This PDF file contains the front matter associated with SPIE Proceedings Volume 8090, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Recovering the three dimensional (3D) surface shape of tissues in minimally invasive surgery (MIS) is important for developing advanced image-guidance and navigation systems. Passive techniques for 3D reconstruction based on computational stereo are limited by the saliency of tissue texture and the view-dependent reflectance characteristics of the scene. Structured lighting provides a viable alternative by projecting known features onto the tissue surface. However, the correspondence problem (distinguishing individual projected features computationally) becomes difficult in tissue due to the presence of occlusions. Furthermore, miniaturisation of a light projection system for use in MIS, while maintaining the required light intensity, is a significant challenge. In this paper, a fibre-based probe is described that projects a spectrally-encoded pattern onto the target surface from its distal end. A dispersed broadband light source is used to project features of varying spectral content. The dominant wavelengths of imaged spots may be deduced from the RGB values of a standard colour camera using an algorithm that locates each colour on a chromaticity diagram. The results show that individual spots of a specified wavelength may be segmented and their centres of mass calculated, despite varying background colour. The probe has also been demonstrated on ex vivo tissue.

Digital holographic microscopy is an interferometric technique enabling the measurement of the quantitative phase shifts induced by cell bodies. We correlate the phase signal measured on neurons with calcium imaging measured by fluorescence on cells loaded with Fluo-4, to monitor responses to glutamate challenges, which provoke well-known calcium increases through activation of various membrane receptors. A very good correspondence can be identified between the two signals, showing the links between the phase signal, being a measure of the intracellular dilution, and the calcium concentration within cells. We then check cell viability by employing propidium iodide (PI), a fluorescent indicator relying on the cell membrane integrity loss to assess cell death. Strong intracellular calcium concentration is indeed known to induce excitotoxic effects, potentially inducing cell death. This enables showing that some cells cannot sustain the calcium saturation identified in our measurements, leading to subsequent cell death.

The 3D structure of light scattering from dark-field illuminated live 3T3 cells marked with 40 nm gold nanomarkers is explored. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. Images are obtained using a novel 3D reconstruction method providing longitudinally undistorted 3D images. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cellular structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis. This characterization is an unambiguous signature of the presence of the gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live cells.

Microorganisms, cells and thin tissue sections are transparent and not visible to view in ordinary microscope. Techniques such as phase contrast and Normarski/Differential interference contrast microscopy transform the phase variation information into intensity distribution to reveal the details of internal structures. Similarly fluorescence microscope uses intrinsic or extrinsic chromophores to reveal specific and hidden details. Advances achieved in recent years have greatly improved the versatility of microscopes to obtain more insightful information about different physiological functions that occur at cellular level. Understanding the cell response, involving both structural and functional changes within the cell, dictates ability to image cell structure and function at the same time. We report a novel optical Fourier phase contrast multimodal optical microscopy technique for real time display of phase and fluorescence features of biological specimens at the same time. It combines the principles of (a) Fourier phase contrast microscopy which exploits monochromaticity, intensity and phase coherence of the laser beam via optical Fourier transform and photoinduced birefringence of dye doped liquid crystal for phase contrast imaging, and (b) common-path multimodal optical microscopy for co-registered imaging of phase and fluorescence features of biological specimens in real time using a single optical path, single light source, and single camera with no requirement of image registration. Further the instrument also enables co-registered imaging of fluorescence and spatial filtering facilitating simultaneous display of structural and functional information. This comprehensive microscope has the capability of simultaneously providing both structural and functional information in a streamlined simplified design and may find applications in high-throughput screening and automated microscopy.

In current report we present synchronized in vivo imaging of tumor vascular network and tumor microenvironment obtained by combined use of Dynamic Light Scattering Imaging, Spectrally Enhanced Microscopy, and Fluorescence Intravital Microscopy. Dynamic Light Scattering Imaging is used for functional imaging of the vascular network and blood microcirculation. Spectrally Enhanced Microscopy provides information regarding blood vessel topography. Fluorescence Intravital Microscopy is used for imaging of tumor microvasculature and tumor microenvironment. These well known modalities have been comprehensively validated in the past and are widely used in various bio-medical applications. As shown here, their combined application has great potential for studies of vascular biology. This multi-modal non-invasive diagnostic technique expands our current capacity to investigate blood microcirculation and tumor angiogenesis in vivo, thereby contributing to the development of cancer research and treatment.

Lung cancer is the leading cause of cancer related mortalities. With current imaging modalities, non-invasive diagnosis of this deadly cancer is difficult due to the lack in specificity. Photonics based optical molecular imaging is a promising alternative for early lung cancer diagnosis because of its molecular based detection for specificity and relatively low cost instrumentation. In particular, gold nanoparticle-based surface enhanced Raman scattering (SERS) probes have shown great promise for disease detection and diagnosis because of its remarkable sensitivity and its ease of engineering for high specificity. Here, a stabilized, biocompatible SERS probe, Raman active phospholipid gold nanoparticles, is synthesized and conjugated to epidermal growth factor to target its receptor, epidermal growth factor receptor (EGFR), a common lung cancer biomarker. This novel nanoparticle encapsulates Raman molecules adsorbed on 60 nm colloidal gold with a phospholipid bilayer coating which show structural and functional stability in serum over 24 hours. Moreover, when conjugated with epidermal growth factor, it specifically detects and localizes EGFR on lung carcinoma cells. We used transmission electron microscopy, Raman and UV spectroscopy to validate the novel nanoparticle's reproducibility and stability. We also validated its molecular specificity and use it as an efficient contrast agent with in vitro confocal reflectance microscopy on lung carcinoma. This novel lipid-based SERS probe provides a viable alternative as a tool for detecting lung cancer biomarkers for non invasive, ultrasensitive and specific diagnoses.

Nanostructure substrates are effective biosensor to spectrally differentiate multiple compounds by Surface-enhanced Raman scattering (SERS). Metal film over nanosphere (MFON) has been demonstrated to exhibit reproducible and predictable Raman enhancement. MFON can be fabricated using an economical process in which polystyrene (PS) nanospheres are self-assembled on a planar solid supports and then followed by metal coating. In this work, we investigate the MFON substrates with bimetallic coating to combine the optical-enhancing and stability features from Ag and Au layers. The SERS responses are then quantified from the resultant bimetallic structures with 2-Naphthalenethiol. We show that the bimetallic substrate of optimal Au/Ag thickness ratio renders SERS enhancement and stability exceeding those of the Au-coated MFON. Compared to Au array, the bimetallic substrate exhibits quasi-bimetallic nanoparticles of surpassing SERS (2.5 times) with enhancement factor determined to be 2×10⁷. As a proof-of-concept for biosensing in microfluidics, SERS nanotag was prepared and tested on the optimized BMFON. In addition, we propose a fabrication scheme to construct MFON with alternating sizes (100nm and 400nm) of nanosphere. At optimal proportional amount, the 100nm-spheres were packed within the gaps between the 400nm-spheres. The resultant morphology renders additional nanogaps that could possibly lead to increment in SERS enhancement.

We present a study of the dynamics of protein aggregation using a common path heterodyne Bloch surface wave sensing scheme. We demonstrate the ability to detect, during thermal incubation, the early events linked to the aggregation of proteins related to conformational diseases. Alzheimer's amyloid-β 1-42 is used to demonstrate the efficiency of the method. A model based on elementary interactions is shown to describe accurately the aggregation process. The described sensing scheme is sensitive to the early events of the aggregation process. is hence proposed as a method for the detection of early stages of the evolution of conformational diseases.

A novel optical platform offering potential for highly integrated polymer-based biophotonic chips is presented, featuring a cladding index that closely matches aqueous samples or biological samples. Applications including evanescent-wave microscopy, surface plasmon-coupled biosensing, and on-chip manipulation of light signals are demonstrated.

There is a growing need for the development of computational models that can account for complex tissue morphology in simulations of photon propagation. We describe the development and validation of a user-friendly, MATLAB-based Monte Carlo code that uses analytically-defined surface meshes to model heterogeneous tissue geometry. The code can use information from non-linear optical microscopy images to discriminate the fluorescence photons (from endogenous or exogenous fluorophores) detected from different layers of complex turbid media. We present a specific application of modeling a layered human tissue-engineered construct (Ex Vivo Produced Oral Mucosa Equivalent, EVPOME) designed for use in repair of oral tissue following surgery. Second-harmonic generation microscopic imaging of an EVPOME construct (oral keratinocytes atop a scaffold coated with human type IV collagen) was employed to determine an approximate analytical expression for the complex shape of the interface between the two layers. This expression can then be inserted into the code to correct the simulated fluorescence for the effect of the irregular tissue geometry.

There is a pressing need for a low cost, passive optical fiber dosimeter probe for use in real-time monitoring of radiation dose delivered to clinical radiation therapy patients. An optical fiber probe using radiochromic material has been designed and fabricated based on the deposition of a radiochromic thin film on a dielectric mirror. Measurements of the net optical density vs. time before, during, and after irradiation at a rate of 500 cGy/minute to a total dose of 5 Gy were performed. Net optical densities increased from 0.2 to 2.0 for radiochromic thin film thicknesses of 2 to 20 μm, respectively. An improved optical fiber probe fabrication method is presented.

A Monte Carlo program for simulation of polarized light propagation in scattering media was developed. By comparing the results of this program (angularly resolved independent Müller matrix elements S11, S21, S34 and S44) with analytical solutions of Maxwell equations, a testing method for Monte Carlo programs simulating polarized light propagation was found. A further goal was to quantitatively point out the differences between solutions of radiative transfer theory and Maxwell theory for polarized light.

A piezoelectric detector with cylindrical shape for photoacoustic section imaging is characterized. This detector is larger than the imaging object in direction of the cylinder axis, giving rise to its integrating properties. Its focal volume has the shape of a slice and the acquisition of signals for one section image requires rotation of an object about an axis perpendicular to this slice. Image reconstruction from the signals requires the application of the inverse Radon transform. It is shown that implementing the Abel transform is a suitable step in data processing, allowing speeding up the data acquisition since the scanning angle can be reduced. The resolution of the detector was estimated in directions perpendicular and parallel to the detection plane. An upper limit for the out of plane resolution is given and section images of a zebra fish are shown.

A large surface area transducer is preferable to be used to detect extremely weak photoacoustic signals in mammography due to its high sensitivity. The lateral resolution is limited by the small acceptance angle of such a transducer. We introduce an excellent material for an acoustic lens used to enlarge the transducer's acceptance angle. Our acoustic characterizations showed that this material has tissue-like acoustic impedance, large speed of sound and low acoustic attenuation. These acoustic properties ensures an excellent acoustic lens material. We further investigated the acoustic irradiation pattern of a 1 MHz, 5 mm x 5 mm single element transducer. Transducer irradiation pattern with and without acoustic lens made from our proposed material and common used lens material are simulated using the Field II program and also the k-wave package. Good agreement has been achieved comparing the simulation results from two different methods. Both simulations show that the proposed material not only enlarged the acceptance angle of the transducer but also minimized the signal loss compared to the common used lens material. We conclude that the proposed material can be used as an excellent acoustic lens for photoacoustic tomography.

A novel technique for the label-free analysis of micro and nanoparticles including biomolecules using optical micro cavity resonance of whispering-gallery-type modes is being developed. Various schemes of the method using both standard and specially produced microspheres have been investigated to make further development for microbial application. It was demonstrated that optical resonance under optimal geometry could be detected under the laser power of less 1 microwatt. The sensitivity of developed schemes has been tested by monitoring the spectral shift of the whispering gallery modes. Water solutions of ethanol, ascorbic acid, blood phantoms including albumin and HCl, glucose, biotin, biomarker like C reactive protein so as bacteria and virus phantoms (gels of silica micro and nanoparticles) have been used. Structure of resonance spectra of the solutions was a specific subject of investigation. Probabilistic neural network classifier for biological agents and micro/nano particles classification has been developed. Several parameters of resonance spectra as spectral shift, broadening, diffuseness and others have been used as input parameters to develop a network classifier for micro and nanoparticles and biological agents in solution. Classification probability of approximately 98% for probes under investigation have been achieved. Developed approach have been demonstrated to be a promising technology platform for sensitive, lab-on-chip type sensor which can be used for development of diagnostic tools for different biological molecules, e.g. proteins, oligonucleotides, oligosaccharides, lipids, small molecules, viral particles, cells as well as in different experimental contexts e.g. proteomics, genomics, drug discovery, and membrane studies.

A high-accuracy biosensor system has been developed to provide rapid detection of biomarker proteins as indicators of ovarian cancer. This photonic detection system is based upon guided-mode resonance sensor technology. The buildup of the attaching biolayer can be monitored directly, without use of chemical tags, by following the corresponding resonance shift with a spectrometer or detector array. Additionally, these high-resolution sensors employ multiple resonance peaks at identical physical location on the sensor surface. Each of these resonance peaks responds uniquely to the detection event, thereby enriching the data set available for quantification. The peaks result from individual, polarization-dependent resonant leaky modes that are the foundation of this technology. Examples are presented for detection of ovarian cancer biomarkers (fibronectin and apoliprotein A-1) in serum and cell culture supernatant, with detection sensitivities to ~20 ng/ml. Minimal nonspecific binding was measured in cell media and serum backgrounds. We also present an example dual-polarization resonance response with corresponding backfitting results that illustrate the capability to distinguish between changes at the sensor surface due to biolayer adhesion and those due to sample background changes.

We recorded eye movements of eight elite junior basketball players. We hypothesized that a more stable gaze is correlated to a better shot rate. Upon preliminary testing we invited male juniors whose eyes could be reliably tracked in a game situation. To these ends, we used a head-mounted video-based eye tracker. The participants had no record of ocular or other health issues. No significant differences were found between shots made with and without the tracker cap, Paired samples t-test yielded p= .130 for the far and p=..900 > .050 for the middle range shots. The players made 40 shots from common far and middle range locations, 5 and 4 meters respectively for aged 14 years As expected, a statistical correlation was found between gaze fixation (in milliseconds) for the far and middle range shot rates, r=.782, p=.03. Notably, juniors who fixated longer before a shot had a more stable fixation or a lower gaze dispersion (in tracker's screen pixels), r=-.786, p=.02. This finding was augmented by the observation that the gaze dispersion while aiming at the basket was less (i.e., gaze more stable) in those who were more likely to score. We derived a regression equation linking fixation duration to shot success. We advocate infra-red eye tracking as a means to monitor player selection and training success.

Time-domain diffuse optical spectroscopy has become a powerful tool to study highly scattering media, mainly in the fields of non-invasive medical diagnostics and quality assessment of food and pharmaceutical products. Up to now this technique has been exploited mostly up to 1100 nm: we extend the spectral range by means of a continuously tunable pulsed laser source at a high repetition rate and a custom InGaAs/InP Single-Photon Avalanche Diode operated in time-gated mode, working up to 1700 nm. The characterization of the system is presented. As a first example of application, we measured the absorption spectrum of collagen powder in the range 1100 - 1700 nm, which could prove useful for breast density assessment.

We report about preliminary results of using upconversion luminophores (UCLs) for tissue imaging. We manufactured luminophores particles of different sizes and tissue-mimicking phantoms for this study. Results of our experiments on imaging correspond well with our Monte Carlo simulations of luminescence detection from an imbedded vessel filled with UCLs.

A novel method for photoplethysmography (PPG) signal detection has been proposed and implemented in a three channel prototype device. The current design is simple, low cost and does not require sophisticated analogue circuits. The prototype was evaluated by physiological measurements and recorded PPG signals from conduit arteries of human subject.

Three channel photoplethysmography (PPG) signal waveform studies of leg conduit arteries during a provocative occlusion test were performed. PPG waveform second derivative amplitude ratio and arterial pulse wave velocity values showed significant correlations with ultrasound (US) reference method of local and regional arterial stiffness (AS), showing the ability to use PPG for AS change quantitative assessment.

Our goal is to provide a cost-effective method for examining human tissue, particularly the brain, by the simultaneous use of functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (NIRS). Due to its compatibility requirements, MRI poses a demanding challenge for NIRS measurements. This paper focuses particularly on presenting the instrumentation and a method for the non-invasive measurement of NIR light absorbed in human tissue during MR imaging. One practical method to avoid disturbances in MR imaging involves using long fibre bundles to enable conducting the measurements at some distance from the MRI scanner. This setup serves in fact a dual purpose, since also the NIRS device will be less disturbed by the MRI scanner. However, measurements based on long fibre bundles suffer from light attenuation. Furthermore, because one of our primary goals was to make the measuring method as cost-effective as possible, we used high-power light emitting diodes instead of more expensive lasers. The use of LEDs, however, limits the maximum output power which can be extracted to illuminate the tissue. To meet these requirements, we improved methods of emitting light sufficiently deep into tissue. We also show how to measure NIR light of a very small power level that scatters from the tissue in the MRI environment, which is characterized by strong electromagnetic interference. In this paper, we present the implemented instrumentation and measuring method and report on test measurements conducted during MRI scanning. These measurements were performed in MRI operating rooms housing 1.5 Tesla-strength closed MRI scanners (manufactured by GE) in the Dept. of Diagnostic Radiology at the Oulu University Hospital.

Photoacoustic imaging is an upcoming technique in the field of biomedical imaging. Our group introduced fiber-based line detectors, which are used to acquire broad-band ultrasonic signals, several years ago. Up to now operating point stabilization of fiber-based line detectors was realized by tuning the wavelength of the detection laser. This is, because of the high costs, not applicable for parallel detection. An alternative stabilization method, the change of the optical path length, is presented in this paper. Changing of the optical path length is realized by stretching the fiber with piezoelectric tubes. Fringe patterns and operation point stabilization of both stabilization schemes are compared. Next, signal detection utilizing a polymer optical fiber in a Mach-Zehnder and Fabry-Perot interferometer is demonstrated, and the influence of the detection wavelength (633nm and 1550nm) is examined. Finally, two-dimensional imaging by utilizing a perfluorinated polymer fiber is demonstrated.

A piezoelectric detection system consisting of concentric rings is investigated for large depth of field photoacoustic imaging. Compared to a single ring, the array with its dynamic focusing capability leads to a reduction of imaging artifacts. Image resolution studies are performed in simulations and in experiments. Detector arrays with four and eight rings were simulated to compare axial and lateral resolution. In simulation an improvement regarding the reduction of Xshaped imaging artifacts for the eight ring detection system in comparison to a four ring detection is presented. To compare the resolution axial and lateral profiles are shown and discussed. Furthermore signal processing methods are demonstrated, such as coherence factor weighting, which improve resolution and further reduce artifacts. To demonstrate the multiple ring detection system in experiment we used a 4 ring detection system and crossed horse hairs as phantom. Different projection images and a 3D image of the phantom are presented.

An analysis of the time-shifting correction in optoacoustic tomographic reconstructions for media with an a priori known speed of sound distribution is presented. We describe a modification of the filtered back-projection algorithm, for which the absorbed optical energy at a given point is estimated from the value of the measured signals at the instant corresponding to the time-of-flight between such point and the measuring points. In the case that a non-uniform speed of sound distribution does exist, we estimate the time-of-flight with the straight acoustic rays model, for which acoustic waves are assumed not to change direction as they propagate. The validity of this model is analysed for small speed of sound variations by comparing the predicted values of the time-of-flight with the ones estimated considering the refraction of the waves. Experimental results with tissue-mimicking agar phantoms with a higher speed of sound than water showcase the effects of the time-shifting of the optoacoustic signals caused by the acoustic mismatch. The performance of the time-shifting correction relates to the optoacoustic imaging of biological tissues, for which the speed of sound variations are usually lower than 10%.

The feasibility of correcting for the effects of acoustic attenuation in optoacoustic tomographic reconstructions obtained with model-based inversion is shown in this work. Acoustic attenuation is a physical phenomenon that takes place inevitably in actual acoustic media and becomes significant at high ultrasonic frequencies. The frequency dependence of acoustic attenuation and the associated dispersion lead to reduction of amplitude and broadening of the optoacoustic signals, which in turn cause, respectively, quantification errors and loss of resolution in the reconstructed images. In this work we imaged an agar phantom with embedded microparticles in three different scenarios, namely with the signals acquired with no attenuation, with the signals collected by placing an attenuating sample in between the phantom and the ultrasonic transducer and with the signals corrected for the effects of acoustic attenuation. The results obtained show that the quantification inaccuracies and the loss of resolution of the images can be partially corrected at the expense of introducing noise at high spatial frequencies due to the amplification of the high frequency components of the noise in the signals.

The reflection and scattering properties of light incident on skin covered with powder particles have been investigated. A three-layer skin structure with a spot is modeled, and the propagation of light in the skin and the scattering of light by particles on the skin surface are simulated by means of a Monte Carlo method. Under the condition in which only single scattering of light occurs in the powder layer, the reflection spectra of light from the skin change dramatically with the size of powder particles. The color difference between normal skin and spots is found to diminish more when powder particles smaller than the wavelength of light are used. It is shown that particle polydispersity suppresses substantially the extreme spectral change caused by monodisperse particles with a size comparable to the light wavelength.

In neurosurgery, navigation is being used to improve surgical orientation by using preoperative images as a roadmap. Skin or bone fiducials couple the image coordinate system to that of the patient's head fixed by the Mayfield clamp. Then the tip of a pointer of another instrument (localization device) can be seen in relation to the image to give the surgeon insight where he/she is in the brain and where the tumor or lesion can be expected in the depth. Drawbacks from current navigation systems are that 1) they only show the actual position of the localization device and thus do not hint whether the surgeon has removed the tumor completely, 2) don't warn when the device is about to hit a critical brain structure, and 3) do not compensate for shifts of the brain during surgery invalidating the pre-operative image data. During the last 5 years we investigated in our hospital whether sound and workflow feedback could improve the surgical resection accuracy and looked how the pre-operative image data could be deformed in real-time using GPU hardware to match the tracked cortical surface to compensate for brain shifts.

We present in this work a method to estimate the distribution of acoustic scatterers within the imaged sample in an optoacoustic tomographic setup and, subsequently, to reduce the artefacts in the tomographic reconstructions due to reflection or scattering events. The procedure to determine the position of the scatterers consists of measuring the scattered waves generated at a point light absorber located in between the transducer and the imaged sample. Such absorber is positioned in a way that the acoustic waves generated at this absorber and scattered within the sample arrive at the position of the transducer after the waves generated within the sample that propagate directly until the measuring point. Then, the signals captured by the acoustic transducer can be used to reconstruct the distribution of acoustic scatterers and to perform the optoacoustic reconstruction itself. Also, the information retrived on the distribution of acoustic scatterers can be used to improve the optoacoustic tomographic reconstructions. For this, we use a modification of the filtered back-projection algorithm based on weighting the signals with the probability that they are not affected by scattered or reflected waves, so that the artefacts in the images due to these acoustic phenomena are reduced. The experimental results obtained with a tissue-mimicking phantom in which a straw filled with air was included in order to cause scattering of the acoustic waves indicate a good performance of the method proposed.

Laser light propagated in semi-transparent turbid media such as biological tissue loses its coherence and polarization. Speckle contrast can be considered as a metric of light coherence. Recently we demonstrated that polychromatic speckle contrast and degree of polarization are useful criteria for skin lesion differentiation. To gain a better understanding of this complex process, we conducted an experiment to measure the speckle contrast and the average degree of polarization of solid skin phantoms with controllable roughness and bulk optical parameters of the order of human skin. The data validated that bulk scattering along with roughness introduce speckle contrast and DOLP reduction. Also we observed that speckle contrast and the average degree of polarization were related to the bulk scattering coefficient. The speckle contrast vs. degree of linear polarization dependence reveals a near linear relationship with the slope varying with the scattering coefficient of the material. The significant difference between slopes for two phantoms with slightly dissimilar optical properties could suggest that this pair of measurements (speckle contrast vs. degree of linear polarization) is highly sensitive to the tissue type and can be potentially used as a parameter for material differentiation.

In this paper we present a digital holographic microscope to track gold colloids in three dimensions. We report observations of 100nm gold particles in motion in water. The expected signal and the chosen method of reconstruction are described. We also discuss about how to implement the numerical calculation to reach real-time 3D tracking.