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Program Book of Abstracts Organized by Lund University, Sweden Technical University of Denmark, Denmark In collaboration with SPIE Preface Lasers, optical methods and instruments based on light interaction with tissues have emerged as powerful techniques for medical diagnostics, monitoring wide spectra of tissue pathology and function, and therapy. In biophysics and biology, optical sensing and manipulation of cells have furthered understanding of basic cell function. Ten years ago, in the beginning of 2002 we decided to organize a graduate school in the field of biophotonics. Our motivation then was to strengthen education and scientific exchange of results and ideas. The five schools were held on the Island of Ven in June 2003, June 2005, June 2007, June 2009, and May 2011, respectively. Since 2007, we collaborate with the Journal of Biomedical Optics on publishing a special section (Selected Topics in Biophotonics) containing invited review papers from lecturers at the school and contributed papers from students at the school, respectively. In time, these special sections, especially the invited review papers, will serve as valuable educational material for students in our field. Since the first school, we have been overwhelmed with the positive feedback from students and lecturers and, therefore, we decided to make the school a bi-annual th event. In June 2013, we open the 6 International Graduate Summer School Biophotonics ‘13. The format of the school is a combination of lectures, student poster presentations – and leisure time. However, the leisure time is also spent studying, discussing, learning and exchanging new scientific ideas. Following the first school held at the Island of Ven, the small island situated between Sweden and Denmark, it became clear to us that this is a perfect setting for our school. By making this choice, we follow in the footpath of the renowned Danish scientist Tycho Brahe (1546-1601) who inhabited the Island of Ven some centuries ago and had his laboratory there. Hence, we decided that this place was perfectly suited for a graduate school combining science and the history of our two countries. th Our purpose with the 6 International Graduate Summer School Biophotonics ‘13 is to provide education for students and young scientists at the highest international level within biophotonics. Moreover, by inviting renowned lecturers from all over the world, the educational program is held in an international atmosphere enhancing exchange of scientific ideas and technological advances within the field of biomedical optics and closely related areas. Biophotonics ‘13 covers the following main areas: • Tissue optics • Optical trapping and their applications in biophotonics • Nanoscopy With Focused Light • Fluorescence life-time imaging • Green Fluorescent Protein and their applications in biophotonics • Biomolecular Imaging With Coherent Raman Scattering Microscopy • Diffusion tomography for biomedical imaging • Optical coherence tomography • Photo-acoustic imaging • Photodynamic therapy • Lasers and their application in medicine We have invited experts, who will give extended presentations reviewing these areas. Moreover, in order to promote scientific exchange of ideas and results, the attendees at the summer school will present their current research projects, results or ideas at three poster sessions. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden i Special issue in Journal of Biomedical Optics Related to Biophotonics ‘13, we are pleased to announce that a special section will appear in Journal of Biomedical Optics (JBO) entitled “Selected Topics in Biophotonics” comprising review papers and contributed papers from the school. Participants at Biophotonics ‘13 are encouraged to submit their manuscript for this special section in JBO, which is scheduled to be published in August 2014 (online following acceptance). We would like to express our gratitude to the lecturers for taking time to teach at the school and spending time with the students, and to the scientific advisory board for their helpful suggestions. We also greatly appreciate the financial support and donations we received from various sponsors making this event possible. We hope that attending the school will be a fruitful and educational experience for all participants. Peter E. Andersen Technical University of Denmark Roskilde, Denmark Stefan Andersson-Engels Lund University Lund, Sweden June 2013 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden ii Organizers Peter E. Andersen Department of Photonics Engineering, Technical University of Denmark, DK-4000 Roskilde, Denmark [email protected] Stefan Andersson-Engels Department of Physics, Lund University, S-221 00 Lund, Sweden [email protected] Organizing committee Peter E. Andersen (DTU), Maria Welling (DTU), Stefan Andersson-Engels (LU), and Camilla Nilsson (LU) Scientific Advisory Committee Darryl J. Bornhop, Chemistry Department, Vanderbilt University, USA Cornelia Denz, Institute of Applied Optics, University of Münster, Germany Kishan Dholakia, School of Physics and Astronomy, University of St. Andrews, Scotland James Fujimoto, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), USA Stefan W. Hell, Dept. of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Germany Joseph Izatt, Department of Biomedical Engineering, Duke University, USA Steven Jacques, Oregon Health & Science University, USA Paul Michael Petersen, Technical University of Denmark, Denmark Brian W. Pogue, Dartmouth College, USA Jürgen Popp, Institute of Photonic Technology, Jena, Germany Eric O. Potma, University of California Irvine, USA Eva Sevick-Muraca, University of Texas, USA Katarina Svanberg, Lund University, Sweden Sune Svanberg, Lund University, Sweden Roy Taylor, Imperial College London, United Kingdom Bruce J. Tromberg, Beckman Laser Institute, University of California, Irvine, USA Hubert van den Bergh, Ecole Polytechnique Fédérale de Lausanne, Institut de l'environnement, Switzerland 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden iii List of Lecturers Professor Kishan Dholakia, University of St. Andrews, Scotland Professor Wolfgang Drexler, Medical University of Vienna, Austria Professor Paul French, Imperial College London, United Kingdom Professor Stefan W. Hell, Max-Planck-Institute for Biophysical Chemistry, Germany Professor Steven Jacques, Oregon Health & Science University, USA Professor Konstantin Lukyanov, Russian Academy of Sciences, Moscow, Russia Dr. Eric O. Potma, University of California, Irvine, USA Professor, MD Katarina Svanberg, Lund University Hospital, Sweden Professor Roy Taylor, Imperial College London, United Kingdom Professor Bruce Tromberg, University of California, Beckman Laser Institute, USA Professor Lihong Wang, Washington University in St. Louis, USA School Location The school is held at the conference center Backafallsbyn on the Island of Ven, Sweden (www.backafallsbyn.se). 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden iv Sponsors of Biophotonics ‘13 The following institutions and organizations support the graduate summer school: • Lund Laser Centre and Lund Medical Laser Centre, Sweden • Technical University of Denmark, DTU Fotonik, Denmark • Danish Optical Society (DOPS), Denmark • NKT Photonics A/S, Denmark • Royal Swedish Academy of Sciences (through its Nobel Institute for Physics), Sweden • SPIE, United States • Thorlabs Sweden AB, Sweden Sponsor of Best Poster Presentation At Biophotonics ‘13 all students present their research or planned research project (depending on the level of the student) during three poster sessions. During these presentations, the lecturers will evaluate the posters and presenters and the best poster is awarded a prize: • SPIE Poster Award 2013 The prize consists of a diploma and a cash award of 400 Euro sponsored by SPIE. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden v Biophotonics ‘13 – Programme Saturday 8 June 2013 9.00−12.00 ...................... Arrival at Biophotonics ‘13 Registration in Lund (meeting point address): Department of Physics, Lund University Sölvegatan 14, Lund, Sweden 12.00 ............................... Light refreshments (sandwiches, fruit, and beverages) 12.45 ............................... Opening: Welcome to Biophotonics ‘13 Peter E. Andersen, Technical University of Denmark, Denmark, and Stefan Andersson-Engels, Lund University, Sweden 13.30−14.00 .................... Royal Swedish Academy of Sciences And Its Activities Professor Anne L'Huillier, Lund University, Sweden 14.30 ............................... Departure for Ven (ferry) The bus ride from Lund University to the ferry is arranged by the summer school. The ferry departs from Landskrona at 16.00. 17.00 ............................... Arrival and check-in at Backafallsbyn on the Island of Ven 19.00 ............................... Dinner 20.30 ............................... Saturday Night Hot Topics Speakers and organizers present themselves and their topic. Sunday 9 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−12.00 ...................... Session 1 – Nanoscopy With Focused Light Professor Stefan Hell, Max-Planck-Institute for Biophysical Chemistry, Germany 8.30−9.10: 9.25−10.05: 10.25−11.05: 11.20−12.00: Lecture I Lecture II Lecture III Lecture IV 12.15 ............................... Lunch 13.30−17.00 .................... Session 2 – Tissue Optics Professor Steven Jacques, Oregon Health & Science University, USA 13.30−14.10: 14.25−15.05: 15.25−16.05: 16.20−17.00: Tissue optics I Tissue optics II Tissue optics III Tissue optics IV 18.00 ............................... Dinner 19.30−22.30 .................... Poster Session I Presentations by students of their research (posters). 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 1 Monday 10 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−11.30 ...................... Poster Session II Presentations by students of their research (posters). 12.00 ............................... Lunch 13.15−16.15 .................... Poster Session III Presentations by students of their research (posters). 17.15 ............................... Dinner 18.30−22.00 .................... Session 3 – Strategies For Cancer Treatment Using Lasers And Photodynamic Therapy Professor, MD Katarina Svanberg, Lund University Hospital, Dept. Of Oncology, Lund, Sweden 18.30−19.10: 19.25−20.05: 20.25−21.05: 21.20−22.00: Strategies for cancer treatment using lasers and photodynamic therapy I Strategies for cancer treatment using lasers and photodynamic therapy II Strategies for cancer treatment using lasers and photodynamic therapy III Strategies for cancer treatment using lasers and photodynamic therapy IV Tuesday 11 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−12.00 ...................... Session 4 – Medical Imaging In Thick Tissues Using Diffuse Optics Professor Bruce J. Tromberg, Beckman Laser Institute and Medical Clinic, University of California, Irvine, USA 8.30−9.10: 9.25−10.05: 10.25−11.05: 11.20−12.00: Medical imaging in thick tissues using diffuse optics I Medical imaging in thick tissues using diffuse optics II Medical imaging in thick tissues using diffuse optics III Medical imaging in thick tissues using diffuse optics IV 12.15 ............................... Lunch Afternoon free for discussions and recreation. 17.15 ............................... Dinner 18.30−22.00 .................... Session 5 – Optical Micromanipulation For Biophotonics Professor Kishan Dholakia, University of St. Andrews, UK 18.30−19.10: 19.25−20.05: 20.25−21.05: 21.20−22:00: Optical micromanipulation for biophotonics I Optical micromanipulation for biophotonics II Optical micromanipulation for biophotonics III Optical micromanipulation for biophotonics IV 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 2 Wednesday 12 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−12.00 ...................... Session 6 – Supercontinuum Light Sources And Lasers Professor J. Roy Taylor, Physics Department, Imperial College, United Kingdom 8.30−9.10: 9.25−10.05: 10.25−11.05: 11.20−12.00: Supercontinuum light sources and lasers I Supercontinuum light sources and lasers II Supercontinuum light sources and lasers III Supercontinuum light sources and lasers IV 12.15 ............................... Lunch Afternoon free for discussions and recreation. 17.15 ............................... Dinner 18.30−22.00 .................... Session 7 – Genetically Encoded Tools For Optical Imaging And Control of Cells And Organisms Professor Konstantin Lukyanov, Laboratory of Molecular Technologies, Russian Academy of Sciences, Moscow, Russia 18.30−19.10: 19.25−20.05: 20.25−21.05: 21.20−22.00: Genetically encoded tools I Genetically encoded tools II Genetically encoded tools III Genetically encoded tools IV Thursday 13 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−12.00 ...................... Session 8 – Multidimensional Fluorescence Imaging And Metrology Professor Paul French, Imperial College London, United Kingdom 8.30−9.10: 9.25−10.05: 10.25−11.05: 11.20−12.00: Multidimensional fluorescence imaging I Multidimensional fluorescence imaging II Multidimensional fluorescence imaging III Multidimensional fluorescence imaging IV 12.15 ............................... Lunch Afternoon free for discussions and recreation. 17.15 ............................... Dinner 18.30−22.00 .................... Session 9 – Optical Coherence Tomography Professor Wolfgang Drexler, Medical University of Vienna, Austria 18.30−19.10: 19.25−20.05: 20.25−21.05: 21.20−22:00: Optical coherence tomography I Optical coherence tomography II Optical coherence tomography III Optical coherence tomography IV 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 3 Friday 14 June 2013 7.00 ................................. Breakfast 8.20−8.30 ........................ Practical notes and announcements 8.30−12.00 ...................... Session 10 – Photoacoustic Tomography: Ultrasonically Breaking through the Optical Diffusion Limit Professor Lihong Wang, Washington University in St. Louis, Department of Biomedical Engineering, USA 8.30−9.10: 9.25−10.05: 10.25−11.05: 11.20−12.00: Photoacoustic Tomography I Photoacoustic Tomography II Photoacoustic Tomography III Photoacoustic Tomography IV 12.15 ............................... Lunch 13.30−17.00 .................... Session 11 – Biomolecular Imaging With Coherent Raman Scattering Microscopy Dr. Eric O. Potma, University of California, Irvine, USA 13.30−13.10: 14.25−15.05: 15.25−16.05: 16.20−17.00: Lecture I Lecture II Lecture III Lecture IV 18.30 ............................... Gala dinner Best Poster Presentation Award sponsored by SPIE Saturday 15 June 2013 Before 9.00 ..................... Check-out 7.30−10.00 ...................... Breakfast 10.15 ............................... Departure for ferry On the mainland, there will be shuttle buses for Lund Train Station and Kastrup Airport, Copenhagen, Denmark 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 4 Poster presentations Poster session I: Sunday 9 June 2013 Tatiana Alexandru, University of Bucharest (Romania) Poster title: Laser Induced Cleavage of Molecules For Application On Biological Targets Simona Bartkova, Technical University of Denmark (Denmark) Poster title: Colonization and Dissemination of luxCDABE-Marked Aeromonas salmonicida In Rainbow Trout Shadi Chreiteh, Technical University of Denmark (Denmark) Poster title: Reflective Pulse Oximetry on Sternum Frédéric Fantoni, CEA-LETI-MINATEC (France) Poster title: Laser Line Scanning Illumination Scheme For The Enhancement of Contrast and Resolution For Fluorescence Reflectance Imaging Gesa Franke, University of Lübeck (Germany) Poster title: High Resolution Holoscopy Richelle Hoveling, University of Amsterdam, Academic Medical Center (The Netherlands) Poster title: Hyperspectral Imaging of The Spatiotemporal Behavior of Chromophores In Aging Bruises Tschackad Kamali, Medical University of Vienna (Austria) Poster title: Multimodal Fourier transform CARS and Spectral Domain OCT using a single ultrafast Ti:Sapphire laser Joao Lagarto, Imperial College London (United Kingdom) Poster title: Development And Application of Compact, Low-Cost Multispectral TimeResolved Fluorometric Fibre-Optic Probes For In Vivo Diagnosis And Study of Disease Jonathan Nylk, University of St Andrews (United Kingdom) Poster title: Wavefront Shaping in Light Sheet Microscopy Uros Orthaber, Optotek d.o.o. (Slovenia) Poster title: Observation of Laser-Induced Cavitation Bubble Dynamics Near A Thin Elastic Membrane With A High Speed Camera Tatiana Pryanikova, Institute of Applied Physics RAS (Russia) Poster title: Influence of Different Kinds of Therapy On The Oxygenation of Experimental Tumor Estimated by Diffuse Optical Spectroscopy Peter Rejmstad, Linköping University (Sweden) Poster title: Optical Monitoring in Neurointensive Care Using Laser Doppler and Reflectance Spectroscopy Emanuel Saerchen, Rowiak GmbH (Germany) Poster title: Physical Investigation of Self-Induced Laser Focus Displacement during Photodisruption with Ultrashort Pulses 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 5 Paulien Stegehuis, Leiden University Medical Center (The Netherlands) Poster title: Discrimination of Benign And Malignant Human Breast Tissue Using Full Field Optical Coherence Tomography Fatma Tümer, Max Planck Institute for the Science of Light (Germany) Poster title: Long-Distance laser Propulsion And Deformation Monitoring of Cells In Hollow-Core Photonic Crystal Fiber Naja Villadsen, Aarhus University (Denmark) Poster title: Laser Manipulation of Optically Trapped Objects Daniel Wangpraseurt, University of Technology, Sydney (Australia) Poster title: Coral Tissue Optics Muhammad Nur Salihin Yusoff, Universiti Sains Malaysia (USM) (Malaysia) Poster title: Optical Reflectance And Morphology of Poly(Vinyl Alcohol) Cryogel Tailored By Rock Salt Behrooz Zabihian, Medical University of Vienna (Austria) Poster title: Towards Multimodal all Optical Detection Photoacoustic Tomography and Swept Source OCT 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 6 Poster session II: Monday 10 June 2013 Maria Carmela Cardilli, Università degli studi di Bari (Italy) Poster title: Self-Mixing Effect In A Multi-Transverse-Mode VCSEL And Polarization Dynamics of The First Order Transverse Modes Jeffrey Cassidy, University of Toronto (Canada) Poster title: FullMonte: Fast 3D Monte Carlo Simulation For Turbid Media Chieh-Li Chen, University of Pittsburgh (United States) Poster title: Signal Normalization Reduced Systematic Differences in Retinal Nerve Fiber Layer Thickness Measurements Between Spectral Domain Optical Coherence Tomography Devices Elizabeth Huynh, University of Toronto (Canada) Poster title: Optically Controlled Pore Formation in Cell-Size Porphyrin Vesicles Wiebke Knoll, Universität zu Lübeck (Germany) Poster title: In vivo Two-Photon Microscopy and UV Laser Nanosurgery of Murine Small Intestine Philipp Krauter, Institut für Lasertechnologien in der Medizin und Meßtechnik (Germany) Poster title: NIR Remission Spectroscopy of Turbid Media Riya Menezes, Institute of Photonic Technology Jena (Germany) Poster title: Raman Spectroscopy - A Fast And Reliable Tool To Detect Secondary Fungal Metabolites Paul O'Mahoney, University of Dundee (Scotland) Poster title: Optical Red Blood Cell Sorting Dino Ott, University of Copenhagen (Denmark) Poster title: Biophotonic Interactions of Individual Nanoparticles Lukasz Paluchowski, NTNU (Norway) Poster title: Dual-Mode Imaging System For Characterization of Wound Surface Sidsel Petersen, Technical University of Denmark (Denmark) Poster title: Ytterbium Doped Fiber Amplifiers Above 1100 nm For Generation of Yellow Laser Light Radoslaw Sadowski, McMaster University (Canada) Poster title: Quantitative Optical Molecular Imaging Eric Seifert, Medical Lasercenter Lübeck (Germany) Poster title: Automatic Irradiation Control By An Optical Feedback Technique For Selective Retina Treatment (SRT) In A Rabbit Model Kristian Sexton, Dartmouth College (United States) Poster title: Pulsed Light Excitation And Image Gating For Fluorescence Guided Surgery In Normal Lighting Conditions Jacob Staley, University of Twente (The Netherlands) Poster title: Photoacoustic Contrast Agents as Acoustic Time-Reversal Sources For Targeted Acousto-Optics 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 7 Idan Steinberg, Tel Aviv University (Israel) Poster title: Frequency Domain Photoacoustic Phase Measurements of the Acoustic Modes in Bone for the Early Detection and Diagnosis of Osteoporosis Peter Van Es, University of Twente (The Netherlands) Poster title: Photoacoustic Imaging of Human and Murine Joints: Towards Assessment of Rheumatoid Arthritis Luka Vidovic, Jozef Stefan Institute (Slovenia) Poster title: Prediction of the Maximal Safe Laser Radiant Exposure on an Individual Patient Basis Based on Photothermal Temperature Profiling Kari Vienola, Rotterdam Eye Hospital (The Netherlands) Poster title: Imaging of Optic Nerve Head With Motion Corrected OCT Using Tracking SLO 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 8 Poster session III: Monday 10 June 2013 Mitra Almasian, University of Amsterdam, Academic Medical Center (The Netherlands) Poster title: i-OCT: Integrated Smart Optics For Low Super Resolution OCT Nico Bodenschatz, Institut für Lasertechnologien in der Medizin und Meßtechnik (Germany) Poster title: Optical Reconstruction in Modulated Spatial Imaging: Frequent Assumptions And Their Validity Lisa Bürgermeister, Fraunhofer Institute for Laser Technology (Germany) Poster title: Physical and Mathematical Modeling of Photodynamic Therapy Andrea Curatolo, The University of Western Australia (Australia) Poster title: Speckle In Optical Coherence Tomography: Simulation And Experiment With A Structured Phantom Ahmed Elmaklizi, Institute for Laser Technology in Medicine and Metrology (Germany) Poster title: Analytical and Numerical Analysis of Electromagnetic Scattering using Gaussian and Focused Beams Adam Glaser, Dartmouth College (United States) Poster title: Optical Imaging And Tomographic Dosimetry of Radiation Beams By The Cerenkov Effect Altaf Hussain, University of Twente (The Netherlands) Poster title: Fluence Mapping Inside The Highly Scattering Medium Using Reflection Mode Acousto-Optics Deepa Kasaragod, University of Tsukuba (Japan) Poster title: Polypyrrole Nanopartcles: A New Contrast Agent At 1300nm And Its Optical Detection Using Photothermal Optical Coherence Tomography Kelsey Kennedy, The University of Western Australia (Australia) Poster title: Probing Elastic Contrast In Human Tissues Using Needle Optical Coherence Elastography Elena Kiseleva, Nizhny Novgorod State Medical Academy (Russia) Poster title: In Vivo Evaluation of The Depolarizing Properties of Collagen By CrossPolarization OCT Kelly Michaelsen, Dartmouth College (United States) Poster title: Effects of Breast Compression on Digital Breast Tomosynthesis Guided Diffuse Optical Spectroscopy Christian Myrtus, University of Lübeck (Germany) Poster title: Analyse And Visualize Ciliary Beat Frequency Ex Vivo Using Spectral Domain Optical Coherence Microscopy Kamilla Nørregaard, University of Copenhagen (Denmark) Poster title: Nanoparticle Mediated Photothermal Therapy and Integrated miRNA Delivery 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 9 Stéphane Perrin, FEMTO-ST (France) Poster title: Optical Coherence Tomography Microsystem For Early Diagnosis of Skin Pathologies Frederico Pimenta, Aarhus University (Denmark) Poster title: Towards the Control and Quantification of Singlet Oxygen Influence in Cellular Mechanisms Ana Rita Ribeiro, University of Porto (Portugal) Poster title: Towards Optofluidic Systems For Single Cell Manipulation And Analysis Hendrik Spahr, University of LÜbeck (Germany) Poster title: Imaging Temperature Distributions of Laser Irradiated Tissue via Phase Sensitive Optical Coherence Tomography Yolanda Villanueva, University of Twente (The Netherlands) Poster title: Determination of The Grüneisen Parameter of Absorbing Liquids Using Photoacoustic Measurements In An Integrating Sphere Chiara Vitelli, Istituto Italiano di Tecnologia (Italy) Poster title: Realization of A Micro-Optical Coherence Tomography (mOCT) Setup For Cell Imaging With Micron Resolution In Tissues 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 10 Abstracts The book of abstracts contains name of the presenting author, title and abstract for the th participants’ poster presentations for the 6 International Graduate Summer School Biophotonics ‘13. Tatiana Alexandru Faculty of Physics, University of Bucharest, Romania [email protected] Laser Induced Cleavage of Molecules For Application On Biological Targets Recent reports show that in the case of multiple drug resistance (MDR) acquired by bacteria and/or malignant tumors, the method which is based on medicine molecules modifications and/or generation of new and stable photoreaction products originating from them by exposure to laser radiation is promising for enhancing the efficiency of the treatments. At the same time the procedure constitutes a new prospective domain in the synthetic chemistry. Possible alternatives to synthesize new drugs, which may optimize the medication and reduce side effects, include the development of photoactivated drug carriers, light activated drugs and their delivery by controlled release of the active form of the drug. The current report presents recent results obtained by the authors in the application of lasers on photosensitive medicines in view of biomedical applications. Mitra Almasian Biomedical Engineering and Physics, University of Amsterdam, Academic Medical Center, The Netherlands [email protected] i-OCT: Integrated Smart Optics For Low Super Resolution OCT Optical coherence tomography (OCT) is a standard used tool in ophthalmology. Although it provides nice cross-sectional images of the retina there is a need from the clinic to improve the resolution to enable better and more direct diagnosis of retinal diseases e.g. Glaucoma (2nd cause of blindness). A convenient benefit of OCT is that the axial resolution depends only on the coherence length of the light source, allowing a depth resolution up to 2 µm. The lateral resolution on the other hand is determined by the optics in the sample arm. For ophthalmologic use the lateral resolution of the OCT signal is hampered by the aberrations introduced by the optics of the eye, which results in a spot size around 20 µm on the retina. The much lower lateral resolution hinders the visualisation of the photoreceptor cells responsible for colour vision, the cones. A high resolution OCT system can improve diagnosis through better visualisation of the structure and physiological function (e.g. blood perfusion and oxygenation) of the retina. The use of adaptive optics (AO) has proven to successfully correct for such aberrations resulting in high resolution images. However a standard AO-OCT system entails practical and economical problems for the use in a clinical setting. In this project we aim to develop a low cost-super resolution OCT system to overcome the practical and economic disadvantages of a conventional AO-OCT set-up . This system will be extended with a tracking mechanism for the temporal motion of the aberrations, resulting in an AO-OCT super resolution at reduced cost and size by to meet the needs of the clinic. Simona Bartkova DTU Veterinary Institute, Technical University of Denmark, Denmark [email protected] Colonization and Dissemination of luxCDABE-Marked Aeromonas salmonicida In Rainbow Trout Aeromonas salmonicida, the causative agent of the disease furunculosis, is one of the major bacterial pathogens in aquaculture throughout the world, affecting many fish species. In Denmark furunculosis causes the greatest problems in sea reared rainbow trout (Oncorhynchus mykiss) production, where outbreaks occur repeatedly, even with A. salmonicida antigen included in the applied commercial vaccines, and thereby cause economically devastating losses in aquaculture. Fish harboring A. salmonicida can moreover be covertly infected and thus not show any symptoms nor can the bacterium be isolated from them, while still being able to shed bacteria into the surrounding environment. One of the most crucial factors for trying to prevent spread of the disease is therefore gaining a deeper understanding on the covert stage of infection, for which early detection of the pathogen and its route of entry and subsequent dissemination in the fish is crucial. One way to of being able to track the bacterial infection progression in the fish is to use in vivo imaging for visualization of the bacterium. For this both the green fluorescence protein (GFP) and bioluminescence i.e. luciferase has been used 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 11 extensively. Nevertheless, luciferase is known to be more sensitive, less toxic and respond faster to changing environments (such as a progression of an infection) than GFP. Using luciferase is thus the most sensible choice for tracking A. salmonicida in trout. The objective of my project is to determine the route of entry and subsequent tissue dissemination of the disease-causing A. salmonicida in Denmark, through in vivo imaging studies. Currently the project is still in its initial stages and certain factors thus remain to be determined. Nevertheless, the general plan is to follow a modified procedure from previous research by Dr Attila Karsi and Dr Mark L. Lawrence, University of Mississippi, where the fish bacterium Edwardsiella ictaluri was tagged with bioluminescence. In short, the plan will be to tag A. salmonicida with a broad host range plasmid vector pAKlux1 that has an inserted luciferase from Photorhabdus luminescens. Through an immersion and cohabitation challenge respectively, rainbow trout will be infected with the bioluminescence tagged A. salmonicida, whereby colonization and subsequent dissemination of A. salmonicida in the trout could be monitored via an IVIS imaging system at several time points. Due to use of bioluminescence tagging, the same rainbow trout could be used for visualization at each time point, although this is one of the factors that remain to be decided. Moreover, the plasmid vector pAKlux1 can be obtained from the laboratory of Dr Attila Karsi through Addgene as agreed upon via communication with Dr Karsi. Transfer of the plasmid into a strain of A. salmonicida will be done via Escherichia coli by conjugation as described by previous research of Dr Karsi. This should result in a constitutive expression of the bacterial luciferase operon in the A. salmonicida and thus a constitutive luminescence allowing detection and quantification of bacteria using the IVIS Imaging System. Nico Bodenschatz Material Optics, Institut für Lasertechnologien in der Medizin und Meßtechnik, Germany [email protected] Optical Reconstruction in Modulated Spatial Imaging: Frequent Assumptions And Their Validity Evaluation of experimental data in spatially modulated imaging aims at spatially resolved reconstruction of optical diffusion and absorption coefficients. This reconstruction is often based on various simplifying assumptions. Among these are the diffusion approximation and assumptions for scattering phase function and anisotropy. Furthermore, surface roughness also influences the captured light intensity and thus alters the experimentally derived optical properties. Analysis of the validity of these frequent simplifying assumptions in spatially modulated imaging is the aim of this study. Thereby, the possible error in the derived optical properties is assessed for various parameter combinations. Lisa Bürgermeister Modelling and Simulation, Fraunhofer Institute for Laser Technology, Germany [email protected] Physical and Mathematical Modeling of Photodynamic Therapy Laser-induced antimicrobial photodynamic therapy (aPDT) is already being used today on a large scale to treat infectious diseases, e.g. periodontitis. How aPDT functions basically in clinical practice is well-known: A photoactive chemical compound, the so-called photosensitizer, is introduced into the infected area where it adheres to the disease-causing bacteria. When a laser with minimal power – in the range of mW – irradiates the photosensitizer, the photosensitizer is excited to higher electronic states by the absorption of laser light, which enables the generation of highly reactive singlet oxygen. The targeted bacteria are oxidized, thus destroyed. Unfortunately physicians cannot tap the potentials of this treatment, as its underlying physical and biochemical processes are not clearly understood. To enable this understanding, mathematical-physical modelling is necessary. In particular, this includes physical and mathematical modelling of how laser radiation propagates in strongly scattering biological tissues and what the kinetics of the photochemical reactions occurring there are. In a case with fixed parameters, the concentrations of the substances involved in the aPDT can be described by a system of ordinary differential equations (ODE). The solution is a time-series depending on treatment parameters and initial concentrations. The treatment success can be calculated and predicted with this ODE system. Using methods of model reduction, we simplify the complexity of the existing aPDT models. This leads to the quantification of the initial concentrations, which lead to good treatment results. We aim at developing an aPDT strategy which guides the treating physician and increases the prediction of the therapy outcome. Our research hypothesis is that spatially distributed intensity and photosensitizer concentration determine each other and thereby influence the extend of the successfully treated area. This area increases in time and its increasing speed determines the irradiation time which is necessary for good aPDT outcomes. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 12 Maria Carmela Cardilli Dipartimento Interateneo di Fisica, Università degli studi di Bari, Italy [email protected] Self-Mixing Effect In A Multi-Transverse-Mode VCSEL And Polarization Dynamics of The First Order Transverse Modes We present an experimental study of a sensor that can be used to simultaneously measure target translations along the optical axis and target rotations in the orthogonal plane. We used a multi-transverse mode Vertical- Cavity surface-emitting laser subject to optical feedback. When the operating conditions preserved the moderate feedback regime, the power along the two polarization modes was modulated according to both target linear displacement and rotation. The simultaneous measurement of both polarization intensity modulations allowed for the independent estimation of the two degrees of freedom of motion. Jeffrey C. Cassidy Electrical & Computer Engineering, University of Toronto, Canada [email protected] FullMonte: Fast 3D Monte Carlo Simulation For Turbid Media Emerging clinical applications including bioluminescence imaging require fast and accurate modelling of light propagation through turbid media with complex geometries. Monte Carlo simulations are widely recognized as the standard for high-quality modelling of light propagation in turbid media, albeit with high computational requirements. We present FullMonte: a flexible, extensible software framework for Monte Carlo modelling of light transport from extended sources through general 3D turbid media including anisotropic scattering and refractive index changes. The problem geometry is expressed using a tetrahedral mesh, giving accurate surface normals and avoiding artifacts introduced by voxel approaches. Input formats from two other popular simulators, TIM-OS (Monte Carlo) and NIRFAST (Finite- Element Method), are accepted to help users integrate the software into their application. We present a discussion of current state-of-the-art algorithms and accel- erated implementations of the modelling problem. Results and performance are compared against existing implementations, showing that FullMonte achieves best-in-class performance for general geometries and material prop- erties using entirely free and open-source libraries. The software uses multithreading, Intel SSE vector instructions, and optimized data structures to achieve high performance. Thorough software design permits a great deal of compile-time flexibility in what information is gathered and how it is stored, with no performance overhead at run time. It also incorporates novel hardware-friendly performance optimizations in the scattering calculation that should be useful for GPU and custom hardware implementations. In addition, control is provided of a new parameter permitting accuracy- performance tradeoffs. Significant performance gains of over 25% can be realized for bioluminescence imaging problems with no measurable result- quality difference. For applications requiring quick rough estimates, even faster run times can be achieved. Since run time is an important limiting factor in the utility of turbid- media Monte Carlo simulation, we also discuss the advantages and limita- tions of both CPU and GPU implementations, with observations important to future advances. Our current work is on creating a custom hardware implementation of the FullMonte algorithm to achieve large-integerfactor speedup and power efficiency gains of an order of magnitude or better. Chieh-Li Chen Department of Bioengineering, University of Pittsburgh, United States [email protected] Signal Normalization Reduced Systematic Differences in Retinal Nerve Fiber Layer Thickness Measurements Between Spectral Domain Optical Coherence Tomography Devices Purpose: Differences in retinal nerve fiber layer (RNFL) thickness measurements between spectral-domain optical coherence tomography (SD-OCT) devices pose significant difficulty in both clinical and research settings where multiple devices are in use. The purpose of this study was to develop and test a novel signal normalization method that reduced systematic differences. Methods: One hundred and nine eyes (36 glaucomatous, 40 glaucoma suspect, and 33 healthy eyes) from 59 subjects were scanned with two SD-OCT devices on the same day (Cirrus HDOCT; Carl Zeiss Meditec, Inc., Dublin, CA; Optic Disc Cube 200x200 scan pattern, and RTVue; Optovue, Fremont, CA; RNFL 3.45 Circle scan pattern). RTVue data had 768 pixels for 2.3 mm scan length on each A-scan, which was oversampled to match with the Cirrus specification 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 13 (1024 pixels for 2.0 mm). Then both the original Cirrus signal and RTVue derived Cirrus signal were normalized in amplitude so that the meaningful signal range was stretched to the full gray scale range of 0 to 255. To compensate signal strength differences, custom high dynamic range (HDR) processing was also applied to images showing significantly poorer image quality than its Cirrus/RTVue counterparts (difference of quality index percentile below 5 percentile or above 95 percentile of the entire set of images.) Conventional global mean circumpapillary RNFL thicknesses were measured automatically using software of our own design and then compared to the original device outputs. Structural equation models were used to analyze the absolute RNFL thickness difference between original device outputs and our software outputs after signal normalization. P<0.05 was considered as statistically significant. Results: The mean absolute difference in RNFL thicknesses between Cirrus and RTVue were significantly different before normalization (10.39 ± 3.98 µm), but not after normalization (5.81 ± 4.06 µm). The absolute difference of RNFL thickness between Cirrus and RTVue was statistically significantly reduced by signal normalization. The median of the absolute difference of the RNFL thickness after normalization was 4.7 µm, which is within the inherent device measurement variability. Conclusion: The reported novel signal normalization method successfully reduced the systematic difference in RNFL thickness measurements between Cirrus and RTVue to the level of the inherent device measurement variability. Enabling direct comparison of RNFL thickness obtained from multiple devices would broaden the use of OCT technology in both clinical and research applications. Shadi S. Chreiteh DTU Nanotech, Technical University of Denmark, Denmark [email protected] Reflective Pulse Oximetry on Sternum This project is about development of a new wearable reflectance mode pulse oximetry sensor for integration in an electronic patch (ePatch), manufactured by DELTA, that can be placed on the chest bone (sternum) of humans. Pulse oximetry is a non-invasive photo-metric technique that provides information about the heart rate and the arterial blood oxygen saturation (SpO2). It has become a standard of care in many areas of clinical medicine. The arterial blood oxygen saturation is determined by measuring the light absorbance of tissue at two different wavelength, usually red (660 nm) and infrared (940 nm), through vascular tissue. The chosen wavelengths are based on the characteristics of the absorption spectra of deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (HbO2). The oxygen saturation is the ratio between HbO2 and Hb and can be calculated from the ratios between the systole and diastole scattered signals at both 660 nm and 940 nm. The ePatch is capable of measuring (electrocardiogram) ECG and saving or sending the measured data. Since the sensor is located on the sternum, it is also possible to measure the respiratory rate with almost the same optical method. Thus by combining pulse oximetry, respiratory rate and ECG a unique product that monitors three important vital signs can be achieved. A device that is able to monitor the mentioned three vital signs wirelessly is needed in Tele-monitoring, at the emergency departments or other hospital wards. Andrea Curatolo Optical + Biomedical Engineering Laboratory, The University of Western Australia, Australia [email protected] Speckle In Optical Coherence Tomography: Simulation And Experiment With A Structured Phantom Speckle, an intrinsic feature of coherent imaging modalities such as optical coherence tomography (OCT), manifests itself as rapid fluctuations of the detected intensity over the spatial extent of the image, conveying a granular texture. The ability to verify theoretical predictions about speckle statistical properties, whether to extract useful information from a speckle pattern or to select the most appropriate speckle reduction techniques, depends on correctly modelling the phenomenon. It also lies in the development of powerful computer simulations on one hand, and contrived tissue targets, where the geometry of scattering is known, on the other. In this work we present a model of OCT image formation under several assumptions: single scattering and negligible attenuation and beam divergence. Under these assumptions the OCT signal can be analysed in terms of linear systems theory with the sample susceptibility as input and a spatially invariant point spread function (PSF) as convolution kernel. The results of this speckle simulation code are corroborated by representing the sample susceptibility of a contrived experimental target, a tissue-mimicking 3D silicone structured phantom produced by replica-molding soft lithography. The combined use of the OCT speckle simulation code and a 3D structured phantom with controlled optical and structural properties shows promising results in terms of verification of statistical properties (first and second order) 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 14 of OCT speckle and proves very useful in the quantification of the speckle contrast/resolution degradation trade-off to evaluate appropriate speckle reduction techniques. Ahmed A. Elmaklizi Material- und Gewebeoptik, Institute for Laser Technology in Medicine and Metrology, Germany [email protected] Analytical and Numerical Analysis of Electromagnetic Scattering using Gaussian and Focused Beams Many modern optical instruments for optical particle characterization and biomedical diagnostics use laser as light sources. Modeling these laser beams as simple plane waves is not valid once the beam waist becomes equal or smaller than the particle under test. In this work, the propagation of light having focused and Gaussian beam profile is investigated both analytically and numerically in the scheme of finite difference time domain. The different phenomena that appear due to the interaction between the incident beam and the scattering media is studied in order to enhance the efficiency of microscopy systems. Frédéric Fantoni DTBAS-STD-LISA, CEA-LETI-MINATEC, France [email protected] Laser Line Scanning Illumination Scheme For The Enhancement of Contrast and resolution for fluorescence reflectance imaging Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to noninvasively monitor fluorescence targeted tumors located below the tissue surface. The drawbacks of this technique are the poor resolution in the axial and lateral directions due to multiple light scattering and background fluorescence decreasing the contrast. The aim of this PhD is to develop new illumination and detection methods to overcome these drawbacks. We propose a novel fluorescence imaging method based on laser line illumination in reflectance geometry. We scan the medium with the laser line and acquire images at each position of the line. We then have access to a large amount of information that we can use in different ways. The first and simplest thing to do is to sum the stack of images to obtain the equivalent of a classical wide-field fluorescence reflectance image. This then serves as the basis we want to enhance in terms of contrast and resolution. Another possible processing is to detect only single stripes of each image located on the excitation line or farther from it. We can also subtract the surrounding signal to the detected stripe, the optimal detection scheme depending on the depth of the object of interest. This allows us to be less sensitive to photons that are scattered multiple times and lead to a blurred image. We have also studied the results obtained with a post-processed structured illumination. To do this, we modulate the sum of the stack of images with a sine wave and then apply the classical processing used in structured illumination to obtain the AC contribution. Finally, we have used the excitation line profile to get an insight on the level of background fluorescence that we want to subtract to the final images to improve the contrast. This technique has been validated with tissue-like liquid phantoms with different levels of background fluorescence. Fluorescent inclusions were observed in several configurations at depths ranging from 1 mm to 1 cm. Our results are compared to those obtained with a more classical wide-field detection scheme. The most recent work done has been the design of a setup which would allow to optically implement the masking detection that will fasten the detection scheme and lead to a real-time enhanced detection of the fluorescence. Gesa Franke Institute of Biomedical Optics, University of Lübeck, Germany [email protected] High Resolution Holoscopy In Fourier-domain optical coherence tomography (FD-OCT) the axial and lateral resolutions are decoupled. The axial resolution is constant over the whole imaging depth and is defined by the center wavelength and the spectral width of the light source. The lateral resolution and depth of focus are given by imaging optics and both are defined by the numerical aperture (NA). While the lateral resolution in the focal plane increases with NA, the focal depth (twice the Rayleigh length) decreases quadratically. In scanning OCT systems out-of-focus photons are rejected by confocal detection. In full-field FD-OCT photons from all depths are detected, but outside the focal depth a degraded lateral resolution causes blurred images. Holoscopy is a new imaging approach combining digital holography and full-field FD-OCT. As in digital holography the interference pattern between light scattered by a sample and a defined reference wave is recorded. The amplitude and phase of the light field backscattered by the sample are encoded in the interference pattern. During reconstruction numerical refocusing is applied, overcoming the limitation of the focal depth. In holoscopy multiple holograms are recorded at different 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 15 wavelengths during the sweep of a tunable light source. The reconstruction process is applied to each hologram with the respective wavelength. As in FD-OCT a one-dimensional Fourier transform with suitable re-sampling is applied along the wave number axis and gives the specific depth information for each scatterer within the sample. Thus a uniform, diffraction limited lateral resolution over the whole measurement depth can be obtained. This advantage of holoscopy has been demonstrated at quite low NA but becomes more significant at high lateral resolutions. In most other optical imaging techniques z-scanning is inevitable for measuring a high-resolution tomographic volume of a sample. In high-resolution holoscopy imaging of deep volumes is possible without z-scanning. The setup is based on a Mach-Zehnder interferometer with a high-resolution microscope objective (NA = 0.75) in the sample arm. The fringe patterns of the interference signal between sample and reference light are recorded with an area detector. With this setup measurements of reflecting and scattering samples with no axial dimension have been demonstrated. For recording volumes of scattering samples the lateral and axial resolution of the setup need to be similar. Thus, for a 0.75 NA objective a tunable light source with a sweeping range of approximately 300nm is required. To provide this, a modified tunable Ti:sapphire laser with sufficient tuning range was implemented into the setup. The Lyot filter in the resonator is rotated by a galvanometric scanner to provide fast automated tuning. So far the feasibility of this light source for high-resolution holoscopy has been demonstrated only with a smaller tuning range. Adam K. Glaser Thayer School of Engineering, Dartmouth College, United States [email protected] Optical Imaging And Tomographic Dosimetry of Radiation Beams By The Cerenkov Effect Since its discovery during the 1930s the Cerenkov effect (light emission from charged particles traveling faster than the local speed of light in a dielectric medium) has been paramount in the development of high-energy physics research. The ability of the emitted light to describe a charged particle’s trajectory, energy, velocity, and mass has allowed scientists to study subatomic particles, detect neutrinos, and explore the properties of interstellar matter. More recently, the phenomenon has found applications in the context of biomedical research through Cerenkov luminescence imaging (CLI), a novel method for tracking β-emitting radionuclides in vivo, as well as fluorescence and absorption spectroscopy of linear accelerator induced light emission for treatment monitoring during radiation therapy. Herein we explore optical imaging of the Cerenkov effect during radiation therapy for dosimetry and quality assurance (QA) of megavoltage x-ray photon beams and present a novel Monte Carlo package capable of simulating the complex radiation induced light transport dynamics for investigatory purposes. The proposed QA technique has several advantages over alternative methods (e.g., ionization chambers, scintillation, and gel dosimetry) including speed and flexibility, and necessitates only water, which serves as a cheap, abundant, and easily standardized tissue equivalent medium. Upon future refinement and improved accuracy the proposed modality may prove to be an important dosimetric tool with both clinical and research applications. Richelle Hoveling Biomedical Engineering and Physics, University of Amsterdam, Academic Medical Center, The Netherlands [email protected] Hyperspectral Imaging of The Spatiotemporal Behavior of Chromophores In Aging Bruises One of the diagnostic factors of child abuse is the determination of the age of inflicted bruises, which is based on the judgment of the physician who compares the color of the bruise to a color chart. This is a subjective method that lacks sufficient accuracy. Research in the department of Biomedical Engineering and Physics proved that the age of bruises is contained in the timevarying areas and concentration distributions of hemoglobin and bilirubin. The combination of this information with a finite element model that includes skin thickness and chromophore diffusivities allowed age determination of specific bruises. Before this technique can be applied in clinical practice, the current finite element bruise model needs further development to improve the accuracy of the method. For practical application of the current finite element model, the model will be expanded by refining the finite element geometry of the skin and chromophore behavior. Hereto, we will include gravity for the better assessment of chromophore diffusivities and implement light propagation physics to determine the influence of the skin’s optical properties on the measured spectra and predict the color appearance of bruises. For the spectroscopic assessment, two measurement systems are used. Localized measurements are performed using a fiber based system and a hyperspectral camera is used for spectral imaging of a larger area. This latter setup enables us to obtain spectral information 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 16 of every pixel in the image and determine the chromophore concentration in different areas in the bruise in one measurement. We will image and determine the potential (an)isotropic diffusion on fresh tissue specimen by injecting hemoglobin and different forms of bilirubin. Noninvasive in vivo measurements will be performed on bruise baring volunteers. To give us better insight in the morphology of the skin and the thickness of the different layers at different locations on the body non-invasive in vivo measurements of the skin at different locations on the body will be performed using Optical Coherence Tomography (OCT) (HSL-2000, Santec, Japan). For the development of tissue equivalent phantoms a mixture of scattering and absorbing media will be used to create phantoms that represent the optical properties of (skin) tissue and the chromophores that are present in the bruise (hemoglobin, bilirubin). The first measurements on volunteers and the development of tissue equivalent phantoms have recently started. Findings will be integrated in the finite element model after which validation studies will be performed. Preliminary results show a difference in the distribution of bilirubin and hemoglobin in a 147 hour old bruise at the volar forearm of a 28 year old female volunteer. Previous exploratory research indicates that the change in bilirubin and hemoglobin areas measured over a certain period of time can serve as an input for the finite element model to determine the age of the bruise. The evaluation of this method will be continued and improved if necessary. Phantom preparations and measurements of bruises on volunteers will be continued together with the refinement of the finite element model. Altaf Hussain MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, The Netherlands [email protected] Fluence Mapping Inside The Highly Scattering Medium Using Reflection Mode Acousto-Optics Optical excitation based imaging modalities, with aim to image structures deep inside the scattering medium, suffer from quantification problem. We propose a methodology to solve the problem of non-invasively mapping the fluence in optically heterogeneous medium without the need of prior knowledge of its optical properties. We present a theoretical model of our concept and provide proof of principle with Monte Carlo simulations. Simulation results show that it is possible to measure the local light fluence in highly scattering medium in absolute terms. Furthermore, we performed an experiment to validate the concept as a strategy to measure local fluence in relative manners. We used reflection mode acousto optics (AO) in our experiment, and showed that with this method we can measure local light fluence (in relative term) in highly scattering medium. Elizabeth Huynh Medical Biophysics, University of Toronto, Canada [email protected] Optically Controlled Pore Formation in Cell-Size Porphyrin Vesicles Efforts to develop self-contained microreactors and artificial cells have been limited by difficulty in generating membranes that can be robustly and repeatedly manipulated to load and release cargo from phospholipid compartments. Here we describe a purely optical method to form pores in a membrane generated from porphyrin-phospholipid conjugates electro-assembled into microscale giant porphyrin vesicles and manipulated using confocal microscopy. The pores in the membrane resealed within a minute allowing for repeated pore formation with precise spatial and temporal control and optical gating to allow selective diffusion of biomolecules across the membrane. Temporal control of pore formation was illustrated by performing sequential DNA hybridization reactions. A biotin-avidin based strategy was developed to selectively attach enzymes to the interior of the vesicle, demonstrating spatial control and the potential of giant porphyrin vesicles as versatile microreactors. Tschackad Kamali Center for medical physics and biomedical engineering, Medical University of Vienna, Austria [email protected] Multimodal Fourier transform CARS and Spectral Domain OCT using a single ultrafast Ti:Sapphire laser Abstract: A FTCARS-OCT system based on a homemade Ti:sapphire laser has been set-up. In a first step interferometric FTCARS has been realized. Based on an ultrashort-pulse Ti:sapphire laser this method provides a simple scheme for obtaining high resolution CARS spectra with a single femtosecond light source. CARS developments with spectral shaping and phase control of ultrashort pulses are replaced by this straightforward and cost-effective method. An approximately 2 mm wide excitation beam is focused with a high NA objective onto a sample 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 17 (acetone, isopropanol, polysterol, oil) to enable the collection of the F-CARS signal with a photomultiplier after re-collimation with a second objective. This geometry only allows the investigation of very thin (300µm) or transparent samples. A spectral OCT is integrated in the setup using the same ultrafast laser. FTCARS and OCT imaging was performed with Polybeads of various diameters. Deepa K. Kasaragod Computational Optics group, University of Tsukuba, Japan [email protected] Polypyrrole Nanopartcles: A New Contrast Agent At 1300nm And Its Optical Detection Using Photothermal Optical Coherence Tomography This paper details a new absorptive type contrast agent, polypyrrole nanoparticles (PPy), for optical coherence tomography (OCT)imaging with good absorption spectra at wavelengths from 700-1300nm and the photothermal based optical detection of its contrast properties using swept source based OCT system at 1300nm and a pump laser source at 975nm. Kelsey M. Kennedy Optical + Biomedical Engineering Laboratory, The University of Western Australia, Australia [email protected] Probing Elastic Contrast In Human Tissues Using Needle Optical Coherence Elastography Optical coherence elastography (OCE) provides images of tissue elasticity on the micro-scale and has potential for several clinical applications, including guidance of tumor resection. However, advancement toward clinical implementation of OCE is currently limited by the technique’s small imaging depth in tissue (1-2 mm), as well as a lack of validation of the elastic contrast generated in OCE. We have overcome the depth limitation of current OCE techniques by developing a method for performing OCE via a needle probe. Our technique, needle OCE, uses an OCT needle probe to perform axial measurements of tissue deformation during needle insertion, and has demonstrated potential for subsurface detection of the boundaries of diseased tissue. In this paper, we present initial needle OCE results in a fresh human mastectomy sample, demonstrating elastic contrast between adipose and tumor tissue. In addition, we have developed a finite element model of tissue deformation in compression OCE as a first step toward better understanding of the generation and interpretation of contrast in OCE images. We show initial results demonstrating excellent agreement between measured and simulated deformation in a tissue phantom. Development of this model provides a foundation for extension to more complex models of tissue deformation, such as that due to needle insertion, which will be essential for characterizing the contrast generated by needle OCE. Elena B. Kiseleva Research Institute of Applied and Fundamental Medicine, Nizhny Novgorod State Medical Academy, Russia [email protected] In Vivo Evaluation of The Depolarizing Properties of Collagen By CrossPolarization OCT Collagen has optical anisotropy at all levels of its hierarchical organization and, consequently, is able to depolarize the polarized light wave. This property is used for high-resolution imaging of collagen by noninvasive optical method - cross-polarization optical coherence tomography (CP OCT), which is widely applicable in clinical diagnostics. The purpose of this study was to develop and test the method of quantitative assessment of the useful signal in the orthogonal CP OCT image reflecting the depolarizing properties of collagen fibers in clinical examples. As a numerical criterion we offered the dimensionless parameter - the integral factor of depolarization (IFD), which represents the ratio of the received power of OCT signal in the original and orthogonal channels, averaged over the area of an image. This factor eliminates the effect of speckle noise, as well as the instrumental noise, that create specific background signal. Quantitative analysis of 162 CP OCT images of the bladder mucosa, obtained with the "OCT 1300-U" device (IAP RAS, Nizhny Novgorod), had been done with a semi-automatic method in the ImageJ program (version 1.43u). Of these, 94 images: healthy volunteers and patients with primary disorders of the bladder mucosa, as an example of an inflammatory or neoplastic process of collagen fibers damage; 68 images of patients with radiation cystitis as an example of radiation induced damage of the collagen fibers. It was shown that IFD offered to quantify the relative useful signal in the CP OCT image objectifies visual characteristics of the image and estimates the depolarizing properties of collagen fibers with different nature of pathology with statistically significant difference (p<0,005). 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 18 Wiebke Knoll Institute for Biomedical Optics, Universität zu Lübeck, Germany [email protected] In vivo Two-Photon Microscopy and UV Laser Nanosurgery of Murine Small Intestine The intestinal epithelium is only protected by a thin layer of mucus that is produced by goblet cells, small lesions occur daily in the human body due to ingestion. These lesions can cause pathogen penetration into the lamina propria, which provokes an immune response. Repeated damage of the mucosal surface barrier often causes diverse intestinal disorders leading to inflammation, uncontrolled immune response and disequilibrium of the homeostasis. Up to now, inflammatory bowel diseases, which cause severe tissue impairments, are not yet understood. In order to gain insight into the dynamics of the highly complex intestinal system, processes in the small intestinal mucosa of mice were studied in vivo over several hours using spectrally resolved two-photon excited autofluorescence microscopy (2PAM). This technique provides a threedimensional imaging with subcellular resolution and allowed a non-damaging discrimination of various cell types and cell organelles that exhibit characteristic autofluorescent spectra. Moreover, to better understand intestinal immune response and healing processes, murine small intestinal epithelium was exposed to laser-induced micro-lesions and investigated in vivo. Another aim in this project is the direct imaging of interactions between bacteria and the intestinal epithelium, using fluorescence-labeled bacteria strains and strains that were expressed in green fluorescent proteins (GFPs). Therefore, the study of local laser-induced lesions is of great interest, in order to understand the significance of accidental leaks for the invasion of bacteria. Philipp Krauter Material Optics, Institut für Lasertechnologien in der Medizin und Meßtechnik, Germany [email protected] NIR Remission Spectroscopy of Turbid Media In process control the knowledge of the quantitative concentration of the ingredients is important. This can be achieved by measuring the remission spectrum and by comparison with calibration spectra. However, this method cannot separate the reduced scattering coefficient from the absorption coefficient, e.g. an unnoticed change of reduced scattering is interpreted as a change in absorption. In contrast, a combination of the spatially resolved reflectance and the total reflectance enables the determination of absorption undisturbed by scattering. At the same time, it delivers a high wavelength resolution. In the following, reduced scattering was determined using the spatially resolved reflectance in the VIS. An empirical description of the wavelength dependence of reduced scattering is given by a power law. In the NIR the reduced scattering is obtained by extrapolating this law. Compared to our recent work the focus of this work is the enhancement of the spectral range of the total reflectance to 450 nm-1700 nm. An optimized data analysis allows fast interpretation of the measured remission spectrum by use of a lookup table. The distribution of light is calculated numerically with the radiative transfer equation. For these calculations, the detection geometry is taken into account. Verification of the method is done by determination of the absorption coefficient of an optical phantom, consisting of a known concentration of polystyrene spheres in water. Finally, the absorption spectrum of butter is shown, representing a possible application of the method. Even though only the total reflectance is regarded here, it is important to remember, that the knowledge of reduced scattering is needed for proper evaluation of absorption. Joao Lagarto Physics, Imperial College London, United Kingdom [email protected] Development And Application of Compact, Low-Cost Multispectral TimeResolved Fluorometric Fibre-Optic Probes For In Vivo Diagnosis And Study of Disease Autofluorescence lifetime (AFL) measurements are used to characterize tissue components and provide label-free contrast and information about structural and metabolic state of tissue without the need for the application of exogenous fluorescent labels and the associated concerns of toxicity and pharmacokinetics. Measurement of AFL has been shown to detect qualitative chemical changes of tissue. This project aims to translate the fluorescence lifetime imaging and measurement technology developed at the Photonics Group to the clinical study and prognosis of cancer, heart disease and osteoarthritis. We present a compact fibre-optic time-resolved spectrofluorometer to be deployed in animal laboratories and clinical settings for extended labelfree studies disease by exploiting tissue AFL to make a systematic study of structural and biochemical changes in tissues and to correlate these with changes in metabolic signals at 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 19 different stages of disease progression. Preliminary results for AFL measurements of cartilage tissue suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents. An ultimate goal of this research is to develop a low-cost, compact and portable fibre-optic fluorometer probe that could be easily replicated and applied in a range of investigations. If clinical efficiency and practicality is demonstrated throughout these studies, such an instrument could have significant potential for commercial development and clinical deployment. Riya C. Menezes Spectroscopy and Imaging, Institute of Photonic Technology Jena, Germany [email protected] Raman Spectroscopy - A Fast And Reliable Tool To Detect Secondary Fungal Metabolites Fungal competition for territory and resources is inevitable in habitats with overlap between the niches of different species or strains. Interactions between wood-decaying basidiomycetes are excellent examples of such competition and the outcome determines the size of territory held by one mycelium, and hence access to nutrients. It is a major impetus of community change, and affects decay rates in woodland ecosystems. Competition is brought about by antagonistic mycelial interactions which elicit mycelial morphology, metabolic, secondary metabolite release and extracellular enzyme pattern changes. The basidiomycete studied, Schizophyllum commune, is a white-rot fungus which is most widely distributed world-wide. The abundance of S. commune can not only be attributed to its incredible mating ability (more than 23,000 different sexes), but also to its superior competition ability. It has been noted that S. commune produces secondary metabolites, especially in the interaction zone or zone of contact with competitor species. The nature of these compounds is unknown and may be the result of the action of the fungal oxidative enzymes in response to stress caused in the face of a stimulans excreted from the competitor. We attempted to elucidate the composition of the compounds produced during these interactions using Raman micro-spectroscopy. The principle is that Raman spectra contain information on molecular vibrations providing a highly specific fingerprint of the molecular structure and biochemical composition of cells and tissues. Spectra are obtained non-invasively without interference from water. The employment of external labels is not necessary and the sample requires little or no preparation. Moreover, the coupling of Raman spectroscopy with microscopy (Raman Micro-spectroscopy) enables high spatial resolution (below ~ 1 µm) and sensitivity. Using particularly resonance Raman spectroscopy, we show the presence of Indigo in the zones of interaction of the fungi. Kelly E. Michaelsen Thayer School of Engineering, Dartmouth College, United States [email protected] Effects of Breast Compression on Digital Breast Tomosynthesis Guided Diffuse Optical Spectroscopy Diffuse optical spectroscopy of the breast has been successfully used to obtain functional information about tissue including hemoglobin, deoxygenated hemoglobin, water, lipid and scattering properties. These metabolic indicators can be altered by disease, leading to detectable changes in near infrared light signals, when measured at multiple wavelengths. However, due to the high propensity of light scattering, the resolution of these techniques is quite low in stand-alone systems. An integrated digital breast tomosynthesis (DBT) and near infrared (NIR) system combines high spatial resolution for identification of anatomic structures with information on the metabolic status of tissue, completely co-registered. NIR is non-ionizing, non-invasive and inexpensive modality that can be easily integrated into existing DBT systems. DBT provides excellent 3D spatial resolution with less superposition artifacts than traditional mammography while maintaining similar clinical workflow procedures. DBT/NIR is a synergistic combination with the potential to decrease the recall rate for breast cancer screening. A first generation prototype DBT/NIR system has been developed at Dartmouth. The system uses eight wavelengths of light from 660-940 nm and is not fiber based, keeping the system cost and complexity to a minimum while taking 46,000 measurements at different sources, detectors and wavelengths in under a minute. However, this time under full mammographic compression is quite long, given that the DBT exam takes under ten seconds. Here we compare the results of optical scans taken at full compression to those at slightly less compression to determine the effects of lower compression on functional tissue markers. Eleven normal subjects have been imaged to date on the DBT/NIR system, at both mild and full compression levels and for both cranio-caudal and medio-lateral oblique views. This study characterizes hemoglobin, oxygen saturation, water and lipid fractions in two segmented tissue types (adipose, and fibroglandular) as determined by full three-dimensional tomographic reconstructions. Comparisons between 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 20 mild and full compression levels effect contrast between the regions for the different chromophores. Changes in contrast between these two normal tissue types may provide insight into the likely effects of compression on contrast between malignant and fibroglandular tissues although addition studies on patients with known malignancy are needed. This analysis assesses the feasibility of decreasing the compression level during the NIR portion of the DBT/NIR exam for future patients. Christian Myrtus Institut of biomedical optics, University of Lübeck, Germany [email protected] Analyse And Visualize Ciliary Beat Frequency Ex Vivo Using Spectral Domain Optical Coherence Microscopy An important topic in lung research is the understanding of mechanisms that continuously clean the airways from inhaled particles by transport of mucus. This transport is maintained by the continuous beating of cilia that are present on airway epithelial cells. To date, the beat frequency of individual ciliated cells is measured as an indicator of mechanical clearance activity. The current frequency analysis is usually performed using Wide Field Microscopy (WFM). The main disadvantages of this technique are the low contrast of the ciliated cells in resulting images and the inability to be adapted to in-vivo diagnosis. One example is the problem, how to adapt the transmission illumination that it can be built in a probe (for example a bronchoscope). This work focuses on the verification of the spectral domain optical coherence microscopy (SDOCM) to be used for ciliary beat frequency analysis. The advantages of SDOCM compared to WFM are the improved contrast and higher resolution. Additionally, there are possibilities to integrate this technology e.g. in an endoscop to bring it into probes. For this purpose a microscope was developed, that is a combination of a SDOCM and a WFM. The integrated WFM acts as a reference system. The results show that the determination of ciliary beat frequency with the SDOCM is possible. Jonathan Nylk Department of Physics and Astronomy, University of St Andrews, United Kingdom [email protected] Wavefront Shaping in Light Sheet Microscopy Light sheet microscopy is a powerful, bio-compatible imaging technique. Although a fast and high contrast method, traditional light sheet microscopes cannot yield high resolution images over a large field of view due to the increasing beam divergence associated with narrowing of the beam waist. Illumination with propagation-invariant Bessel light modes has shown promising results to circumvent this issue, however, the extended transverse structure associated with propagation-invariant beam types causes contrast reduction and additional photo-damage. The use of beam shaping techniques, such as Bessel beam generation and more general wavefront shaping methods, can be advantageous to light sheet microscopy. Light sheet microscopy with any type of illumination beam suffers from sample based aberrations and scattering. The light sheet thickness and uniformity, which ultimately limit the image quality, are degraded in the presence of such aberrations. To extend the imaging capabilities of light sheet microscopy at depth in turbid specimens, in situ wavefront correction may be used to correct for aberrations in the illumination light sheet at a point of interest. In trials with scattering tissue phantoms the correction method gave intensity enhancements of ~2 times and reductions in sheet thickness by ~3 times, in all cases yielding a light sheet of diffraction-limited thickness. The use of novel beam types for light sheet microscopy has been demonstrated primarily with Bessel beam illumination but there has been little investigation into the how the properties of these beams impact on the image quality. By analysing the modular transfer function for different beam types, a detailed study of the beam properties and their impact on image quality is accomplished. From this analysis it is seen that Bessel beam illumination in light sheet microscopy can yield uniform resolution over a larger field of view due to the propagation-invariance of the Bessel beam, although a greater extent of propagation-invariance of the Bessel beam reduces the attainable resolution. Kamilla Nørregaard Niels Bohr Institute, University of Copenhagen, Denmark [email protected] Nanoparticle Mediated Photothermal Therapy and Integrated miRNA Delivery Conventional cancer therapies are highly invasive, limited by tumor accessibility, and patients typically suffer from severe side effects. Here we will investigate a novel strategy that combines thermal cancer therapy with microRNA (miRNA) silencing of cancer specific genes. By irradiating intracellular gold nanoparticles (AuNPs) with near-infrared light (NIR) we wish to 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 21 accomplish remote thermally assisted release of AuNPs from cellular endosomes, as well as systematic release of miRNA from AuNPs. This dual strategy is particularly promising since AuNPs are easily taken up by living cells and are promising as biomedical drug carriers or as local heat inducers. Plasmonic nanoparticles are strong photoabsorbers, and when irradiated at their resonance frequency the absorbed energy is converted into heat leading to elevated temperatures in the surrounding medium. AuNPs can be tailored to absorb and scatter light in the NIR region by modifying their size, shape and surface properties. NIR is preferable for cancer therapies since it has the lowest absorption and highest penetration depth in biological material. In addition, because of their surface properties AuNPs can easily be decorated with anticancer targeting agents, such as miRNA, and used for delivery. Functional miRNA can be released from the AuNPs by controlled heating, and silence or destructing specific pathogenic genes and consequently down regulating their encoded proteins. However, to obtain specific delivery of the miRNA the AuNPs must be taken up by the cancer cells, and escape the endosomes to prevent enzymatic degradation of the miRNA. Hence, we are tracking the AuNPs when taken up by MCF-7 cancer cells via the endosomal pathway and aiming to find a method to trigger externally controlled endosomal escape and miRNA release from the AuNPs. More precisely, we will use a Leica SP5 confocal microscope with an optical trap based on a 1064 nm laser beam to monitor the time-dependent transport of AuNPs encapsulated in fluorescently tagged endosomes by subcellular colocalization experiments. Subsequently, we will trigger release from a single endosome by laser induced membrane rupture. For the purpose of elucidating delivery of miRNA, we will use AuNPs coated with ~20 nm “carrier” miRNA and loaded with a complementary miRNA tagged with a dye. Hence, upon laser induced endosomal membrane rupture we expect to be able to reach temperatures that will also be sufficient to release the dye tagged miRNA from the “carrier” miRNA. This release can be monitored by a decrease of the fluorescent signal from the dye tagged miRNA. Provided with information on both the kinetics of the transport of encapsulated AuNPs and the release characteristic, i.e. how release depends on laser power and exposure time, we can control and systematically release the miRNA into the cytosol. When the miRNA are released into the cytosol they can mediate their therapeutic effect before fusion and degradation in the lysosomes. Due to their extraordinary physical, chemical, and optical properties and biocompatibility, we expect plasmonic nanoparticles to have great potential as delivery agents, and in therapeutic and diagnostic applications. Paul O'Mahoney Institute of Medical Science and Technology, University of Dundee, Scotland [email protected] Optical Red Blood Cell Sorting Cell based therapies are becoming a reality and hold great promise in applications as wide ranging as eye surgery and blood transfusion. In the latter, large numbers of industrially generated red blood cells need to be monitored and/or sorted to remove the risk of the teratomas which are associated with non-fully differentiated stem cells. Because of the volumes of cells involved, and their subsequent in vivo use, a passive monitoring/sorting approach is needed which does not rely on cell labelling. We have previously shown that optical techniques have the ability to sort according to size-dependant polarisability: i.e. between different particles depending on their intrinsic properties, i.e. size, shape, and refractive index. We exploit this passive sorting ability to sort stem cell derived RBC models made of both inert and biological content: i) using polymer spheres and ii) using an HL60 cell line. The latter is a cell model being used to develop the scaling up RBC production. Hence, the results of the HL60 experiments can be used as a good indicator of how red blood cells will perform under similar conditions. In initial experiments, an optical line trap was used to sort different sizes of polymer particles with reasonable success. A more sophisticated optical landscape has now been constructed and will be the basis for future experiments. Uros Orthaber Research and development, Optotek d.o.o., Slovenia [email protected] Observation of Laser-Induced Cavitation Bubble Dynamics Near A Thin Elastic Membrane With A High Speed Camera The presented research focuses on ophthalmic application of laser, more precisely on wellestablished surgical procedure known as posterior capsulotomy. This is a procedure where opacification occuring on the posterior capsule of the intraocular lens in the eye is removed by laser. A highly focused laser beam triggers rapid ionization in the intraocular environment which leads to plasma formation. Its subsequent expansion produces a shock wave that propagates spherically from the point of optical breakdown. Vaporization of the liquid in the focal volume leads to the formation of a cavitation bubble. The bubble contains significant amount of the laser 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 22 pulse energy, which is released during the bubble collapse. Combination of all the mentioned effects following the optical breakdown is called photodisruption. Mechanisms of photodisruption are responsible for the rupture of posterior capsule in the surgical procedure. Behavior of the cavitation bubble near a thin elastic membrane has been studied by recording the bubble-membrane interaction with a high speed camera. Cavitation bubble was generated as a result of a laser produced optical breakdown. Laser pulse energy and the distance between the bubble and the membrane was varied, leading to the observation of different regimes of bubble-membrane interaction. It has been discovered that at fixed energy the time of the membrane rupture is roughly independent of the distance between bubble and the membrane. It has been demonstrated that in the case of laser focus coinciding with the membrane surface, the bubble has little damaging effects. By increasing the distance between the bubble and membrane the rupturing becomes more pronounced, while at larger distances interaction becomes weaker. In order to produce maximum damage on the membrane, the laser focus should be shifted away from it. The whole picture of the interaction was obtained by recording events on both sides of the membrane. Membrane bending is followed by the jet intrusion, whereby the remainings of the bubble penetrate to the other side forming a conical shape which is eventually reduced. Results of presented work are of great importance for further improvements of the posterior capsulotomy surgery. Strong indications exist that by shortening the pulse duration it is possible to obtain same disruptive effects at lower energy, which can reduce unwanted damage to surrounding intraocular structares and thus improve safety, while also making the operation more efficient. Dino Ott Niels Bohr Institute, University of Copenhagen, Denmark [email protected] Biophotonic Interactions of Individual Nanoparticles In recent studies, it has been shown that optically trapped nanoparticles can serve as ultrasensitive probes for acoustic signals [Ohlinger et al., “Optically trapped gold nanoparticle enables listening at the microscale”, PRL (2012)]. Notably, Fourier spectral analysis of the dynamics of an optically trapped particle even allows to sense periodic processes that would normally be hidden behind Brownian motion. This capability allows for unique biophysical insights into the interplay between cell biology and cell mechanics. We present a dual-beam optical tweezers setup which is capable of both exerting a well-defined force via optically generated pressure waves and simultaneous detection of low amplitude pressure waves with high temporal resolution. Cross-talk, i.e. the incomplete separation of the detectable position signals of the individual particles, would typically limit the precision of this optical system. To circumvent this problem, a novel cross-talk eliminating detection method based on spatial filtering was established. Biophotonic applications of the presented setup include the investigation of the periodic beating of bacterial flagella and the response of mechanosensitive cells, e.g. auditory cells, to a mechanical stimulus of well-defined magnitude. Lukasz A. Paluchowski Dept. of Electronics and Telecommunications, NTNU, Norway [email protected] Dual-Mode Imaging System For Characterization of Wound Surface Evaluation of wound bioburden and wound severity is an important prognostic factor in optimal wound care. However, complete wound assessment should also include information about the depth, volume, length, width and surface area of a wound as well as its extent in time. While vascular and molecular aspect of wound assessment by using spectroscopic methods has been already investigated by our group, the main goal of this work is to study possible improvement of wound diagnosis by analyzing additional parameters like wound surface and wound geometry. In this project a dual-mode vision system to collect optical properties, shape and volume of chronic skin ulcers is under development. This system combines the functionality of 3D stereo-photogrammetry and 2D imaging spectroscopy. A high resolution pushbroom hyperspectral camera and monochromatic video frame camera are mounted on the same scanning system. Stereo images can be acquired with different baselines by controlling the position of the camera on the translation stage only in the scanning direction. Image processing algorithms have been implemented to combine the functionality of imaging spectroscopy and stereo-photogrammetry. In the first stage a Digital Surface Model (DSM) of the wound surface is computed after the rectification. The resulting DSM of normal skin surrounding the wound is then used to reconstruct the top surface above the wound and thus estimate the wound volume. Additionally, the hyperspectral image is co-registered to the monochromatic frame image. This allows for metric measurements of parameters delivered from spectroscopic and statistical analysis (e.g. tissue oxygenation, pigmentation, classification). Simultaneous analysis of the hyperspectral data and the surface model give a promising, new, non-invasive tool for 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 23 characterization of chronic wounds. Changes of the wound surface can indicate the rate of wound healing. Furthermore, the spectroscopic analyses can, if desired, be limited to a certain depth of interest, like the wound bed or wound border. Further work will concentrate on automated image acquisition and implementation of real time analysis. Stéphane Perrin Micro Nano Sciences & Systems, FEMTO-ST, France [email protected] Optical Coherence Tomography Microsystem For Early Diagnosis of Skin Pathologies Cutaneous cancer is nowadays the most commonly diagnosed type of cancer. Its early diagnosis increases the chances of successful treatment. Existing OCT systems can perform non-invasive 3D optical biopsies of skin, improving patient’s quality of life. Nevertheless these bulk systems are expensive, only affordable for Hospital and hence not sufficiently employed by physicians and dermatologists as an early diagnosis tool. VIAMOS project aims to improve this situation developing a handheld, low-cost and multifunctional Optical Coherence Tomography microsystem. It will enable doctors to perform a painless and earlier detection of skin pathologies such as melanoma and non-melanoma cancers. The goal of this project is to benefit from advanced MOEMS technologies proper to FEMTO-ST Institute (French National Centre for Scientific Research), enabling a new generation of miniature OCT instruments. This OCT system will allow getting an axial resolution and a lateral resolution equal to 5μm with a total penetration of 600μm. Thanks to array interferometer, filed of view of the system reaches to 8 x 8 mm². The main objective of my thesis work is to create the Optical Coherence Tomography microsystem, including the optical setup, software development (motion control, data acquisitions, processing, graphical user interface…), and validation (clinical trials on patient and volunteers) in collaboration with hospital. Sidsel R. Petersen DTU Fotonik, Technical University of Denmark, Denmark [email protected] Ytterbium Doped Fiber Amplifiers Above 1100 nm For Generation of Yellow Laser Light Ytterbium-doped photonic crystal fiber amplifiers are investigated as amplifiers for wavelengths above 1100 nm. The Ytterbium gain spectrum reaches from approximately 900 nm to 1200 nm and to realize an amplifier above 1100 nm the light generated below 1100 nm by spontaneous emission must be filtered out of the core, to avoid amplified spontaneous emission and parasitic lasing. In the fibers considered in this work a distributed spectral filter is incorporated in the fiber cladding, allowing for filtering of spontaneous emission, while the light propagates in the fiber core. Thereby the maximum gain is achieved at wavelengths above 1100 nm. The yellow light regime can be reached by frequency doubling of the amplified light, a regime which is of high interest for the medical industry, since it can be utilized for removal of birthmarks and treatment of blood vessel disorders. Frederico M. Pimenta Department of Chemistry, Aarhus University, Denmark [email protected] Towards the Control and Quantification of Singlet Oxygen Influence in Cellular Mechanisms Singlet oxygen, O2(a ∆g), the first electronic excited state of molecular oxygen, has been known in the community for over 80 years. Even though singlet oxygen can be produced in a variety of ways, the most used is by electronic energy transfer from an organic molecule (commonly referred to as photosensitizer) to ground state oxygen. Despite this method of generating singlet oxygen being widely used in photoinitiated cell death (e.g. photodynamic therapy) the ability to quantify the amount of singlet oxygen necessary to trigger any cell response is still limited by several things: (1) photobleaching and sensitizer re-localization (2) knowing the sensitizer concentration in the irradiation domain and (3) control over the local environment immediately surrounding the sensitizer. In short, a better mechanistic understanding of the roles played by singlet oxygen in cell death is limited to a dosimetry problem: controlling and quantifying the amount of singlet oxygen that perturbs a cell. To provide solutions to this rather complicated issue, two approaches have been examined. First, capitalizing on the fact that singlet oxygen produced outside a cell is also cytotoxic, a hydrophilic dendrimer-encased membraneimpermeable sensitizer was used to generate an extracellular population of singlet oxygen upon spatially localized two-photon irradiation. Through the use of this sensitizer and this approach, it is now possible to better control the singlet oxygen dose in selected microscope-based time1 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 24 and space-resolved single cell experiments. Afterwards, taking the knowledge obtained from this first study, specifically the control over the immediate environment surrounding the sensitizer, a protein designated “miniSOG” (for mini Singlet Oxygen Generator), was tested to ascertain if it could in fact be used as an intracellular sensitizer. MiniSOG is an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2 genetically engineered by Roger Tsien. In contrast to claims made by those who developed miniSOG, we show that this protein-encased FMN is actually a very poor singlet oxygen photosensitizer; enclosing FMN in this protein facilitates the photosensitized production of oxygen-dependent radicals (Type I chemistry) at the expense of singlet oxygen production (Type II chemistry). Nonetheless, efforts are being made to further improve MiniSOG by specific mutations that will increase singlet oxygen formation, effectively decreasing radical chemistry. The results presented here not only show the much-needed development of standards for the intracellular production of singlet oxygen, but also provide the important tools for the development of well-controlled sensitizers suitable for use in heterogeneous environment such as a mammalian cell. Tatiana Pryanikova Department for Radiophysical Methods in Medicine, Institute of Applied Physics RAS, Russia [email protected] Influence of Different Kinds of Therapy On The Oxygenation of Experimental Tumor Estimated by Diffuse Optical Spectroscopy Tumor oxygen state is considered to be one of the key factors influencing the disease prognosis and treatment effectiveness. Studying of the dynamics of oxygenation of tumor and normal tissues influenced by the different kinds of therapy (radiotherapy, radiomodifying agents) are necessary for optimization of dose and time parameters of the treatment. Diffuse Optical Spectroscopy (DOS) makes it possible the noninvasive determination of the tissue oxygen status based on information on the local changes in the optical parameters, and visualization of metabolic processes in the region of interest. DOS allows reconstruction of two-dimensional distribution of main tissue chromophores, that characterize the processes of oxygen supply (oxygenated hemoglobin) and oxygen consumption (deoxygenated hemoglobin), as well as the blood oxygen saturation level (StO2) that indirectly reflects the tissue oxygenation. The aim of the study was to investigate the experimental tumor oxygen status using DOS in vivo during tumor growth and under different types of treatment. Experiments were performed using white outbreed male rats on DOS setup (Institute of Applied Physics RAS, Russia). Plyss lymphosarcoma (PLS) was transplanted subcutaneously into the right lower third of the abdominal wall. Prior to investigation the animals were anaesthetized and placed in a cuvette filled with an immersion liquid of known optical parameters. PLS tumors were irradiated with single dose of 10 Gy using Co60 unit and studied by DOS before (7th day of tumor growth) and repeated every 24 hours after irradiation for 96 hours. A direct measurement of pO2 of tumor tissue was used as a method of verification of DOS results in three irradiated animals. For modification of tissue oxygenation hemorheologic agent pentoxifylline (Ptx) was used. Pentoxifylline was administered intraperitoneally at a dose of 10 mg/kg. The first DOS study was performed before drug administration on the 5-7th day of tumor growth. Further observations were conducted randomly with the interval of 15, 30 or 60 min for 4 hours. Radiation-induced changes of LSP oxygenation demonstrated a biphase character. 24 hours after irradiation the level of StO2 was decreased comparing to the initial level, and comparing to non-irradiated tumor. 48 hours after irradiation there was a statistically significant increase oxygen saturation comparing to non-irradiated tumor, comparing to non-irradiated tumor, which persisted during the next 24 hours; in later terms saturation had been gradually reduced and reached a control level in 96 hours after irradiation. Comparison between the tumor tissue oxygenation data obtained by the DOS and direct pO2 measurements demonstrated high correlation coefficient between two methods. Ptx caused increase of StO2 level in the tumor zone in 15 min after injection due to increase of oxyhemoglobin concentration. The increase of saturation was maintained up to 100 minutes after administration. In normal tissue Ptx did not effect the level of the oxygenation. DOS allows providing information on tumor oxygen state and its dynamics in the course of of therapy. The differences in the nature of changes of tumor’s oxygenation under influence of different types of treatment have been demonstrated. Peter Rejmstad Department of Biomedical Engineering, Linköping University, Sweden [email protected] Optical Monitoring in Neurointensive Care Using Laser Doppler and Reflectance Spectroscopy The high mortality rates of patients that experience secondary bleeding linked to traumatic brain injury (TBI) and the risk of delayed ischemia in patients with subarachnoid hemorrhage (SAH) motivate the development of new monitoring methods in neurointensive care. To gain more 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 25 information about the continuous cerebral events that occurs following brain damage an optical monitoring system using combined laser Doppler perfusion monitoring (LDPM) and diffuse reflectance spectroscopy (DRS) is implemented. LDPM gives information about the microcirculatory blood flow (perfusion) and level of reflected light in the sample volume. The DRS technique makes use of a spectrometer that collects the reflected light that has been affected by scattering or absorption. The collected spectra can be used to extract the chromophore content in the sample through multi-linear regression analysis and known absorption spectra of relevant chromophores. The optical monitoring system including software was first tested on skin and later evaluated during brain tumor resection operations. The gathered data was analyzed in order to distinguish the differences in blood flow and optical properties among the tissue types; white matter, gray matter and tumor tissue. Results were collected using a fiber optic probe containing four adjacent optical fibers, placed in cerebral tissue. Gathered data included microcirculatory perfusion with reflected light alongside diffuse reflectance spectroscopy data from which chromophore content could be extracted resulting in tissue oxygenation values. The laser Doppler measurement could discriminate between cerebral gray and white matter by looking at the reflected light intensity. The perfusion was found to be higher in gray tissue compared to white, in agreement with literature. However the relative perfusion values between gray and tumor tissue needs further investigation to elucidate their relation. The DRS results from the evaluation during tumor operations imply that tumorous and gray brain tissues have similar levels of oxygen saturation. Lower oxygen saturation values were recorded in the tumor border while the lowest values were found in white brain tissue. The work includes evaluation of an optical monitoring system, intended for use in neurointensive care, that records online perfusion data and can be used to collect spectroscopic information to assess chromophore information in a post analyzing step. Data collected during the evaluation phase of the optical monitoring system points out similarities and differences in perfusion and oxygenation values between various cerebral tissue types. The optical system has a promising ability to monitor patient status and help to gain more understanding about the cerebral circulation following brain damage. Ana Rita S. R. Ribeiro Physics and Astronomy, University of Porto, Portugal [email protected] Towards Optofluidic Systems For Single Cell Manipulation And Analysis Laser manipulation or optical trapping, as it is also known, refers to techniques for mechanical control of small objects, with sizes ranging from nanometers to micrometers, using the radiation force. This phenomenon was discovered by Arthur Ashkin in 1970. Optical Tweezers technology has been used in numerous directions, namely in the manipulation and sorting of small particles, or as a force sensor. In addition, Optical Tweezers are usually useful in the fields of biology, biochemistry and biophysics, where they are used to manipulate and interrogate individual cells or even molecules. The use of Optical Tweezers typically depends of bulk optical system, including conventional light microscopes combined with objectives with high numerical apertures. However, these types of setups are frequently expensive and also take up a lot of space. Nevertheless, Optical Tweezers systems can also be realized using optical fibers or other guided wave platforms, which reveal relevant advantages as low cost, miniaturized setups and flexibility. Optofluidics is a multidisciplinary area, which integrates both optics and fluids in the same platform, and which has been developed in order to provide techniques for sensing and analysis of biological samples. Additionally, these setups are designed to work in the micro scale, what makes them very useful to study and characterize single cells. However, optofluidic setups lack ability in the manipulation of particles/cells. Therefore, they become a much powerful tool when combined with fiber optical tweezers systems, allowing accurate sensing, and characterization of micron sized bodies. My PhD research aims to develop low cost optoelectronic systems for manipulation and monitoring of single cells. Therefore, this work addresses the problem of manipulating and sensing of small particles using both optical tweezers and optofluidic devices. Concerning to optical tweezers, a simulation program of the optical trapping forces will be presented, and also some results will be discussed. In addition, an implementation of fiber optical tweezers will be briefly presented. Furthermore, an optofluidic chip for red blood cell study (refractometric and spectroscopic measurements) will be presented. Further improvements of the setup will allow both manipulation and characterization of cells in the same platform. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 26 Radoslaw Sadowski Medical Physics and Applied Radiation Sciences, McMaster University, Canada [email protected] Quantitative Optical Molecular Imaging Bioluminescence and fluorescence tomography are used in optical molecular imaging to visualize tumours in small animals. Cone-Beam Computed Tomography (CBCT) integrated with an optical imaging system is described to perform 3D optical molecular imaging on optical phantoms. The work attempts to co-register CBCT images with optical images using a projective transformation to perform 3D optical tomography. Emanuel Saerchen Ophthalmic Laser Surgery, Rowiak GmbH, Germany [email protected] Physical Investigation of Self-Induced Laser Focus Displacement during Photodisruption with Ultrashort Pulses The usage of lasers in medicine gains more and more popularity in public. For instance, the huge advantage of femtosecond-laser-surgery is dominated by the precise cutting technique at the focal position with limited thermal influence of surrounding tissue. The cutting process is achieved by laser-induced photodisruption to create micro-cavities. This principle was already extensively investigated for single pulses. However, several phenomena of fs-laser processing are still not entirely understood, when using multiple pulses with variable parameters. Focusing the laser inside the material and scanning the laser spot for planar dissection results in a periodic, step-like structure oriented in the axial direction of the laser beam with variable step height up to 100 µm. The influence of laser parameters as pulse energy, pulse-to-pulse distance, numerical aperture and repetition rate was investigated for single pulses, applied lines and planes. Therefore, the laser was focused inside the copolymer Hydroxy-Ethyl-Methacrylat (HEMA). The resulted height of the step-like structures was measured for evaluation. Apparently, the parameter pulse-to-pulse distance has the major effect on self-induced focus displacement. A decrease of the pulse-to-pulse distance below 50 nanometers (nm) resulted in an exponential increase of step height on pulse distance up to 40 µm. Furthermore, increasing pulse energy enhances the effect. For constant pulse distance of 0.13 nm the step height and width increases from ca. 7 to 16 µm with increasing laser pulse energy from 80 to 240 nJ. The size of step height and width increases consistently. Those periodic structures are unwanted for any application in the field of precise laser cutting. Since the focus displacement occurs for two neighbored laser lines too, the effect is no pulse-to-pulse interaction. A local refractive index change around the laser focal spot is assumed as micro-scale reason for the focal displacement of those macroscopic unwanted structures. Digital-holographic microscopy was used for relative refractive index measurement of single pulse fs-laser application. A single applied femtosecond laser pulse with 210 nJ pulse energy revealed a central relative refractive index change of 0.005 in HEMA. The measured refractive index change per pulses was also used for numerical ray tracing simulation with Zemax. A microscopic focus displacement caused by a single local refractive index profile was shown. First clues for microscopic investigation of macroscopic unintended structures were revealed. The experiments indicate the importance to gather the triggering parameters to suppress this phenomenon. Since, fs-laser treatment should remain a fast and precise treatment. Eric Seifert Medical Lasercenter Lübeck, Medical Lasercenter Lübeck, Germany [email protected] Automatic Irradiation Control By An Optical Feedback Technique For Selective Retina Treatment (SRT) In A Rabbit Model Purpose: Selective retina therapy (SRT) is a laser method which targets the retinal pigment epithelium (RPE) with repetitive microsecond laser pulses, while causing no thermal damage to the neural retina, the photoreceptors as well as the chorioidea. Microbubbles arising at the melanosomes inside the RPE cells are the origin of selective RPE cell death. This makes SRT to a potential treatment for several retinal diseases. Beneficial effects on Central Serous Retinopathy (CSR) and diabetic macula edema (DME) have already been shown. The pigmentation variation of the RPE makes the treatment procedure impossible without additional feedback techniques, because the required laser pulse energy to achieve selective lesions varies with it. Furthermore, there is just a low therapeutic window of two times the laser pulse energy of angiographic visibility until the SRT loses its selectivity. This is why dosimetrycomponents designed to detect signals correlated with RPE-cell damage are a mandatory element in SRT devices. Optical techniques to detect micro bubble formation based on the evaluation off the backscattered light (“reflectometry”) just need to analyze one single pulse to 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 27 accomplish this. This makes it possible to build an SRT system able to increase the pulse energy with every pulse and to turn off the laser irradiation automatically (“automatic dosimetry”). Our purpose was to evaluate the safety, selectivity and healing of the retinal lesions by using an automatic dosimetry technique for SRT based on the evaluation of reflected light during irradiation. Methods: Ten eyes of Chinchilla Bastard rabbits were treated with SRT using a Q-switched NdYLF laser (wavelength: 527 nm, pulse duration: 1.7 µs, repetition rate: 100Hz, max. number of pulses in a burst: 30, retinal spot diameter: 120µm, typical maximum energy: 85µJ to 100µJ). The pulse energy has been increased with every pulse within a burst by 3% of the maximum energy. The signals are extracted from the backscattered light and analyzed by an algorithm which calculates a final value for each pulse within 2ms. In the experimental treatments of the rabbit eyes the laser irradiation has been ceased as soon as the reflectometry value was larger than the chosen threshold value. Results: Typical fundus photographs obtained 1 hour after irradiation showed that all lesions produced by SRT with automatic turnoffs were not visible ophthalmoscopically at all energy levels used. The lesions could be detected only by angiography. Fundus examination showed focal pigmented areas due to healing status and no fluorescein leakage after 7 days. OCT images revealed an inner retina continuity. By 3 weeks, histology showed selective RPE damage sparing photoreceptor continuity without inner retinal effects and focally proliferated. Conclusions: SRT controlled by an automatic dosimetry system based on analyzing reflected light to detect micro bubble formation achieves selective targeting of the RPE. Reflectometry is a reliable noncontact technique to monitor RPE disintegration and can serve as real-time dosimetry control during SRT. Kristian J. Sexton Thayer School of Engineering, Dartmouth College, United States [email protected] Pulsed Light Excitation And Image Gating For Fluorescence Guided Surgery In Normal Lighting Conditions Optical imaging may prove to have its greatest clinical impact in the area of fluorescence guided surgery. In order for this impact to be realized fluorescence guided surgical systems must both prove efficacy and be integrated into the operating room. Outlined is a surgical imaging system that utilizes the gating capabilities of an intensified CCD as well as high powered pulsed lights to allow for imaging in normal room light. This system also has the potential to allow real time spectral imaging due to the fast rate of acquisition. Together these two features may provide a superior technology moving forward in this field. Hendrik Spahr Institute of Biomedical Optics, University of LÜbeck, Germany [email protected] Imaging Temperature Distributions of Laser Irradiated Tissue via Phase Sensitive Optical Coherence Tomography The thermal processes taking place during retinal photocoagulation treatment can be visualized via phase sensitive OCT with high lateral as well as temporal resolution. We demonstrate that the observed effects are caused by thermal expansions and that the underlying temperature distribution can be estimated from the recorded OCT data. Therefore the reversible thermal expansion of laser irradiated multilayered silicone phantoms is investigated. The silicone samples are irradiated by a photocoagulation laser (ZEISS Visulas 532s) and the thermal expansion is measured by a commercially available high speed Fourier domain OCT system (Hyperion Spectral Radar, Thorlabs GmbH, Munich, Germany). A comparison between the measured temperature distribution (and its temporal evolution) to Monte Carlo simulations of the photon propagation inside the sample and analytical solutions of the heat equation show good accordance. Also in measurements of retinal photocoagulation in ex vivo porcine eyes and in vivo rabbit eyes, that are done using the above mentioned OCT system and photocoagulation laser as well as a setup for optoacoustical temperature measurements attached to a laser slitlamp, the temperature distribution can be visualized by measuring the thermal expansion. As one would expect, the highest temperature increase is observed in the retinal pigment epithelium (RPE), that absorbs the biggest part of the incoming laser power. The temporal evolution of this expansion is strongly correlated to the optoacoustically measured temperature increase. For a quantitative comparison, first of all the thermal expansion coefficients and the temperature dependence of the refractive index for retinal tissue have to be determined. This can also be done very accurately via phase sensitive OCT by imaging the optical and geometrical thickness of a tissue sample in the temperature range of interest. Between 30°C and 40°C the thermal expansion of retinal tissue is reversible and about three times larger than 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 28 the one of water. At higher temperatures irreversible and highly anisotropic tissue alterations occur, that cause a strong increase of the thermal expansion. These effects can possibly be used for an OCT based real time dosimetry system for retinal photocoagulation treatment. For in vivo measurements a robust motion correction is necessary. It is based on a separation of physiological global movement of the retina due to breathing and heartbeat from the differential movement inside the retina due to thermal expansion. In in vivo studies on rabbit eyes, physiological reactions to the laser irradiation are observed, that did not occur in ex vivo porcine eyes. Therefore, the investigations will soon be continued in a clinical study on photocoagulation in human eyes. Jacob Staley MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, The Netherlands [email protected] Photoacoustic Contrast Agents as Acoustic Time-Reversal Sources For Targeted Acousto-Optics To overcome speed of sound aberrations of the acoustic focus in acousto-optic imaging we utilize the time-reversal invariance characteristics of the lossless wave equation, extending time-reversal acoustics (TRA) to time-reversal photoacoustics (TRPA) for targeted measurements. Paulien L. Stegehuis Surgery and Radiology, Leiden University Medical Center, The Netherlands [email protected] Discrimination of Benign And Malignant Human Breast Tissue Using Full Field Optical Coherence Tomography Introduction: Despite many technological developments, accurate detection and imaging of human tumors intraoperative remains difficult and time consuming. During surgery it is extremely important that borders of the excised specimen do not contain tumor cells, since these positive margins are associated with an increased risk of local recurrence of the primary tumor. To remove the whole tumor and achieve clear margins, surgeons still rely mainly on visual assessment and palpation. In breast conserving surgery (BCS), this results in positive margins found in 20-40% of patients. Full field OCT: We propose an optical coherence tomography (OCT) principle which is able to image with microscopic resolution tissue specimens intraoperative: full field OCT (FF-OCT). OCT detects back-reflected light in a tissue sample; an interference pattern arises when light returning from the sample and an oscillating reference mirror have traveled the same distance. This allows determination of the depth from which the light is reflected in the tissue sample. Areas in the sample exhibiting great reflectivity will consequently generate higher interference, creating a larger signal than areas with lower reflectivity. The combined signal is collected by the detector, and an OCT image, typically cross-sectional, can be created. FF-OCT is different from conventional OCT in several aspects. First, en face images are made instead of crosssectional images. Moreover, a field of view of 0.8 x 0.8 mm is illuminated and reconstructed without need for scanning, and larger fields are obtained by stitching the images automatically. Second, with FF-OCT images with a high resolution of ~ 1 µm in 3D, an isotropic resolution is achieved. Margin assessment and future applications: With its high resolution and non-invasive, nondestructive nature and because tissue preparation, coloring and staining is not needed, we think FF-OCT will be a good technique to examine tissue specimens. Currently, the FF-OCT device used (light-CTTM device, LLTech, Paris, France) is not yet fast enough for intraoperative purposes, and also the measurements of the scanned tissue are limited. However, when these issues are resolved, this technique has the potential to become a powerful tool to substantially improve the surgical treatment of cancer patients, enabling direct feedback to surgeons on the excised tissue. Because of the great clinical relevance of resection margins in BCS, in the first clinical study, we will focus on breast margin assessment. If successful, this technique can easily be used for the complete resection of other tumor types. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 29 Idan Steinberg Faculty of Engineering; Department of Biomedical Engineering, Tel Aviv University, Israel [email protected] Frequency Domain Photoacoustic Phase Measurements of the Acoustic Modes in Bone for the Early Detection and Diagnosis of Osteoporosis Osteoporosis is a major public health problem worldwide. It is extremely widespread, has a catastrophic impact on patients life expectancy and quality and has an overwhelming related healthcare costs. Early diagnosis of patients at risk of fracture, but who have not yet sustained a fracture, can substantially reduce the healthcare costs and improve patient's lives. The risk of osteoporotic fracture depends not only on the bone mineral density, measured in clinical practice using the Dual-energy X-ray Absorptiometry (DXA) method, but also on the bone microstructure and functional status. In addition DXA is costly and involves ionizing radiation. Attempts to develop alternatives to DXA using quantitative ultrasound technology achieved limited success due to their insensitivity to bone functionality. Pure optical method, fail as well due to tissue scattering. Thus, there is an unmet need for a non-invasive, non-ionizing and costeffective method to detect the disease based on its pathological expressions. We propose a hybrid multispectral photoacoustic measurement that has great advantages over pure ultrasonic or optical methods as it allows deducing: a) bone functionality from the bone absorption spectrum and b) bone resistance to fracture from the characteristics of the ultrasound propagation. Here we describe a single experiment to demonstrate the feasibly of such photoacoustic method to differentiate between naïve and demineralized bone. To this end, a single wavelength, phase measurements were performed on fowl bone sample. We used amplitude modulated, fiber coupled laser diode at 830 nm to excite acoustic signals in a distal location along the bone. The excitation position is scanned along the bone while the acoustic response is measured proximally. As phase accumulation is highly non-linear with the changes in distance, a multimodal phasor based model is presented to account for such behavior. We demonstrate that frequency domain, multimodal phase analysis can yield the phase velocities and relative amplitude of two significant acoustic modes. This process was repeated for multiple acoustic frequencies. Theoretical results are shown to predict experimental results very well. However, there is great variability in the esimated speed from freqncy to frequncy. This can be explained by both numerical inaccuracies due to the fitting of a complicated model as well as the extreme dispersion as predicted by theoretical models. The bone is then soaked in mild acetic acid to simulate the effect of osteoporosis and all measurements were performed again for comparison. It is shown that bone demineralization is accompanied by significant changes in the speeds of the acoustic mode and in their relative amplitude. To conclude, Frequency domain photoacoustic measurements of bone parameters were demonstrated over multiple acoustic frequencies. We have shown that the measurements of phase of the photoacoustic signal in the modulation frequency revels the existence of fast and slow modes which propagate in the bone. The speed of each mode and their relative amplitude convey biomechanical information regarding the bone strength. It was shown that such method has a potential to provide important information regarding the bone status. Fatma Tümer Russell Division, Max Planck Institute for the Science of Light, Germany [email protected] Long-Distance laser Propulsion And Deformation Monitoring of Cells In HollowCore Photonic Crystal Fiber In optical tweezers, radiation forces near the focus of a laser beam allow trapping and micromanipulation of particles and cells. In hollow-core photonic crystal fibre (HC-PCF), light propagates in a single, non-diffracting optical mode, allowing particles to be trapped laterally at the center of the core. The radiation forces are highly uniform along the fibre, enabling guidance and propulsion of particles over long distances. In the past years, our group has shown that µmsized particles can be controllably launched into and propelled by the fundamental mode in both fluid-and air-filled HC-PCF. This system may have important applications in biomedicine, as it makes possible the laser propulsion of cells over distances of 10s of cm through a liquid-filled HC-PCF. In these recent experiments, individual red blood cells were optically trapped at the center of the core, several microns away from the glass interface, thus eliminating adherence effects and external perturbations. The velocity of the cells was monitored by Doppler velocimetry, and dynamic changes in velocity at constant optical powers up to 350 mW indicated strong stress-induced changes in the cell morphology. In future work we plan to integrate HC-PCFs into microfluidic circuits, allowing high-throughput biomechanics experiments. This will allow us to develop biomechanical assays capable of measuring the mechanical properties at the single-cell level. Such assays would allow detailed measurements 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 30 of the changes in physical properties during cellular processes such as differentiation, progression of disease and oncogenics. Peter Van Es BioMedical Photonic Imaging, University of Twente, The Netherlands [email protected] Photoacoustic Imaging of Human and Murine Joints: Towards Assessment of Rheumatoid Arthritis Rheumatoid arthritis is a chronic and progressive disease that affects about 1% of the population. Inflammation and proliferation of synovial tissue leads to degradation of the cartilage and subchondral bone. Hypoxia is suggested to contribute significantly to the disease progression and maintenance by promoting angiogenesis. Angiogenesis results in a significant increase in the blood vessels density in and around the synovial membrane. This makes Photoacoustic imaging is a good candidate for the assessment of rheumatoid arthritis because the hemoglobin, which is abundantly present in the inflamed synovial membrane, is an intrinsic chromophore that highly absorbs the visible and near-infrared light that is typically used for photoacoustic imaging. Currently two projects have started. The first is on photoacoustic joint imaging on healthy finger joints with a reflection-mode geometry with the goal to assess inflammatory arthritis. The second project focusses on ex-vivo imaging of mouse knees in a CTmode geometry with top illumination. Imaging in human subjects still proves difficult without more sophisticated reconstruction algorithms because acoustic reflections from tendons and bone obscure the signals of the synovial membrane. Imaging mouse knees seems to be promising in detecting blood vessels around the knee joint but the imaging device and reconstruction algorithms should be improved first. Luka Vidovic Complex Matter, Jozef Stefan Institute, Slovenia [email protected] Prediction of the Maximal Safe Laser Radiant Exposure on an Individual Patient Basis Based on Photothermal Temperature Profiling Efficancy of several laser procedures in dermatology and aesthetic surgery is compromised by the risk of permanent side effects, such as dyspigmentation or scarring. Strong absorption in epidermal melanin can cause overheating of superficial layer of skin and hence limits applied radiant exposure in the treatment. Although the concentration and distribution of melanin in skin exhibit large inter- and intra-patient variation, the applied laser radiant exposure is currently selected based on subjective visual impression and expert opinion of the treating physician. Our motivation is to predict the maximal safe radiant exposure on an individual patient basis based on photothermal temperature profiling. Pulsed photothermal radiometry (PPTR) allows noninvasive determination of laser-induced temperature profiles in strongly scattering layered samples (e.g. skin). PPTR measures transient change of IR emission from tissue following the short laser pulse. The temperature depth profiles can then be reconstructed by solving the inverse problem of heat diffusion and blackbody emission using a custom iterative algorithm. We show that such information can be used to derive rather accurate predictions of the maximal safe radiant exposure (Hmax) on an individual patient basis. We use a dedicated numerical model of heat transfer and protein denaturation dynamics to compute the predicted level of epidermal thermal damage (Ω) at the applied radiant exposure. Finally, we establish a quantitative relationship between the predicted thermal damage (Ω) and clinically observed severity of injury in a former clinical study of Verkruysse et al. of laser injuries induced by 3 ms laser pulses at 755 nm. The quality of correlation enables us to define the thermal damage threshold value and thus objective determination of the maximal safe radiant exposure (Hmax) on individual patient basis. This approach may increase safety and efficiency of dermatologic laser treatments in the near future. Kari V. Vienola Rotterdam Ophthalmic Institute, Rotterdam Eye Hospital, The Netherlands [email protected] Imaging of Optic Nerve Head With Motion Corrected OCT Using Tracking SLO Fixational eye movements remain a major cause of artifacts in optical coherence tomography (OCT) images despite the increases in acquisition speeds. One approach to eliminate the eye motion is to stabilize the ophthalmic imaging system in real-time. In our research project, an experimental OCT instrument was combined with an active image-based eye tracking system to compensate for eye motion in OCT imaging. The OCT instrument was a phase-stabilized optical frequency domain imaging (OFDI) system operating at a center wavelength of 1040 nm and the eye tracker was an 840 nm scanning laser ophthalmoscope (SLO). Retinal tracking was 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 31 performed using real-time analysis of the distortions within SLO frames. OFDI had axial resolution of 4.8 µm (6.5 µm in air) and the theoretical spot-size on the retina was calculated to be 13.7 µm. Eye motion was reported at a rate of 960 Hz and motion signals were inverted to correction signals and used to keep the OCT scanning grid locked on the same retinal target throughout the measurement. In the case of a tracking lock failure (e.g. blink or large saccade), the tracker signaled the OFDI system to rescan corrupted B-scans immediately stepping back 10 B-scans and holding the position until signal was valid again. The achieved tracking bandwidth was 32 Hz due to an internal time lag of the hardware. The combined system allowed visualization of the optic nerve head (ONH) and the lamina cribrosa with negligible artifacts from eye motion. The measured residual motion in the OCT B-scans was 0.32 minutes of arc (~1.6 µm) in a human eye, which is in a good agreement with the residual motion measured from the model eye. Four volumes from the same location were registered together to visualize the different depths of the retina with a high signal-to-noise ratio. The pore structure was clearly visible up to 430 µm from the bottom of the ONH cup. Tracking OCT can be advantageous for routine clinical use, but also for patients who have weakened fixation capabilities due to a disease, age or recent trauma in the eye. Naja Villadsen Department of Chemistry, Aarhus University, Denmark [email protected] Laser Manipulation of Optically Trapped Objects We present two specific examples where optically trapped particles are manipulated with a femtosecond (fs) and a nanosecond (ns) laser, respectively. The ns laser has a low repetition rate and will result in a kicking movement away from the centre of the trap, as opposed to the fs laser which has a high repetition rate and will result in a pushing movement. In both applications the dislocation is observed along the direction of pushing while a synchronised observation of the intensity is made. The results reveal viscoelastic characteristics of the media. The experiments are made with a unique counter propagating optical trap [1]. Central for the setup are two objectives of relatively low NA, giving a large working distance. The large working distance enables manipulation and detection orthogonal to the trapping beams. High speed three dimensional detection occurs through two CMOS cameras and a newly installed position sensitive detector (PSD), which increases the time resolution from 1 kHz to 250 kHz. For the fs pushing - because of viscoelastic effects of the fluid surrounding the particle, a time delay will appear between the pushing force and the resulting position of the particle. The intensity of the pushing laser is manipulated to get a sinusoidal force, for which the time delay can be analysed either through Fourier analysis or through cross correlation. In both cases the time delay fits the theoretical prediction for a sinusoidal force, confirming that the pushing force of a fs laser with high repetition rate resembles a CW-laser source. Furthermore, knowing the time delay, one can post-eliminate the Brownian motion, thus enabling a more precise determination of the relationship between the power of the pushing laser and the resulting displacement of the trapped particle. Now for the ns kicking. By synchronizing the kick to the detection system and by repetition of the kick, the Brownian nature of the particle can once again be post-eliminated to reveal the underlying deterministic dynamic [2]. For the ns laser with low repletion rate it is the relaxation after a kick that appears. With the new PSD the time resolution can be improved we hope to get a time resolution below the impulse relaxation time for our system to get an even closer look at the relaxation. A high power trap mode and the possibility of side-view observations of trapped objects with cameras, enables viscoelastic observation of these trapped objects. Among these, observations of cell stretching reveal cellular characteristics, with the application of detecting deceased cells. As the setup is best suited for objects larger than three microns it is ideal for handling cell size objects. All of our current experiments have application within microrheology with the focus on either the trapping media or the trapped objects. [1] T. B. Lindballe, M. V. Kristensen, et al. , Journal of the European Optical Society-Rapid Publications 6 (2011). [2] T. B. Lindballe, et al. , Opt. Express 21, 1986 (2013). Yolanda Villanueva MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, The Netherlands [email protected] Determination of The Grüneisen Parameter of Absorbing Liquids Using Photoacoustic Measurements In An Integrating Sphere A method for determining the Grüneisen parameter of absorbing liquids is presented. An integrating sphere is used as a platform for accurate and simultaneous detection of optical and photoacoustic signals. Calibration of the setup is done using aqueous ink solutions. The method is validated using human blood sample. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 32 Chiara Vitelli Center of Life Nanoscience at La Sapienza, Istituto Italiano di Tecnologia, Italy [email protected] Realization of A Micro-Optical Coherence Tomography (mOCT) Setup For Cell Imaging With Micron Resolution In Tissues Our project is devoted to the realization of a micro-OCT (ƒÝOCT) setup for the study of cellular and subcellular tissues structures, achieved by the use of a supercontinuum laser source, able to provide bandwidth of 250nm at 800nm and 1300nm wavelength, corresponding to an axial resolution equal to 1.2ƒÝm and 3ƒÝm in air, respectively. A first application will be related to cardiovascular research, in particular we will address the identification of endothelial cells lining coronary arteries, whose discrimination is important on the one hand for the identification of artheriosclerotic plaques, on the other hand is crucial for the analysis of stent implantation. Daniel Wangpraseurt Department of Environmental Sciences, University of Technology, Sydney, Australia [email protected] Coral Tissue Optics Light quantity and quality are among the most important factors determining the physiology and stress response of symbiotic corals. Yet, almost nothing is known about the light field that coral photosymbionts experience within their coral host, and the basic optical properties of coral tissue are unknown. We used fibre optic microsensor for scalar irradiance and field radiance to directly measure vertical and lateral light gradients within coral tissues. Our results revealed the presence of steep light gradients with PAR (photosynthetically available radiation) decreasing by about one order of magnitude from the tissue surface to the coral skeleton. Upper coral tissue layers are characterised by scalar irradiance maxima of ~200% of the incident irradiance, whilst deeper tissue layers, e.g. ~1000 µm into aboral polyp tissues, harbor optical microniches, where only ~10% of the incident irradiance remains. When corals were illuminated with a laser beam (636 nm) intense scattering of both tissue and skeleton was observed. Scalar irradiance measurements within the coral tissue showed that light was laterally transported up to 15–20 mm away from the beam. We conclude that the optical microenvironment of corals exhibits strong lateral and vertical gradients of scalar irradiance, which are affected by both tissue and skeleton optical properties. Our results imply that populations of photosymbionts inhabit a strongly heterogeneous light environment and highlight the presence of different optical microniches in corals; an important finding for understanding the photobiology and stress response of coral symbionts. Muhammad Nur Salihin Yusoff School of Physics, Universiti Sains Malaysia (USM), Malaysia [email protected] Optical Reflectance And Morphology of Poly(Vinyl Alcohol) Cryogel Tailored By Rock Salt Poly(vinyl alcohol) (PVA) is widely used as tissue-mimicking phantom in magnetic resonance imaging (MRI) and ultrasound studies by cross-linking of its monomer. Freeze-thawing is one of the cross-linking techniques in which the polymer obtained is called PVA cryogel (PVA-C). The chemical and mechanical properties of PVA-C have been extensively studied. However, in biomedical optics it is still new and not much explored yet. One of the challenges of using PVAC in optical studies is its apparent whiteness, which gives scattering effect. In this study we evaluate to what extent a rock salt with 2.5 %, 5 % and 7.5 % concentrations can alter the optical reflectance and morphology of PVA-C. Besides that, the effect of PVA-water ratios of 11, 1-2 and 1-3 (without rock salt) on PVA-C characteristics was also analysed. The optical reflectance of PVA-C samples across 380 – 780 nm of wavelengths was measured using Jaz Spectrometer equipped with HL 2000 Tungsten Halogen light source (Ocean Optics). Our results show that, adding rock salt to PVA can alter the optical reflectance of PVA-C and gives an advantage of a smoother structure. However, the amount of rock salt is only limited to concentration below 7.5 %. At this concentration, immediate coagulation occurred during the stirring process forming a PVA hydrogel. While different concentrations of rock salt below this limit did not give much variation on optical reflectance of PVA-C. Besides that, using different PVA-water ratio during preparation can also alter the optical reflectance of PVA-C but its effect is not consistent across the wavelengths. The different of PVA-water ratios have no significant effects on PVA-C structure obtained. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 33 Behrooz Zabihian Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria [email protected] Towards Multimodal all Optical Detection Photoacoustic Tomography and Swept Source OCT Optical Coherence tomography (OCT) is a well establish noninvasive biomedical imaging modality that is based on back scattered laser light incident on a sample. Since the introduction of the Fourier Domain OCT (FD-OCT), performance of OCT imaging systems is constantly improving. Newer sources with higher speed and better sensitivity are realized. With OCT one could obtain depth resolved morphological information of the sample under study with penetration depth of 1-2 mm. Swept Source OCT (SS-OCT) uses a coherent narrow band light source that sweeps wavelengths across a certain bandwidth. On the other hand, Photoacoustic Imaging (PAI) combines optical absorption contrast with ultrasonic spatial resolution. PAI is based on the detection of ultrasound waves produced by thermoelastic expansion of absorbers. In this technique, short pulsed laser beam is used to excite the sample so that the endogenous chromophores selectively absorb the laser energy and therefore cause the thermoelastic expansion. Photoacoustic Tomography is one approach to PAI in which the sample is illuminated light in full-field manner. 6th International Graduate Summer School Biophotonics ‘13 8-15 June 2013 • Island of Ven • Sweden 34