After Biophotonics '09
6-13 June 2009, Ven, Sweden
Best Poster Presentation Award 2007
Lakshmi Sampath
Baylor College of Medicine
Molecular Physiology and Biophysics
Houston, TX, USA
lsampath@bcm.edu
Poster title: Non-invasive in-vivo detection of HER2 overexpression in breast cancer using dual-labeled trastuzumab-based imaging agent
Abstract: Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in cancer owing to its participation in signaling pathways regulating cellular proliferation, differentiation, motility and survival. In this work, we have exploited the extracellular binding property of Herceptin (trastuzumab), an anti-HER2 monoclonal antibody, to design a diagnostic imaging agent that is dual-labeled with 111-Indium, a gamma emitter, and a near-infrared fluorescent dye, IRDye 800CW, to detect HER2 overexpression in breast cancer cells. Methods: Fluorescence and confocal microscopy were used to determine the molecular specificity of DTPA-trastuzumab-IRDye800CW in-vitro in SKBr3 (HER2 +) and MDA-MB-231 (HER2 -) breast cancer cells. SKBr3 cells were incubated with IRDye 800CW; or pre-treated with Herceptin or human IgG followed by DTPA-trastuzumab-IRDye800CW and examined under a fluorescence microscope. For in-vivo characterization, athymic nude mice bearing SKBr3-luc subcutaneous (s.c.) xenografts, were injected with the dual-labeled imaging agent intravenous (i.v.), and imaged using SPECT and near infra-red (NIR) fluorescence imaging at 24 h and 48 h. Tumor-bearing mice were also injected i.v. with Herceptin 24 h before following up with 111In-DTPA-trastuzumab-IRDye800CW. Non-specific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and 111In-DTPA-IgG-IRDye800CW. Results: DTPA-trastuzumab-IRDye800CW binds significantly higher to SKBr3 cells compared to MDA-MB-231 cells. Confocal imaging revealed that this binding occurs predominantly around the cell membrane. Competitive binding studies using excess Herceptin prior to incubation with DTPA-trastuzumab-IRDye800CW abolished this binding affinity while pre-treatment with non-specific IgG did not alter binding. In-vivo imaging of SKBr3-luc xenografts mimicked the binding affinities observed in-vitro. Nuclear and optical imaging show strong binding of 111In-DTPA-trastuzumab-IRDye800CW in the tumor region compared to the contralateral muscle region. The tumor-to-muscle ratio (TMR) decreased in mice pre-treated with Herceptin, and further reduced in mice injected with IRDye 800CW and 111In-DTPA-IgG-IRDye800CW. Ex-vivo imaging of dissected organs confirm the same. Finally, co-registering H&E stains with autoradiography from tumor tissue slices indicate that 111In-DTPA-trastuzumab-IRDye800CW binds only in the viable region of the tissue and not necrotic areas. Conclusion: Dual–labeled 111In-DTPA-Trastuzumab-IRDye800CW may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.
Lakshmi Sampath (right) at her award-winning poster at Biophotonics '07. From left: Profs. Stefan Andersson-Engels and Peter E. Andersen.
Honorable Mentioning
Nicole Prent
University of Toronto
Physics and Institute for Optics
Toronto, Canada
n.prent@rogers.com
Poster title: Investigating mitochondrial activity during muscle contractions with harmonic generation microscopy
Abstract: Laser scanning harmonic generation microscopy is used to image cellular and subcellular biological structures in vivo, and is particularly useful for dynamic investigations of these structures. When high intensity light is under tight focusing conditions, such as in a high numerical aperture microscope objectives, nonlinear optical signals are produced from the interaction of matter with light. Of particular interest are the second harmonic generation (SHG) and third harmonic generation (THG) processes, however, absorptive processes, such as, multiphoton excitation fluorescence (MPEF) can also be utilized in the nonlinear microscope. There are many naturally occurring autofluorescing molecules that can be used for biological imaging, such as the dinucleotide NADH, which is a key member in the electron transfer chain in mitochondria. This research focuses on determining the functional dynamics of cellular processes using a combination of simultaneously acquired harmonic generation and MPEF detection. Although, fluorescence microscopy is a useful imaging technique it is accompanied by heat dissipation in the sample, resulting in devastating effects on living systems. Although high laser scanning rates reduce the exposure time, the signal still decreases over time (bleaching). Another way to reduce sample heating is to employ parametric processes such as SHG and THG, since they involve only virtual electronic states where energy is not transferred into the medium. There are many naturally occurring structures that exhibit harmonic generation effects, and hence, do not require dyes or external additives that can potentially disrupt the normal functionality of the system. SHG is efficiently generated in noncentrosymmetric media, including collagen bundles and striated muscle. Conversely, the third harmonic signal is enhanced by the presence of interfaces, such as, biological membranes including cell walls. THG enhancement from mitochondria and chloroplasts is attributed to their multilayered membranous structure, which theoretically depends on the spacing and relative optical properties of the layers, and can hence be used to probe the functional properties of these organelles. The sensitivity of the harmonics to media properties and the noninvasive nature of harmonic generation is ideal for studying cellular dynamics over extended periods of time. Dynamic imaging of contracting myocytes has been performed with a multimodal nonlinear microscope, which is capable of simultaneous detection of MPEF, SHG and THG. The contraction of sarcomeres was monitored by observing the distances between anisotropic bands, which are indicated by efficient SHG signal. In contrast, characteristic rows of mitochondria aligned along myofibrils were visualized with THG. The enhancement of the third harmonic signal is thought to arise from the multilayer arrangement of the mitochondrial crista. Time lapse series of 2D optical slices of a myocyte revealed third harmonic intensity fluctuations “flickering” similar to the previously observed fluorescence flickering of tetramethylrhodamine methyl (TMRM) labeled mitochondria. Flickering THG signals could result from variations in the crista spacing and changes in the transmembrane potential that are associated with mitochondrial activity. Correlations between the sarcomere nanocontractions and the mitochondrial activity are being investigated to determine the functional dynamics of mitochondria during muscle contraction.
Honorable Mentioning
Emilie Krite Svanberg
Lund University
Anaesthesiology and Intensive Care
Lund, Sweden
emilie.krite_svanberg@med.lu.se
Poster title: Physiological effects on skin and skeletal muscle studied with three noninvasive optical techniques
Abstract: Physiological processes in skin and skeletal muscle can be studied clinically with three different noninvasive optical measurement techniques, Near-Infrared Spectroscopy (NIRS, Laser Doppler Imaging (LDI) and Tissue Viability Imaging (TVI). With NIRS near-infrared light (700-1000nm) reaches the underlying muscle tissue through the skin and is there absorbed by oxygenated hemoglobin. The spectral change in the back-scattered light will therefore reflect the saturation of the tissue. Using LDI the perfusion of the skin can be calculated from the frequency shift in the continuous laser light when it is reflected by red blood cells in motion, the so called Doppler shift. TVI uses polarisation spectroscopy to measure changes in the blood volume of the skin by calculating the concentration of hemoglobin. The polarised light, which is reflected by the skin surface is blocked out. In this study NIRS, LDI and TVI are used to study saturation, perfusion abd blood volume in skin and skeletal muscle during different physiological situations. Continous measurements with NIRS, LDI and TVI were made on the forearm in 16 healthy young volonteers under seven different physiological conditions (I: change of position; II: cooling; III: heating; V: venous/arterial occlusion and reactive hyperaemia; VI: dynamic handgrip exercise; VII: dynamic handgrip exercise with arterial occlusion. Before and after each situation physiological stability was verified (control situation: rest, normal body temperature, no occlusion). Arterial and venous blood samples (Hb, pO2, lactate, BE, pH) were taken to study possible changes under those different physiological conditions (II-VII). The results are currently being analysed. The design of the study has made possible unique parallell evaluation of three different noninvasive optical techniques under seven different physiological situations in the same individual during careful physiological monitoring. Saturation of muscle tissue has never before been studied with NIRS during regional hypotermia and the study also provides information on the correlation between the saturation of the muscle tissue and the perfusion in the overlying hypothermic skin studied with LDI and NIRS. Clinically, TVI has to a very small extent been used to study blood volume and has never been correlated against LDI, a clinical reference method in this context. The study therefore gives us information about potental fields of use, clinical as well as scientific, for TVI, NIRS and LDI.
Honorable Mentioning
Ozzy Mermut
McMaster University-Juravinski Cancer Center, and National Optics Institute
Medical Physics, and Biophotonics
Quebec City, Canada
ozzy.mermut@ino.ca
Poster title: Frequency domain, time-resolved and spectroscopic investigations of photosensitizers encapsulated in liposomal phantoms
Abstract: A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes. These liposomal phantoms are efficient and selective photosensitizer drug delivery vesicles, although their effects on the photochemical properties of the photosensitizer are not well characterized. The physical and chemical properties of liposomes can be highly tailored, making them suitable tissue and cell-like model systems. The liposomes employed in this study (both blank and photosensitizer-containing) were characterized using dynamic light scattering, scanning electron microscope, optical fluorescence microscope, flow cytometry and spectrofluorometry. The fluorescence decay of the encapsulated photosensitizer, a tetrasulfonate (MePcS4), a disulfonated metallophthalocyanine and, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function of the liposome’s physical properties, such as size-scale (0.1µm to 1 µm), size distribution, degree of lamellarity, concentration and the photosensitizer spatial confinement. The ionic strength of the solution, and the chemical properties of the liposome, and photosensitizer were varied to study these effects on fluorescence decay of the capsulated photosensitizer. The emission decay of PDT-encapsulated liposomes in deoxygenated environments, relevant to pathway I phototoxicity, was also probed in the frequency-domain. Fluorescence lifetime measurements were performed using the broadband frequency-domain instrument as well as a time-domain system for comparison and confirmation. Measurements on model photosensitizers in bulk solution were obtained to verify and compare the consistency of the two systems. To examine spectral shifts related to the photosensitizer encapsulation and confinement, or the formation of photosensitizer aggregates within the liposomes, spectrophotometric measurements were also acquired.
Constantin Ungureanu
University of Twente
Biomedical Technology Institute
Twente, The Netherlands
c.ungureanu@tnw.utwente.nl
Poster title: Contrast agents for photoacoustic imaging
Abstract: Photoacoustic imaging can deliver images based on optical absorption of tissue with high ultrasound-like resolution. This technique may have potential in breast cancer detection. To improve the sensitivity of the detection process, contrast agents can be used. Gold nanorods with their strong optical absorptions in the near-infrared are potential contrast agents in photoacoustic imaging. We present our results of synthesis and characterization of gold nanorods using experiments and computer simulations. The results of our experiments recommend these particles for applications where high optical absorption is needed. These applications can be not only in photoacoustic imaging but also for photothermal therapy. Performing photoacoustic spectroscopy of gold nanorods, we validate the theoretical model used to simulate optical properties of these particles.
Honorable Mentioning
Anders Gunnarsson
Lund University
Department of Physics
Lund, Sweden
anders.gunnarsson@ftf.lth.se
Poster title: Real-time single-molecule detection on random arrays for biosensing applications using total internal reflection fluorescence
Abstract: A novel biosensor assay was developed based on the spontaneous self-assembly of biotinylated copolymer, Poly(L-lysine)-g-poly(ethylene oxide) (PLLgPEG), streptavidin and biotinylated DNA onto a negatively changed silicon dioxide surface. The created interface is highly inert to various biomolecules achieving nearly zero background. Total internal reflection fluorescence (TIRF) microscopy enables single DNA molecule detection at 10 fM concentrations without using any form of microfabricated device. Detection is accomplished by introducing rhodamine-labeled 100 nm vesicles carrying one bivalent cholesterol-based coupled DNA duplex. The target strand acts as a mediator for tethering the fluorescently labeled lipid vesicles to the polymer-modified surface where each binding event, monitored in real-time using TIRF, corresponds to a single DNA target detection. This novel approach offers great potential since it enables studies of biomolecular interactions on a single molecule level, and in real time. Various biomolecules besides DNA can be probed with the assay, including soluble and membrane proteins utilizing the lipid bilayer of the vesicles. Not only achieving ultra-sensitive detection, probing single molecules interactions also provides possibilities to extract kinetic information by analyzing binding fluctuations. The biosensor assay holds great promise of achieving even lower detecting limits using various microfluidic devices and further optimization of the surface chemistry.
Henrik Sørensen
Technical University of Denmark
Risø National Laboratory, Optics and Plasma Research Dept.
Roskilde, Denmark
henrik.schioett.soerensen@risoe.dk
Poster title: Chip based in situ interferometric method for detection of cardiac markers
Abstract: Myocardial infarction constitutes a global health problem. Out of 52.2 million deaths worldwide in 2001, cardiovascular disease accounts for 25 to 45 percent of deaths depending on the country [1]. A myocardial infarction occurs when one of the coronary arteries becomes severely or totally blocked. When the heart muscle does not receive oxygenated blood, it will die in the affected areas. As cardiac myocytes die, proteins are introduced into the blood stream, some of which are known as the cardiac or coronary biomarkers. Currently cardiac markers are analyzed individually during the first critical hours after patients are admitted to the hospital. There are several critical factors involved in determining the best suitable treatment of a patient, one important way is by investigating the concentration level and activity of the cardiac markers, which includes Troponin I, Troponin T, Creatine Kinase MB fraction, and Myoglobin. These are all present in the blood stream at increased levels after myocardial infarction and are measured at 30 to 700 ng/ml for Myoglobin (Cardiac Reader from Roche Diagnostics). As this molecule has a molecular weight of 18 kDa, the detection limit can be directly correlated to the previously obtained data with other proteins on the proposed Back Scattering Interferometry (BSI) system [2]. Conservative estimates suggest that the presently available sensitivity can be enhanced by five orders of magnitude. By using model systems the final goal is to do a complete decisive analysis on whole blood at near-to real-time speed with unprecedented sensitivity in a bedside-like environment. Back Scattering Interferometry (BSI) is a refractive index measuring technique based on light interacting with a microfluidic channel. In this method the physical variable being measured is the change in refractive index with time, which can be caused by numerous bulk properties or solute interactions. The unique optical train employed in BSI allows near real-time quantification of solutes at attomole levels and within detection volumes of tens of picoliters. The fabrication of such a device is done using silicon based microfabrication. The typical length scale is 100 µm in diameter with sub-micron accuracy. The nontrivial task of fabricating the structures is made possible by using procedures developed by the groups at Risø National Laboratory and Vanderbilt University [3]. Modeling of the optical system shows that the sensitivity of the fringe pattern increases as the angular range moves towards 0°, i.e. directly backscattering and shown not to be dependent on channel dimension. The expanded model has been used to describe molecular binding occurring on the microstructure wall [4]. Here is presented label-free molecular interaction results, performed on clinical relevant cardiac markers in free solution in single use polymer flowchips. In order to enter medical clinics the flow chip has to be able to handle full blood. If present in the measurement volume the blood cells will perturb the binding signal significantly, thereby devaluating the diagnostic power of the sensor. A potential technology to be used for the separation task has been shown by the Tegenfeldt group [5].
[1] J. Mackay and G Mensah, “Atlas of Heart Disease and
Stroke,” WHO, ISBN: 92 4 156276 5, Sept. 2004
[2] D. Bornhop, Vanderbilt University, “Label-Free Technique for the Study
of Molecular Interactions,” NanoTech 2005, Montreux, 200
[3] H.S. Sørensen, J.C. Latham, D.A. Markov, P.E. Andersen, D.J. Bornhop,
N.B. Larsen, “Fabrication of polymer flow chips with capillary-like geometry
for an interferometric sensor,” Manuscript in preparation for submission to
Lab-On-A-Chip.
[4] H.S. Sørensen, N.B. Larsen, J.C. Latham, D.J. Bornhop, P.E. Andersen,
“Highly sensitive biosensing based on interference from light scattering in
capillary tubes, Appl. Phys. Lett. 89, 151108, 2006
[5] J. P. Beech, Jonas O. Tegenfeldt, University of Lund, “Elastic
Deterministic Lateral Displacement Devices - Stretching the Limits of
Separation,” NanoTech 2005, Montreux, 2005 "
Best Poster Presentation Award 2005
Sarah Martin
School of Physics and Astronomy
University of St. Andrews
St. Andrews, Scotland
Poster title: Fluorescence Resonance Energy Transfer (FRET) studies of protein interaction
Sarah Martin at her winning poster: Hamamatsu Photonics Best Poster Award 2005.
Honorable Mentioning
Maria Dienerowitz
Institut für Theoretische Festkörperphysi
University of Karlsruhe
Karlsruhe, Germany
Poster title: Theoretical analysis of negative index materials
Maria Dienerowitz at her winning poster
Honorable Mentioning
Jennifer E Hastie
Institute of Photonics
University of Strathclyde
Glasgow, Scotland
Poster title: Visible VECSELs: Novel laser sources for biophotonics research
Jennifer Hastie at her winning poster.
Honorable Mentioning
Joey C Latham
Chemistry, Biophysics, and Molecular Physiology
Vanderbilt University
Nashville (TN), USA
Poster title: Interferometric approach to monitoring the binding of small Heat Shock Proteins (sHSP) associated with cataracts and ischemia
Joey Latham (middle) at his winning poster.
Joey is surrounded by organizers Peter E. Andersen (left)
and Stefan Andersson-Engels (right).
Honorable Mentioning
Ekaterina Sergeeva
Optical methods in random media imaging
Institute of Applied Physics of the Russian Academy of Science
Nizhny Novgorod, Russia
Poster title: Estimation of physical and biological limitations for multiphoton fluorescence microscopy in biological objects
Ekaterina Sergeeva displaying her diploma for her poster prize
Best Poster Presentation Award 2003
Hannah Melville
School of Physics and Astronomy
University of St. Andrews
St. Andrews, Scotland
Poster title: Bessel beams and spatial light modulator technology for biophotonics
Download poster (PDF, 2.3 Mb)
