, J. J.Wang1, J. N. Coleman1, G. S. Duesberg2,

J. F. Donegan1 and H. Z. Zhang1

1School of Physics and CRANN, Trinity College Dublin, Dublin 2, ROI 2School of Chemistry and CRANN, Trinity College Dublin, Dublin 2, ROI

We have demonstrated both controllable modification and sub-nanometre metrology of nanomaterials using a helium ion beam, with graphene used as an example [1]. The sub-nanometre He+ probe of the helium ion microscope (HIM) facilitates controllable defect production with extremely high spatial resolution. Raman spectroscopy was used to quantify the disorder which can be introduced into the graphene as a function of helium ion dose. The ion dose required to safely image graphene was established to be on the order of 1013 He+/cm2, while graphene became highly defective at doses over 5 x 1014 He+/cm2. The effect of the presence of a substrate was also investigated. The unique, enhanced edge sensitivity of HIM imaging was evidenced by comparison with the more widely established SEM and TEM techniques. A freestanding graphene ake was imaged in HIM, SEM and TEM. The strong dependence of secondary electron yield on incidence angle provides greater edge contrast in HIM than the other techniques investigated. This effect coupled with the efficient generation of secondary electrons by the sub-nanometre helium ion beam make helium ion microscopy ideally suited to graphene imaging. A HIM image of the edge of a graphene ake with the intensity profile overlaid is shown in figure 1. The extent of the beam induced sample modification can be tuned and exploited by following the guidelines set out in this work. The use of helium ions for high-contrast nanoscale imaging was also shown to be superior to other similar techniques.

Figure 1: HIM image of the edge of a graphene ake with the intensity profile overlaid. References: 1. D. Fox, Y. Zhou, A. O'Neill, S. Kumar, J.J.Wang, G. Duesberg, J. Donegan, and H.Z. Zhang. Helium ion

microscopy of graphene: beam damage, image quality and edge contrast, 2013, Nanotechnology, 24 (In Press) Featured Article.


Does the structure of the pericentriolar material control checkpoint responses to DNA damage?

Alicja K. Antonczak1, Yifan Wang1, Maurice Barrett1, Aslak Rouaze1, Laurence Pelletier2 and Ciaran G. Morrison1

1Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway,

Ireland 2Samuel Lunenfeld Research Institute, University of Toronto, Canada

Centrosomes, the principal microtubule-organising centers of animal somatic cells, consist of two centrioles, barrel-shaped microtubule assemblies that are embedded in a poorly-characterised pericentriolar material (PCM). Emerging data suggest that the PCM is not an amorphous assembly of proteins, but actually a structured complex around the centrioles. Pericentrin (Pcnt) is a major PCM protein that is involved in all centrosomal processes. Mutations in PCNT have been connected to several human disorders, including primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. We and others have observed that pericentrin ensures appropriate mitotic spindle activity. Here we describe how pericentrin controls the PCM and influences the response to DNA damage. Using structured illumination microscopy, we examined the PCM during the normal cell cycle and upon ionising radiation (IR) treatment in chicken DT40 cells. PCM volumes increased greatly in response to DNA damage, with a marked loss of PCM scaffolding structure. This PCM response was dependent on functional checkpoint kinase 1 (Chk1), and was exacerbated by the loss of the pericentrin interactor, Mcph1/ Brit1. We also examined Chk1 activation in PCNT-deficient or overexpressing cells, and found that pericentrin acts as a positive regulator of nuclear Chk1 activity after DNA damage. Our current experiments are focussed on whether Chk1 activation requires its centrosomal localisation and on whether PCM expansion regulates Cdk activity and checkpoint activation. Funding: EMBO Long-Term Fellowship (AKA), JCS Travelling Fellowship, Science Foundation Ireland PI award 10/IN.1/B2972.


Mesenchymal stem cell cilia: Characterising novel pathways and components

James A. L. Brown, Tapesh Santra, Sharon A. Glynn, Peter Owens, Frank P. Barry Currently there is intense interest in the use of mesenchymal stem cells (MSC) for therapeutic interventions in many diseases and conditions. MSC are multipotent cells residing in the bone marrow niche and are responsible for renewing key cell lineages. A crucial aspect facilitating the therapeutic use of MSC is their ability to migrate to sites of injury and secrete therapeutic factors. To accelerate the therapeutic use of MSC we must understand how MSC sense their environment and respond. Primary cilia (non-motile) are a sensory organelle present on almost every non-replicating cell. Primary cilia transduce information about the extracellular environment into cells, triggering signalling cascades that have profound effects on development, cell cycle, proliferation, differentiation and migration. MSC migrating to sites of injury are likely to encounter hypoxic conditions, therefore we investigated the effect of oxygen tension on MSC cilia. We characterised in vitro MSC culture conditions examining cilia induction, numbers and length. We found oxygen tension significantly affected the production and induction of cilia. We also identified a novel cilia localising protein in MSC. Investigating the effects of chemotactic migration on cilia orientation, we observed no alignment of cilia to the direction of migration. Finally, using systems biology we identified links between migration and ciliation signalling pathways, characterising the novel role of HSP90 and PI3K signalling on ciliation. These findings significantly enhance our current understanding of chemotactic mediated MSC behaviour and advance our understanding of both MSC biology and the processes regulating cilia.

MSC Cilia: Cilia stained in red, centrosomes in green, DNA in blue.


Probing the limits of rigidity sensing with nanoscale patterning of substrate elastic modulus

Manus Biggs1,2; Ryan Cooper1; Teresa Fazio1; & Shalom Wind1.

1 Network of Excellence for Functional Biomaterials, National University of Ireland, Galway. 2 Department of Applied Physics and Applied Mathematics, Columbia University, NY, USA.

The bulk rigidity of the cellular environment directly influence multiple cellular processes through mechanotransductive pathways1. Cellular adhesion, an important modulator of differential cell function2, is significantly modulated by the substratum rigidity, via the activation of integrins, influencing the frequency and reinforcement of focal adhesion formation3. Tissues however present a wide spectrum of discrete rigidities, due to the compound nature of tissues and their components. We describe a novel system driven by focused electron-beam for the fabrication of soft (350 kPa) planar PDMS substrates, which present ordered submicron scale differential rigidity. Substrates were shown to undergo a linear increase in rigidity with E-beam dose, up to 3.5 MPa, facilitating the patterning of relatively soft substrates with micron and submicron spots of significantly increased elastic modulus (1 order of magnitude). Human mesenchymal stem cells (MSC) cultured on patterned rigidities were observed to form focal adhesions that co-localised with the stiffer spots in a modulus dependent manner (Fig. 1). However, focal adhesion co-localisation was reduced by decreasing the spot diameter. Focal adhesions formed on regions of increased modulus were associated with an increase in paxillin recruitment, indicating a role in force-mediated focal adhesion reinforcement. Our studies suggest that cellular rigidity sensing and focal adhesion reinforcement is initiated on discrete regions of modulated rigidity measuring from 0.5 - 1 µm, and that a 3x increase in elastic modulus is required to initiate differential focal adhesion on discrete regions of altered rigidity. Tissue present as heterogeneous rigidity composistions. Micro and nano-patterned rigidities are powerful tools for microscopical analysis of focal adhesion formation and subcellular mechanotransductive processes.

Funding: MB is a Science Foundation Ireland SIRG fellow (11/SIRG/B2135). The work was funded by the NIH Common Fund Nanomedicine Programme (PN2EY016586).

References 1. Engler, A.J., et al. Cell 126, 677-689 (2006). 2. Evans, N.D., et al. Eur Cell Mater 18, 1-13; discussion 13-14 (2009). 3. Fu, J., et al. Nat Methods 7, 733-736 (2010).

Figure 1: (A) MSC cultured on heterogeneous rigidity. Green actin, Blue paxillin, Red nucleus. (B) Co-localisation analysis of focal adhesions as quantified by Mander’s coefficient.

https://staffmail.nuigalway.ie/exchange/[email protected]/Inbox/Re:%20MSI%20abstract.EML/MSI%20Symposium%202013.docx/C58EA28C-18C0-4a97-9AF2-036E93DDAFB3/MSI%20Symposium%202013.docx?attach=1#_ENREF_1




Spatial variation of microtexture in linear friction welded Ti-6A1-4V

Yina Guo1, Moataz M. Attallah2, Yulung Chiu2, Hangyue Li2, Simon Bray3 and Paul Bowen2

1Material and Surface Science Institute, University of Limerick, Ireland

2School of Metallurgy and Materials, University of Birmingham, B15 2TT, UK 3Rolls-Royce plc, PO Box, Derby DE24 8BJ, UK

The spatial variation of the microtexture across various weld regions of linear friction welded Ti-6A1-4V alloy has been investigated using electron backscattered diffraction (EBSD). The pole figures and the misorientations distribution curves showed that the weld is composed of four zones with different textural characteristics which are strongly related to the amount of transformed β phase in microstructure. The deformation in the primary α grains has a negligible effect on the texture development. Keywords: Linear friction welding, titanium alloys, microstructure, electron backscattered diffraction (EBSD)


Micro-environment interface: Macromolecular crowding meets cell-sheet engineering

Abhigyan Satyam1, Pramod Kumar1, Fan Xingliang1, Yury Rochev1, Lokesh Joshi2, Héctor Peinado3, David Lyden3, Benjamin Thomas4, Brian Rodriguez5, Michael Raghunath6, Abhay

Pandit1 and Dimitrios Zeugolis1

1Network of Excellence for Functional Biomaterials (NFB), NUI Galway, Galway, Ireland. 2Alimentary Glycoscience Research Cluster, NUI Galway, Galway, Ireland.

3Departments of Pediatrics, Cell and Developmental Biology, Weill Cornell Medical College, New York, USA. 4Central Proteomics Facility, Sir William Dunn Pathology School, Oxford University, Oxford, UK.

5Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin, Ireland. 6Department of Bioengineering, National University of Singapore, Singapore.

Advancements in polymer chemistry enabled a scaffold-free technology termed Tissue Engineering by Self-Assembly1. Very few commercial products have arisen from this technology so far. This is largely due to the prolonged culture times required to grow implantable tissue stabilised by extracellular matrix (ECM). For example, generating of corneal stromal tissue requires 84 days2 and small blood vessels take 18 weeks3. It was recently shown that macromolecular crowding (MMC) enhances the deposition and stabilizing this deposited ECM 4-6. Here, we assessed the potential of combining MMC technology with a Tissue Engineering by Self-Assembly approach. Human primary fibroblasts [lung (WI-38), skin (WS-1)] were cultured under various MMC conditions (100μg/ml dextran sulphate; 37.5mg/ml Ficoll™ 70 and 25mg/ml Ficoll™ 400; 75μg/ml carrageenan)] in a range of fetal bovine (FBS) and human (HS) serum concentrations (0.0-10%). ECM deposition was verified by SDS-PAGE, immunocytochemistry (ICC) atomic force microscopy (AFM), scanning electron microscopy (SEM) and mass-spectrometry. The influence of crowders on cell morphology, cell viability and metabolic activity were evaluated using phase-contrast microscopy, Live/Dead® and AlamarBlue® assays respectively. NIPAM based thermo-responsive polymers were used to facilitate detachment of ECM-rich cell sheets. Densitometric analysis of SDS-PAGE demonstrated that MMC significantly increase type-I collagen deposition (p<0.0001) at all tested serum concentrations and the maximum deposition was observed in 2 days with 0.5% serum (figure-1). ICC, AFM and SEM further confirmed enhanced deposition of fibrillar ECM in presence of MMC (figure-2, a-h). Phase-contrast microscopy, Live/Dead® and AlamarBlue® assays demonstrated that the cellular morphology, viability and metabolic activity respectively were not affected by crowded environments. Thermo-responsive coating with 65% NIPAM: 35% N-tert-butylacrylamide facilitated detachment of ECM rich cell-sheet from culture (figure-2, a-d). Complementary ICC for mass spectrometry validation confirmed the enhanced deposition of collagens (III, IV, V, VI) and other ECM molecules (laminin, fibronectin, hyaluronic acid, decorin, lysyl oxidase), without changing collagen-VII, elastin, fibrillin-1, transglutaminase-2, α-smooth muscle actin, epithelial keratin, tubulin, chondroitin sulphate, keratin sulphate, heparin sulphate, aggrecan, biglycan, CD248 and IL-10.


Modulation of the in vitro microenvironment with macromolecular crowding enhances extracellular matrix deposition even under low serum supplementation and it facilitates the production of intact ECM rich cell-sheets when combined with a novel thermo-responsive polymer coating. Supporting figures:

References:

1. Peck M. et al., Mat. Today, (2011),14:218-24 2. Vrana N. et al., IOVS (2008),49:5325-31 3. L'Heureux N. et al., Nat. Med., (2006),12:361-65 4. Lareu R. et al., FEBS Letter, (2007),581:2709-14 5. Cigognini, D. et al., Drug Discovery Today, (2013), in press 6. Zeugolis D.I. & Satyam A. European Patent, (2011), 11169176.2-2401

Keywords: Macromolecular crowding, Extracellular matrix, Cell-sheet technology Acknowledgments: Authors would like to thank Science Foundation Ireland (Grant-09/RFP/ENM2483 and 07/IN1/B031) and SFI ETS-Walton award for financial support.


Secondary electron contrast mechanism and layer determination for graphene

Yangbo Zhou, Robert O’ Connell, Daniel Fox and Hongzhou Zhang

School of Physics and CRANN, Trinity College Dublin, Dublin 2

Graphene has attracted numerous interests since its discovery and has revealed great potential applications in the fields of nano-devices. In a broad sense, graphene may refer to multilayered structures as well as the more strict definition of single-layer hexagonal network of carbon atoms. The properties of graphene depend on its thickness and the determination of graphene thickness becomes important to its device applications. Recent research indicates that secondary electron (SE) contrast in the scanning electron microscope (SEM) may provide an accurate and scalable method for graphene metrology, while the mechanism of graphene SE contrast is unclear and quantitative results are absent. In this work, we investigated the SE contrast of graphene by using the signals generated in both SEM and the latest Helium Ion Microscope (HIM). The measured SE contrast varied with the energy of the primary electron beam and the graphene layer thickness. A contrast reversal was observed at about 2keV and the layer contrast was optimized at about 5keV (See Fig. 1). As shown in Fig. 2, for a given beam energy a linear relationship between the SE contrast and the thickness was revealed, indicating that the graphene thickness can be identified up to 10 layers from the SE images. The SE contrast excited with a 30 keV He+ beam in HIM was similar to that in SEM at a beam energy below 0.5keV (Fig. 3). The SE contrast observed in HIM and low voltage SEM can be attributed to the SE emission from graphene itself, while under high-energy electron beam excitation the SE contrast of multilayer graphene were dominated by both the graphene surface work function and SE attenuated from the underneath substrate. A theoretical model has been developed which allows the extraction of graphene work function and doping level from the measured SE contrast. The linear layer dependent SE contrast also offered an effective method to determine the graphene layer thickness [1, 2]. Reference 1. H. Hiura, H. Miyazaki, and K. Tsukagoshi, Appl Phys Express 3 (2010). 2. V. Kochat et al., Journal of Applied Physics 110 (2011).

Keywords: graphene, secondary electron contrast, layer determination.

Figures: (1) SE contrast for a 1-5 layer graphene flake at voltages from 0.5KV to 15KV. (2) Normalized SE intensity for 1-7 Layer graphene flake at 3KV (3) HIM image of a 1-7 Layer graphene flake.


TTPCK targets elements of mitotic spindle and induces cell cycle arrest in prometaphase

Zsolt Fábián and Howard O. Fearnhead

Regenerative Medicine Institute, NUI Galway The serine protease inhibitor N-α-tosyl-ε-phenylalanyl chloromethyl ketone (TPCK) has been long used in studies of cellular processes including apoptosis. Depending on the experimental conditions, TPCK either induces or inhibits changes associated with apoptosis but there has been little progress in identifying the relevant targets for TPCK. Our group recently showed that the largest subunit of the RNA polymerase II is one of the intracellular targets of TPCK. The complex effects of TPCK on apoptosis, however, suggested the existence of additional apoptosis-relevant targets in cells. Using our unique polyclonal anti-tosyl antibody, here we report the identification of the mitotic spindle as another intracellular target for TPCK. We also provide data that TPCK-mediated labelling of the mitotic spindle correlates with cell cycle arrest in prometaphase.


MAPK activation-reduced nuclear diffusion of HIF-1α in hypoxic U-2 osteosarcoma cells

Zsolt Fábián, Sivaramakrishnan Ramadurai, Heinz-Peter Nasheuer and Frank Barry

Regenerative Medicine Institute, NUI Galway At the heart of the molecular machinery orchestrating the biological responses to hypoxia are the hypoxia inducible factors (HIF). Active HIFs are basic helix-loop-helix transcription factors that act as heterodimers of one alpha and beta subunits constitutively expressed in most of the cells. In normoxia, however, HIF-1α is removed from cells by proteasomal degradation initiated by prolyl hydroxylase enzymes (PHDs). Under hypoxic condition, PHDs become inactive that leads to the accumulation of the functional HIFs in the cell. Once stabilized, active HIFs alter the transcriptional pattern to make cells adapt to the hypoxic milieu. Here we show the use of U-2 osteosarcoma cells expressing eGFP-tagged HIF-1α for real time fluorescent correlation spectroscopy to measure the diffusion of the GFP-tagged HIF-1αunder hypoxia. We found that HIF-1α is further regulated by the activity of the MAPK signalling pathway by the reduction of diffusion of HIF-1α within the nucleus, an indicator molecular complex formation. Our data suggest that the effect of the MAPK pathway on post-translational regulation of HIF-1α may be important to evoke prompt adaptive cellular response to acute hypoxia.


Raman micro-spectroscopy for rapid screening of oral squamous cell carcinoma

Luis Felipe CS Carvalho1, Franck Bonnier1, Kate O´Callaghan2, Jeff O´Sullivan2, Stephen Flint2, Hugh J. Byrne1, Fiona M. Lyng3,4

1Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8

2Dublin Dental School and Hospital, Trinity College Dublin, Dublin 2 3Radiation and Environmental Science Centre, Focas Research Institute, Dublin Institute of Technology, Kevin

Street, Dublin 8 4School of Physics, Dublin Institute of Technology, Kevin Street, Dublin 8 Ireland

Oral cancer has one of the highest incidences of cancer. Timely diagnosis can however be crucial for the patient health and prognosis. While oral cancers don’t have high mortality incidences index, the morbidity is exactly the opposite, because any surgical procedure can cause significant aesthetic and functionality effects. Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful for early and non-invasive diagnosis. In this study, we evaluate the different Raman spectral signatures of TR146 oral squamous cell carcinoma (buccal mucosa) and primary human oral epithelial cells (normal cell), collected from the nucleoli, nucleus and cytoplasm. Data were collected using a Raman micro spectroscopy system using a 532nm diode laser as excitation source. In addition, we use principal component analysis (PCA) as a statistical method to discriminate the spectra. The results show good distinction between the cancer and normal cells using only the first and second principal components (PCs) in each site studied (nucleoli, nucleus and cytoplasm). The sensitivity and specificity index were respectively determined to be: nucleoli 95% and 85%, nucleus 95% and 90%, cytoplasm 75% and 80%. The loading plot of the PCs showed significant variations related to nucleic acid and proteins for nucleoli and nucleus sites and variation in vibrational modes of lipids for cytoplasmic areas. However, the discrimination in this latter region is not as good as the results from the nucleoli and nucleus because of the greater cytoplasm heterogeneity. In conclusion, we can argue that Raman micro spectroscopy can be used to detect the differences between oral cancer cells and normal oral epithelial cells. This technique may provide a rapid screening method and have potential use in early and non-invasive diagnosis of oral squamous cell carcinoma. References:

1. Vargis E. et al. Near-infrared Raman microspectroscopy detects high-risk human papillomaviruses. Translational Oncology. 2013 5(3), 172-79.

2. de Carvalho, LFCS. et al. Spectral region optimization for Raman-based optical biopsy of inflammatory lesions. Photomedicine and Laser Surgery. 2010, 28: 111-7.

3. Bonnier F. et al. Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy. Analyst. 2010 Dec;135(12):3169-77.

4. Swain RJ. et al. Assessment of cell line models of primary human cells by Raman spectral phenotyping. Biophys J. 2010 Apr 21;98(8):1703-11.

5. Keller MD et al. Detecting temporal and spatial effects of epithelial cancers with Raman spectroscopy. Dis Markers. 2008 ; 25(6): 323–337.

Keywords: oral cancer, Raman spectroscopy, optical biopsy


In-situ micromechanical testing of composite materials under scanning electron microscope

D.J. Mortell, D.A. Tanner and C.T. McCarthy

Mechanical, Aeronautical and Biomedical Engineering, University of Limerick

There is significant interest in composite materials for use in structures in the aviation industry and other high performance applications due to the high stiffness and low weight properties of the material. A Jeol JSM-5600 Scanning Electron Microscope (SEM) has been used, in conjunction with a Deben micromechanical tester, to determine failure characteristics in composite materials subjected to flexural loading. This system allows observation of failure events on the microscale in real-time. Load and displacement data is extracted during testing and, using the SEM video and still imaging capabilities, stresses associated with failure events can be determined. This testing technique has been used before in similar studies of human bone tissue [1], and investment cast TiAl Alloys [2]. The failure mechanisms that have been observed in composite materials with this system are microcrack initiation (Figure 1), transverse crack growth, delamination (Figure 2) and catastrophic failure of the specimens. The material characteristics investigated in current research are the transverse crack initiation loads in a composite material, and the lengths of the corresponding delaminations as a function of the applied flexural load. Image stitching techniques have been used in post processing of visual data from the SEM to produce images of significantly higher resolution than is capable by default with the Jeol JSM-5600 SEM. Using this technique a much greater area can be viewed in a single image after post processing. This method provides a better visual representation of the extent of damage within the composite material during mechanical testing. This imaging technology along with the micromechanical tester can be used to develop a greater understanding of the damage mechanisms which lead to catastrophic delamination in a composite material.

Figure 1: Microcrack growth Figure 2: Delamination resulting from

microcrack growth References:

1. Koester, K. J., Ager, J. W. & Ritchie, R. O. 2008. The true toughness of human cortical bone measured with realistically short cracks. Nature Materials, 7, 672-677.

2. Chen, Y.-F., Zheng, S.-Q., Tu, J.-P., Xiao, S.-L., Tian, J., Xu, L.-J. & Chen, Y.-Y. 2012. Fracture characteristics of notched investment cast TiAl alloy through in situ SEM observation. Transactions of Nonferrous Metals Society of China, 22, 2389-2394.

Keywords: SEM, micromechanical testing, composite materials


Using lectin and glycan microarrays to investigate the ‘cross-talk’ between host and commensal bacteria

Benoit Houeix, Marie LeBerre, Michelle Kilcoyne, Marian Kane and Lokesh Joshi

Glycoscience Group, National Centre for Biomedical Engineering Science (NCBES)

National University of Ireland, Galway

The human gastro-intestinal tract (GIT) is an elaborate ecosystem specific to an individual. It involves inter- and intra-relationships between the bacteria composing the microflora and between the host and the microbes. Commensal bacteria are the subject of increasing interest for their influence on host biology and commercial value as potential probiotic formulas. According to the WHO, probiotic bacteria are ‘live microorganisms which, when administered in adequate amounts, confer a health benefit on the host’. The idea of a communication system or a ‘crosstalk’ between the host and the microbiota has emerged after bacterial-derived molecules, such as polysaccharide A in Bacteroides fragilis, were shown to be essential for host immune system maturation (1). Probiotics are seen as temporary residents of the GIT but there is increasing interest in their adherence and colonisation of the gut and effector molecules they produce. Studies to date on adhesion structures, such as pili, and immunogenic molecules, such as polysaccharides, have been mainly concerned with human pathogens, as potential candidates for vaccines, rather than commensals. Although a highly specific system has been reported between the intestinal cell receptors (glycoproteins acting as pattern recognition receptors) and adhesins (glycan-binding proteins) of pathogenic bacteria, evidence is only beginning to emerge that similar structures of attachment exist between probiotic strains and their host cells with distinctive epitopes (2). Recent advances in technology such as glycomics databases, synthesis of glycoconjugates and microarrays have provided new tools to study lectin-glycan interactions. Here, we present lectin microarrays as a high-throughput method to profile surface carbohydrate structures of commensal strains and their EPS. We also demonstrate the potential of neoglycoconjugate arrays to detect adhesin ligands using two strains of E.coli producing two different types of pili clearly visible with TEM. References:

1. Mazmanian SK, Round JL, & Kasper DL (2008) A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453(7195):620-625.

2. Kline KA, Fälker S, Dahlberg S, Normark S, & Henriques-Normark B (2009) Bacterial Adhesins in Host-Microbe Interactions. Cell Host & Microbe 5(6):580-592.


Peptide modified nanostructured surfaces for single platelet capture

Kellie Adamson1, 2, Marc Devocelle2, Niamh Moran3, Robert J. Forster1, Tia E. Keyes1

1School of Chemical Sciences, Dublin City University, Dublin 9 2Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, Dublin 2

3Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2 Although the process of platelet activation is well-characterized biochemically, the affect of physical and spatial cues in the microenvironment on platelet adhesion and spreading remain unclear. Understanding and controlling platelet interaction with surfaces is important across a range of domains including diagnostics and medical implants. For example, platelet adhesion directly or through adsorbed protein on medical implants is important in dictating hemocompatibility as it can lead to thrombus formation. Whereas intact platelet capture in which the activation status of the platelet is controlled may be useful in a diagnostic device. This study exploited scanning electron and confocal fluorescence microscopy to examine the interactions of whole human platelets with highly ordered gold nanocavity arrays. We explored the effect of different cavity dimensions and surface functionality on platelet capture and activation status by comparing with influence of surface chemistry on platelet capture on planar gold surfaces. The arrays are prepared from microlithography via electrochemical deposition to produce gold coated planar and cavity surfaces. Monolayers of the αIIbβ3 integrin platelet protein sequence GRGDS were self-assembled at the surfaces to investigate the spatial guidance of platelet binding and spreading at the single cell level. Spatiotemperal adhesion control was investigated using the widely reported bio-inert alkane and poly(ethylene)glycol (PEG) platelet binding inhibitors at varying RGD/alkane and RGD/PEG ratios. The effect of cavity size on platelet binding and platelet morphology was also investigated, comparing gold planar surfaces approximately 100 nm thick to three different microcavity array sizes; 5 μm, 1.5 μm and 1 μm. In addition, the ability to selectively capture single intact platelets using these microcavity arrays was also investigated. Confocal fluorescence microscopy and scanning electron microscopy confirmed platelet adhesion and spreading for all gold surfaces. Platelet binding and spreading was found to be greatly influenced by the surface geometry with the platelets ability to bind and spread decreasing with decreasing microcavity size. For example, platelets bound and retained the ability to spread fully onto 5μm cavity arrays (Figure 1), a length consistent with that of maximally extended filopedia and also present granule secretion, a process not seen for the planar surfaces or smaller cavity sizes. CD62P selectin and phalloidin staining was carried out to study the activation status and actin localization across the varying surfaces. Using this staining technique the activation status and actin content of platelets was found to differ greatly depending on the surface chemistry and surface functionality. The ability to control platelet binding and spreading as presented in this study is of significant value biomedically and may be useful for example in the design of medical implantations.


Figure 1: Scanning electron microscopy images presenting (i) extended filopedia and (ii) fully spread platelets bound to RGD coated 5 μm microcavity array surfaces. 5kV accelerating voltage was used in all cases.


Optimisation of high content screening platform for label-free quantification of nanoparticle uptake and colocalization studies

Bogumiła Reidy, Sergio Anguissola, Iseult Lynch and Kenneth A. Dawson

Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin

In recent years, nanoparticles’ (NPs) interactions with cells, tissues and living organisms has gained a lot of attention, and many studies on this issue have been performed and published. The uptake of NPs by cells can be assessed using different imaging techniques, but the quantification of NP uptake, which is important for toxicological and other results interpretation (i.e dose-response), is often lacking. There are also methods which give an insight into sub-cellular localisation of internalised NPs, and their colocalization with specific organelles, but usually only limited number of cells can be analysed, and the particles must also be optically labelled. High Content Screening (HCS)/High Content Analysis (HCA) can be described as the process of extracting and understanding multi-parameter data from high-throughput subcellular imaging [1]. Comparing to the methods typically used in NP uptake and sub-cellular localization analysis (flow cytometry, confocal microscopy, transmission electron microscopy), HCS has several important advantages. Firstly, it is a high-throughput method, widely used for cytotoxicity and other studies both in industry and in academia [1-3]. The analysis is based on images, and provides both quantitative and qualitative data on different parameters, as well as their spatial distribution, simultaneously. As many samples can be acquired with exactly the same settings, the obtained data sets can be easily compared with each other. The method can also utilise internal reflectance allowing a much broader range of NPs to be assessed than by optical imaging methods. Depending on the staining approaches used, HCA can be further exploited to also provide insights into uptake mechanisms, impacts of NPs on morphology and metabolism etc. in a time and dose-dependent manner. Colocalization applications can be used to obtain relevant statistical parameters. In this study we established a reliable method of studying both fluorescent and metallic NPs uptake via the HCS platform and compare it with some well-established methods (mainly based on flow cytometry). Secondly, we compared the usability of the HCS platform for colocalization studies (colocalization of NPs with organelles) to the established method based on confocal microscopy and subsequent image analysis (which is widely used in NPs internalisation and colocalization studies). Keywords: nanoparticles, High Content Screening, uptake, colocalization, label-free nanoparticle quantification. References: 1. Thomas, N., High-content screening : A decade of evolution. J Biomol Screen 2010, 15, 1-9. 2. Jan, E., Byrne, S. J., Cuddihy, M., Davies, A. M., Volkov, Y., Gun’ko, Y. K., Kotov, N. A., High-content

screening as a universal tool for fingerprinting of cytotoxicity of nanoparticles. ACS Nano 2008, 2, 928-938. 3. Damoiseaux, R., George, S., Li, M., Pokhrel, S., Ji, Z., France, B., Xia, T., Suarez, E., Rallo, R., Mädler, L.,

Cohen, Y., Hoek, E. M. V., Nel, A., No time to lose—high throughput screening to assess nanomaterial safety. Nanoscale 2011, 3, 1345–1360.


b

Figure 1: Uptake of 80 nm silver NPs by A549 cells, assessed on HCS using reflected light. Raw image (a) and colocalization protocol applied (b).


Investigating crossing fibers in white matter using ex vivo human diffusion MRI and polarized light microscopy

Laurena Holleran

NUI, Galway


Poster Presentation Abstracts

1. Optimization of TK promoter sequence for fluorescence protein (FP) fusion studies specially Fluorescence Correlation Spectroscopy (FCS)

R. Ali, S. Ramadurai and H.P. Nasheuer

Systems Biology Ireland and Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.

Promoter region plays an important role in the expression of fluorescent protein (FP) fusions to investigate the subcellular localization and distribution of proteins. Over expression of FPs is problematic for highly sensitive imaging methods such as fluorescence correlation spectroscopy (FCS) and one of its variant fluorescence cross correlation spectroscopy (FcCS). Therefore, to overcome this problem, several deletion mutants of TK promoter were constructed (TKe, TKeΔSS, TKmTSC & TKm2ST) and introduced into the gateway (GW) system which enabled us to efficiently transfer DNA-fragments between plasmids of interest. The TK promoter relies primarily on “CCAAT”, “SP1” and “TATA” elements found farther upstream. Almost 2/3 of the promoter activity is maintained by a region of around 150 base pairs of sequence upstream of the transcription initiation region (1). Therefore, we used this region to design our deletion mutants. The developed TK mutants were inserted in both GFP- and mcherry-GW plasmids. These plasmids were further tested for expression in U2OS and HeLa CCL2 cell lines. Here we show that deletion mutants, TKmTSC and TKm2ST are most suitable for our FCS applications (fig. 1). In the next step, TK deletion mutants developed in this study will be transferred into vectors carrying genes of proteins of interest and fluorescent tagged protein expression will be analyzed after transfection using FCS.

Tkm2ST Figure 1: U2OS cells transfected with (a) GW-GFP-TKm2ST plasmid DNA and (b) GW-GFP-TKmTSC plasmid DNA. References:

1. Arcot, S.S., E.K. Flemington, and P.L. Deininger, The human thymidine kinase gene promoter. Deletion analysis and specific protein binding. J Biol Chem, 1989. 264(4): p. 2343-9.

2. Elson, E.L., 40 years of FCS: how it all began. Methods Enzymol, 2013. 518: p. 1-10. Keywords: FCS, TK promoter, GW System


2. The use of SEM cathode luminescence imaging to reveal inner structure in geological specimens

H. Bagshaw1, G. Kenny2 and Prof. B. Kamber2

1Centre for Microscopy and Analysis, University of Dublin, Trinity College, Dublin 2

2Department of Geology, University of Dublin, Trinity College, Dublin 2

In some samples, electron bombardment stimulates light emission via Cathode Luminescence (CL). In a non-metallic material, incident electrons excite valence band electrons into the normally empty conduction band. Relaxation of these energetic electrons may lead to the emission of photons.[1] Cathode Luminescence images of zircons often show details related to the growth process of the mineral which are not visible using SE and BSE imaging because CL emission is partially dependent on variations in composition (usually involving rare earth elements). CL images of zircon grains are used to characterise different types of zircon and also for selection of areas for further analysis, including U-Pb dating by Laser Ablation Inductively Coupled Plasma Spectrometry (LA-ICP-MS) studies. CL images show zoning effects in zircons which give clues about the history of the material. Each subsequent crystallisation, irradiation (and subsequent damage), then recrystallization leads to the formation of specific\distinct zones. Comparison to know samples using the ‘Atlas of Zircon Textures’ by Fernando Corfu[2] allows identification of zonal regions and growth processes in zircons. We will show examples of CL imaged zoned structures when compared to SE images and some compositional data taken from specific zones to show elemental variation in the same crystal due to this zonal growth methodology. References:

1. S. J. B. Reed, (2005) Electron Microprobe Analysis and Scanning Electron Microscopy in Geology, Cambridge University Press.

2. F. Corfu et al. (2003) ‘Atlas of Zircon Textures’. Reviews in Mineralogy and Geochemistry January 2003 v. 53 no. 1 p. 469-500

Keywords: SEM, Cathode Luminescence, Geology, Zircon.

Figure 1: Comparison of a Secondary Electron Image (a) and a Cathode Luminescence Image (b) of the same region. Cylindrical pit in centre of grain is result of LA-ICP-MS analysis (see text).


Figure 2: Cathode Luminescence image of a Zircon grain showing zonal growth patterns


3. Resist based helium ion lithography

Alan Bell

Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, Dublin 2,

Republic of Ireland Helium Ion Microscopy (HIM) is a recent addition to the charged particle microscope family. HIM works under the same principle as an SEM, with the critical difference being incident electrons replaced with helium ions. Owing to its atomically sharp source and the larger momentum of helium ions compared to electrons, a sub-nanometre probe size is realized. HIM offers many advantages over standard SEM, namely, better resolution (0.35 nm), higher secondary electron yield giving higher surface detail, the ability to control charging of insulating samples without the need for a sputtered conductive coating as well as the ability to sputter specimen atoms and modify the material. Results from Helium Ion lithography will be presented. A relatively new resist, SML, exhibits reduced forward scattering and an almost non-existent proximity effect. This allows for the fabrication of ultra-small and dense arrays of nano sized features. The increased secondary electron yield from Helium Ions, compared to electrons, results in increased sensitivity in the resists investigated. Negative tone HSQ data will also be presented.


4. Analysis of proinflammatory and anti-inflammatory macrophages within cell loaded scaffolds in a transection model of spinal cord injury

Niall Bergin (1), Nicolas Madigan (2, 3), Kerry Thompson (1), Timothy O’Brien (2), Anthony

Windebank (3), Siobhan McMahon (1)

(1) Discipline of Anatomy, School of Medicine, NUI Galway, (2) Regenerative Medicine Institute, NUI Galway

(3) Department of Neurology, Mayo Clinic, Rochester, MN, USA

Traumatic spinal cord injury results in complete tissue destruction and loss of neurologic function below the level of the lesion in 40% of patients in Ireland. The injury is characterized by a primary traumatic phase and a secondary inflammatory phase. Tissue engineering, using biodegradable polymer scaffolds loaded with different cell types, offers potential to rebuild neural tissue through the glial scar and to reestablish functional connections. Much remains to be elucidated about the complex interactions of the immune system before we can expect successful modulation of cells in the inflammatory phase following spinal cord injury. In this study we determined the expression of macrophages within a polymer scaffold comprising seven channels, which were loaded with matrigel, mesenchymal stem cells (MSCs) or schwann cells. Scaffolds were implanted within transected rat spinal cord and harvested after four weeks. We hypothesised that there would be a difference in the pattern of proinflammatory and anti-inflammatory cells present in MSC and schwann cell loaded OPF scaffolds in comparison to control (matrigel) scaffolds. Immunohistochemical staining for macrophages was carried out on paraffin embedded sections using the pro-inflammatory (M1) marker CD86 and the anti-inflammatory (M2) marker CD206. Fluorescence microscopy and Image J analysis were carried out. There were significant results for comparisons between matrigel and schwann cell treated groups for M2 staining but no significant findings emerged in comparisons of M1 staining between the groups. The schwann cell treated group showed significantly lower amounts of M2 staining compared to M1. It was also observed that there was a degree of co-localisation between MSCs and macrophages within channels. Macrophage infiltration was seen within every animal group, at varying intensity. Given the tendency towards pro-inflammatory effects of macrophages at 4 weeks post-injury, our results suggest increased inflammation associated with cell loaded scaffolds at 4 weeks post-injury.


5. The role of centrosome clustering in survival of breast cancer cells

Danielle Gaffney and Emer Bourke

Discipline of Pathology, Clinical Sciences Institute, National University of Ireland Galway, Galway, Ireland. Centrosomes are the principle microtubule organising centres in somatic cells. Amplification of centrosome number (≥3) is observed in genetically unstable cells such as tumour cells, cells with mutations in DNA repair/checkpoint genes as well as cells exposed to DNA damage. Centrosome amplification is thought to be a mechanism evolved by the cell to drive excessively damaged cells into mitotic catastrophe and death. However, despite a high incidence of amplified centrosomes, cancer cells continue to divide unimpeded, indicating the activation of mechanisms which evade death by mitotic catastrophe. One such mechanism is centrosome clustering whereby assembly of multiple centrosomes into two poles permits formation of a normal bipolar spindle. In the current study, we examined a range breast cancer cell lines representing various intrinsic molecular subtypes (luminal and basal) for prevalence of centrosome amplification and centrosome clustering in absence and presence of irradiation---induced DNA damage. Basal levels of centrosome amplification were elevated in all breast cancer lines compared to non---transformed breast epithelial control line, MCF10A and were seen to increase in all cell lines following DNA damage induced by γ---irradiation. The extent of amplification correlated with tumour grade, with HCC1937 cells (representing the highest grade, basal subtype) showing the highest percentage of cells with abnormal centrosome number (± irradiation). Integrin Linked Kinase (ILK) has previously been shown to be involved in the process of clustering, suppression of mitotic catastrophe in cells with multiple centrosomes and promotion of cancer cell survival. To examine the role of clustering in breast cancer cell division, clustering was blocked using ILK inhibitor, QLT0267, and immunofluorescent microscopy used to examine the incidence of cells entering mitosis with more than two poles. An increased percentage of multipolar mitotics in all breast cancer cell lines compared to MCF10A in response to ILK inhibition suggests that the mechanism of centrosome clustering is employed in human breast cancer cells to allow cells with multiple centrosomes to complete mitosis in a bipolar manner.


6. Vascular Tissue Engineering: Endothelial Cell-Mediated Effects on Extracellular Matrix Synthesis by Arterial Smooth Muscle Cells in Culture

Brophy M.J., Flanagan T.C.

School of Medicine and Medical Science, University College Dublin, Dublin, Ireland

The mechanical strength of tissue-engineered vascular grafts has been promoted by methods that enhance extracellular matrix (ECM) synthesis by seeded cells, including bioreactor conditioning/growth factor supplementation. Endothelial cells are an important component of such grafts due to their anti-thrombogenic properties, but there are conflicting data in the literature concerning their effect on ECM synthesis by arterial smooth muscle cells (SMCs). The present study aims to determine whether or not an overlying layer of endothelial cells, and/or their secretions, enhances or inhibits ECM production by co-cultured SMCs, which may determine an optimal time-point for graft endothelialisation. For 2-D analysis, commercially-available human umbilical artery SMCs (HUASMCs) were cultured either alone, in direct contact within human umbilical artery endothelial cells (HUAECs), or exposed to secretions from HUAECs using co-culture well inserts. For preliminary 3-D analysis, HUASMCs were embedded within fibrin gels and were cultured either alone, with a surface monolayer of HUAECs, or in HUAEC-conditioned medium. All cultures were maintained for 12 days and ECM development (type-I collagen, elastin, fibronectin) was analysed using immunofluorescence microscopy. In addition, collagen and elastin production was quantified using spectrophotometric assay. In 2-D culture, it was observed that HUASMCs and HUAECs in direct cell contact produced an increased amount of elastin (~7.6%) when compared to HUASMCs cultured alone, while collagen production was significantly reduced (~29%). These results were corroborated by immunofluorescence microscopy. Preliminary results from 3-D fibrin gel culture showed a 26.8% increase in elastin production in the group containing HUASMCs and a surface monolayer of HUAECs compared with HUASMCs cultured alone, while collagen production in 3-D gel constructs was also significantly reduced in this group (~39%), mirroring the 2-D results. Strikingly, a clear potent relationship between both cell types was observed, with significant contraction of gels in this group alone to a fraction of their size after 3 days in culture. The results of the present study indicate that seeding of ECs onto fibrin-based vascular grafts should be completed toward the end of the in vitro conditioning period to allow for adequate deposition of collagen and elastin proteins by seeded SMCs and to prevent premature tissue contraction. Acknowledgement: This project was funded by the National Children’s Research Centre. MJB also acknowledges funding through a HRB Summer Student Scholarship.


7. CoCu Nanoalloys: from discs to rings

Yanhui Chen†, Felim Vajda†, Robert O’Conell†, Jiandi Lin‡, Wolfgang Schmitt‡ and Hongzhou Zhang†

†School of Physics and Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity

College Dublin, Dublin 2, Republic of Ireland ‡School of Chemistry, Trinity College Dublin, Dublin 2, Republic of Ireland

Cobalt copper composites have been widely used for energy storage systems, electrochromic thin films, magneto resistive devices and heterogeneous catalysis1. The properties of alloys vary with the concentrations of the constituent elements; At the nanoscale the morphology of the nanoalloys may affect their properties dramatically due to the high surface to volume ratio and its high sensitivity to the morphology2. In this work we aim to control the morphology of Co-Cu nanoalloys via a hyderthmal method and study their formation mechanisms. For a typical synthesis cobalt and copper ions with different ratios of concentration were mixed and then dissolved in deionized water. A small amount of N2H4·H2O and a moderate amount of NaOH were added to the solutions in sequence. This solution was then heated at a temperature of 105oC - 175oC for nine hours in a sealed Teflon-lined autoclave. After heating the precipitate was collected from the inner wall of the autoclave and the product was then washed by distilled water. Characterization was carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1(a) shows the SEM image of CoxCu1-x(OH)2 nanodisc prepared at room temperature (i.e., 20oC). After reaction in the autoclave for nine hours CoCu alloy nanorings are formed, as shown in Fig. 1b. Figure 1(c) shows the composition variation before and after reaction. It indicates an increase of copper when the shape of the nanoalloy changes from discs to rings. In conclusion, crystalline CoxCu1-x(OH)2 nanoring and nanodisc were synthesized by using a hyderthermal method. A stress corrosive process was proposed to explain this shape transformation.

Figure 1: SEM images of (a) nanodiscs formed in room temperature, (b) nanorings formed in 175oC; and (c) composition variation between discs and rings, the insets are High Angle Annular Dark Field images of the samples.


Keywords: CoCu alloys; nanorings; morphology control, Electron Microscopy References:

1. N. M. Deraz; et al. nt. J. Electrochem. Sci., 2013, 8,2682 2. B.Varghese et al, Advanced Functional Materials, 2007, 17, 1932

Acknowledgements: The work is supported by Science Foundation Ireland under Grant 07/SK/I1220a.


8. Localisation, quantification and theragnostic properties of bioconjugated nanosensitisers in an in vitro cancer cell model

Jennifer Connolly1, Hai Ming Fan1, Vijay Raghavan1, Peter Dockery2, Kerry Thompson2,

Malini Olivo1

1Nanobiophotonics and Imaging group, National University of Ireland Galway, Galway, Ireland 2Anatomy, National University of Ireland Galway, Galway, Ireland

The development of bioconjugated nanosensitisers that simultaneously facilitate cancer therapy and diagnosis (theragnosis) is an area of considerable current interest. Here we report the use of novel nanogold based photosensitisers (nanosensitisers) as optical diagnostic probes and as potential photodynamic and photothermal cancer therapeutic agents using an in vitro cancer cell model. Such multi-modal nanosensitisers were constructed by layering photosensitisers (Hypericin, Chlorin e6) onto multi-branched gold nanoparticles. Nanosensitisers were also conjugated to tumour-specific antibodies to confer cancer-cell specificity. A number of cell lines were cultured in vitro with various concentrations of nanosensitisers. Cellular uptake and intracellular localisation of nanosensitisers was assessed using confocal fluorescence microscopy and transmission electron microscopy. In order to assess the photodynamic therapeutic potential of the nanosensitisers, treated cells were exposed to light of appropriate wavelength and fluence, and cytotoxicity was examined using the 3-(4,5- dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. It was observed that nanosensitisers were taken up by cells and internalized within a 24-hour period. While the intracellular target of nanosensitisers appeared to be cell and photosensitiser- type-dependent, it was found to localise primarily to intracellular organelles. Nanosensitiser cytotoxicity was observed at high treatment levels (>5 μM; p<0.05). Significant photodynamic- mediated cell death was observed in the nanomolar concentration range (p<0.05). While preliminary, results from this study suggest biocompatibility of these novel gold nanosensitisers in vitro, and highlight the potential of such novel nanosensitisers as multifunctional optical agents for cancer diagnosis and therapy.


9. The role of C-Nap1 in centrosome duplication and DNA damage

Anne-Marie Flanagan and Ciaran Morrison

Centre for Chromosome Biology, National University of Ireland, Galway

C-Nap1 is a linker protein that acts as a tether the two developing centrosomes in S phase and the mature centrioles in G1 (1). The phosphorylation of Nek2 permits the dissociation of this linker at the onset of M phase (2). We wish to investigate the activities of C-Nap1 throughout the cell cycle. We are also interested in the impact of DNA damage on the centriolar linker, with particular emphasis on C-Nap1. To explore this question, we are targeting C-Nap1 in the hyper-recombinogenic chicken DT40 cell line. We are knocking in a C-terminal tag; an Auxin Inducible Degron (AID) (3) linked to GFP. We have generated a homozygous mutant in DT40 chicken cells, which expresses the double tagged form of the protein. The AID tag should allow the selective degradation (via the proteasome) of C-Nap1, facilitating the investigation of the role of the protein in the centrosome. Immunofluorescence microscopy experiments, using the attached GFP tag, show the mutant causes no ill effects on cell viability, the protein localises to the centrosome and still functions correctly though mitosis.

References:

1. S. Bahe, Y.D. Stierhof, C.J. Wilkinson, F. Leiss, E.A. Nigg. Rootletin forms centriole-associated filaments and functions in centrosome cohesion. J. Cell Biol., 171(2005), pp. 27 - 33.

2. A.M. Fry, T. Mayor, P. Meraldi, Y.D. Stierhof, K. Tanaka, E.A. Nigg. C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2. J. Cell Biol., 141(1998), pp. 1563 - 1574.

3. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods., 12(2009), pp. 917 – 922.


10. Histological and molecular observations of the vascular development on the chick embryos after cadmium exposure

Anna Kaskova---Gheorghescu and Jennifer Thompson

School of Medicine and Medical Science, University College Dublin, Dublin,

Ireland Cadmium (Cd) is teratogenic in chick embryos following treatment in shell---less culture. Is has been noted previously that chicks were vulnerable to Cd teratogenesis between Hamburger---Hamilton (H---H) stages 13 and 18. The vascular endothelium is a primary target of Cd toxicity at a variety of molecular levels, including cell adhesion molecules, metal ion transporters and protein kinase signaling pathways resulting in abnormal angiogenesis. The purpose of this study was to examine vascular development following Cd treatment in chick embryo and in the extra---embryonic membrane. Embryos incubated for 48 hours to H---H 13 were explanted according to Dugan’s method and treated with 50 µl of 50 µmol CdAc. 50 µl of equimolar sodium acetate was given to controls. Test solutions were dropped onto the blastodisc using a micropipette and entered the embryo by a process of diffusion. Following treatment, embryos were returned to the incubator. At 8 hours after treatment, embryos were dissected and fixed in 4% paraformaldehyde---PBS, dehydrated through ascending concentrations of ethanol, cleared in xylene, impregnated with molten wax for 6 hours, then orientated and blocked out in paraffin. Transverse sections were cut at 3 µ using the rotary microtome, then mounted on polysine slides for hematoxylin and eosin (H&E) staining. All of the Cd---treated embryos had stunted growth when compared to the control embryos (weight Cd---group=0.01g±0.003 vs. CTRL---group=0.02g±0.008; p<0.001 at 48+8 hours). Chick extra---embryonic membranes exposed to Cd exhibit defects in vascular development. Vascular network and branching pattern of the omphalo---mesenteric vessels in Cd exposed embryos were not as intricate as that of control embryos. Further observations revealed reduced and delayed vascular development in Cd treated embryos. Controls showed normaldevelopment with ramified vascular network with numerous capillary branches emerging from segmental vessels present in the extra---embryonic membrane. Our results demonstrate that Cd exposure clearly impairs vascular development. Cd treated embryos revealed delayed growth with abnormal vascular development embryonically and extra---embryonically.


11. Microscopy analysis of nickel superalloy joint microstructures and high--- temperature braze flowability using different braze compositions

Eoin Hinchy

Design and Manufacturing Technology, University of Limerick

Nickel superalloy turbine components are extensively used in the hot section of aero and land based gas turbine engines. Non---uniform cooling of turbine components causes thermal stresses in the alloy, which result in thermal fatigue cracking during service life. Cracks greater than 250µm in width are healed using a two---part braze mixture. A high---melt superalloy powder is mixed with a low--- melt braze powder. The low---melt material contains boron which, acts as a melting point depressant (MPD) – lowering the melting point of the braze below that of the substrate. The brazed component is heated in a vacuum furnace above 1200˚C where the presence of the MPD causes the braze to melt. High temperature diffusion of boron MPD from the braze into the substrate causes the melting temperature of the liquid braze to increase above the bonding temperature – resulting in isothermal solidification. Excessive boron within the joint however can form detrimental brittle boride secondary phases, drastically weakening the joint. It is imperative that the braze completely fills cracks, while also producing a joint which is free from brittle boride phases. This study uses a novel technique to study the effects of high---melt to low---melt ratio on high temperature braze flowability and the formation of brittle boride eutectic phases. Three separate ratios of high---melt superalloy to low---melt braze material were examined using scanning electron microscopy and backscattered electron detection. Backscattered electron imaging has the ability to clearly highlight boride phases within the nickel matrix. The ratios of high---melt to low---melt used were 80:20, 50:50 and 20:80. The excessive high---melt sample showed very poor melting and joint filling, with the least number of detrimental secondary phases. The 50:50 sample showed good filling with the formation of more secondary phases within the braze material. The excessive low---melt sample showed excellent filling, with the disadvantage of a very high concentration of boride phases in the braze material and diffusion zone.


12. An investigation of digital holographic microscopy

Ellen Keenan and Nicholas Devaney

School of Physics, NUI, Galway In Digital Holographic microscopy (DHM) in transmission mode, the interference pattern between light which has propagated through the sample and a coherent reference beam is recorded on a digital detector. Typically, a laser is focused onto a pinhole and then propagated through the sample and onto a detector. The image can be subsequently back-propagated numerically to reconstruct the sample in 3D. We have built a simple DHM setup in order to investigate its performance as a function of different experimental parameters including the pinhole size, distance between the pinhole and sample and distance between sample and detector. We back-propagate assuming Fresnel diffraction, and use a sharpness measure to detect the distance at which the sharpest images are reconstructed. We have tried applying this to image regions and stitching the sharpest reconstructions together. For thin samples this can give an ‘all-in-focus’ image. For thick samples we are able to reconstruct 3D representations of the sample using the reconstructed images over a range of propagation distances. We have used a USAF resolution target for the tests, and by optimizing the parameters we are able to resolve 228 cycles per mm. We have also examined some simple biological specimens.

Figure 1: Hologram of USAF resolution Test Target imaged using the 5 μm pinhole (left) and reconstructed image (right).

Figure 2: 2 3D representation of a housefly leg.


13. Modification of ZnO nanowires using a focused helium ion beam

Abbas Khalid, Yanhui Chen, Daniel Fox, Yangbo Zhou and Hongzhou Zhang

School of Physics and CRANN, Trinity College Dublin, Dublin 2, Rep. of Ireland We report the modification of Zinc Oxide nanowires using the helium-ion microscope. Zinc oxide (ZnO) nanowires were grown by a vapour-liquid-solid process. A 30-keV focused helium (He+) ion beam irradiated the nanowires with varying doses in the range of 1013 ions/cm2 to 1016 ions/cm2. The morphology of the irradiated nanowires were characterised by using Scanning Electron Microscopy (SEM), Helium Ion Microscopy (HIM), and Transmission Electron microscopy (TEM). The shape of the nanowire remained unchanged at a dose up to 1016 ions/cm2 in SEM/HIM images, while crystal defects induced by the irradiation can be identified in TEM. The effects of the modification were further investigated by using photoluminescence (PL) and cathodoluminescence (CL). Room temperature PL spectra of as-grown ZnO nanowires are dominated by near band edge (NBE) emission and a broad green band, while the He+ irradiated nanowires showed a significant decrease in the NBE emission and suppression of the green band as well. The decrease in NBE emission was confirmed in spatially-resolved CL measurement, and a blue shift of approximately 9 nm in green band of the irradiated ZnO nanowire was identified. The origin of the blue shift needs however further investigation [1]. Our results demonstrate that we can perform nanoscaled modification on a nanostructure using the HIM and the structural modification can be analysed in a TEM. The development of an in-situ CL system allows spatially-resolved optical measurements and can be taken concurrently with secondary electron images, each highlighting different information in the sample analysing the potential for introducing defects by He+

radiation.

References:

1. Fan H.J., BertramF., DadgarA., ChristenJ., Krost A. and Zacharias M. Self-assembly of ZnO nanowires and the spatial resolved characterization of their luminescence. Nanotech., 15,1401–1404(2004).

Keywords: Zinc Oxide Nanowire, Helium ion microscope, Cathodoluminescence, Transmission electron microscope, Photoluminescence, defects.


14. Application of macromolecular crowding in cell sheet technology for the development of human corneal fibroblast cell sheets

Pramod Kumar1, Abhigyan Satyam1, Xingliang Fan1, Estelle Collin1, Brian Rodriguez2, Yury Rochev1, Michael Raghunath3, Simon Dillon4, Abhay Pandit1 and Dimitrios Zeugolis1

1Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland,

2Conway Institute of Bimolecular & Biomedical Research, University College of Dublin, Dublin, Ireland, 3Tissue Modulation Laboratory, National University of Singapore, Singapore,

4BIDMC Proteomics Canter, Harvard Medical School, Boston, USA

Despite efficacious in-vitro and in-vivo results in cell based therapy, very few products have been commercialised. This limited translation from laboratory to clinic has been attributed to the prolonged culture time required to develop an implantable tissue-like structure1. For example, cultured primary human corneal fibroblasts (HCFs) need 35-84 days in culture for the development of a thinner than the native tissue implantable material. Recent studies have shown that, macromolecular crowding (MMC), the addition of macromolecules to culture media, not only enhances deposition of extracellular matrix (ECM), but also facilitates cell phenotype maintenance2,3. Herein, we evaluate the influence of MMC on HCFs culture and ECM deposition.

HCFs cultured under MMC (100μg/ml dextran sulphate (DxS); 37.5mg/ml Ficoll™ (Fc) 70 and 25mg/ml (Fc) 400; and 75μg/ml Carrageenan (CR)). ECM deposition was analysed by SDS-PAGE, immunocytochemistry and proteomic analysis. RT-PCR used to assess changes in gene expression. An intact HCFs cell sheet was developed using NIPAAM/n-tert butyl acrylamide thermal responsive polymer. The ECM rich cell sheet was characterized for transparency, ECM characterization and cell sheet detachment.

Densitometric analysis of SDS-PAGE (Figure 1) shows that MMC significantly increased collagen I deposition (p<0.05). No change in collagen deposition was observed among the various crowders at day 6. ICC analysis confirmed the enhanced deposition of various collagens. Fibronectin also expressed in fibrillar pattern (Figure 2). The deposited matrix appears to be highly ordered. Proteomic and genomics analysis also confirmed the increase in total ECM proteins and phenotype maintenance (results not shown). Finally ECM rich transparent HCFs cell sheets (Figure 3) were developed using thermal responsive polymers.

MMC significantly accelerates corneal specific ECM deposition, whilst maintaining cell phenotype. Thermal responsive co-polymer allowed the production of transparent HCFs cell sheets.

References:

1. Proulx S. et al., Mol.Vis., (2010),16:2192-201

2. Lareu R. et al., FEBS Lett., (2007),581:2709-14

3. Zeugolis D. et al., Drug Dis. Today (2013)

Keywords: Macromolecular crowding, Human corneal fibroblasts, Extracellular matrix, Cell sheet technology


Acknowledgements: The authors would like to thank the College of Engineering and Informatics, NUI, Galway, SFI (Grant-07/IN1/B031 & 09/ RFP/ENM2483) and SFI- ETS Walton award for financial support.

Figure 1: Figure 2: Figure 3:


15. Far-field absorption nano-spectroscopy

Mahendar Kumbham1, Ning Liu1, Nordine Hendaoui2, Isabel Pita1, Syed A. M. Tofail1, André Peremans2 and Christophe Silien1

1Department of Physics and Energy, and Materials and Surface Science Institute, University of Limerick, Ireland

2Centre de Recherche en Physique de la Matière et des Rayonnements, Facultés Universitaires Notre Dame de la Paix, Namur, Belgium

Absorption micro-spectroscopy in the mid-infrared (mid-IR) is of growing interest for spatially resolved chemical analysis of materials and of biological samples, for the development of new materials, for failure analysis in industrial processes, and for medical diagnostics. Far-field imaging affords three-dimensional visualization and observation of buried features but the spatial resolution remains limited by diffraction at the very best to a dimension equivalent to half the wavelength used. We are investigating a novel scheme of far-field absorption nano-spectroscopy based on a differential pump-probe operation where the pump spatial profile is modulated between a vortex and a Gaussian shape. Sub-diffraction resolution of the probe absorption at the center of the vortex is possible when the pump energy is set above the saturation threshold of the probed transition. We have studied the scheme from a theoretical point of view to highlight reasonable expectations in terms of mid-IR compatible optical components, point-spread functions, spatial resolution, signal sensitivity, influence of pump-probe delay, and sample damage threshold [1, 2]. Our study shows that probing the absorption of CH stretch modes at 3.5 μm is possible at a resolution of ca. 250 nm. The super-resolution scheme is currently developed in our laboratory on the basis of a visible to mid-IR tunable optical parametric amplifier and home-built confocal microscope. We will discuss our theoretical findings and the most recent advances with the experimental demonstration of this novel nano-spectroscopy scheme. References: 1. Silien, C., Liu, N., Hendaoui, N., Tofail, S. A., & Peremans, A. (2012). A framework for far-field infrared

absorption microscopy beyond the diffraction limit. Optics express, 20(28), 29694-29704.

2. Isabel, P., Hendaoui, N., Liu, N., Kumbham, M., Tofail, S. A., Peremans, A., and Silien, C. (2013). High resolution imaging with differential infrared absorption micro-spectroscopy. Accepted for publication in Optics Express.

Keywords: micro-spectroscopy, diffraction limit, sub-diffraction, far-field, nanoscale imaging


16. Reorganisation of the cytoskeleton after exposure to natural and synthetic hormonal

compounds

Emma Mc Mahon1, Kerry Thompson1, 2, Peter Dockery1, 2

1Anatomy, NUI Galway, Ireland. 2Centre for Microscopy and Imaging, NUI Galway, Ireland

In recent years documented incidences of animal and human exposure to certain compounds known as endocrine disrupting chemicals (EDCs) have been related to a rise in developmental and reproductive problems. EDCs such as Bisphenol A (BPA), a plasticiser and synthetic estrogen, and Genistein, a phytoestrogen derived from soya beans, can act by mimicking the activity of naturally occurring estrogen which can lead to the irregular activation of genomic or non-genomic cellular signalling pathways. Estrogens have been identified in playing a role in influencing the signalling pathways that control cytoskeletal reorganisation. As a consequence of this reorganisation, this may then lead to alterations in cell morphology, structure and function. The aim of this study was to examine the effects of the above EDCs on the reorganisation of the actin and microtubule cytoskeleton in the endometrial epithelial KLE cell line, using various microscopy and image analysis techniques. Initially, cell viability (live/dead assay) and cell migration (time-lapse microscopy) were investigated to assess whether these EDC treatments were harming the cells. Immunofluorescence staining and confocal microscopy were next carried out to assess alterations in cytoskeletal content throughout the depth of the cell population under observation. Experiments used 3 concentrations of BPA and Genistein (1nM, 10nM & 100nM) along with appropriate controls (n=5 per treatment group). The anti-estrogen fulvestrant (1μM &1nM concentrations), was also investigated to assess whether any other non-classical pathways were being employed (n=5 per treatment group). Statistical analysis of the data revealed significant differences in overall area of actin filaments after treatment with 1nM BPA and 1nM and 10nM Genistein. No significant differences were recorded in total area or area per cell of microtubules after treatments and inhibitors. Finally cells cultured on coverslips were processed for Scanning Electron Microscopy (SEM) and cytoskeletal components were isolated and exposed to allow the visualization of the delicate fine structure.


17. A biosensor to visualise centrosomal separase activity

Lisa Mullee and Ciaran Morrison

Centre for Chromosome Biology, National University of Ireland, Galway The cell cycle describes the series of events by which cells duplicate their DNA and divide for growth or renewal. This vital process is tightly regulated in order to detect and repair DNA damage, thus ensuring cell division can proceed. The caspase-like protease plays a major role in the appropriate segregation of DNA prior to cell division. Recent evidence suggests that separase activity is altered in response to DNA damage and that it plays a role in the duplication of the centrosome, the primary microtubule organising centre (MTOC) of animal cells. It has been proposed that separase cleaves centriolar cohesion during mitosis, licensing the centrosome for duplication in the next cell cycle. We wish to define the timing, location, and rate of separase protease activity during the cell cycle and after DNA damage. To achieve this, we have developed a separase activity biosensor to analyse centrosome-localised separase function in vivo (Fig. 1). This biosensor is based on the physiological separase substrate, Scc1. Scc1 is a subunit of the cohesin complex which encircles the duplicated chromosones, preventing sister chromatid segregation prior to anaphase. The intact biosensor is composed of an Scc1 sequence sandwiched between a green fluorescent protein (GFP) and a red fluorescent protein (mCherry) that is anchored to a localizing protein tag. In the presence of active separase, the biosensor is cleaved, which can be detected by fluorescence microscopy. As localisation tags, we have used Centrin4, Chibby (both centrosome-localising) and Histone H2B (chromatin-localising). We have analysed cell cycle progression and DNA damage responses in stably-transfected cell lines that express different variants of our biosensor. Preliminary analyses demonstrate the cleavage of the chromatin-localised biosensor during anaphase and after DNA damage. We are now focusing on the regulation of this cleavage, with the goal of testing the roles of separase after DNA damage.

Figure 1: Schematic of the centrosome-localized biosensor in the presence of active separase


18. Understanding carbon contamination in a helium ion microscope

Robert O’ Connell, Yanhui Chen, Yangbo Zhou, Daniel Fox, and Hongzhou Zhang

School of Physics and CRANN, Trinity College Dublin, Dublin 2, Ireland The secondary electron (SE) dopant mapping in the Helium-ion microscope (HIM) is plausible for addressing the metrology challenge in the miniaturisation of semiconductor devices[1,2], while it can be dramatically modified by contamination induced during the imaging. Understanding the generation and the effect of the contamination is of importance. Little work has been done in the HIM, but extensive knowledge has been attained in the scanning electron microscope (SEM), a similar charged-particle microscope. In this work, we aim to understand the mechanism of contamination formation in HIM and its impact on the dopant contrast by comparing with that established in SEM. The contamination was built on the surface of a polished silicon wafer in a FEI SEM working at 1.6 kV and a 30 kV HIM respectively. Arrays of squares (5 µm, 6 µm) were irradiated with varying beam doses in the two microscopes. SE images of these contamination regions were then acquired in the SEM with a 5-keV beam. The height of the contamination was measured by using an Atomic Force Microscope (AFM). SEM images of the contamination built in the HIM and SEM are shown as the insets in Fig. 1 (a) and (b) respectively. The electron-induced contaminated regions (inset in Fig 1(b)) are all darker than the ‘clean’ silicon surface, while some He+-induced contaminated regions (inset in Fig 1(a)) are brighter than the background. The abnormal bright regions in the HIM-irradiated area (Fig 1 (a) indicated by the arrows) maybe due to the topographical modification induced by excessive He+ ion injection and the swelling of the substrate [3].

Figure 1: Contamination height growth profiles and resulting SE contrasts are indicated above for HIM (a) and SEM (b). Inset SEM Images of contaminated regions formed in HIM (a) and SEM (b). The SEM contrast and the height of the contaminated regions are drawn against the dose in Fig.1. It can be seen that a small number of scans (~20) in SEM produced a thin layer of contamination (c.a. 1 nm), which is efficient in blocking the SE emission. The contamination height grew almost linearly with the dose. The height of the HIM induced increases with the scan numbers initially, drops at 60 scans, and increased


again at 130 scans. The final increase is most likely due to the swelling of the substrate, corresponding to the abnormal brightness observed in the SEM image. In summary, the formation of contamination in HIM and SEM has been investigated by measuring the height of the contaminated area as a function of the irradiation dose and analysing the SE contrast of the contaminated region. In SEM, the larger the electron dose the higher the contamination and the larger the contrast. The contrast saturates at higher doses. In HIM, the contrast and the height seem not correlate with the dose monotonically, and this might be due to the sputtering and the substrate swelling induced by the heavier helium ions. References:

1. M. A. E. Jepson, B. J. Inkson, C. Rodenburg, and D. C. Bell, EPL 85 46001 (2009).

2. M. A. E. Jepson, et al, Microscopy and Microanalysis, 2011. 17(4):p637-642.

3. R Livengood, et al, Vacuum Society, [DOI:10.1116/1.3237101]


19. Protein-protein interaction in living cell using fluorescence cross-correlation spectroscopy

Sivaramakrishnan Ramadurai, Chiara Saladino, Jens Rauch, Walter Kolch and Heinz Peter

Nasheuer

School of Natural Sciences and System Biology Ireland, National University of Ireland Galway, Galway;

Conway Institute for Biomedical sciences, University College Dublin, Dublin, Ireland

Quantifying interactions and dynamics of biomolecules are crucial for understanding important biomolecular interactions in living systems. Recent developments in fluorescent-based imaging assays will allow us to study the molecular interaction in a biological pathway at single molecule resolution. Fluorescence microscopy and spectroscopy are often used in systems biology to study temporal and the spatial distributions of biological processes throughout an intact living cell. Fluorescence correlation spectroscopy [FCS] is a fluorescent-based single molecule technique which measures fluorescent fluctuations in a tiny observation volume, defined by confocal optics. Correlating fluctuations signals provides quantitative data on both mobility and number of molecules in the observation volume. Fluorescence cross correlation spectroscopy [FCCS] is an extension of the FCS principle and measures interactions or dynamic co-localization of two molecules, by spatiotemporal coincidence of their fluorescence signals [1]. When distinctly labeled molecules interact to each other, they synchronously diffuse through the observation volume and induce a cross-correlation signal, the amplitude of which gives information on the level of interaction or dynamic co-localization. FCCS, quantitative imaging technique, will be used to study the interaction of proteins in the Mitogen-activated protein kinase pathway, a key pathway involved in cancer development [2]. This approach can potentially accelerate the achievement of a systems level understanding of biological complexity. The integration of FCCS and modelling that can enable new advances in oncology and other fields in the biomedical sciences. References:

1. Bacia K. And Schwille P. (2003) A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy, Methods, 29: 74—85.

2. Matallanas D.,Birtwistle M., Romano D., Zebisch A., Rauch J., van Kriegsheim A, Kolch W. (2011) Raf family kinases: old dogs have learned new tricks, Genes Cancer, 2(3): 232—60.

Keywords: Fluorescence (cross) correlation spectroscopy (FCCS)


20. An in vivo FRAP assay reveals distinct roles for intracellular transport and disease modules in regulating diffusion of Joubert Syndrome-associated ARL-13 at ciliary

membranes

Anna A.W.M. Sanders and Oliver E. Blacque

School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland

Cilia are evolutionarily conserved microtubule based organelles extending from the surface of most cells serving important sensory and signalling functions. Defects in cilia cause a variety of disorders with overlapping phenotypes, termed ciliopathies. Because cilia house many different channels, receptors, and signalling molecules, great efforts are underway to understand how proteins are targeted to and compartmentalised within cilia. One important pathway is intraflagellar transport (IFT), a bidirectional motility along ciliary axonemes required for ciliogenesis and cilia maintenance. Also important is a specialised region at the base of cilia called the transition zone (TZ), which acts as a physical and molecular barrier to protein and vesicle transport. Numerous Meckel-Gruber syndrome (MKS), Joubert syndrome (JS), and Nephronophthisis (NPHP) ciliopathy proteins localise at the TZ and are thought to regulate barrier functions. In our lab we investigate the molecular basis of ciliary protein transport in sensory cilia of the nematode, Caenorhabditis elegans. One on-going project focuses on how Joubert Syndrome ARL-13/ARL13B is restricted to cilia and recently we found that in IFT and MKS/NPHP gene mutants, ARL-13 abnormally accumulates at the periciliary membrane immediately beneath the TZ. However, it was unclear if these accumulations were due to a leaky TZ diffusion barrier or because of defects in active transport mechanisms that drive ciliary entry and/or retention. To address these questions, we developed an in vivo fluorescence recovery after photobleaching (FRAP) assay to examine ARL-13 exchange kinetics between ciliary and periciliary pools in various loss-of-function mutant worms. Here we report that in worms with disrupted MKS/NPHP or IFT genes, ARL-13 diffuses between the ciliary and periciliary compartments. ARL-13 recovers faster and to a greater degree in MKS/NPHP mutants than in IFT mutants. We also found that IFT genes limit ARL-13 diffusion at ciliary membranes. Our findings suggest that MKS/NPHP and IFT genes are differentially required for establishing a transition barrier to ARL-13 diffusion; IFT genes limit ARL-13 diffusion at the ciliary membrane and may perform active transport roles in regulating ciliary entry and/or retention of ARL-13.

Keywords: cilia, ciliopathies, FRAP


21. Elucidating the cardioprotective role of SPARCL1 in ex vivo model of myocardial infarction

Ismail Siti, E. Farrell, M. Creane, M. Harte, M. Murphy, T. O’Brien, F. Barry

Regenerative Medicine (REMEDI), National University of Ireland in Galway (NUIG), Galway, Ireland

There is great interest in mesenchymal stem cells as pharmacological mediators in tissue repair. Paracrine effects, exerted through the release of biologically active factors, are the key mechanisms mediating MSC-based therapy in many diseases. Using an engrafted myocardial infarction model several factors abundantly secreted by MSCs were identified. In this study, we focus on one novel and promising therapeutic factor known as SPARCL1. SPARCL1, is an extracellular matrix glycoprotein and has been described in many cellular processes. This is the first study describing the cardio-protective effect of SPARCL1. The aim of the study was to elucidate the role and potential mechanism of SPARCL1 in cardio protection and MSC survival. The cardiomyocytes/MSC injury model was established by exposing rat primary neonatal cardiomyocytes and bone marrow derived MSC to a hypoxic environment (0.5% oxygen) in the absence of serum and oxidative-stress induced injury by hydrogen peroxide. Viability, cytotoxicity and caspase activity were monitored to measure the cyto protective effects of SPARCL1. Genes with potential cardio-protective effects were analysed by quantifying at mRNA and protein levels. Using the in vitro model of oxidative stress, SPARCL1 treatment resulted in significant increase of cell viability and a decrease in apoptosis for up to 6 hours post treatment. These observations were confirmed by gene and protein expression which demonstrated elevated expression of anti apoptotic genes Bcl-2, and reduction of anti fibrotic genes, Col1 and Col3. SPARCL1 protein also exhibits pro-angiogenic properties by increasing tubular and capillary formation in an in vivo matrigel angiogenesis assay. Interestingly, the capillary formation was significantly higher in lower dose (100ng/ml) compared to the higher dose (400ng/ml) indicating a threshold effect. SPARCL1 treatment during induced oxidative stress demonstrated a significantly increased expression of pAKT compared to the non-treated group, indicating the involvement of PI3K/AKT pathways in the survival of cells in oxidative stress injury. In this study, we demonstrate that SPARCL1 protected cardiomyocytes via inhibition of apoptosis and reduced fibrosis. These findings supported the hypothesis that SPARCL1 is a promising candidate for therapies aimed at reducing myocardial injury and resulting cardiac dysfunction. This work highlights the new perceptive that therapeutic effects are mediated via the secretome and will overcome current limitation of cells delivery in cardiac regenerative therapy. References:

1. Mirotsou, M., Zhang, Z., Deb, A., Zhang, L., Gnecchi, M., Noiseux, N., Mu, H., Pachori, A. & Dzau, V. (2007) Secreted frizzled related protein 2 (Sfrp2) is the key Akt mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. Proc Nat. America Society USA, 104, 1643-1648.

2. Gnecchi, M., Zhang, Z., Ni, A. & Dzau, V.l (2008) Paracrine mechanisms in adult stem cell signaling and therapy. Cire Res, 103, 1204-1219.

3. Gnecchi, M., He, B., Noiseux, N., Liang, O.D., Zhang, L., Mu, H., Melo, L.G., Pratt, R.E., lngwall, lS. & Dzau, V. T. (2006) Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. Faseb J, 20, 661-669.


22. Elastogenic potential of neonatal versus adult arterial smooth muscle cells for

tissue-engineered cardiovascular grafts

Strauther S., Clarke F. and Flanagan T.C.

School of Medicine & Medical Science, University College Dublin, Dublin, Ireland A significant challenge lies in cellular generation of elastin networks within tissue engineered vascular grafts and heart valves. It is known that elastin production declines during normal post-natal growth, suggesting that autologous cells isolated from post-natal patients may not be suitable for generating elastin-rich grafts. The present study aimed to evaluate the hypothesis that neonatal arterial smooth muscle cells (SMCs) are more elastogenic in in vitro culture than adult arterial SMCs. Commercial SMC lines from umbilical artery (HUASMCs) and adult coronary artery (CASMCs) were seeded in 24-well plates (10,000/well) and maintained in culture for up to 18 days. Each cell population was cultured with or without TGF-β1 supplementation (2ng/ml). Elastin deposition was quantified in each culture using a colorimetric assay (Fastin) after 1, 6, 12 and 18 days. Elastin, fibrillin-1 and lysyl oxidase synthesis were also analyzed at the same time points using immunocytochemistry. Quantitative analysis demonstrated significantly enhanced deposition and processing of elastin into the cell layer over 18 days by UASMC cultures (28.2±5.7 μg/well) compared to CASMCs (16.5±1.2 μg/well). TGF-β1 supplementation significantly increased elastin synthesis in UASMC cultures by ~15%, although decreased elastin synthesis in CASMCs. No notable differences in staining pattern or intensity of elastin assembly proteins were observed between the cell populations using immunocytochemistry. The results of the present study suggest that UASMCs may be more a suitable cell source for the development of tissue-engineered vascular grafts or heart valves than adult arterial cells based on their elastin-synthesizing potential.


23. Using total internal reflection fluorescence microscopy (tirfm) for studying protein-surface interactions

Przemyslaw M. Zarskia and Alan G. Ryder

Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland Galway, Ireland. Many fundamental features of protein-surface interactions can be difficult to measure and understand, in particular information about protein structural changes during adsorption processes. This prompts questions about protein folding/unfolding, denaturation, orientation, stability, conformation and activity when interacting with surfaces. In many cases, the lack of information results from the fact that high spatial resolution microscopy methods often do not provide and chemical restitution tools are not always available. The Nanoscale Biophotonics Laboratory (NBL) has over the past couple of years developed a programme of research to develop a range of analytical methodologies based on fluorescence spectroscopy, coupled with confocal microscopy [1-3]. Here we use TIRFM coupled with fluorescence spectroscopy to develop novel analytical methods that can yield quantitative data on protein-surface interactions. The TIRF illumination method coupled with a 405 nm excitation source should enable selective excitation in a ~50 nm zone above the surface thus minimising fluorescence signal from the bulk solution. In addition the TIRFM system is fitted with a low noise EMCCD camera for measurements of the weak fluorescence signals from protein monolayers. Currently we are developing a range of suitable protocols and standards which will help facilitate the generation of very reproducible TIRFM data. We are starting to develop a hardware solid state standard for penetration depth measurements, a solution state standard to validate measurement methods and liquid systems. The final standard is a protein standard based on fluorescently labelled Bovine Serum Albumin (BSA) which we aim to use as a standard control measurement to validate experimental protocols. The final hardware development step will involve integrating a fluorescence lifetime measurement (FLIM) mode using Time Correlated Single Photon Counting with 405 and 470 nm excitation sources. Once these are completed we will apply TIRFM-FLIM to the detailed study of protein interactions with polymer coated surfaces. References:

1. Togashi, D. M., Ryder, A. G., Biophysical Chemistry 2010, 152, 55-64. 2. Togashi, D. M., Ryder, A. G., Heiss, G., Colloids and Surfaces B: Biointerfaces 2009, 72, 219-229. 3. Togashi, D. M., Ryder, A. G., Experimental and Molecular Pathology 2007, 82, 135-141.

Keywords: Total Internal Reflection Fluorescent Microscopy, protein-surface interaction, BSA, TIRFM-FLIM


Delegate List First Name Last Name Affiliation Email Kellie Adamson Dublin City University [email protected]

Sergey Alexandrov NUI Galway [email protected]

Rizwan Ali NUI Galway [email protected] Alicja Antonczak NUI Galway [email protected] Heath Bagshaw Trinity College Dublin [email protected] Valerie Barron NUI Galway [email protected] Alan Bell Trinity College Dublin [email protected] Niall Bergin NUI Galway [email protected] Manus Biggs NUI Galway [email protected] Alex Black NUI Galway [email protected] Emer Bourke NUI Galway [email protected] Gerry Brennan Queen's University Belfast [email protected] Megan Joanne Brophy University College Dublin [email protected] James Brown NUI Galway [email protected] George Burke University of Ulster [email protected] Hugh Byrne Dublin Institute of Technology [email protected] Luis Felipe Carvalho Dublin Institute of Technology [email protected] Yanhui Chen Trinity College Dublin [email protected] Jennifer Connolly NUI Galway [email protected] David Cottell MSI [email protected] Dermot Daly Trinity College Dublin [email protected] Nicholas Devaney NUI Galway [email protected] Ian Dobbie University of Oxford [email protected] Peter Dockery NUI Galway [email protected] Helen Dodson NUI Galway [email protected] Zsolt Fábián NUI Galway [email protected] Tom Flannagan University College Dublin [email protected] Danny Fox Trinity College Dublin [email protected] Yina Guo University of Limerick [email protected] Eoin Hinchy University of Limerick [email protected] Laurena Holleran NUI Galway [email protected] Richard Horobin University of Glasgow [email protected] Benoit Houeix NUI Galway [email protected] Siti Ismail NUI Galway [email protected] Anna Kaskova Gheorghescu University College Dublin [email protected] Ellen Keenan NUI Galway [email protected]

mailto:[email protected]


Abbas Khalid Trinity College Dublin [email protected]

Pramod Kumar NUI Galway [email protected]

Mahendar Kumbham University of Limerick [email protected]

Yvonne Lang NUI Galway [email protected]

Martin Leahy NUI Galway [email protected]

Claire Masterson NUI Galway [email protected]

Katya Mc Donagh NUI Galway [email protected]

Emma Mc Mahon NUI Galway [email protected]

Siobhan McMahon NUI Galway [email protected]

Laoise McNamara NUI Galway [email protected]

Daniel Mortell University of Limerick [email protected]

Lisa Mullee NUI Galway [email protected]

Mohd Hairulhisyam Ngatiman NUI Galway [email protected]

Robert O'Connell Trinity College Dublin [email protected] Tiina O'Neill University College Dublin [email protected] Peter Owens NUI Galway [email protected] Dmitri Papkovsky University College Cork [email protected] Tatiana Perova Trinity College Dublin [email protected] Sivaramakrishnan Ramadurai NUI Galway [email protected]

Jeremy Rees Cambridgenanoscience Ltd [email protected] Bogumila Reidy University College Dublin [email protected] Alan Ryder NUI Galway [email protected] Anneke Sanders University College Dublin [email protected] Abhigyan Satyam NUI Galway [email protected] Dimitri Scholz University College Dublin [email protected] Dalibor Soukup NUI Galway [email protected] Alanna Stanley NUI Galway [email protected] Martin Steer MSI [email protected] Eva Sweeney NUI Galway [email protected] Kerry Thompson NUI Galway [email protected] Felim Vajda Trinity College Dublin [email protected] Fiona Weafer NUI Galway [email protected] Katarzyna Welzel University College Dublin [email protected] Tony Wheatley NUI Galway [email protected] Dimitrios Zeugolis NUI Galway [email protected]

mailto:[email protected]

NATIONAL UNIVERSITY OF IRELAND, GALWAYsymposia.microscopy.ie/MSI2014/Doc/Programme_MSI2013.pdf · 2016-04-11 · MSI2013 | National University of Ireland, Galway, 21st- 23rd August

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