Group photo of the Biophotonics '15 class.

Best Poster Award at the
International Graduate School Biophotonics

Winners 2003–2015

Best Poster Presentation Award 2015

Sandra Tamosaityte (2nd from right) receives the best poster presentation award 2015 from Katarina Svanberg.

Sandra Tamosaityte
Vilnius University
Faculty of Physics, General Physics and Spectroscopy
Vilnius, Lithuania

Poster title: Vibrational spectroscopy based imaging techniques provide biochemical information of effects of alginate hydrogel implants in spinal cord injury model

Abstract: Spinal cord injury (SCI) leads to a cascade of complex biochemical changes of nervous tissue, which, taken together, result in intrinsic disability of damaged nervous tissue to regenerate. Therefore, new strategies for SCI regeneration-promoting treatments are in focus of the research for regenerative therapies. One promising approach is the implantation of biopolymer hydrogels in the damaged area to provide a permissive environment for neuronal growth. To evaluate the effects of soft Ca-alginate hydrogel implantation in rat models of SCI at one and six months after the induced injury, we applied label – free vibrational spectroscopy imaging. In total we investigated 28 rats, which were divided into groups of seven control and seven experimental animals for each time point after the surgery. Infrared (IR) imaging, based on intensities of lipids, collagen and alginate hydrogel specific IR spectral bands enabled to address and semi – quantify the effects of alginate hydrogel for demyelination and fibrotic scarring as well as to monitor the alginate hydrogel implant. The semi – quantification of IR data showed the positive effects of the alginate implant to preserve the nervous tissue and a reduction of scarring in the chronic stage of SCI. The hydrogel remained in the lesion up to six months after the implantation and was permissive to tissue growth. Raman spectroscopic imaging followed by cluster analysis allowed to get straightforward distinction between alginate hydrogel implant and tissue as well as to assess the extension of the gross tissue damage. Coherent anti-Stokes Raman (CARS) microscopy served for combining chemical information (addressing lipids) with the morphology of the tissue at subcellular level to reveal the bundles of myelinated axons immediately located at the border of implant and several isolated axons in the implant. The results demonstrate the potential of alginate hydrogel implants in future regenerative strategies as well as application of vibrational spectroscopy based techniques to evaluate the effects of SCI treatment strategies on biochemical level.

Second Prize 2015

Jacqueline Andreozzi (right) receives the second prize for the best poster presentation award 2015.

Jacqueline Andreozzi
Dartmouth College
Thayer School of Engineering
Hanover, NH, USA

Poster title: Exploring Cherenkov Imaging in the Radiotherapy Clinic: Applications and Challenges

Abstract: The goal of this research is to examine the potential for Cherenkov imaging to provide beam tracking and surface dosimetry feedback to clinicians in real-time, during radiotherapy treatment on a patient-specific, day-to-day basis. When the high energy photons or particles from medical linear accelerators travel through dielectric media such as human tissue, a spectrum of optical photons is emitted via the Cherenkov Effect. Imaging these Cherenkov photons on the surface of the patient thus provides an optical analog for the otherwise invisible treatment beam.

Using an Intensified Charge Coupled Device (PIMAX4 1024i, Princeton Instruments, Trenton, NJ, USA) placed 1–5 meters from the patient, images can be acquired at frame rates up to 12 fps. Image acquisition is carried out in either the proprietary software provided by the camera manufacturer, LightField (Princeton Instruments, Trenton, NJ, USA), or in LabVIEW (National Instruments, Austin, TX, USA). Each image acquisition is triggered by the beam pulses from the linear accelerator, and microsecond time-gating allows for isolation of the Cherenkov emission signal, even with ambient room lights at modest levels for patient comfort and safety.

Image analysis of the Cherenkov profile (treatment beam shape), and of pixel intensities (relative surface dose) provide unique information for each treatment session which can be compared to a treatment standard for each patient. While image analysis is currently carried out as post-processing in MATLAB (The Mathworks Inc., Natick, MA, USA), efforts are being made to perform this processing in real-time using both hardware and software solutions.

So far, two patients undergoing high dose rate total skin electron therapy (HDTSe-), and two patients receiving volumetric modulated arc therapy (VMAT) in the head and neck region have been imaged in our second clinical trial. The HDTSe- patient images prompted further study of the treatment geometry with regards to the position of the gantry head (source of the radiation beam). Cherenkov imaging offered a novel method of rapid beam field analysis that provided 2-dimensional information of the entire imaged surface, as opposed to the point measurements provided by ionization chambers or thermoluminescent dosimeters.

The two VMAT patients have provided extremely dynamic data sets, where the treatment beam is constantly changing in shape and source location (since the gantry head is arcing around the patient continuously during treatment). Beam edge tracking, as well as correlation between Cherenkov intensity and predicted surface dose, are currently being examined in these data sets. Correction factors for tissue composition at the Cherenkov photon source are being investigated to quantify both relative surface doses across the treatment region, as well as possible absolute surface dosimetry at any given point.

Preliminary results are promising, and indicate several possible applications of Cherenkov imaging during radiotherapy. With regards to this clinical trial, Cherenkov imaging has demonstrated its capacity to assess large-scale beam uniformity, and begun to pave the way towards more quantitative roles in treatment verification and surface dosimetry.

Second Prize 2015

Jan Philip Kolb (2nd from left) receives the second prize for the best poster presentation award 2015.

Jan Philip Kolb
Universität zu Lübeck
Institute of Biomedical Optics
Lübeck, Germany

Poster title: Ophthalmic ultra-widefield MHz-OCT-imaging with up to 100° field of view

Abstract: We use Optical Coherence Tomography (OCT), to create 3D ultra-widefield images of the human retina. Three imaging modes are tested: Single shot imaging with 85° field of view (FOV) as well as with 100° and stitching of five 60° images to a 100° mosaic. We employ a MHz-OCT system based on a 1060 nm Fourier domain mode locked (FDML) laser with a depth scan rate of up to 1.68MHz. Imaging over such a wide FOV poses several challenges including optical aberrations, pupillary and ciliary shadowing, the curvature of the eye and the high number of required depth scans. These issues are discussed and solutions to most of them are presented. Moreover, we investigate the origin of an angle dependent signal fall-off which we observe towards larger imaging angles. It is present in our 85° and 100° single volume images, but not in the mosaic. Our results suggest that 100° FOV OCT should possible with current swept source OCT technology.

Second Prize 2015

Abel Swaan (2nd from right) receives the second prize for the best poster presentation award 2015.

Abel Swaan
Academic Medical Center
Biomedical Engineering and Physics
Amsterdam, The Netherlands

Poster title: A novel optical biopsy probe design combining elastic scattering spectroscopy and optical coherence tomography

Abstract: Prostate cancer is usually diagnosed by transrectal ultrasound guided biopsies. Unfortunately, this has a low sensitivity and the tendency to understage. As a result, patients undergo multiple series of biopsies; 10–20 are taken in a single series and follow-up series can be necessary.

Guiding biopsies with real time information on location and possible grading of suspected lesions could reduce the amount of biopsies and increase diagnostic accuracy. Optical Coherence Tomography (OCT) and Elastic Scattering Spectroscopy (ESS) offer structural, physiological and biochemical information of tissue in vivo.

We will develop a needle based probe that integrates OCT and ESS. However, some challenges need to be addressed. First, ESS and OCT operate at different wavelengths (350–1000 vs. 1290–1350 nm) and employs multimode and single mode fiber. Second, most ESS-probes are stationary and forward-looking, whereas the design of our approach performs a rotating side-firing pullback.

To overcome these challenges, we designed a GRIN lens and prism based side-fire aperture in the fiber tip that support both ESS and OCT. The fibers from the OCT and ESS are coupled into a double clad fiber (DCF) by a double clad fiber coupler (DCFC). In the Fiber Optic Rotary Joint (FORJ), the static DCF and the rotary DCF are coupled using two GRIN lenses. To test the feasibility off this design, we simulated ray propagation in the probe using Zemax with promising results. Based on this, we are going to realize the first prototype from which preliminary results will be shown at the conference.

Second Prize 2015

Xinwen Yao (2nd from right) receives the second prize for the best poster presentation award 2015.

Xinwen Yao
Columbia University
Electrical Engineering
New York, NY, USA

Poster title: High-Resolution Spectral Domain Optical Coherence Tomography (SD-OCT) System Optimized for Myocardial Imaging

Abstract: There are a large range of diseases and therapies of the heart that can benefit from the information provided by a high-resolution, real time imaging modality. Previous works have indicated that heterogeneity in the myocardial tissue microstructures may contribute to the increased risk of life-threatening cardiac diseases such as arrhythmia. We present a high-resolution SD-OCT system that has an axial resolution of 2.89 µm and is designed for an imaging depth of 1.2 mm in air, which correspond to 2.06 µm and an imaging depth of 0.71 mm in human heart tissues (n ≈ 1.4). It can be employed to study myocardial fiber orientations and 3D mapping of the Purkinje fiber network.

The spectrometer of the SD-OCT system is customized to accommodate a light spectrum with FWHM bandwidth of 160 nm centered at 811 nm with a spectral resolution of 0.11 nm on a 2048-pixel CCD camera. The focusing optics of the spectrometer is designed and optimized to achieve the best fringe visibility, characterized by the modified transfer function, for different wavelengths on the imaging plane. For the OCT imaging system, dispersion mismatch is minimized by adopting the same optics in the two optical arms. It is further compensated numerically during post-processing, where Gaussian spectral shaping is performed to suppress the side lobes in the coherence envelope so as to improve axial resolution as well as image quality. The axial resolution (2.89 µm in air) is calculated as the FWHM of the point spread function obtained from the Fourier transform of gaussian fitted envelop of the interference fringes. The 6-dB sensitivity fall-off range is measured to be around 1 mm by vertically translating a flat mirror in the sample arm.

Cross-sectional images were acquired from five hearts. Three human hearts were obtained from the National Disease Research Interchange (NDRI) Tissue Bank within 48 hours of donor death. Two swine hearts were acquired through Columbia University’s tissue sharing program. In addition, two fresh human breast tissue samples were obtained from the Molecular Pathology Department at Columbia University Medical Center (CUMC) within 12 hours of resection. The tissue specimens from human and swine hearts were dissected from two atriums, two ventricles, and ventricular septum respectively upon delivery and imaged from the endocardium side immediately after dissection. The penetration depth is measured to be 0.6 ± 0.15 mm from 5 right ventricular septum specimens, which is comparable to images acquired from a commercial SD-OCT system at 1300 nm.

In the future, we will use the system to study myocardial fiber orientations in the presence of remodeling such as infarction and 3D mapping of the Purkinje fiber network. We will also further improve the system performance by optimizing the spectral resolution as well as the bandwidth of the spectrometer. The lateral resolution, which is depended on the diffraction limited spot size produced by the imaging lenses, can be enhanced over an extended distance by incorporating diffraction-free beams or numerical correction while maintaining sufficient image penetration.

Best Poster Presentation Award 2013

Best Poster Award ceremony (left to right): Peter E. Andersen,
Idan Steinberg (2nd prize), Kelsey Kennedy (2nd prize),
Kari Vienola (1st Prize), and Katarina Svanberg (SPIE)

Kari V. Vienola
Rotterdam Eye Hospital
Rotterdam Ophthalmic Institute
Rotterdam 3021 XW, The Netherlands

Poster title: Imaging of Optic Nerve Head With Motion Corrected OCT Using Tracking SLO

Abstract: 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 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.

Second Prize 2013

Kelsey M. Kennedy
The University of Western Australia
Optical + Biomedical Engineering Laboratory
Crawley 6009, Australia

Poster title: Probing Elastic Contrast In Human Tissues Using Needle Optical Coherence Elastography

Abstract: 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.

Second Prize 2013

Idan Steinberg
Tel Aviv University Faculty of Engineering
Department of Biomedical Engineering
Tel Aviv 6997801, Israel

Poster title: Frequency Domain Photoacoustic Phase Measurements of the Acoustic Modes in Bone for the Early Detection and Diagnosis of Osteoporosis

Abstract: 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 cost-effective 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.

Best Poster Presentation Award 2011

Tracy Liu
University of Toronto / Ontario Cancer Institute
Medical Biophysics
Toronto M5G 1L7, Canada

Poster title: Specific Activation of Photodynamic Molecular Beacons: An Image -Guided Therapeutic Approach for Vertebral Metastases

Abstract: Breast cancer is the second leading cause of cancer-related death in women. Approximately, 80% of patients with advanced cases will develop spinal metastases. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. In contrast, photodynamic therapy (PDT) in combination with vertebroplasty presents an attractive alternative due to its minimally invasive and selective nature. PDT destroys cells when light activates a photosensitizer (PS) in the presence of oxygen. However, current PSs are limited by their non-specific accumulation in normal tissues. We developed photodynamic molecular beacons (PMBs) comprised of a disease-specific linker, a PS and a quencher as a potential solution. PMBs are activatable PSs that provide another level of PDT selectivity and unparalleled image-guidance. PMBs are in a 'dormant' state until transformed into an 'awaken' state by a disease-specific trigger. Once activated, PMBs restore photodynamic activity and fluorescent image-guidance. Since highly metastatic breast cancers have been shown to over-express matrix metalloproteinases (MMP), a MMP-triggered PMB (PPMMPB) has potential application in image-guided treatment of vertebral metastases. Human breast carcinoma cells, MT-1 were used to model the metastatic behaviour of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PPMMPB in MT-1 cells and establishes the specific activation of PPMMP7B in the vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of PMBs. Preliminary PDT studies demonstrate the specific destruction of metastatic tumours while preserving healthy, non-target tissue. However, the limited tissue penetration of optical imaging and the silent nature of intact PMB prevented evaluation of the in vivo beacon distribution and pharmacokinetics. By radiolabeling the beacon, PET imaging and biodistribution studies evaluated the pharmacodynamics and pharmacokinetics of PPMMPB in vivo. With the emergence of PMB, we reveal an unprecedented level of PDT selectivity and multi-modality imaging for the clinical management of vertebral metastases.

Honorable Mentioning

Ashley Laughney
Dartmouth College
Thayer School of Engineering
Hanover 3755 NH, United States

Poster title: Guided multi-spectral assessment of pathology in surgically resected breast cancers using spatially modulated imaging

Abstract: Near-infrared (NIR) spatially modulated light is used to guide local spectral assessment of surgically resected breast tissues for identification of residual disease. Breast conserving therapy is the standard of care for early breast cancers (Stages 0-II) and resection adequacy is determined post-operatively by microscopic evaluation of pathology in small, representative pieces of the surgical margin. The strongest risk factor for local recurrence and mortality is a positive resection margin (tumor cells on ink). Therefore, if a margin is positive or close, the patient is advised to undergo re-excision surgery to achieve clear margins. In this decision process, pathology sampling is a surrogate for margin status, and wide field of view imaging techniques are needed to accurately assess important prognostic factors like disease extent, multi-focality and heterogeneity. Modulated Imaging (MI) is a non-scanning technique that can rapidly sample wide-field optical properties with sensitivity to sub-millimeter absorption and scattering features. Its spectral maps are used here to identify suspicious lesions in the surgical field for dense spectral sampling using a dark-field in situ scanning spectroscopy platform designed to image thick tissue samples at a spatial resolution sensitive to the diagnostic gold standard, pathology. MI employs spatial frequency domain sampling and model-based analysis of the spatial modulation transfer function to interpret a tissue's absorption and scattering parameters at depth. Multi-spectral measures quantify chromophore absorption and the reduced scattering coefficient by use of a power law with exponential attenuation due to the dominant visible chromophores, dominated by oxygenated and deoxygenated hemoglobin among others. The spectroscopy platform employs a scanning-beam, telecentric dark-field illumination and confocal detection to image fields up to 1 cm2 with a broadband source. The broadband reflectance signal is confined to a 100?m diameter pixel, so that multiple scattering effects are minimized and simple empirical models may be used to parameterize the spectra. Both systems were validated independently in phantom and murine studies. Ongoing work focuses on assessing the combined utility of these systems to identify cancer involvement in excised breast tissue specimens, particularly in the margins of resected breast tumors. Automated classification of scattering parameters extracted from highly localized tissue volumes according to pathology has already demonstrated diagnostic success. Guidance provided by MI will improve tissue sampling. Furthermore, combination of multi-spectral MI with localized broadband spectroscopy will expand upon the dynamic range of recovered optical properties because two light-transport mechanisms are probed.

Honorable Mentioning

Patrik Lundin
Lund University
Physics Department
22363 Lund, Sweden

Poster title: Diode laser absorption spectroscopy for medical applications

Abstract: Diode lasers have, thanks to properties like tunability, low cost and easy operation, been widely applied for sensing gases. The unique, spectrally sharp light absorption properties of gases make their identification and concentration quantification possible with the help of tunable lasers. A standard procedure to measure a gas concentration is to send collimated light through an open, well-defined path, where the gas is present. The absorbance due to the gas will then, together with the path-length, give the concentration through the Beer-Lambert law. It has also been shown that even gas situated in scattering media can be quantified in this way ? with the help of a few tricks though. This approach has shown to be implementable in many situations, e.g., within medicine. The main focus in this field has so far been to study human sinuses, to search for blocked cavities. Recently, plans are made and a trial performed to extend this research to measure the oxygenation in the lungs of neonatal children. As these organs are among the last to develop during maternity, it is of great importance to perform surveillance of their function in newly born children. If one could avoid harmful x-ray radiation, and still monitor the spatial distribution of oxygenation in the lungs, a lot would be earned. During 2011, continued studies will be performed on measuring oxygen concentration and pressure in the human mastoids with tunable laser spectroscopy, as well as the oxygenation in the lungs of newly borns. To better allow for these studies, the potentials of a change of laser wavelength was investigated with the help of Monte Carlo simulations, time-of-flight spectroscopy and diode laser absorption spectroscopy.

Honorable Mentioning

Adi Sheinfeld
Tel-Aviv University
School of Electrical Engineering, Faculty of Engineering
Tel-Aviv 69978, Israel

Poster title: Photoacoustic Doppler mapping of flow

Abstract: Simultaneous spatial and spectral mapping of flow is desired for early detection and monitoring of many vascular diseases. One approach for imaging flow in blood vessels is via the Photoacoustic Doppler (PAD) effect. Photoacoustic (PA) imaging in general is based on measurement of the acoustical waves generated when modulated light is absorbed by a medium under test. PA vascular imaging offers exceptional contrast due to the high optical absorption of hemoglobin in the visible and near-IR spectral ranges relative to the surrounding tissue. The differential absorption of oxygenated and deoxygenated hemoglobin can also be used for estimating the oxygen saturation level. Therefore, another important application of PAD is simultaneous measurement of flow and oxygen saturation for estimation of oxygen consumption. Photoacoustic Doppler measurement makes use of the frequency shift which is observed when flowing light-absorbing particles are illuminated by sinusoidally modulated light. This Doppler frequency shift is proportional to the velocity. In this work we present a novel technique for PAD measurement which allows simultaneous characterization of flow and axial position, using tone-burst modulation of the optical signal and time-gated spectral analysis of the detected acoustical waves. The modulation technique is designed to optimize the PA response in systems with limited peak power, such as systems based on laser diodes. Using laser diodes for photoacoustic excitation is highly desirable due to their small size, low cost and availability in a wide range of wavelengths. In addition, they allow flexible control over the waveform parameters, such as the pulse repetition frequency and central frequency, which determine the limitations on the measurable velocities. In order to allow time-resolved spectral characterization of the measured volume, the intensity of the optical excitation was modulated by repetitive tone-bursts signals at the central frequency of the ultrasonic transducer. To find the Doppler shift as a function of time, time-gated spectral analysis was implemented on the received PA response, yielding maps of the velocity profile vs. axial position. We also proposed a coded modulation technique for SNR improvement based on filling both the time and the frequency domains with repetitive sequences of tone-bursts at equally spaced frequencies. Averaging the responses after the proper scaling and shifting resulted in a marked SNR improvement. The optical excitation was generated using a combined pair of directly modulated 830nm laser diodes. The acoustical receiver comprised an ultrasonic transducer, an RF amplifier and a data acquisition card. Experimentally, the method was tested in a blood vessel phantom made of a transparent tube with flowing suspension of micron-scaled carbon particles immersed in water. Velocities ranging from few mm/sec up to 200mm/sec were accurately estimated, demonstrating the potential use of the method for mapping of blood flow in the vascular system. The system was also used for mapping spatial flow irregularities by inducing local narrowing in the tube and scanning the response around the narrowing. We also experimentally tested the SNR improvement achieved by using the interleaved tone-bursts modulation.

Honorable Mentioning

Teresa Torzicky
Medical University of Vienna
Center for Medical Physics and Biomedical Engineering
Vienna 1090, Austria

Poster title: Retardation and depolarization imaging by polarization sensitive optical coherence tomography at 840 nm and 1030 nm

Abstract: A polarization sensitive spectral domain optical coherence tomography (PS-OCT) instrument working at a central wavelength of 1030 nm was developed and used for imaging of the ocular fundus. 2D and 3D data sets of the fovea centralis and the optic nerve head region of healthy human volunteers were acquired. Additionally the distribution of depolarization was calculated based on Stokes vector analysis to generate degree of polarization uniformity (DOPU) images, which allow a better identification and segmentation of depolarizing layers like the retinal pigment epithelium (RPE). Intensity, retardation and DOPU images acquired with the PS-OCT working at 1030 nm were compared with images taken with a PS-OCT working at 840 nm. Both systems provided tissue specific contrast for retinal pigment epithelium and birefringent tissues like, sclera, lamina cribrosa and retinal nerve fiber layer. Intensity images acquired with the 1030 nm set up showed, as expected, a higher penetration depth and therefore better visualization of the choroid and the sclera. Beside the RPE the sclera could be seen in the retardation images of the fovea region taken with the 1030 nm system. The RPE could be well observed in the DOPU images acquired with both systems. In the retardation images of the optic nerve head the birefringent scleral ring was clearly observable.

Honorable Mentioning

Sarah Unterkofler
Max Planck Institute for the Science of Light
Russell Division
Erlangen 91058, Germany

Poster title: Single Cell Mechanics in Liquid-Filled Hollow-Core Photonic Crystal Fibers

Abstract: Recently, we have demonstrated that micron-sized glass particles can be transported inside the core of liquid-filled hollow-core photonic crystal fiber (HC-PCF) over distances of tens of centimeters by radiation pressure. Particles can also be held stably against a fluidic counterflow. This method is found to be a unique way of studying drag forces induced by a liquid flow around single particles in a microfluidic channel. Currently we are building bridges to biology by introducing single human cancer cells into HC-PCF. Such soft dielectrics will deform under the combined action of optical and drag resp. shear forces. Our approach could act as a novel tool to investigate cellmechanical behavior in which the acting forces are known precisely due to the highly symmetric arrangement and shielding from surrounding disturbances. The cell's mechanical behavior is a strong indicator of its state of health. Hence, with this interdisciplinary approach within the emerging fields of biophotonics and optofluidics we hope to contribute interesting insights into cell mechanics and medical treatment of diseased states.

Best Poster Presentation Award 2009

Sebastian Berning received the Best Poster Award 2009
(Profs. Stefan Andersson-Engels (left)) sponsored by
Thorlabs Sweden AB.

Sebastian Berning
Max Planck Institute for Biophysical Chemistry
Göttingen 37077, Germany

Poster title: Developing STED microscopy for deep imaging

Abstract: In the last decade, a number of techniques have been developed in order to overcome the fundamental resolution limit in far-field microscopy imposed by diffraction. All of these techniques rely on non-linear photophysical properties in fluorescence microscopy to make sure that only a small, strongly localized fraction of the flurophores within a diffraction-limited excitation volume is able to contribute to the fluorescence signal at a time, for instance by switching the fluorescence of the surrounding fluorophores reversibly off. In Stimulated Emission Depletion (STED) microscopy, this kind of switching is achieved with a second laser beam enclosing the focal excitation spot with a doughnut-shaped illumination pattern exhibiting a point of zero intensity at its centre. The wavelength of this STED-beam is chosen such as that it will suppress the fluorescence anywhere within the excitation area but at this point of zero-intensity through the mechanism of stimulated emission. In this way, impressive resolutions in the range of a few tens of nm have been routinely demonstrated in a multitude of biological samples. What remains challenging is the imaging of deeper layers of samples with a low refractive index close to that of water, as commonly found in in-vivo experiments. In this case, using a high-NA oil-immersion microscope objective will introduce spherical aberrations causing the resolution advantage of STED over confocal microscopy to significantly decrease with increasing depth. As a first step in the development of a new STED microscope specially designed for deep imaging applications, different objectives were therefore systematically screened for their ability to maintain good imaging properties over a large depth range in aqueous mounting media. The results are very promising, as it could be shown that STED microscopy is applicable for these samples even at depths exceeding 100 microns if certain precautions are taken. The overall complexity of the optical setup could at the same time be kept on a well-manageable level.

Link to extended summary

Honorable Mentioning

Torsten Frosch
University Jena
Physical Chemistry
Jena 07743, Germany

Poster title: Raman spectroscopic investigation of drug-target-interactions

Abstract: Malaria is one of the most devastating infectious diseases on earth and resistances against key drugs like chloroquine arise on a global scale. However, the molecular mode of action of those drugs is not well understood. It is believed that the quinoline class of antimalarials acts by interfering with the detoxification process of the hemoglobin digestion by-products in the red blood cell state of the plasmodium’s asexual life cycle. In this contribution - the high sensitivity and selectivity of UV resonance Raman microscopy is demonstrated by a structural investigation of different drugs under physiological conditions. The protonation of the weak base chloroquine is crucial for the efficiency of the drug and has been modelled by means of DFT calculations. Also the new, promising active agent dioncophylline A was localized in situ in very low concentrations in the tropical liana Triphyophyllum peltatum. This was possible by exploiting the advantages of UV resonance Raman spectroscopy, namely: 1) the spectral separation of the Raman spectra from overlapping fluorescence signals, 2) the resonance enhancement and 3) the intrinsic enhancement of the scattering by using UV wavelengths. A convincing mode assignment of the resonance Raman spectra of the drugs was possible by means of a combination of NIR Raman spectra and DFT calculations. Also the biological target - the Malaria pigment hemozoin - was structurally investigated. Hemozoin was localized in Plasmodium falciparum infected red blood cells by means of Raman microscopy in the visible range, in resonance with the iron porphyrin. These in situ results were compared with spectra of extracted hemozoin as well as with synthesized ß-hematin. Importantly it was proofed that ß-hematin is the synthetic analogue of the malaria pigment and even more it was possible to distinguish between different morphologies of this structure. Also the dimeric unit cell of the malaria pigment was calculated for the first time. Those DFT calculations of the Raman spectra and the atomic displacements assisted much in the assignments and the understanding of the experimental results. The interactions of the drugs and hematin were studied. A dramatic change of the depolarization ratio of the v19-mode appeared in the polarization resolved Raman spectra. Those inverse polarized mode of hematin is very sensitive for symmetry lowering – in our case the docking of the chloroquine drug. To be able to detect the very small wavenumber shifts which occur due to the weak interactions, a novel device for Raman differences spectroscopy (RDS) was designed, that allows for detection of wavenumber shifts down to 1/100 of the linewidth. With help of this new RDS device we were able to monitor weak interactions of chloroquine and hematin for the first time. Once more the DFT calculations of the normal modes turned out to be very helpful for an interpretation of the experimental findings.

Honorable Mentioning

Anita Gollmer
University of Aarhus
Department of Chemistry
Aarhus 8000 C, Denmark

Poster title: Detection of singlet oxygen using fluorescent chemical traps in sub-cellular domains of a single cell

Abstract: Singlet oxygen (1O2), the lowest excited electronic state of molecular oxygen, plays a major role in many chemical and biological processes, e.g. in photodynamic therapy. Several methods have been established to detect 1O2. Due to the experimental detection limits of these existing methods, we would like to introduce fluorescence probes which are chemical traps to detect 1O2 in single cells. Fluorescence probes are known to be excellent sensors for biomolecules, being sensitive, fast-responding and capable of affording high spatial resolution via microscope imaging. Chemical traps which are normally almost non fluorescent, can react with 1O2 forming a fluorescent endoperoxide. Measuring its fluorescence or monitoring the decrease in the amount of the trap provides a powerful tool to detect 1O2 with a higher sensitivity. Another promising advantage is that lower amounts of 1O2 are required to detect a signal. The fluorescence quantum yield of the endoperoxides are much higher than the phosphorescence quantum yield of 1O2. Therefore, the cells are less in risk of being damaged under unnecessarily harsh experimental conditions. The next step will be to specifically localize a photosensitizer and a trap into different parts of a single cell using, for example, protein labeling techniques. With this method we want to achieve a greater control of the generation and decay of 1O2 inside a cell.

Honorable Mentioning

Pier-Anne Lachance
University of Texas Health Science Center
Institute of Molecular Medicine Center for Molecular Imaging
Houston 77030 TX, United States

Poster title: Validation of IRDye800 conjugated peptides imaging agent targeted to integrin α9 for optical imaging of lymphangiogenesis

Abstract: The lymphatic system is a major component of the circulatory system which functions are fluid balance, lipid absorption, and a site for immune surveillance. Due to the importance of the lymphatic’s functions for tissue homeostasis, pathologies which involve lymphatic dysfunctions are diverse and include lymphedema, inflammation, obesity, and cancer. The process of forming new lymphatic vessels is called lymphangiogenesis. There is a need to understand the mechanism and the role of lymphangiogenesis in diseases. Herein, there is a need for developing a molecular imaging agent which could image the process in vivo. The α9 integrin subunit has been linked to lymphangiogenesis through its direct interaction with the vascular endothelial growth factor family and the hepatocyte growth factor. In addition, α9 integrin is a critical in lymphatic vessel development, suggested by the lethality of α9 knockout mice. We believe that expression and affinity of α9 integrin are change during lymphangiogenesis to promote lymphatic cells proliferation and migration. Therefore, α9 integrin is a potential molecular target for imaging of lymphatic remodeling. In this study, we first would like to determine if integrin α9 is a marker for lymphangiogenesis. To answer this question, we are using in vivo and in vitro assay to determine the role and level of expression of integrin α9. We are in the process of developing an integrin α9 inducible tissue specific knockout mouse to assess the role and mechanism of integrin α9 in lymphangiogenesis. We have synthesized and labeled two peptides containing the MLDG motif and three peptides with the EIDGIEL motif, which are known to bind to the integrins α9. The peptides were conjugated to IRDye800 for near-infrared optical imaging. We have performed fluorescent microscopy studies validating binding of the conjugated peptides to integrin α9 using Caco-2 cells, and CHO-K1 cells overexpressing α9 subunit. Further characterization of the peptides specificity, affinity, and stability in vitro shows that these peptides are candidates for imaging of α9 integrin. This is the first step in developing and validating a new peptide agent for in vivo lymphangiogenesis imaging.

Honorable Mentioning

William Lo
University of Toronto
Department of Medical Biophysics
Toronto M5G 2M9, Canada

Poster title: Hardware-accelerated MC Simulation for PDT Treatment Planning using FPGAs and GPUs

Abstract: Monte Carlo (MC) simulations are being used extensively in the field of medical biophysics, particularly for modeling light propagation in tissues. The high computation time for MC limits its use to solving only the forward solutions for a given source geometry, emission profile, and optical interaction coefficients of the tissue. However, applications such as photodynamic therapy treatment planning or image reconstruction in diffuse optical tomography require solving the inverse problem given a desired dose distribution or absorber distribution, respectively. A faster means for performing MC simulations would enable the use of MC-based models for accomplishing such tasks. To explore this possibility, a digital hardware design of an MC simulation based on the Monte Carlo for Multi-Layered media (MCML) software was implemented on multiple field-programmable gate arrays (FPGAs). The hardware performed the MC simulation approximately 80 times faster on a development platform called the TM-4 with four Stratix I FPGA chips (20x per chip) compared to MCML executed on a 3-GHz Intel Xeon processor based on Pentium 4 technology. By migrating to the modern DE3 board, a 42-fold speedup was achieved with one Stratix III device. For comparison, MCML was also implemented on a multi-GPU system with 2 NVIDIA GTX280 graphics cards comprising 480 cores. This approach led to a 113x speedup (56.5x per device) compared to the same Intel processor. However, compared to a 3-GHz Intel Xeon 5160 processor with better CPU architecture, the performance gap was narrowed by 1.5 times. Both the FPGA-accelerated and GPU-accelerated MCML implementations were validated with a skin model and the isofluence lines generated closely matched those produced by MCML in software. The development process will also be discussed to highlight the key differences between FPGA-based custom hardware design and GPU-based CUDA programming for high performance computing.

Honorable Mentioning

Tilman Schmoll
Medical University Vienna
Center of Biomedical Engineering and Physics
Vienna 1090, Austria

Poster title: In-vivo Assessment of Photoreceptor Response with Functional Optical Coherence Microscopy

Abstract: Probing the retina with flicker light of defined frequencies allowed to offset the detection for intrinsic signals from proband motion artifacts as well as blood flow. In addition the fast imaging sequence capability of FDOCT is promising for the assessment of fast physiologic changes within retinal structures. For the present study two measurement protocols are evaluated: first, taking fast tomogram series across a flickered region, and then constructing via frequency analysis and bandpass filtering a functional OCT tomogram similar to fMRI. The second protocol consists of a fast local A-scan series at 17kHz rate with 1Hz flicker. “Light-on” time is 250ms. “Lights off” time is 750ms. 500ms before “light-on” is used for calculating the baseline. Finally the average over 5 cycles is taken. A clear negative response is found at the outer photoreceptor segment for both “light-on” and “light-off” edge. The response appears to be stronger for the “light off” edge. The shape of the responses is analysed and might eventually be used in linear regression models to enhance the sensitivity of our fOCT approach.

Best Poster Presentation Award 2007

Lakshmi Sampath (right) at her award-winning poster at Biophotonics '07. From left: Profs. Stefan Andersson-Engels and Peter E. Andersen.

Lakshmi Sampath
Baylor College of Medicine
Molecular Physiology and Biophysics
Houston, TX, USA

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.

Link to extended summary

Honorable Mentioning

Best poster award and honorable mentioning award winners (from left): Henrik Schiøtt Sørensen, Constantin Ungureanu, Nicole Prent, Lakshmi Sampath (Best Poster Award), Emilie Krite Svanberg, Ozzy Mermut, and Anders Gunnarsson.

Nicole Prent
University of Toronto
Physics and Institute for Optics
Toronto, Canada

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

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

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.

Honorable Mentioning

Constantin Ungureanu
University of Twente
Biomedical Technology Institute
Twente, The Netherlands

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

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.

Honorable Mentioning

Henrik Sørensen
Technical University of Denmark
Risø National Laboratory, Optics and Plasma Research Dept.
Roskilde, Denmark

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 at her winning poster: Hamamatsu Photonics Best Poster 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

Abstract: The discovery of the genetic structure of the green fluorescent protein (GFP) in the jellyfish Aequorea Victoria (1992) enabled rapid improvements in the tools available for fluorescent labelling of proteins in vitro and in vivo. Furthermore, the introduction of mutations into GFP has allowed the generation of fluorescent proteins with altered spectral properties (e.g. cyan CFP and yellow YFP), that facilitate the use of fluorescence resonance energy transfer (FRET) to study molecular interactions. FRET results from non-radiative coupling of two fluorophores within 10-100Å of each other, making it an ideal assay for studying the interactions between tagged proteins.

In this work, we report the FRET signal of the binding between the small ubiquitin-like modifier SUMO-1 and the enzyme Ubc9. Ubc9 catalysed SUMO modification of proteins is important in many biological processes, including intracellular transport. Complexes of CFP-SUMO and YFP-Ubc9 were expressed and their fluorescence spectra recorded. FRET efficiency was measured in mixtures of CFP-SUMO-1 with increasing amounts of YFP-Ubc9 to determine the affinity of SUMO-1 to Ubc9. Relevant controls were performed with CFP, YFP, Ubc9 and SUMO-1. From these results we determined the association constant Kd, and find that it compares well with data from conventional isothermal calorimetry (ITC). Furthermore, this new technique enables the study of two proteins in the presence of further interacting proteins, with a prospective application in high-throughput screening of potential drugs. The versatility of FRET-based techniques is also expected to yield fresh insight into the specificity of protein modification by SUMO and other ubiquitin-like proteins.

Honorable Mentioning

Maria Dienerowitz at her winning poster

Maria Dienerowitz
Institut für Theoretische Festkörperphysi
University of Karlsruhe
Karlsruhe, Germany

Poster title: Theoretical analysis of negative index materials

Abstract: Negative-index or left-handed materials (LHM) are characterised by simultaneously negative permeability μ and permittivity ε. The theoretical idea has already been established in 1968 when Veselago [1] first proposed that electromagnetic waves traveling through these media exhibit unique properties such as inverted Snell’s law and a reversed Doppler effect. Unfortunately, LHM do not occur in nature and only recently Smith et al. [2,3] succeeded in fabricating an effective negative index of refraction of a composite at radio frequencies.

The possibility to tailor the permittivity ε and especially the permeability μ in this new class of meta-materials allows to obtain magnetic activity at almost any desired frequency range and is also expected to support surface plasmons, even of magnetic nature. They should make valuable contributions to the fields of sensor development, magnetic resonance imaging and spectroscopy [4].

We discuss the problems that constrain the desired new effects at optical frequencies. Furthermore we present new ideas how to minimise absorption that originates from resonances and manufacturing tolerances of the underlying nano-scale size substructures. In addition, we analyse different theoretical models to achieve negative magnetic and dielectric response and demonstrate the possibilities to create LHM at optical frequencies.

1. V. G. Veselago, Sov. Phys. Usp. 10 (1968) 509
2. R. A. Shelby, D. R. Smith, S. Schultz, Science 292 (2001) 77
3. C. G. Parazzoli et al., Phys. Rev. Lett. 90 (2003) 137401
4. M. C. K. Wiltshire et al., Science 291 (2001) 849

Honorable Mentioning

Jennifer Hastie at her winning poster.

Jennifer E Hastie
Institute of Photonics
University of Strathclyde
Glasgow, Scotland

Poster title: Visible VECSELs: Novel laser sources for biophotonics research

Abstract: Vertical external cavity surface emitting lasers (VECSELs) are an important new category of semiconductor laser with all the advantages of conventional edge-emitting lasers and vertical cavity surface emitting lasers (VCSELs), but with none of the disadvantages that come with electrical injection and micro-cavities. They are capable of high power in circularly symmetric, TEM00 output beams with tuneable output wavelength. They have the potential to replace a number of gas and dye lasers in many biophotonics research applications due to the fact that they can match their performance in a much more efficient and compact packages with greater wavelength flexibility.

In this work, two different areas of biophotonics research have been targeted with two distinct embodiments of a red VECSEL; a conventional, high power, tuneable VECSEL, and a unique high power microchip VECSEL array.

The technique of photodynamic therapy (PDT) requires a monochromatic source, generally at red wavelengths, for selective and efficient excitation of specific photosensitizers. For PDT research purposes, high power and wavelength tuning is essential and so far the only red lasers widely available for this function have been dye lasers. The ideal solution is a compact, tuneable solid-state laser with high beam quality at red wavelengths, performance which can now be provided by VECSEL technology. In this work, high-power continuous-wave operation at red wavelengths was achieved with a VECSEL based on the GaInP/AlGaInP/GaAs material system. Output power of 0.4W was obtained in a linearly polarized, circularly symmetric, diffraction-limited beam. The output spectrum of the laser was tuned over a 10nm range around 674nm.

A diamond heatspreader, bonded to the red VECSEL wafer, was mirror-coated for 1% transmission at the laser wavelength to achieve a plane-plane laser cavity with volume ≪1mm3. Simultaneous pumping with three separated input beams achieved lasing from three discrete areas of the microchip to produce 95mW per beam. Each beam had a Gaussian profile with M2 < 1.2. These characteristics are attractive in chip-based biosystems, where optical tweezers can be used for multiple experiments performed in parallel. The electrically-driven VCSELs currently used in this application suffer low trapping strength due to limited output power. A microchip VECSEL array has no such limitations on power, and the multiple output beams can be brought to a tight focus with arbitrary array spacing depending on the chosen imaging lens.

The next challenge within this research is to work closely with biophysicists and biologists to develop these lasers further for demonstrations of the above techniques.

Honorable Mentioning

Joey Latham (middle) at his winning poster.
Joey is surrounded by organizers Peter E. Andersen (left)
and Stefan Andersson-Engels (right).

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

Abstract: Members of a family of proteins termed, small heat shock proteins (sHSP), exhibit chaperone activity. sHSP’s play important roles in physiological processes underlying aging, stress, and apoptosis. Mutations, either inherited or age related, can inhibit the chaperone function of these proteins. Currently, there are 10 sHSP’s identified in humans.

Hsp 27 and crystallins are both members of the heat shock protein family. Hsp 27 has been found to be abundant in muscle tissue where it plays a role in protection against ischemia. The crystallins account for ~90% of the dry weight of the human lens as well as its transparency and refractive nature. These crystallin proteins help maintain the function of the lens by preventing aberrant protein aggregation. Mutations to the crystallins have been related to cataract formation.

The world health organization (WHO) estimates over 180 million people are visually disabled, with 40-45 million individuals deemed to be blind [1]. Cataracts, opacities in the lens of the eye, account for over 50% of the earth’s populace of blind persons with this number expected to double by the year 2020 [2,3]. In the United States, approximately 50% of people between the ages of 60-74 have cataracts with that percentage rising to over 70% for citizens 75 or older. During the late 1990’s 1.35 million operations (currently the only effective treatment for cataracts is surgical removal) where performed per year in the U.S. resulting in a cost of 3-4 billion dollars [4].

The robust and copious use of interferometry can be found scattered throughout the scientific community. From its use for vascular pathology and noninvasive biopsy in optical coherence tomography (OCT), to astronomical telescopes that have produced the highest resolved images ever in space exploration, the simple nature of measuring the interference of coherent waves has produced a technique that is both highly sensitive and widely applicable.

A detection methodology based on backscattering interferometry [5] has been used in universal solute analyses (e.g. thermometry [6], polarimetry [7], and electrophoresis [8]) carried out in microfluidic devices with nanoliter to picoliter detection volumes at high sensitivity. A micro-interferometric backscatter detector (MIBD), consisting simply of a coherent light source, a microfluidic channel, and a phototransducer will be described here for its use in monitoring the chaperone function of the sHSP’s. The use of this interferometric sensor will allow us the opportunity to monitor the binding efficacy of these proteins against a destabilized lysozyme non-invasively and label-free. Furthermore, the high sensitivity of the instrument will enable us to perform these investigations at near cellular concentrations.

1. World Health Organization. Global initiative for the elimination of avoidable blindness.
2. World Health Organization. Blindness and visual disability: major causes worldwide.
3. World Health Organization. Management of cataract in primary health care services.
4. E. P. Steinberg, J. C. Javitt, P. D. Sharkey, Arch. Ophthalmol 111, 1041-1049 (1993).
5. D. J. Bornhop, Appl. Opt. 34, 3234-3239 (1995).
6. K. Swinney, D. J. Bornhop, Electrophoresis 23, 613-620 (2002).
7. D. J. Bornhop, J. Hankins, J. Analytical Chemistry 68, 1677-1684 (1996).
8. Z. Wang, K. Swinney, D. J. Bornhop, Electrophoresis 24, 865-873 (2003).

Honorable Mentioning

Ekaterina Sergeeva displaying her diploma for her poster prize

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

Abstract: We report on experimental and theoretical study of limitations on deep imaging of biological tissue structure using multiphoton fluorescence microscopy (MFM) imposed by scattering of short NIR pulses in optically turbid medium. The potentialities of MFM as the tool of in vivo imaging biological objects with subcellular resolution are restricted by scattering of femtosecond laser pulses and the intensity of phototoxic reactions in biotissue. Small-angle scattering causes beam defocusing which results in decrease of lateral resolution of the method. Another consequence of scattering is so-called photon multiple passage effect, when photons reach the point of interaction by random ways having covered different distances and acquiring different delays. This effect is mostly important for non-absorbing media when the path of a photon is not limited by the length of absorption, so photons may obtain large relative lags. In the case when a femtosecond pulse propagates in scattering medium multiple passage effect causes the pulse widening due to delay of "snake" photons from ballistic ones, which may result in up to several times extension of the initial pulse width. Increasing the imaging depth by raising the laser power may result in phototoxic damage of biological objects since multiphoton fluorescence efficiency from the medium is the power function of energy flux density in the excitation laser beam. These limitations were analyzed on the example of two-photon fluorescence microscopy by considering the following problems: creating an adequate theoretical model of scattering of ultrashort laser pulse and developing the methods for estimation of phototoxic effect in biological objects.

To describe intensity distribution in turbid medium with strong forward scattering and short pulse modification in such medium the analytical model based on solution of radiative transfer equation under small-angle diffuse approximation was proposed which considers photon dispersion over travel pathlengths. To verify the developed theoretical approach, we have experimentally studied temporal structure of collimated femtosecond laser pulse scattered by a layer of an optically turbid model medium with controlled concentration of micron-sized spherical beads. Temporal structure of a scattered 50-fs pulse was measured by the means of nonlinear optical gating at noncollinear second harmonic generation. Experimental results proved reliability of the proposed analytical model.

To investigate phototoxicity of high-intensive pulsed radiation in biological tissue we used blood plasma from laboratory rats. The specimens of biological liquid were irradiated by trains of 50-fs Ti:sapphire laser pulses with peak intensity up to 1 TW/cm2. After irradiation a morphological analysis of blood plasma was performed by observing the structure of a dried plasma droplet. Obvious structural changes were observed in the part of a dried droplet containing organic fractions of biological liquid. The performed experiments indicated that potentiality and period of recovery of the droplet structure depend on the imposed intensity of femtosecond NIR radiation which may serve as a proof of photodamaging effect of femtosecond light on living objects.

Best Poster Presentation Award 2003

Hannah Melville at her winning poster

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

Abstract Optical forces allow us to trap and manipulate microscopic particles for a multitude of purposes. In optical tweezing small microscopic particles are drawn to the highest intensity region of a light beam where they are held by the light and can be manipulated by moving the beam. This methodology can be applied to exciting studies in biology including measuring the elasticity of DNA and blood cells, manipulating chromosomes and studies of molecular motors. For advanced future studies novel light beams and extended trapping patterns hold the key to achieving the next generation of results. Bessel beams can be used to create more complex tweezing systems. Bessel beams are light beams whose wave vectors lie on a cone, this property means that the beam does not undergo diffractive spreading as it propagates and also that it can self-regenerate after an obstacle is placed in its path. Using these properties it has been possible to trap and manipulate a number of different particles simultaneously at different points along the beam. Bessel beams have applications in biological guiding and creating arrays of biological material. These and other complex light beams can be created using glass holograms made using microfabrication techniques to etch patterns onto the glass. However, a spatial light modulator (SLM) is a device that allows us to create and dynamically control new, more complex, light fields without the need for microfabrication. An SLM consists of an array of liquid crystals, each of which can be individually addressed so as to allow us to sculpt or tailor any light beam hitting its surface. As such we can create novel light beams in a much simpler manner than before. This potentially offers a new level of control, allowing us to manipulate and manoeuvre biological matter in a manner not previously seen.

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