Microscopical Society of Ireland

33rd Annual Symposium 2009

Astra Hall,Student Centre University College Dublin August 26-28, 2009

Programme & Abstract Booklet

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Kindly Sponsored By:

33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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Officers of the Society 2009

PRESIDENT: Dr. Alan Ryder Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland, Galway.

IMMEDIATE PAST PRESIDENT: Dr. Gerard Brennan School of Biological Sciences, Medical Biology Centre, Queen’s University, Belfast.

HONORARY SECRETARY: Dr. Tom Flanagan School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, Belfield, Dublin.

HONORARY TREASURER: Dr. Fiona Lyng FOCAS Institute, Dublin Institute of Technology, Kevin St., Dublin.

COMMITTEE MEMBERS: Prof. Martin Steer, Department of Botany, University College Dublin, Belfield, Dublin. Dr. David Cottell, Electron Microscopy Laboratory, Agriculture & Food Science Centre, University College Dublin, Belfield, Dublin.

Alexander Black, Department of Anatomy, National University of Ireland, Galway. Dr. George Burke, Nanotechnology & Integrated BioEngineering Centre, Faculty of Engineering, University of Ulster, Jordanstown.

Dr. Ian Fairweather, School of Biological Sciences, Medical Biology Centre, Queen’s University, Belfast.

Dr. David Tanner, Department of Manufacturing & Operations Engineering, University of Limerick.

Dr. Brendan Wilkins, Department of Anatomy, National University of Ireland, Galway. Chris O’Kane (Post-Graduate Member), Nanotechnology & Integrated BioEngineering Centre, Faculty of Engineering, University of Ulster, Jordanstown.

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Local Organising Committee

Dr. David Cottell, Electron Microscopy Laboratory, Agriculture & Food Science Centre, University College Dublin, Belfield, Dublin.

Dr. Tom Flanagan, School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, Belfield, Dublin.

Prof. Martin Steer, Department of Botany, University College Dublin, Belfield, Dublin. Ms. Tiina Toivonen, Electron Microscopy Laboratory, Agriculture & Food Science Centre, University College Dublin, Belfield, Dublin.

E-Mail: [email protected] Symposium Homepage: http://www.nuigalway.ie/msi/symposium.htm

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- The support of the following companies is greatly appreciated –

GOLD SPONSORS Company Name: FEI Company Company Address: Achtseweg Noord 5, P.O. Box 80066, 5600 KA, Eindhoven, The Netherlands Phone No. +44 (0) 114 234 0100 Fax No. +44 (0) 7836 645 492 Contact Name(s): David Beamer Contact E-Mail: [email protected] Web Address: www.fei.com

SILVER SPONSORS Company Name: Hitachi High-Technologies Europe GmbH Company Address: Whitebrook Park, Lower Cookham Road, Maidenhead, Berkshire SL6 8YA, UK Phone No. +44 (0) 1628 585 200 Fax No. +44 (0) 1628 585 391 Contact Name(s): Patrick Marks Contact E-Mail: [email protected] Web Address: www.hht-eu.com Company Name: HORIBA Scientific Company Address: 2 Dalston Gardens, Stanmore, Middlesex, HA7 1BQ, UK Phone No. +44 (0) 20 8204 8142 Fax No. +44 (0) 20 8204 6142 Contact Name(s): Adrian Knowles Contact E-Mail: [email protected] Web Address: http://www.horiba.com/uk/scientific/products/microanalysis

BRONZE SPONSORS Company Name: Andor Technology Company Address: 7 Millenium Way, Springvale Business Park, Belfast, BT12 7AL, UK Phone No. +44 (0) 28 90 237126 Fax No. +44 (0) 28 90 310792 Contact Name(s): Sarah McAllister Contact E-Mail: [email protected] Web Address: http://www.andor.com Company Name: JEOL (UK) Ltd. Company Address: JEOL House, Silver Court, Watchmead, Welwyn Garden City, Hertfordshire, AL7 1LT, UK Phone No. +44 (0) 1707 377117 Fax No. +44 (0) 1707 373254 Contact Name(s): Jason Dalby, Sales Executive Contact E-Mail: [email protected] Web Address: http://www.jeoluk.com

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Information for Delegates Dear Delegate, It is a pleasure to welcome you to the 33rd Annual MSI Symposium, 2009 at the Astra Hall, Student Centre, University College Dublin (please refer to map overleaf, ref. 11). Student Prizes • A prize of €250 will be awarded for the best student oral presentation in Life Sciences. • A prize of €250 will be awarded for the best student oral presentation in Material Sciences. • A prize of €250 will be awarded for the best student poster presentation in Life Sciences. • A prize of €250 will be awarded for the best student poster presentation in Material Sciences. These prizes will be awarded by Dr Alan Ryder, President of MSI, at 12:50 on Friday, 28th August 2009. Please be aware that the winning students must be present to receive the prize, otherwise it will be retained by MSI. Scientific Programme This year, oral presentations will be 15 mins plus 5 mins discussion. Poster presentations will be for 3 mins. The progamme is quite busy so all participants should be aware that the chairpersons of sessions will rigorously enforce the time limits. Trade Exhibition The trade exhibition will be adjacent to the lecture hall and poster display in Astra Hall. All delegates are urged to interact as much as possible with the commercial personnel who are extremely knowledgeable of microscopy and are eager to help in all manner of ways. In addition, MSI is much indebted to the trade for their continued sponsorship over 33 years and recognises that without that level of support it would be difficult to exist as the thriving Society that it is. Symposium Breaks Coffee and tea will be available at the programmed times in the trade exhibition area. Lunch on Thursday 27th August is included in your registration fee and will be served in Cafe Sport in the nearby Sports Centre between 12:50 – 14:00 (map ref. 62). Please note the cost of drinks is not included. Symposium Dinner The symposium dinner will be a BBQ in the Stillorgan Park Hotel on the N11, and will take place on the evening of Thursday 27th August. Delegates are advised to take the Nr. 46A, 47, 84x or 145 bus from outside the Montrose Hotel (across the dual carriageway from N11 entrance to UCD, see map below). The journey time is about 10 mins and the frequency of 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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the buses is also at least once every 10 mins. There is a stop just before the Stillorgan Park Hotel served by each of these routes. The BBQ will be served at about 7:45pm and will be followed by a table quiz at approximately 10pm. The quiz team this year is made up of Paola Maderna, Emma Borgeson and Federico Fenaroli, all of whom will be happy to advise delegates (during the BBQ) of the rules and regulations of the quiz. The bar will be open on arrival serving a complimentary cocktail and will remain open until about 12:45am Friday.

We hope that you will find the conference to be a stimulating and rewarding experience and that above all you will enjoy it. MSI Organising Committee On behalf of MSI.

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Scientific & Social Programme Wednesday, August 26, 2009 *All lectures will take place in the Astra Hall, UCD Student Centre. 11:00 13:00

REGISTRATION. Trade stand and poster setup in the Astra Hall.

Session 1 Chair: Dr. Tom Flanagan (UCD). 13:15

Welcome Address: Dr. David Cottell

13:30

Keynote 1 Advanced Imaging Approaches in Nanomedicine. Yuri Volkov, Department of Clinical Medicine, Trinity College Dublin, Ireland.

14:30

Delegate Dispersion Artefacts in FTIR Spectra of Single Biological Cells. 1 Hugh J. Byrne, Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.

14:50

Delegate Towards Improvements in the Efficacy of the Diagnostic Approach to 2 Primary Cilia Dyskinesia in Ireland. Tiina H. Toivonen, The Electron Microscopy Laboratory, & UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

*

15:10

Delegate Comparison of Efficacy of Several Microscopic Techniques in 3 Characterisation of Structure and Composition of Highly Dispersed Sludges. Anna V. Piterina, Department of Chemical and Environmental Sciences, Material and Surface Science Institute, & Centre for Applied Biomedical Research (CABER),University of Limerick, Limerick, Ireland.

15:30 16:00

COFFEE BREAK: Astra Hall

Session 2 Chair: Prof. Martin Steer (UCD). 16:00

Keynote 2 Foundation Principles of the Interaction of Nanoparticles with Cells. Anna Salvati, Centre for BioNano Interactions, University College Dublin, Belfield, Dublin 4, Ireland.

17:00

Delegate Scanning Electron Microscopy on Feeding Appendages of Endemic 4 Shrimp from Lake Tanganyika, Africa. Marjolein Kamermans, School of Natural Sciences, Zoology Building, Trinity College, University of Dublin, Dublin 2.

*

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17:20

Delegate An Investigation into the Bioactivity of Porous Silicon Nanosponge 5 Particles. Edward Chadwick, Materials & Surface Science Institute, & Department of Manufacturing and Operations Engineering; University of Limerick, Limerick, Ireland

**

17:40

18:00 19:00 19:00 21:00

Delegate The Use of the Polarizing Microscope by Victorian Microscopists. 6 Stephen F. Lowry, School of Biological Sciences, University of Ulster, Cromore Road, Coleraine, BT52 1SA MSI Annual General Meeting WELCOME WINE RECEPTION: Astra Hall

* - denotes Student presentation (Life Sciences). ** - denotes Student presentation (Materials Science).

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Thursday, August 27, 2009 08:30 09:45

LATE REGISTRATION.

Session 3 Chair: Dr. Alan Ryder (NUIG). 10:00

Keynote 3 Genome-Wide Imaging and Analysis of Membrane Traffic Pathways. Jeremy C. Simpson, School of Biology and Environmental Science, University College Dublin, Ireland.

11:00 11:30 11:30

COFFEE BREAK: Astra Hall Delegate Nanostructural Origin of Hierarchical Organization in Bone: a 7 Microscopic Study. Yuqi Zhang, Materials and Surface Science Institute (MSSI), University of Limerick, Limerick.

*

11:50

Delegate Microstructural Analysis of Pt/VACNT Nanostructured Electrodes. 8 Navneet Soin, NIBEC, University of Ulster, Shore Road, BT37 0QB, Northern Ireland

**

12:10

Delegate Microscopy Investigations of Surface Engineered Coatings Prepared by 9 Nitrocarburising. Dale A. Molloy, School of Mechanical and Aerospace Engineering, Queen’s University of Belfast, Ashby Building, Stranmillis Road, Belfast, BT9 5AH.

**

12:30

Delegate Defocus Contrast Image of Hexagonally-Ordered Mesoporous 10 Material. David A. Tanner, Materials & Surface Science Institute, & Department of Manufacturing and Operations Engineering, University of Limerick.

12:50 14:00

LUNCH: Café Sport, Sports Centre; included in registration fees.

Session 4 Chair: Dr. David Cottell (UCD). 14:00

Delegate Analysis of the Interaction between Human A549 cells and Silica 11 Nanoparticles Using Electron Microscopy. Federico Fenaroli, Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4.

*

14:20

Delegate Strain Determination Using Convergent Beam Electron Diffraction 12 (CBED). Vishnu Mogili, Materials & Surface Science Institute, & Department of Manufacturing and Operations Engineering, University of Limerick, Limerick, Ireland.

**

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14:40

Delegate Identification of Signalling Events Involved in Radiation Induced 13 Bystander Effects. Fiona M. Lyng, Radiation and Environmental Science Centre (RESC), Dublin Institute of Technology, Dublin 8.

15:00

Delegate Development of Nanoscale Temperature Responsive Culture Surfaces for Cell Culture and Recovery. 14 Maria Nash, School of Chemistry, National University of Ireland, Galway, Galway, Ireland.

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15:20 16:00

COFFEE BREAK: Astra Hall

16:00 - Keynote 4 State of the Art Light and Electron Microscopy for Cell Biology. Gareth Griffiths, Dept Molecular Biosciences, University of Oslo, Norway 17:00 18:00 19:30 late

Poster Session: all student presenters are to be ready to present their work to the judges. Each presentation will be 3 mins. SYMPOSIUM DINNER & TABLE QUIZ: Stillorgan Park Hotel

* - denotes Student presentation (Life Sciences). ** - denotes Student presentation (Materials Science).

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Poster Session - Thursday, August 27, 2009 (17:00 -18:00) Life Sciences: Poster 1 A New Dimension in Study of Living Cells by Raman Spectroscopy. Franck Bonnier, Focas Research Institute, Dublin Institute of Technology (DIT), Kevin Street, Dublin 8, Ireland. Poster 2 Raman and FTIR Imaging of Prostate Cancer – a Correlation to Common Diagnostic Biomarker. Kelvin Poon, Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Camden Row, Dublin 8, Dublin, Republic of Ireland. Poster 3 Microstructure Analysis of Process Cheese by Using Cryo-Scanning Electron Microscopy and Light Microscopy. Mamdouh El-Bakry, UCD Institute of Food and Health, Belfield, Dublin 4.

*

Poster 4 Carbon Sources Affect the Growth and Development of Burkholderia cepacia Complex (Bcc) Biofilms Cultured in Continuous Flow Cells. Sarah Kennedy, Centre for Microbial Host Interactions (CMHI), Institute of Technology Tallaght, Dublin.

*

Poster 5 Using TEM Analysis of Primary Cilia in C. elegans to Understand the Molecular Basis of Ciliary Disease (Human) Gene Function. Katarzyna Kida, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. Poster 6 Glaucomatous Human Lamina Cribrosa Cells Show Increased Lipofuscin Accumulation. Neil Docherty, University College Dublin School of Medicine and Health Sciences, University College Dublin. Poster 7 Cervical Cancer Cell Lines Investigation Using Raman Microspectroscopy. Kamila Ostrowska, RESC, Focas Institute, Dublin Institute of Technology, Dublin, Ireland.

*

Poster 8 Interaction of Quantum Dots with Cellular Systems. Lorenzo Salford, Focas Research Institute, Dublin Institute of Technology, Dublin 8.

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Poster 9 Dorso-ventral Hippocampal Contributions to Learning in the Rat. Darren Scully, UCD Conway Institute, University College Dublin. Poster 10 Cell Death Responses in Bystander Cells Exposed to Signals from Irradiated Cells. Kishore Kumar Jella, RESC, Dublin Institute of Technology, Kevin St, Dublin 8.

*

* - denotes Student presentation.

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Materials Science: Poster 11

** Poster 12

**

Analysis of Phosphorous Diffused Tungsten Silicide Using Raman Spectroscopy. David Adley, Department of Electronic and Electrical Engineering, Trinity College Dublin, Dublin. FTIR Micro-spectroscopy for Investigation of Optical Properties of Grooved Silicon: Comparison with Simulation by Scattering Matrix Method. Sergey Dyakov, Department of Electronic and Electrical Engineering, Trinity College Dublin, Ireland.

Poster 13

Phase Mapping of Naturally-Aged Uncapped InN Quantum Dots. Juan Gabriel Lozano, Dpto de Ciencia de los Materiales e Ing. Metalúrgica y Q. I., Universidad de Cádiz, 11510, Puerto Real, Cádiz, Spain.

Poster 14

Microcopy Investigations of Surface Engineered Coatings Prepared by Selective Plating. Dale Molloy, School of Mechanical and Aerospace Engineering, Queen’s University of Belfast, Ashby Building, Stranmillis Road, Belfast, BT9 5AH.

** Poster 15

** Poster 16

**

Microscopy and Spectroscopic Characterization of Acid-Treated SingleWalled Carbon Nanotubes. Dania Movia, School of Chemistry/CRANN, Trinity College Dublin, College Green, Dublin 2, Ireland. Raman Investigation of Different Polytypes of SiC Layers Grown on Si and SiC Substrates. Joanna Wasyluk, Department of Electronic and Electrical Engineering, University of Dublin, Trinity College, Dublin 2, Ireland.

Poster 17

Spectroscopic and Hemometric Approaches to Radiobiological Analyses. Aidan Meade, School of Physics, Dublin Institute of Technology, Dublin, Ireland.

Poster 18

AFM and FTIR Micro-Spectroscopy Analysis of Two-Dimensional Silicon Photonic Crystals Victor Ermakov, Department of Electronic and Electrical Engineering, University of Dublin, Trinity College, Dublin 2, Ireland.

**

** - denotes Student presentation.

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Friday, August 28, 2009 Session 5 Chair: Dr. David Tanner (UL). 10:00

11:00 11:30

Keynote 5 High Resolution Electron Microscopy Applied to Nanostructured Oxides – Structure-Function Relationships and Characterization. Colm O’Dwyer, Department of Physics, & Materials & Surface Science Institute, University of Limerick, Limerick, Ireland. COFFEE BREAK: Astra Hall

11:30

Delegate Image Analysis/Interpretation Framework for Studying the Kinetics of 15 Anti-Apoptotic Response of Cancer Cells to Proteasome Inhibition Therapy. Tytus Bernas, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland, & Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland

11:50

Delegate Border Patrol: a Fundamental Study of Interface Formation in Polymer 16 Nanocomposites using Nano-Thermal Analysis (nanoTA). Gordon Armstrong, Materials & Surface Science Institute, University of Limerick, Castletroy, Co. Limerick.

12:10

Delegate TEM Analysis of Epithelial Junction Structure in the Normal Margins 17 and Stricture Sites of Ileo-Caecal Resections for Fibrostenosing Crohn’s Disease. Michael F. Cunningham, UCD Conway Institute of Biomolecular and Biomedical Sciences, UCD School of Medicine and Medical Sciences, & Surgical Professorial Unit, St. Vincent’s University Hospital, Dublin 4.

12:30

Delegate Functional Autologous Tissue-Engineered Vascular Grafts in the 18 Systemic Circulation: A Microscopic Analysis Tom Flanagan, School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, & Helmholtz Institute for Biomedical Engineering, Aachen University, Aachen, Germany.

12:50

Award of Student Prizes: Dr. Alan Ryder (President, Microscopical Society of Ireland). Each student must be present to receive the award, otherwise it will be retained by the MSI.

13:00

Closing Remarks. Dr. Alan Ryder.

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Abstracts – Keynote Speakers Keynote 1 - Advanced Imaging Approaches in Nanomedicine. Yuri Volkov Department of Clinical Medicine, Trinity College Dublin, Ireland. [email protected] Rapid development of nanotechnology consistently increases the likelihood of human contact with environmentally presented and engineered nanomaterials, i.e. the tiny objects ranging in size from one to several hundreds of nanometres and featuring an extreme diversity in shapes and physico-chemical properties (inorganic silica, carbon, metal oxide, inert metal nanoparticles and core/shell structures, polymeric particles and spheres, nanorods, nanotubes, nanowires, dendrimers, liposomes and complex derivatives of all the above, to name but a few). Phagocytes, epithelium in the lungs and gastrointestinal tract as well as cells of the cardiovascular system are the primary candidates to encounter these nanomaterials in real life situations. Such encounters may cause specific functional responses, including triggering of the intracellular signaling cascades and immune reactions, apoptotic and direct toxic effects. However, there is still very little definitive systematic information about the consequences of interactions of nano-scale objects with human cells and tissues of diverse origin and therefore safety-related issues are high on the agenda in the emerging scientific area of nanomedicine. On the other hand, optimistic expectations are associated with the opportunities of using the nanoparticles as a new class of drug delivery systems, arising from the fact that the finite, but tunable size of the engineered nanostructures used as drug delivery vehicles can impose very precise nano-scale drug distribution barriers at the level of cells, tissues and entire organism thereby eliminating undesirable side effects pertinent to most contemporary medicines. High content imaging and analysis approach in combination with live cell confocal imaging, atomic force microscopy, fluorescence lifetime imaging and Raman spectrometry provides a unique integrated technological toolkit for visualization and physical characterization of nanoparticle-cell interactions at the levels of uptake, intracellular transport, subcellular and organelle targeting of nanoparticles. An exceptional aspect of this approach is that it is possible to identify individual cell, as well as population responses associated with nanoparticle exposure, as in this way subtle effects on small groups of cells within the whole, which could be averaged out by routine screening, are fully registered and elucidated. We will provide here an overview of such multi-platform cell and subcellular imaging application scenarios for investigations of safety and intracellular distribution of nanomaterials with promising biomedical application potential in live human phagocytes and cells of non-phagocytic origin. Supported by the Health Research Board of Ireland, Science Foundation of Ireland SRC BioNanoInteract and EU FP-6 Consortium NanoInteract.

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Keynote 2 - Foundation Principles of the Interaction of Nanoparticles with Cells. Anna Salvati, Iseult Lynch, and Kenneth Dawson Centre for BioNano Interactions, University College Dublin, Belfield, Dublin 4, Ireland. [email protected] The importance of understanding the interactions between nanoscale materials and living matter has begun to be appreciated in recent years. Thanks to their size (10-100nm), nanoparticles give the opportunity to identify and study specific interactions both at cellular and at tissue level. The underlying rationale is both real and durable. Nanoparticles of less than 100nm can enter cells and those of less than 35 nm can pass the blood brain barrier. These are fundamental size scales of biological relevance that will ensure that engineered nanoscience will impinge on biology and medicine for many decades. Likely, the uptake and trafficking of nanoparticles in contact with biological fluids is greatly affected by the proteins and other biomolecules that associate to their surface, and the protein corona taken from the surrounding biological milieu will constitute the real identity of nanoparticles exposed to cells. The pace of advance is extraordinary. This arena of research not only opens up new directions in nanomedicine and nanodiagnostics, but offers the chance to implement nanotechnology in a safe and responsible manner, addressing the concerns of nanosafety in parallel with the development of applications. Fundamental in this scenario is the ability to follow (and ultimately control) the localisation in time and space of nanoparticles inside cells, in a quantitative and reproducible manner. This has been achieved by the combination of electron microscopy and fluorescence based techniques, including fluorescence microscopy and live cell imaging of single nanoparticles inside the cell, thus enabling us to study nanoparticle uptake from the early entry to their final localisation. Keynote 3 - Genome-wide Imaging and Analysis of Membrane Traffic Pathways. Jeremy C. Simpson School of Biology & Environmental Science, University College Dublin, Ireland. [email protected] Mammalian cells have a highly complex internal architecture consisting of membranebounded organelles each with defined functions. Organelles within the secretory and endocytic pathways however do not function in isolation, but are able to exchange material through the highly dynamic process of membrane traffic. A more complete inventory and understanding of the membrane traffic machinery is an essential prerequisite to improved drug design and targeting. We have applied high-throughput systematic subcellular localisation, genome-wide gene downregulation by RNA interference, and automated high content screening microscopy as approaches to more comprehensively discover the protein machinery involved in cellular trafficking pathways. These experiments have revealed a wide

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diversity of previously unidentified membrane traffic regulators, and an unexpected high degree of inter-dependence with basic cellular processes. Keynote 4 - State of the Art Light and Electron Microscopy for Cell Biology. Gareth Griffiths Department of Molecular Biosciences, University of Oslo, Norway. [email protected] In the first part of this presentation I will cover the main EM techniques that are important for molecular cell biology. An important conceptual breakthrough in EM was the introduction of cryo EM and the concept of vitrification by Jacques Dubochet and colleagues in the early 1980’s. This approach made it possible to avoid fixatives and other chemicals that had long been necessary for most EM methods. Two different cryo EM approaches have been developed, namely single particle methods and a sectioning method referred to as Cryo EM of Vitrified Sections (CEMOVIS). I will show examples of these methods, as well as briefly cover the method of tomography, that is being increasingly used in conjunction with cryo EM, and with conventional plastic embedding to provide three-dimensional models of biological specimens. I will also cover the importance of using light microscopy (LM) in conjunction with EM for cell biology. The use of both methods together is especially important for interpreting the results of immuno-labeling studies, an approach that still require the use of chemical fixatives in almost all cases. In the second part of the presentation I will focus on the use of LM and EM in our recent work on phagocytosis, addressing two systems, model latex beads and Mycobacteria, including M.tuberculosis. For LM live cell video microscopy will be described in order to reveal striking dynamic process of transient assembly of actin by latex bead phagosomes in GFP-actin expressing mouse macrophages. The subsequent part will address the use of different EM sectioning approaches to visualize different mycobacteria in macrophage phagosomes and will conclude that the best sectioning method to monitor the native structure of free mycobacteria and mycobacteria within phaagosomes is CEMOVIS. Keynote 5 - High Resolution Electron Microscopy Applied to Nanostructured Oxides – Structure-Function Relationships and Characterization. C. O’Dwyer Department of Physics, and Materials & Surface Science Institute, University of Limerick, Limerick, Ireland. [email protected] One-dimensional nanomaterials, such as nanotubes, nanowires, and nanobelts or nanoribbons have attracted considerable attention in the past decade because of their novel and useful physical properties leading to numerous applications. Although the majority of research and development has been based on carbonaceous and compound semiconducting nanostructures, attention is now being directed to transition metal 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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nanostructures based on their oxides which, due to their versatile chemical properties often modulable by changes in the oxidation state in the metal co-ordination sphere, can lead to a variety of products and tuneable materials. Incorporating such nanostructures into known device configurations could improve on current designs while potentially allowing further functionality by exploiting nanoscale electronic and photonic properties of suitable materials. This talk will outline recent findings by our group concerning the structure-function relationships of a range of metal oxide nanostructures: vanadium oxide, zinc oxide, and indium tin oxide. Starting from the laminar V2O5 xerogel numerous two-dimensional organic-inorganic VOx intercalation products have been obtained. Many of these nanostructures may be obtained in quantities on the order of grams. Structure related properties such as scrolling to form nanotubes, nanoscale actuation, charge storage for batteries and self-assembly will be addressed. In parallel, nanostructures designed for optical emission and absorptive charge storage have garnered obvious interest in recent times, resulting from a drive to research new charge storage architectures and advanced solar cell designs. In this section, both ZnO and ITO nanowires will be shown to exhibit excellent optical properties. TEM was used in all cases to determine the nanostructure phase and relation to opto-electronic characteristics. ITO nanowires, grown as branched layers, will be shown to be fully transparent and conductive contact layers, optimized for application as fully transparent contacts in the visible to nearinfra red region for silicon-based light emitting devices (LEDs). For ZnO, this talk will outline how high resolution microscopy was used to probe the structure and growth of the Al-coated ZnO nanowires and related that information to their optical properties. These structures were grown by catalyst-free chemical vapour deposition (CVD). After removal from the growth substrate by sonication, a second mirror facet was obtained as an effect of the cleavage along the (0001) crystallographic plane, thus leading to an enhanced axial Fabry-Perot resonator. The resulting nanostructures form the lowest threshold core-shell UV nanolasers reported to date.

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Abstracts – Oral Sessions Delegate 1 - Dispersion Artefacts in FTIR Spectra of Single Biological Cells. Bassan P.1, Byrne H.J.2, Lee J.1, Bonnier F.2, Clarke C.2, Dumas P.3, Gazi E.4, Brown M.D.4, Clarke N.W.4,5,6, Gardner P.1 1. School of Chemical Engineering and Analytical Science, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. 2. Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. 3. Synchrotron SOLEIL, L'Orme des Merisiers,BP48 - Saint Aubin,91192 Gif-sur-Yvette Cedex, France. 4. Genito Urinary Cancer Research Group, School of Cancer and Imaging Sciences Paterson Institute for Cancer Research, University ofeManchester, Wilmslow Road, Manchester, UK, M20 4BX, UK. 5. Department of Urology,The Christie NHS Foundation Trust, Manchester, UK, M20 4BX, UK. 6. Department of Urology, Salford Royal NHS Foundation Trust, Salford, UK, M6 8HD, UK. [email protected] Fourier transform infrared (FTIR) spectroscopy has been demonstrated as a potential tool for disease diagnostics in tissue and in its microscopical form, for cellular analysis. The use of synchrotron sources has achieved subcellular resolution thus promising a powerful technique for the analysis of the biochemical origin of disease. Spectra of a single cell are seen to vary significantly with position on the cell, however, showing a distorted derivativelike lineshape in the region of the optically dense nucleus making assignment of changes difficult. Using model systems, it is demonstrated that the so-called dispersion artefact can be understood in terms of reflection and scattering contributions and that their physical origin can be extracted from the inherent bio chemical variations in the cell. Rather than being an artefact, the contributions are ubiquitous in spectra of spatially and chemically inhomogeneous samples but a true understanding of their origin enhances the interpretation of the microscopic data. Delegate 2 - Towards Improvements in the Efficacy of the Diagnostic Approach to Primary Cilia Dyskinesia in Ireland. Toivonen, T. H.1, Docherty, N.2, O'Riordan, D.3, Elnazir, B.4 and Cottell, D. C.1 1. The Electron Microscopy Laboratory and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. 2. School of Medicine and Medical Sciences, University College Dublin, Belfield, Dublin 4, Ireland.

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3. School Of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland. 4. The Adelaide and Meath Hospital, Dublin Incorporating the National Children’s Hospital, Tallaght, Dublin 24, Ireland. [email protected] Primary cilia dyskinesia (PCD) is an autosomal recessive inherited disorder manifested by ultrastructural defects and/or function of cilia. The main clinical features of PCD are chronic infection of the upper and lower respiratory tract, bronchiectasis, otitis media, situs inversus in 50% of patients, fertility reduction and a risk of ectopic pregnancy. In short, PCD elicits significant pathology in all of the ciliated systems in humans. The diagnosis of PCD is most commonly made by co-relating clinical evidence with ciliary ultrastructural defects found by transmission electron microscope (TEM). However, in doubtful cases the diagnosis should be secured by measuring the cilia beat frequency (CBF) and cilia beat pattern (CBP). For this the most reliable technique is to culture airway epithelial cells and after re-growth of the cilia perform CBF and CBP. Using this method all secondary abnormalities can be excluded while all primary abnormalities, both structural and functional are revealed, thus, PCD may be differentiated from acquired cilia dyskinesia (ACD). The long term aim of this study is to find a reliable method of culturing human (ciliated) epithelial cells. In the present study primary cultures of ciliated tracheal epithelial from mouse were cultured on collagen coated semipermeable membranes at an air-liquid interface. After 2 weeks cells were harvested, identified as epithelia by TEM and were found to be bereft of cilia. After 4 weeks of culture cilia had not yet regenerated. In the next phase of the work it is hoped to increase the culture time, regenerate the cilia and carry out CBF and CBP analysis co-related with ultrastructure. Delegate 3 - Defocus Contrast Image of Hexagonally-Ordered Mesoporous Material. Tanner, D. A.1,2, Nakahara, S.1,3, Hudson, S.1,4, Magner, E.1,4, Redington, W.1 and Hodnett, K.1,4 1. Materials & Surface Science Institute, 2. Department of Manufacturing and Operations Engineering, 3. Department of Physics, & 4. Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland. [email protected] A wide variety of mesoporous silica materials have been explored in recent years for possible applications in drug delivery, catalysis and sensors. Most recently, we characterized a hexagonally-ordered mesoporous silica material using conventional transmission electron microscopy (TEM) [1]. This material contained three characteristic image features described as wide/narrow parallel lines and hexagonal honeycomb structures. These images show very weak amplitude contrast at zero focus ( ∆f ≈ 0 ) but exhibit strong phase contrast by defocusing ( ∆f ≠ 0 ). These image features, therefore, were found to vary sensitively with 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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the sign/amount of defocus, ∆f . To further understand the behaviour of these images with focussing, we computer-simulated the images of the wide/narrow parallel lines using the defocus contrast theory [2,3]. In the present theory, we considered only the effect of defocusing on the images, thus ignoring the effect of spherical and chromatic aberrations. With this assumption, the wave function, ψ D ( x) , at the exit of a specimen can be simplified to yield in a one-dimensional integral form as: (1 − i ) K exp i∆fK ∞ exp iφ ( x ') exp  iK x − x ' 2  dx ' ψ D ( x) = ( )∫ [ ) ]  ( π∆f 2  2 ∆f  −∞ The phase shift, φ , can be expressed in terms of the mean inner potential, V0 , of a material and its thickness, t, along the electron-beam direction as φ = π tV0 / λ E . A structural model for this image simulation was made for two projections along the symmetry directions, which give rise to images described as wide and narrow parallel lines. References [1] S. Hudson, D. A. Tanner, W. Redington, E. Magner, K. Hodnett, and S. Nakahara, Phys. Chem. Chem. Phys. 8 (2006) p. 3467. [2] K. Fukushima, H. Kawakatsu, and A. Fukami, J. Phys. D: Appl. Phys. 7, (1974) p. 257. [3] S. Nakahara, J. Electrochem. Soc. 129 (1982) p. 201C. Delegate 4 - Scanning Electron Microscopy on Feeding Appendages of Endemic Shrimp from Lake Tanganyika, Africa. Kamermans, M. and Donohue, I. School of Natural Sciences, Zoology Building, Trinity College, University of Dublin, Dublin 2. [email protected] Our study focuses on elucidating the processes of diversification of the endemic shrimp species from the ancient Lake Tanganyika, which is one of the most species-rich lakes on Earth. The aim of this part of our study is to investigate whether feeding appendage structures have evolved to facilitate dietary selectivity among species. Morphological differentiation of feeding appendages between closely related species would result in diversification and eventually possibly in speciation. To reveal possible morphological adaptations of feeding appendages to certain food sources, both the feeding appendage morphology and dietary preferences were examined. Morphological structures of the feeding appendages were studied by Scanning Electron Microscopy (SEM). SEM photographs indicate substantial morphological diversity among the species studied. As shrimp tissue reflects the consumed food sources over time, Stable Isotope Analysis (SIA) was done on the tissue of a number of shrimp species to examine their feeding preferences. SIA suggested a degree of trophic partitioning, providing indications of potentially specialised diets among the different shrimp species. There was, however, also considerable overlap in consumed food sources among the species, suggestive of more generalist feeding strategies. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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Results from both the SEM and SIA work will be discussed in the context of the phylogenetic relatedness of the Tanganyikan shrimp. The phylogeny provides indications of the evolutionary history of the feeding appendage structures. More knowledge about the morphology of the feeding appendages within this group of shrimp species will contribute significantly to the understanding of diversification within this shrimp group, and therefore evolutionary processes in general. Delegate 5 - An Investigation into the Bioactivity of Porous Silicon Nanosponge Particles. Chadwick, E.1,2, Clarkin, O.1,2 and Tanner, D. A.1,2 1. Materials & Surface Science Institute, & 2. Department of Manufacturing and Operations Engineering; University of Limerick, Limerick, Ireland. [email protected] A ‘biomaterial’ is a non-living material used in a medical device which is intended to interact with biological systems[1]. A prerequisite of any biomaterial is the formation or growth of hydroxyapatite on the surface of the material once in contact with body fluids[2]. Many studies into hydroxyapatite growth on various materials when in contact with simulated body fluid (SBF) are currently being assessed. These studies not only establish a general method for determining the bioactivity of that material but coincidently lead to designing of new biocompatible materials for applications such as bone substitutes and grafts or tissue engineered scaffolds. Porous structures, in general, have been found to permit fast deposition of HA layers with strong bonds to the surface and good osteointegration when implanted[3]. In 1995, Canham first introduced the idea of porous silicon as a biomaterial with a range of experiments involving microporous silicon and SBF and ultimately proved that the microporous films of silicon could induce hydroxyapatite growth both on themselves and neighbouring areas of bulk silicon[1]. In this particular study, porous silicon nanosponge particles of different grades are being tested for their ability to form an apatite layer on the surface of the particles with the SEM EDX results indicating the relative calcium and phosphate peaks indicative of hydroxyapatite growth. Acknowledgement: The authors would like to thank Paula Olsthoorn at the MSSI for useful discussions and continuous support during this work. [1] Canham, L.T., Bioactive silicon structure fabrication through nanoetching techniques. ADVANCED MATERIALS-DEERFIELD BEACH-, 1995. 7: p. 1033-1037. [2] Pastor, E.M., E. Parkhutik, V. Curiel-Esparza, J. Millan, M.C., Influence of porous silicon oxidation on its behaviour in simulated body fluid. phys.stat.sol. (c) 4, No. 6, 2136-2140, 2007. [3] Pramatarova, L., et al., Porous silicon as a substrate for hydroxyapatite growth. Vacuum, 2004. 76(2): p. 135-138.

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Delegate 6 - The Use of the Polarizing Microscope by Victorian Microscopists. Lowry, S.F. School of Biological Sciences, University of Ulster, Cromore Road, Coleraine, BT52 1SA [email protected] We tend to think of the Victorian era as drab and austere. This is, however, far from true and the art of the era was bright and vibrant. This quest for vibrancy and colour was also evident in the collections of many amateur microscopists and in the popular exhibitions of projected microscopical specimens. Specimens were collected for their intrinsic beauty as well as out of scientific interest. The strict delineation between artistic and scientific endeavours which we encounter today was not a factor in the minds of the Victorians and they were open to viewing scientific objects as works of art. Many of the wealthy professional gentlemen of the time were the proud owners a microscope and would show off their latest specimens as after dinner entertainment. The polarising microscope or ‘polariscope’, which had been invented in 1811, led to a marked increase in these pursuits. An industry sprung up to supply these treasures and both microscopes and prepared microscope slides were retailed, and in many cases produced, by opticians. Notable among these were Dancer, Cole, Enoch and Topping. Many substances have the ability to rotate the plane of polarised light and hence become visible between crossed polarising filters. The colours of these specimens are enhanced by the use of the polarizing microscope or ‘polariscope’. The colours produced can be further enhanced by the use of wave retarding filters. The filters used by Victorian microscopists consisted of monoclinic crystals of gypsum (calcium sulphate) and were referred to as selenites. The most commonly used was the red selenite or ‘sensitive tint’ filter. This produced a delicate rose red background effect as well as modifying the colours of the actual specimen. Other selenites in common use created green or blue backgrounds. consisted of monoclinic crystals of gypsum (calcium sulphate) and were referred to as selenites. The most commonly used was the red selenite or ‘sensitive tint’ filter. This produced a delicate rose red background effect as well as modifying the colours of the actual specimen. Other selenites in common use created green or blue backgrounds. Delegate 7 - Nanostructural Origin of Hierarchical Organization in Bone: a Microscopic Study. Zhang, Y.1, Piterina, A. V.1,2, Dickinson, C.1 and Tofail, S. A. M.1 1. Materials and Surface Science Institute (MSSI), University of Limerick. 2 . Centre for Applied Biomedical Research (CABER), University of Limerick. [email protected] The building blocks of bone vary in dimensions from a few nm to tens of cm and form a complex, hierarchical structure. Microscopic properties, such as microporous structures with open and closed pore systems, modelling and remodelling and the mechanical 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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behaviour of bone are believed to be a consequence of its hierarchical organisation. While much is known about the behaviour of bone at the macroscopic scale, its behaviour at micro and nano length scales is less understood and is an active area of research. In this study, we combined microscopic, nanoscopic and spectroscopic techniques to characterise the hierarchical organisation of bone (Fig 1a). For this purpose, EDA/ formic acid extractions from fresh bovine femoral compact bone were performed to remove one of the structural units: the inorganic matrix (apatitic, Fig 1b,d) and the organic matrix (collagenous, Fig 1c,e). The observation of the structural alteration in the total morphology of bone, as a result of the loss of building blocks (apatite or collagen), was made by Laser Scanning Confocal Microscopy (LSCM). The surface morphology of the residues was studied and the amount of apatite and collagen residues was determined by a range of analytical techniques, e.g. FESEM, Micro Raman spectroscopy and FT-IR Spectroscopy. The extraction treatment did not effect the hierarchical organisation and a macroscopic, rigid entity was maintained both in the demineralised (collagen) and the de-proteinated (apatite) states. In addition, electron diffraction and TEM revealed the defects in the nanostructure of the apatite (Fig 1f).

Figure 1: Compact bone organisation (a-e) as seen in confocal microscopy: (a) Raw bone with 100% collagen and apatite (b) 100% Apatite (c) 100% Collagen (d) Organic matrix with apatite residues (e) Inorganic matrix with collagen residues (f) TEM image of textured apatite extracted from bone.

Delegate 8 - Microstructural Analysis of Pt/VACNT Nanostructured Electrodes. Soin, N., Roy S.S. and McLaughlin J.A.D. NIBEC, University of Ulster, Shore Road, BT37 0QB, Northern Ireland. [email protected] We have deposited platinum (Pt) nanoparticles in the size range 3-5 nm over vertically aligned carbon nanotube (VACNT) arrays for Polymer Electrolyte Membrane (PEM) fuel cell electrodes via a DC sputtering system. Interaction of graphitic surfaces with noble metals is very weak and in order to obtain a homogeneous dispersion of electrocatalyst particles it is necessary to understand their formation mechanism. The Pt/VACNT nanostructures were characterized using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) among other techniques. The length of VACNT arrays was ~ 20 μm with a diameter of 60-80 nm (Fig. 1a) as observed in the SEM. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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TEM analysis showed variable particle density along nanotubes length with cluster formation near tip ends closest to the plasma while discrete particles were obtained towards the tube ends (Figs. 1b and 1c). Energetic particle induced defects are maximum on tip ends nearest to plasma and hence the observed high density of Pt particles, along with structural damage to tips. Large difference between cohesive energy and adsorption enthalpy, along with the mobility of transition metal atoms promotes cluster formation observed towards middle length of CNTs (Fig. 1b inset). XPS analysis was used to evaluate the chemical changes post Pt deposition. Raman analysis showed an increase in the ID/IG ratio from 0.97 to 1.07 further corroborating the structural changes observed by TEM. X-ray diffraction study provided an average Pt particle size of 4.7 nm matching well with size obtained from TEM images. Above studies confirm well dispersed Pt nanoparticle distribution over VACNTs which can be further used as fuel cell electrodes.

a)

b)

c)

20 nm Fig. 1a) SEM image of Pt/VACNT array, b) TEM images of Pt covered tip, inset shows clusters towards middle length, c) individual Pt nanoparticles towards the end of CNT.

Delegate 9 - Microscopy Investigations of Surface Engineered Coatings Prepared by Nitrocarburising. Molloy, D. A., Malinov, S. and Crudden, D. School of Mechanical and Aerospace Engineering, Queen’s University of Belfast, Ashby Building, Stranmillis Road, Belfast, BT9 5AH, UK. [email protected] Microcopy techniques where used to investigate the effect of secondary heating on the microstructure and composition of surface engineered coatings. Nitrocarburising was applied to mild steel substrates. The coated samples were reheated using two heating methods, namely continuous and isothermal heating to temperatures of 500°C and 750°C, respectively. Optical and scanning electron microscopies were used to investigate the microand nanostructure of the heat treated coatings. In addition energy dispersive X-ray analysis was applied to investigate the local composition changes and phase transformations taking place during secondary heating. The microscopy analysis showed that the secondary heating caused diffusion within the coating itself. This diffusion redistribution caused a number of phase transformations and changes in the surface layers. Formations of new sub-layers at 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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different conditions were observed. The compound layer in the nitrocarburised samples was found to be destroyed at temperatures above 750°C. Delegate 10 - Comparison of Efficacy of Several Microscopic Techniques in Characterisation of Structure and Composition of Highly Dispersed Sludges. Piterina, A. V. 1,2, Barlett, C. D 3, and Pembroke, J.T. 1 1. Department of Chemical and Environmental Sciences, Material and Surface Science Institute, University of Limerick, Limerick, Ireland. 2. Centre for Applied Biomedical Research (CABER), University of Limerick, Limerick, Ireland. 3. Centre for Sustainability, Sligo Institute of Technology, Sligo, Ireland. [email protected] Aerobic thermophilic digestion (ATAD) is one of the most advanced technologies for the tertiary treatment of wastewaters and produces a pathogen-free effluent. However, produced sludge is highly dispersed with poor settling and low compaction capacities. This alterations in the quality of the final effluent influenced the economy of the process is disposal. Several studies have shown that the settling and the compaction properties of the sludge are directly related to floc structures and their chemical, physical and biological constituents. Imaging techniques have been extensively used to evaluate morphological properties of sludge within various types of bioengineering system and bioreactor and serve traditional monitoring tools for monitoring of a treatment succession and failures. In present study, morphological parameters and structural components (EPS, bacteria, inert organic matter) of ATAD sludge were obtained with application of several microscopic techniques (electron, conventional phase-contrast optical, laser scanning confocal microscopy). In addition, optimal procedure for sample preparation for such analysis has been developed in order to achieve maximum information on this unique thermophilic habitat. Delegate 11 - Analysis of the Interaction between Human A549 cells and Silica Nanoparticles Using Electron Microscopy. Fenaroli, F.1, Salvati, A.1, Lynch, I.1, Cottell, D.2 and Dawson, K1. 1. Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4. 2. Electron Microscopy Laboratory, University College Dublin, Belfield, Dublin 4. [email protected] The importance of understanding the interactions between nanoscale objects and biological systems has become very significant in recent years. As a result of their small size, nanoparticles can theoretically enter almost everywhere in cells, and a deep understanding of the mechanisms of their interactions with cells would open up new opportunities in nanomedicine and nanodiagnostics.

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Our aim in this work is to try to understand the mechanisms by which nanoparticles are taken up by cells, and how they are processed by the cellular endocytic machinery. As a model system we chose fluorescently labelled silica nanoparticles (50 and 100nm) and our study focuses on their uptake and trafficking in lung tumoral epithelial (A549) cells. For this purpose we used a combination of different techniques, including fluorescence microscopy, flow cytometry and electron microscopy. The results obtained with the different techniques have been integrated and compared. In particular EM analysis of ultrathin sections, unlike conventional light microscopy, allows us to follow the uptake of nanoparticles from the very early events, and to visualise the exact localisation of single nanoparticles in acell. The results suggest a rapid internalisation of nanoparticles through vesicles (within 10 minutes). Increasing the time of exposure results in a larger amount of nanoparticles accumulated in the cells. A higher concentration of nanoparticles in the cell medium also results in a higher number of nanoparticles accumulating inside the cell. The combined data obtained from electron microscopy and fluorescence microscopy with antibody staining suggests that the final localisation of the silica nanoparticles seems to be in the lysosomal compartment. Delegate 12 - Strain Determination Using Convergent Beam Electron Diffraction (CBED). Mogili, V.1,2, Tanner, D. A.1,2 and Nakahara, S.1,3 1. Materials & Surface Science Institute, 2. Department of Manufacturing and Operations Engineering, & 3. Department of Physics University of Limerick, Limerick, Ireland. [email protected] Since the 1970’s, by following the now famous prediction known as Moore’s law (Gordon E. Moore – Co-Founder of Intel), the number of transistors on a single integrated circuit doubles approximately every 18 months. As a result, in semiconductor technologies, the complexity of devices continues to increase with the push towards smaller feature sizes. This leads to strain that comes from the incorporation of a wide range of dissimilar materials [1], and also in the formation of dislocations and stacking faults that cause degradation in electronic performance [2]. Hence the experimental measurement of stress and strain in materials at nanometer resolution with high-sensitivity has become a crucial issue [3]. CBED is regarded as a sensitive and complicated technique within Transmission Electron Microscopy (TEM) analysis [4]. It provides information from an area as small as 10-100 nm 2 with an accuracy of 0.2% which is an optimal value for strain analysis [5]. The main limitations of the technique are imposed by specimen preparation and complexity in interpreting the results. To ensure precise interpretation of the results in this project, CBED is currently being undertaken on a TEM calibration sample known as MAG*I*CAL©. This sample has been accurately manufactured to have 10nm alternating layers of silicon and a silicon-germanium alloy. CBED for foil thickness measurements and zone axis patterns for symmetry analysis have been obtained so far (see Figs. 1 and 2). An accurate determination of the TEM voltage and a precision measurement of the strain present in silicon/silicongermanium layers are currently being undertaken. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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I would like to thank Dr. Calum Dickinson for useful discussions and continuous support during the course of this study.

Fig.-1.

Fig.-2.

References: [1] A. Steegen and K. Maex, Materials Science & Engineering R-Reports 38, 1 (2002) [2] L. F. Tz. Kwakman, D. Delille, M. Mermoux, A. Crisci, and G. Lucazeau, ALTECH, Paris (2003). [3] J.M.Zuo, Ultramicroscopy 41(1992) 211-223 [4] DB Williams and CB Carter, Transmission Electron Microscopy: A Textbook for Materials Science. Plenum Press, N.Y (1996). [5] J. Spence and J.Zuo, Electron Microdiffraction, Plenum Press, New York, (1992). Delegate 13 - Identification of Signalling Events Involved in Radiation Induced Bystander Effects. Lyng F.M.1, Howe O.1 and McClean B.2 1. Radiation and Environmental Science Centre (RESC), Dublin Institute of Technology, Dublin 8. 2. St Luke’s Hospital, Rathgar, Dublin. [email protected] It is now well accepted that radiation induced bystander effects can occur in cells exposed to medium from irradiated cells. The study of the signaling events involved in these bystander effects will help in the understanding of the life or death response of the bystander cells. Signalling pathways leading to apoptosis, such as calcium, MAP kinase, mitochondrial and reactive oxygen species (ROS) signalling have been shown previously. The aim of this study was to follow the bystander cells in real time following addition of medium from irradiated cells and to elucidate the sequence of events in the responding cells. Human keratinocyte cells (HaCaT cells) were irradiated using a Cobalt-60 teletherapy source. One hour after irradiation the medium was harvested and filtered. This irradiated cell conditioned medium (ICCM) was then added to parallel HaCaT cells. Cell signalling events were followed in real time in living cells using fluorescent probes. The sequence of very early signaling events was determined in the responding bystander cells. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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Delegate 14 - Development of Nanoscale Temperature Responsive Culture Surfaces for Cell Culture and Recovery. Nash, M.1,2, Carroll, W.1, O’ Connell, C.3 and Rochev, Y.2 1. School of Chemistry, National University of Ireland, Galway, Ireland. 2. National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. 3. National Centre for Laser Applications, National University of Ireland, Galway, Ireland. [email protected] Traditional methodologies employed for cell harvesting using proteolytic enzymes degrade cell surface receptors, consequently impairing subsequent function. In aqueous solution poly-N-isopropylacrylimide has a lower critical solution temperature of 32°C. It is this phenomenon that is exploited in temperature controlled cell harvesting. It has been shown that pNIPAm surfaces are generally non-conducive to reasonable cell growth. This issue has been overcome using two different approaches; Okano et al. have grafted pNIPAm onto tissue culture plastic using electron beam polymerisation to yield an ultra-thin layer of pNIPAm. It is hypothesised that a low concentration of grafted polymer density allows for seeded cells to interact with the underlying substratum. Alternatively, the addition of an over-layer of cell adhesion promoting proteins onto a pNIPAm film negates pNIPAm’s adverse effect on cell adhesion. This body of work seeks to offer a more simple and economic protocol to fabricate pNIPAm coatings for cell harvesting. We propose that coating thickness has a significant bearing on how well cells adhere and proliferate. This research focused on the biocompatibility of coatings deposited using two methods; the solvent casting technique was used to yield films of 4-5 µm thickness and spin coating film deposition to yield sub-micron thick films. Since film thickness seems to have an effect on cell response, it is imperative that this parameter can be evaluated. Sub-micron film thickness cannot be evaluated trivially and a system of thin film appraisal was developed. AFM, SEM-3D-MeXTM and profilometry analysis estimated the film thickness of the spin coated films to be approximately 100 nm. Cells were seeded on the fabricated films and cell growth was monitored using phase contrast microscopy. Cell detachment was optically assessed using time-lapse microscopy and phase contrast microscopy. Delegate 15 - Image Analysis/Interpretation Framework for Studying the Kinetics of AntiApoptotic Response of Cancer Cells to Proteasome Inhibition Therapy. Bernas, T.1,2, Ramapathiran, L.1, Duessmann, H.1, Rehm, M.1, Concannon, C. G.1 and Prehn, J. H. M.1 1. Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland. 2. Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland. [email protected]

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Proteasome inhibitors (PI) are approved by FDA as treatment agents for a number of malignancies. However, efficiency of PI may be affected by their simultaneous activation of pro-survival and pro-apoptotic signalling pathways. Previous studies in our laboratory demonstrated induction by PI of several cell survival regulators, including pro-apoptotic BH3-only protein, PUMA, and pro-survival Hsp70. It should be noted that the acquisition of cellular resistance to chemotherapeutic regimes is a major bottleneck in cancer therapy. Therefore, we used high throughput microscopy to monitor increase of GFP-tagged Hsp70 on cellular level in large populations of cells subjected to several kinds of treatments. In order to implement this task we developed an adaptative algorithm for segmentation of cells in time-lapse microscopy images. The images of cells isolated in single time frames were linked using a linear assignment problem approach to provide cell tracking. Kinetics of Hsp70 increase was represented with a set of orthonormal polynomials. Simultaneously cell viability was monitored. The relationship between the kinetics, cell death and treatment was established using linear discriminant analysis. Application of this integrated imaging, processing and analysis workflow made it possible to address several biological questions regarding cellular response to PI in qualitative and quantitative way. Delegate 16 - Border Patrol: a Fundamental Study of Interface Formation in Polymer Nanocomposites using Nano-Thermal Analysis (nanoTA). Armstrong, G.1, Dickinson, C. 1, Ruether, M.2 and Walton, D.3 1. Materials & Surface Science Institute, University of Limerick, Castletroy, Co. Limerick. 2. School of Chemistry, Trinity College Dublin, College Green, Dublin 2. 3. CMB Science Ltd, Technocentre, Coventry University, Puma Way, Coventry CV1 2TT, UK. [email protected] Carbon nanotube (CNT)/polymer composites have received much attention for their remarkable physical properties, but their large-scale use in industry has yet to be achieved. Typically, CNTs are chemically modified (“functionalised”) to render them compatible with polymers. Good nanotube/polymer interaction is vital to achieve the improvements in mechanical, thermal and electrical properties expected of nanocomposites in engineering and electronics applications. In this study, a novel atomic force microscopy (AFM) technique - nano-thermal analysis (nanoTA) – was applied to examine polymer/CNT interaction in model nanocomposites consisting of functionalised multi-walled carbon nanotubes (MWNT) dispersed in a polymer thin film. During nanoTA experiments, the sample was imaged using a combination of tapping mode and high-temperature AFM (HT-AFM) to locate the polymer/MWNT interface. Heat was then applied locally to the area of interest via the probe tip and the local thermomechanical response measured. These experiments allowed the different domains present within the nanocomposite to be identified and provided information on changes in thermal transitions relative to the bulk polymer, degree of crystallisation and changes in thermal conductivity and mechanical properties within the sample.

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It is expected that the Border Patrol study will provide new insights into the nature of polymer/CNT interface formation and how best to functionalise nanotubes to achieve optimal nanocomposite properties that meet specific application requirements. Acknowledgement: This work was conducted under the framework of the INSPIRE programme, funded by the Irish Government's Programme for Research in Third Level Institutions, Cycle 4, National Development Plan 2007-2013. Delegate 17 - TEM Analysis of Epithelial Junction Structure in the Normal Margins and Stricture Sites of Ileo-Caecal Resections for Fibrostenosing Crohn’s Disease. Cunningham, M. F.1, Docherty, N. G.1, Toivonen, T.2, Cottell, D. C.2 and O'Connell, P. R.1 1. UCD Conway Institute of Biomolecular and Biomedical Sciences UCD School of Medicine and Medical Sciences and Surgical Professorial Unit St. Vincent’s University Hospital, Dublin 4. 2. Electron Microscopy Laboratory, University College Dublin, Dublin 4, Ireland. [email protected] Introduction: Upwards of 1/3 of patients with Crohn’s Disease develop fibrostenosing disease (FCD) characterised by intestinal stricture formation. Intestinal resection is rarely curative and recurrence is most commonly encountered proximal to the site of anastomosis. The impact of the length and disease status of the resection margin on recurrence rates remains a topic of debate in the surgical management of FCD. Impairment of epithelial barrier function as a result of ultrastructural deterioration of epithelial junctions may be of importance in the development of recurrent lesions. Whether the morphometric features of junctional complexes in “normal margins” are disrupted has not previously been investigated. Aim: To quantify tight junction and adherens junction integrity at the internal stricture site and normal margins of resected terminal ileum from patients with FCD. Methods: Transmural biopsies from the terminal ileum of three FCD patients undergoing ileo-caecal resection for FCD were fixed and processed for TEM quantification of the epithelial junctional length and diameter. A total of 5 junctional complexes from 3 distinct mid crypt regions were measured for each normal adjacent (Nadj) and strictured (S) region of all specimens. Results: In all samples from S sites, morphometric assessment of tight and adherens junctions revealed striking disruptions in their structural integrity. In all corresponding Nadj segments studied, measurements of tight junction and adherens junction structure were within a range of values coherent with normal structure. Conclusion: In this small study, ultrastructural quantification of epithelial junctional complexes from “normal” resection margins demonstrates evidence of intact junctional structure. This suggests that there is no preexisiting mucosal epithelial barrier disruption proximal to the anastomotic site at the time of surgery.

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Delegate 18 - Functional Autologous Tissue-Engineered Vascular Grafts in the Systemic Circulation: A Microscopic Analysis. Koch, S.1, Flanagan, T.C.1,2, Sachweh, J.S.3, Tschoeke, B.1, Tanios, F.1, Schnoering, H.3, Tolba, R.H.4, Schmitz-Rode, T.1, Jockenhoevel, S.1 1. Department of Applied Medical Engineering, Helmholtz Institute for Biomedical Engineering, Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany. 2. School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, Belfield, Dublin, Ireland. 3. Department of Paediatric Cardiac Surgery, University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany. 4. Institute for Laboratory Animal Research, University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany. [email protected] Introduction: Tissue engineering approaches are being investigated to construct living, autologous implantable cardiovascular structures, which have a post-implantation capacity for growth and remodeling. Our group is focusing on the development of implantable autologous cardiovascular prostheses, by combining biodegradable scaffolds with an autologous fibrin cell carrier material. The current study reports the pre-clinical application of fibrin-based vascular grafts in a large animal model. Method: For the construction of vascular grafts, an autologous mixed cell population was expanded from ovine carotid artery. Vascular grafts were cast in customized moulds by combining a cell/fibrinogen suspension (10x106 cells/ml) with a thrombin/CaCl2 solution to initiate polymerization and cell encapsulation around a supporting mesh. The constructs were subsequently conditioned for 3-4 weeks in vitro in a bioreactor system (pulsatile perfusion). Vascular grafts (n=6) were then surgically interposed in the carotid artery. Tissue structure and remodeling were examined in all constructs after 1, 3 and 6 months in vivo using microscopical techniques. Results: Implanted grafts remained patent and exhibited sufficient mechanical properties for periods of at least 6 months in vivo. Histological staining demonstrated excellent tissue development within the grafts, with no evidence for calcification. Remodeling of the grafts occurred post-implantation, with the deposition of extracellular matrix proteins, such as type I collagen, and resorption of the initial fibrin scaffold components. Scanning electron microscopy demonstrated a confluent layer of endothelial cells on the blood-contacting luminal surface of the grafts, while transmission electron microscopy demonstrated viable cells and mature collagen bundles throughout the tissue. Discussion: Small-calibre vascular grafts based on a fibrin gel scaffold can remain intact and patent in the systemic circulation for at least 6 months, and undergo extensive remodeling in vivo. Conclusion: Fibrin is a promising autologous scaffold material for the development of implantable grafts for the replacement of diseased blood vessels. This multi-centre research project was financially supported by the European Union Sixth Framework Programme (Project STRP 013633, BioSys). 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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Abstracts – Poster Sessions Life Sciences Posters Poster 1 - A New Dimension in Study of Living Cells by Raman Spectroscopy. Bonnier, F., Meade, A. D., Knief, P., Bhattacharya, K., Lyng, F. M. and Byrne H. J. Focas Research Institute, Dublin Institute of Technology (DIT), Kevin Street, Dublin 8, Ireland. [email protected] The study of single cells with infrared and Raman spectroscopy is an emergent topic in the field of “biospectroscopy” [1]. The main advantage of Raman spectroscopy is the weak signal from water, which offers the potential to record spectra from living cells using an immersion lens. Normally, the confocality of the Raman microspectrometer reduces the spectral contribution of the optical substrates that are used during measurements on optically thin single cells. Until now, quartz has been preferred due to its weak signal but also for its surface properties which allow cells to attach and grow, in comparison to CaF2 or ZnSe [2]. Nevertheless, dissimilarities between different optical substrates within in vivo cell culture result in morphological and functional changes in the cell [3]. In order to create an experimental model closer to the real conditions encountered by the cell in vivo, 3-D collagen gels have been used as a substrate for spectroscopic study of living cells. In this paper we demonstrate the advantages and potential of such a model for Raman spectroscopy using 785nm as source. The Alamar Blue fluorescence assay demonstrated an increase in cell viability of up to 20% of A549 and HaCaT cells in the 3-D collagen matrix in comparison to quartz windows. An additional advantage of the use of collagen gel instead of quartz windows is a significant improvement in multivariate data analysis. We have observed through the measurement of various Z-profiles through various cells that collagen gels give the same background spectral contribution regardless of Z position, making background removal in data pretreatment more efficient. The density of the collagen gel appears too low to contribute significantly to the spectra recorded, with the result that it is rendered invisible spectrally. Thus the background contributions are reduced to that of the water based solution (medium/NaCl) used to keep the cells alive during the experiments. References [1] R. J. Swain and M. M. Stevens, Biochemical Society transactions 35(Pt 3), 544-549 (2007). [2] F. Draux, P. Jeannesson, A. Beljebbar, A. Tfayli, N. Fourre, M. Manfait, J. Sule-Suso and G. D. Sockalingum, The Analyst 134(3), 542-548 (2009). [3] A. D. Meade, F. M. Lyng, P. Knief and H. J. Byrne, Analytical and bioanalytical chemistry 387(5), 1717-1728 (2007).

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Poster 2 - Raman and FTIR Imaging of Prostate Cancer – a Correlation to Common Diagnostic Biomarker. Poon, K. W. C.1, Howe, O.1, Byrne, H. J.2 and Lyng, F.M.1 1. Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Camden Row, Dublin 8, Dublin, Republic of Ireland. 2. FOCAS Institute, Dublin Institute of Technology, Camden Row, Dublin 8, Dublin, Republic of Ireland. [email protected] Annually, over 670,000 men are diagnosed with prostate cancer worldwide, accounting for approximately one quarter of all diagnosed cancer cases in men[1]. The advent of the PSA (Prostate Specific Antigen) blood test has revolutionised early detection of prostate cancer, however, it is unable to distinguish between benign growths and aggressive cancerous forms. Definitive diagnosis is only possible through a biopsy of the prostate, from which a skilled histopathologist is required to observe changes related to tissue/cell ultrastructure based on the universally accepted Gleason tumour grading system. This system focuses on a variety of complex transformations in the prostate glandular morphology known to occur during the progression of the cancer. However, Gleason scoring suffers from intra/inter observer subjectivity, with studies showing 70.8% of prostate cancer biopsies being misgraded [2]. Raman spectroscopy has been shown to discriminate between normal and pathological tissue samples initially processed by formalin fixation and paraffin embedding (FFPE)[3]. As tissue samples sourced from hospitals and medical institutions use FFPE as the standard processing technique, it is important to correlate spectroscopic studies with such protocols. Results will be presented regarding the capability of Raman spectroscopy to distinguish between FFPE tissue sections of normal, benign prostate hyperplasia (BPH) and prostate cancer in combination with multivariate analysis. The results will also be correlated with various immunohistochemical stains such as AMACR (α-methylacyl-CoA racemase), PSA (Prostate Specific Aantigen) and their relationship to existing data regarding aberrant G2 radiosensitivities in patients.[4] References [1] Cancer Research UK CancerStats Key Facts: Prostate Cancer. http://info.cancerresearchuk.org/cancerstats/types/prostate/ (25/05/2009). [2] Lattouf, J.-B.; Saad, F., Gleason, BJU International, 90, 694-698 (2002). [3] Lyng, F. M.; Faoláin, E. Ó.; Conroy, J., et al., Experimental and Molecular Pathology, 82, 121-129, (2007). [4] Howe, O.; O'Malley, K.; Lavin, M., et al., Radiation Research, 164, 627-634, (2005).

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Poster 3 - Microstructure Analysis of Process Cheese by Using Cryo-Scanning Electron Microscopy and Light Microscopy. El-Bakry, M., Duggan, E., O’Riordan, E.D. and O’Sullivan, M. UCD Institute of Food and Health, Belfield, Dublin 4. [email protected] Important functional properties of process cheese (texture and meltability) are known to be influenced by microstructure, especially fat globule size and distribution which in turn is affected by the emulsifying salts (ES) used in manufacture. This study examines the effect of reducing ES and NaCl on the microstructure and functionality of process cheese. Process cheese (50% moisture) was manufactured by mixing water, vegetable oil, rennet casein, NaCl and ES at 50°C, heating to 80°C until a homogeneous mass was formed followed by citric acid addition. The standard formulation had 1.5% NaCl and 1.4% ES. Cheese microstructure was assessed by cryo-scanning electron microscopy (cryo-SEM) and light microscopy (LM) in conjunction with ImageJ image analysis software. Cryo-SEM was done by sublimating (-85°C/5min) and gold coating (-180°C/2min) the cryofixed cheese blocks, which were subsequently examined at an accelerating voltage of 10 kV and a magnification of 5007500×. In LM, 8µm cryosections were sequentially stained for fat (red) and protein (green) by immersion in oil red 0 (30 seconds) and fast green (1 min) prior to examination by 10×, 20× and 40× lens. Cryo-SEM micrographs showed smooth surfaced spherical fat globules which were uniformly distributed in a continuous protein matrix. LM images were consistent with those of cryo-SEM. In comparison to the standard formulation, the average fat globule size systematically decreased from 18 to 8µm and increased to 29µm, at 40% reduced ES and 0% NaCl respectively. The fat globule size was highly correlated with hardness (R2=-0.9), as measured by textural analysis, and empirically determined meltability (R2=+0.9). Thus both LM and cryo-SEM were very useful techniques to assess the fat globule size and hence to predict the process cheese texture and meltability at various formulations. Poster 4 - Carbon Sources Affect the Growth and Development of Burkholderia cepacia Complex (Bcc) Biofilms Cultured in Continuous Flow Cells. Kennedy, S.1, Heller, S.2, Eberl, L.2, Caraher, E.1 1. Centre for Microbial Host Interactions (CMHI), Institute of Technology Tallaght, Dublin. 2. Department of Microbiology, Institute of Plant Biology, University of Zürich, Switzerland. [email protected] Biofilms are microbial communities that irreversibly attach to innate and living surfaces such as living tissue, medical devices, water treatment facilities or each other. They are embedded in a matrix of extracellular polymeric substances and are inherently resistant to antibiotic therapies and are associated with the chronic nature of some infections for example Cystic Fibrosis (CF). The Burkholderia cepacia complex (Bcc) is a group of gram negative bacteria comprised of at least 17 species that cause infections in CF patients. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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These Bcc strains can grow as biofilms within the CF lung which may lead to increased resistance to antibiotics and decreased protection from host defences. The objective of this study was to determine the effects of different carbon sources, present in media, on the growth and development of Bcc biofilms cultured in flow cells. Analysis was carried out using confocal laser scanning microscopy. Biofilms of two fluorescently labelled Bcc strains, B. cenocepacia H111:pBBR,gfp and B. vietnamensis LMG 10929:gfp,B were grown over three to four days in flow cells at a flow rate of 0.5 rpm. Each strain was grown in either Modified FAB medium supplemented with 0.1 mM Sodium Citrate, 0.1 mM D-Xylose or 0.1 mM Malic Acid or diluted (1:20) Luria Bertani (LB) broth. It was found that B .cenocepacia H111:pBBR,gfp formed biofilms regardless of carbon source, with “cauliflower” structures forming in Modified FAB media with 0.1mM Sodium Citrate and D-xylose and diluted LB (1:20). However, B. vietnamensis LMG10929:gfp did not form biofilms with Sodium Citrate, Malic Acid or D-xylose. Microcolonies did form in diluted LB. In conclusion, these findings suggest that selection of media and carbon source for the optimum growth and development of Bcc biofilms is a strain dependent process. Poster 5 - Using TEM Analysis of Primary Cilia in C. elegans to Understand the Molecular Basis of Ciliary Disease (Human) Gene Function. Kida, K.1, Toivonen, T.2, Cottell, D.2 and Blacque, O. E.1 1. UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. 2. The Electron Microscopy Laboratory, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. [email protected] Our work is focused on the study of cilia in the model organism Caenorhabditis elegans. Primary cilia are microtubule-based organelles responsible for chemo-, mechano- or photo sensing, as well as developmental signaling in mammals. Defects in cilium function underlie numerous diseases including polycystic kidney disease and retinitis pigmentosa, as well as multisymptomatic disorders such as Bardet-Biedl syndrome and Joubert Syndrome. In our laboratory, we employ C. elegans sensory cilia as a model system for investigating the molecular basis of human ciliary disease. One approach has been to employ TEM to investigate the ultrastructure of amphid cilia in C. elegans mutant alleles of human ciliary disease gene homologues. Here we will present the methodologies used to perform TEM on C. elegans sensory cilia and discuss the ultrastructural phenotypes that we can observe such as missing or severely shortened axonemes, grossly enlarged cilia, abnormal accumulation of electron dense material within ciliary axonemes, as well as defects in microtubules. We will also demonstrate how such phenotypes can help us to understand the molecular functions of individual ciliary disease genes and how different disease genes functionally interact.

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Poster 6 - Glaucomatous human lamina cribrosa cells show increased lipofuscin accumulation. McElnea, E. M.1,2, Docherty, N. G.1,3, Wallace, D. M.1,2, Quill, B.1,2, Irnaten, M.1,2, Farrell, M.4 and O’Brien, C. J.1,2 1. University College Dublin School of Medicine and Health Sciences, University College Dublin. 2. Institute of Ophthalmology, Mater Misericordiae University Hospital. 3. Department of Physiology, Trinity College Dublin. 4. Neuropathology, Beaumont Hospital, Royal College of Surgeons in Ireland. [email protected] Introduction: Disease associated alterations in the phenotype of glial cells of the lamina cribrosa are implicated in extracellular matrix remodelling of the optic nerve head (ONH) in glaucoma. Mitochondrial dysfunction and oxidative stress may play important roles in the emergence of this altered cellular phenotype. Lipofuscin is an intralysosomal, non-degradable, autofluorescent macromolecule which accumulates in the peri-nuclear region of cells with oxidative stress induced elevations in the rate of mitochondrial turnover. Aim: To compare levels of lipofuscin-like material in lamina cribrosa cells from normal donor eyes (NLC) and from glaucomatous donor eyes (GLC). Methods: Post-confluent cultures of NLC and GLC were examined by TEM and the number and size of peri-nuclear lysososomes per high powered field (x 20,000) recorded. Cells were also stained with Sudan Black B, to assess peri-nuclear lipophilic body number and size. Perinuclear bodies were examined by live cell fluorescence microscopy and cellular autofluorescence quantified using flow cytometry (emission at 563-607nm) Results: The number of peri-nuclear lysosomes was increased in GLC (11.1 +/- 3.8 v 4.2 +/3.7, p = 0.002). A similar observation was made using Sudan Black B staining on number ( 22.10 +/- 3.57 v 13.77 +/- 5.66, p = 0.07) and size of aggregates ( pixel counts-2023.6 +/611.23 v 862.8 +/- 74.23, p= 0.04 ). Perinuclear lysosomes were found to be autofluorescent and an increase in whole cell autofluorescence was observed in GLC. Conclusion: We present evidence supportive of increased lipofuscin formation being a characteristic of lamina cribrosa cells derived from glaucomatous donors. The persistence of this phenomenon in vitro suggests that permanent alterations in mitochondrial function and oxidative stress may be of importance in ONH remodelling in glaucoma. Poster 7 - Cervical Cancer Cell Lines Investigation Using Raman Microspectroscopy. Ostrowska, K.M.1, Malkin, A.2, Meade. A.3, O’Leary J.J.4, Martin C.4, Byrne H. J.3 and Lyng F. M.1 1. RESC, Focas Institute, Dublin Institute of Technology, Dublin, Ireland. 2. School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland. 3. Focas Institute, Dublin Institute of Technology, Dublin, Ireland. 4. Department of Pathology, Coombe Women & Infants University Hospital, Dublin, Ireland. 33rd Annual Symposium, Microscopical Society of Ireland August 26-28, 2009

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[email protected] Over the last years the number of applications of vibrational spectroscopy for biological systems has increased remarkably. Raman spectroscopy shows great potential for the nondestructive analysis of biological samples delivering specific biochemical information. Our study presents results of the investigation of four cervical cancer cell lines using a Raman spectrometer operating at a wavelength of 532nm. The cervical cancer cell lines included: HPV negative C33A cells, SiHa cells - containing 1-2 copies of integrated HPV16, HeLa cells having integrated HPV18 (10-50 copies per cell) and the CaSki cell line containing in the region of 60 - 600 copies of HPV16. An integrated biochemical cell fingerprint of the proteins, nucleic acids, carbohydrates and lipids has been recorded from each cervical cell line. The Raman peak assignments characteristic to each cell line are presented and discussed. A search for potential spectral markers for each cell line has been performed. The differences between characteristic to each cell line spectra were noted mainly in the protein levels, but also in nucleic acid and lipid levels. In order to present highly objective discrimination technique, the multivariate statistical method Principal Component Analysis (PCA) has been employed. The principal component scatter plot and loadings are presented and discussed. PCA shows very good separation of the cervical cancer cell lines indicating HPV concentration dependence. Results obtained in this study validate Raman spectroscopy as a reliable method for investigating biochemical features of biological systems. Poster 8 - Interaction of Quantum Dots with Cellular Systems. Salford, L., Meade, A., Garcia, A., Bonnier, F., Lyng, F.M. and Byrne, H.J. Focas Research Institute, Dublin Institute of Technology, Dublin 8. [email protected] Quantum dots (QDs) are a diverse class of engineered nanomaterials that have great potential for use as imaging, diagnostic and drug-delivery agents because of their intense and photostable fluorescence. Advances in the field of nanotoxicology, however, have recently identified potential risks and hazards associated with QDs exposure. The main purpose of our research is to investigate the capabilities of a synergistic study with different techniques: cytotoxicity assays, confocal microscopy and vibrational spectroscopy. With the combination of these techniques we hope to understand the mechanisms of the QDots interaction with biological systems. Fluorescence and Confocal Microscopy shows that internalization of the QDs occurs within 1 hour exposure and the QDs are possibly retained in lysosomes. This is confirmed by colocalization of green LysoTracker probes and 625QDs. The Lysotracker probes are fluorescent acidotropic probes for labeling acidic organelles in live cells. Using an excitation wavelength of 785 nm we were able to combine two-photon excitation fluorescence emission of QDs (emission at 625 nm) at the anti-Stokes side with the Stokes Raman signal of a single cell.

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This study demonstrates preliminarily the possibility to use QDs as lysosome Raman tags. QDs localize in the lysosomes and due to their intense fluorescence their presence in the cell can be detected with both confocal and Raman microscopy. Raman microscopy offers a means to both localise (image) and study the chemical interaction between the particle and its biological environment. Poster 9 - Dorso-ventral Hippocampal Contributions to Learning in the Rat. Scully, D., Fedriani, R., Murphy, K.J. and Regan, C.M. UCD Conway Institute, University College Dublin. [email protected] Lesion studies have suggested afferent/efferent pathways distributed through the dorsoventral axis of the hippocampus to process task-specific information for long-term memory consolidation. As evidence for neuroplastic and ultrastructural change that accompanies memory consolidation within these distinct pathways is lacking, we investigated the transient increase in hippocampal granule cell neural cell adhesion molecule polysialylation (NCAM PSA) and synaptic density throughout its dorso-ventral axis following training in spatial learning and avoidance conditioning paradigms. Given the rat hippocampus is a Cshaped structure with an overhanging dorsal lip, we sectioned the brain region in two directions. Firstly, we advanced in a rostro-caudal manner using coronal sections. Secondly, using the same brain tissue, we used horizontal sections to proceed in a dorso-ventral direction. These analyses were combined to describe the dorso-ventral distribution of both the neuroplastic marker and ultrastructural synaptic density following two independent learning paradigms. Inter-animal consistency was maintained by defining the rostro-caudal and dorso-ventral co-ordinates by reference to bregma as described in a rat brain atlas. To investigate the role of the hippocampus in spatial memory, we used the water maze paradigm. We found significant increases in NCAM PSA (33.62±1.43 vs. 28.36±1.13 cells per unit area; p