This PDF file contains the front matter associated with SPIE Proceedings Volume 9540 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Microcavity array sensor has been developed for biomedical objects identification. Experimental data on detection and identification of variety of biochemical agents, such as proteins, microelements, antibiotic of different generation etc. in both single and multi-component solutions analyzed on the light scattering of whispering gallery mode optical resonance are represented.

The analysis of fast temporal changes of the human retina can be used to get insight to normal physiological behavior and to detect pathological deviations. This can be important for the early detection of glaucoma and other eye diseases. We developed a small, lightweight, USB powered video ophthalmoscope that allows taking video sequences of the human retina with at least 25 frames per second without dilating the pupil. Short sequences (about 10 s) of the optic nerve head (20° x 15°) are recorded from subjects and registered offline using two-stage process (phase correlation and Lucas-Kanade approach) to compensate for eye movements. From registered video sequences, different parameters can be calculated. Two applications are described here: measurement of (i) cardiac cycle induced pulsatile reflection changes and (ii) eye movements and fixation pattern. Cardiac cycle induced pulsatile reflection changes are caused by changing blood volume in the retina. Waveform and pulse parameters like amplitude and rise time can be measured in any selected areas within the retinal image. Fixation pattern ΔY(ΔX) can be assessed from eye movements during video acquisition. The eye movements ΔX[t], ΔY[t] are derived from image registration results with high temporal (40 ms) and spatial (1,86 arcmin) resolution. Parameters of pulsatile reflection changes and fixation pattern can be affected in beginning glaucoma and the method described here may support early detection of glaucoma and other eye disease.

The present paper shows a method for pulse waveform extraction using laser speckle contrast analysis. An experimental apparatus was assembled, using a coherent light source and a digital video camera to record time varying speckle patterns emitted from the radial artery. The speckle data were analysed by computing the speckle pattern contrast on a sequence of video frames. The speckle pulse wave signal was then compared with a photoplethysmographic signal both time and frequency domain. A total of thirty data-sets were acquired from 10 individuals. Subjects heart rate was identified with a root mean square error of 1.3 beats per minute. Signals similarity was evaluated using spectral coherence with an overall mean coherence of 0.63. Speckle contrast analysis is a newly commercialized technique to monitor microvascular blood flow. However, these results demonstrate the ability of the same technique to extract pulse waveform information. The inclusion of this feature in the current speckle devices is only associated with a slightly change in the signal processing techniques and video acquisition parameters but can be very useful in clinical context.

We present an improved Laser speckle imaging approach to investigate the cerebral blood flow response following function stimulation of a single vibrissa. By synchronising speckle analysis with the cardiac cycle we are able to obtain robust averaging of the correlation signals while at the same time removing the contributions due to the pulsation of blood flow and associated tissue adaptation. With our inter-pulse correlation analysis we can follow second-scale dynamics of the cortical vascular system in response to functional brain activation. We find evidence for two temporally separated processes in the blood flow pattern following stimulation we tentatively attribute to vasodilation and vasoconstriction phases, respectively.

Raman Spectroscopy has become an important technique for assessing the composition of excised sections of bone, and is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material and to date little is known regarding its diffuse scattering properties which are important for the development and optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic medical applications; for instance on the detection of bone tumors or areas of infected bone.

Optical clearing technique was applied to the problem of OCT imaging of articular cartilage and subchondral bone. We show that optical clearing significantly enhances visualization of articular cartilage and cartilage-bone interface. The effect of different clearing agents was analyzed. For the clearing, iohexol solution and propylene glycol (PG) were used. Clearing was performed in vitro at room temperature by immersion method. Cylindrical osteochondral samples (d=4.8mm) were drilled from bovine lateral femur and stored in phosphate-buffered saline at -20°C until clearing. Monitoring of clearing process was performed using high-speed spectral-domain OCT system providing axial resolution of 5.8μm at 930nm. Total duration of experiment was 90-100min to ensure saturation of clearing. We have shown that iohexol solution and PG are capable to optically clear articular cartilage enabling reliable characterization of cartilagebone interface with OCT. Being a low osmolarity agent, iohexol provides minimal changes to the thickness of cartilage sample. Clearing saturation time for the cartilage sample with the thickness of 0.9 mm measured with OCT is of 50 min. However, less than 15 min is enough to reliably detect the rear cartilage boundary. Alternatively, PG significantly (60%) reduces the cartilage thickness enabling better visualization of subchondral bone. It was observed that PG has higher clearing rate. The clearing saturation time is of 30 min, however less than 5 min is enough to detect cartilage-bone interface. We conclude that iohexol solution is superior for OCT imaging of cartilage and cartilage-bone interface, while PG suits better for subhondral bone visualization.

We report on the deep-tissue imaging using novel upconversion nanoparticles (UCNPs) β-NaYF₄:Yb³⁺ ,Tm³⁺ (excitation wavelength: 975 nm, detection wavelength: 800 nm) and glycerol as an optical clearing agent to enhance imaging from under 6-mm-thick porcine muscle tissue samples. We show that improvement of luminescent label visualization is caused by transforming of the diffuse label-emitted light into the direct component. This results in the increase in visibility (ratio of the sum and difference of the maximal and minimal intensity) of the label and increase in maximal signal intensity thus making the combination of the phosphors and optical clearing promising for precise detection of tissue-embedded labelled inhomogeneities.

We combine pulsed photothermal radiometry (PPTR) depth profiling with diffuse reflectance spectroscopy (DRS) measurements for a comprehensive analysis of bruise evolution in vivo. While PPTR enables extraction of detailed depth distribution and concentration profiles of selected absorbers (e.g. melanin, hemoglobin), DRS provides information in a wide range of visible wavelengths and thus offers an additional insight into dynamics of the hemoglobin degradation products. Combining the two approaches enables us to quantitatively characterize bruise evolution dynamics. Our results indicate temporal variations of the bruise evolution parameters in the course of bruise self-healing process. The obtained parameter values and trends represent a basis for a future development of an objective technique for bruise age determination.

Interest in the interaction between laser light and biological samples has gained momentum in recent years, particularly in neurobiology, where there is significant potential to stimulate neurons with infrared laser light. Despite recent reports showing the application of infrared light for neurostimulation, the underlying mechanism is still unknown. The two main hypotheses are based on thermal or electrostatic mechanisms. Here, a novel optical method is presented to make temperature measurements in human neural cells under infrared laser excitation (λ=800nm) using the dye Rhodamine B (RhB). The measurement of temperature is based on the property of RhB, a fluorescent dye whose fluorescence intensity decreases linearly with increases in temperature. We present and detail the setup and measurement procedure that has temporal resolution of few milliseconds, based around a fluorescent live-cell imaging microscope used for cellular microfluorimetry experiments.

We have engineered nano-vesicles, derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETS) since in response to NIR photo-excitation they can generate heat or emit fluorescent light. Using biochemical methods based on reduction amination, we have functionalized the surface of NET with antibodies to target specific biomolecules. We present results that demonstrate the effectiveness of NETs in targeted imaging of cancer cells that over-express the human epidermal growth factor receptor-2 (HER2).

Full morphometric data analysis and 3D rendering of Red Blood Cells (RBCs) is provided by means of Digital Holography (DH) in combination with Optical Tweezers (OT). The proposed method is compared with a geometrical model of RBC in order to evaluate its accuracy and tested for many kinds of RBCs, from healthy ones with double-concavity to that with abnormal shapes. Applications in diagnostics are foreseen.

In this paper, we present a new approach for three-dimensional reconstruction and biovolume estimation of some species of diatoms. An optofluidic platform, composed by an optical tweezer and holographic modulus, is employed to retrieve several holograms at different angular positions, which are processed by the shape from silhouette algorithm to estimate the 3D shape of the cells.

Wavefront shaping techniques have recently evolved as a promising tool to control the light distribution in optically-scattering media. These techniques are based on spatially-modulating the phase of an incident light beam to create positive interference (focusing) at specific locations in the speckle pattern of the scattered wavefield. The optimum phase distribution (mask) of the spatial light modulator that allows focusing at the target location(s) is determined iteratively by monitoring the light intensity at such target. In this regard, optoacoustic (photoacoustic) imaging may provide the convenient advantage of simultaneous feedback information on light distribution in an entire region of interest. Herein, we showcase that volumetric optoacoustic images can effectively be used as a feedback mechanism in an iterative optimization algorithm allowing controlling the light distribution after propagation through a scattering sample. Experiments performed with absorbing microparticles distributed in a three-dimensional region showcase the feasibility of enhancing the light intensity at specific points. The advantages provided by optoacoustic imaging in terms of spatial and temporal resolution anticipate new capabilities of wavefront shaping techniques in biomedical optics.

During minimally invasive surgery the visual (3 dimensional) and mechanical (haptic) feedback is restricted or even non-existing, which imposes a serious loss of important information for decision making. Information about the mechanical properties of the biological tissue helps the surgeon to localize tissue abnormalities (benign vs. malign tissue). The work described here is directed towards assisting the surgeon during minimally invasive surgery, which in particular relates to the segmentation and navigation based on the recovery of mechanical properties. Besides the development of noninvasive elastographic measurement techniques, a reliable constitutive FE-model of the organ (describing its mechanical properties) is generated resulting in a further improvement of the segmentation and localization process. At first silicon phantoms, with and without foreign bodies have been generated for the purpose of testing the transfer of information (delivery and processing of data). The stress-strain curve was recorded and embedded in the FE-model (Arruda-Boyce). Two dimensional (2D) displacement maps have experimentally been obtained from the phantom, which were in good agreement with the FE simulation.

A micro-structured fiber-based system for identification and collection of fluorescent particles is demonstrated. An optical fiber probe with longitudinal holes in the cladding is used to retrieve fluorescent particles by exerting microfluidics forces. Laser induced fluorescent (LIF) is carried out by the fiber probe and an optical setup. When a particle with a previously chosen fluorescence wavelength is identified, a vacuum pump is activated collecting the particle into a hole. Green and red fluorescent polystyrene particles were detected and selectively retrieved.

We present an optical concept to visualize nerves during surgical interventions. The concept relies on the anisotropic optical properties of the nerves which allows for specific switching of the optical reflection by the nervous tissue. Using a low magnification polarized imaging system we are able to visualize the on and off switching of the optical reflection of the nervous tissue, enabling a non-invasive nerve specific real-time nerve visualization during surgery.

Holographic scanning microscopy - novel technique both in laser scanning microscopy and digital holographic microscopy allow multimodal approach to cell and tissue investigation in biomedical applications promising new advantages (quantitative phase imaging, superresolution, computerized tomography), but regular reconstruction leads to incorrectness. Analysis of light propagation through the schematics allows to offer reconstruction procedures depending on recording conditions.

Our aim is to optically monitor the delivery of the chemotherapy drugs for brain tumours, particularly used in the central nervous system (CNS) lymphoma therapy. In vivo monitoring would help to optimize the treatment and avoiding unnecessary medications. Moreover, it would be beneficial to be able to measure which of the multi-regimen drugs actually do penetrate and how well into the brain tissue.

There exist several potential optical measurement techniques to be utilised for the purpose. The most desired method would allow the detection of the drugs without using optical biomarkers as a contrast agent. In this case, for non-invasive sensing of the drug in the brain cortex, the drug should have a reasonably strong optical absorption band somewhere in the range between 600 nm and 1700 nm, and not directly coincident with the strong bands of haemoglobin or water. Alternatively, mid-infrared (MIR) range has the potential for invasive drug monitoring techniques.

In this paper, we report the optical properties of several chemotherapy drugs used in CNS lymphoma therapy, such as rituximabi, cyclophosphamide and etoposide. We measured their transmittance and reflectance spectra in near-infrared (NIR) range, particularly 900 nm − 2500 nm, to be considered when choosing the in vivo monitoring method to be developed. The absorption and scattering coefficients were retrieved from the measurements and applying Beer’s law. For the measurement of the sum of total transmission and reflection in NIR range we used integrating sphere with spektralo to enable calculation of the scattering coefficient.

Understanding of the complex interactions of molecules at biological interfaces is a fundamental issue in biochemistry, biotechnology as well as biomedicine. A plethora of biological processes are ruled by the molecular texture of cellular membrane: cellular communications, drug transportations and cellular recognition are just a few examples of such chemically-mediated processes. Tip-Enhanced Raman Scattering (TERS) is a novel, Raman-based technique which is ideally suited for this purpose. TERS relies on the combination of scanning probe microscopy and Raman spectroscopy. The basic idea is the use of a metalled tip as a sort of optical nano-antenna, which gives place to SERS effect close to the tip end. Herein, we present the application of TERS to analyze the surface of Bacillus subtilis spores. The choice of this biological systems is related to the fact that a number of reasons support the use of spores as a mucosal delivery system. The remarkable and well-documented resistance of spores to various environmental and toxic effects make them clear potentials as a novel, surface-display system. Our experimental outcomes demonstrate that TERS is able to provide a nano-scale chemical imaging of spore surface. Moreover, we demonstrate that TERS allows differentiation between wilde-type spore and genetically modified strains. These results hold promise for the characterization and optimization of spore surface for drug-delivery applications.

The Ulm University of Applied Sciences is investigating a technique using visible optical radiation (405 nm and 460 nm) to inactivate health-hazardous bacteria in water. A conceivable application could be point-of-use disinfection implementations in developing countries for safe drinking water supply. Another possible application field could be to provide sterile water in medical institutions like hospitals or dental surgeries where contaminated pipework or long-term disuse often results in higher germ concentrations. Optical radiation for disinfection is presently mostly used in UV wavelength ranges but the possibility of bacterial inactivation with visible light was so far generally disregarded. One of the advantages of visible light is, that instead of mercury arc lamps, light emitting diodes could be used, which are commercially available and therefore cost-efficient concerning the visible light spectrum. Furthermore they inherit a considerable longer life span than UV-C LEDs and are non-hazardous in contrast to mercury arc lamps. Above all there are specific germs, like Bacillus subtilis, which show an inactivation resistance to UV-C wavelengths. Due to the totally different deactivation mechanism even higher disinfection rates are reached, compared to Escherichia coli as a standard laboratory germ. By 460 nm a reduction of three log-levels appeared with Bacillus subtilis and a half log-level with Escherichia coli both at a dose of about 300 J/cm². By the more efficient wavelength of 405 nm four and a half log-levels are reached with Bacillus subtilis and one and a half log-level with Escherichia coli also both at a dose of about 300 J/cm². In addition the employed optical setup, which delivered a homogeneous illumination and skirts the need of a stirring technique to compensate irregularities, was an important improvement compared to previous published setups. Evaluated by optical simulation in ZEMAX® the designed optical element provided proven homogeneity distributions with maximum variation of ± 10 %.

Detecting pre-cancer in epithelial tissues such as the cervix is a challenging task in low-resources settings. In an effort to achieve low cost cervical cancer screening and diagnostic method for use in low resource settings, mobile colposcopes that use a smartphone as their engine have been developed. Designing image analysis software suited for this task requires proper modeling of light propagation from the abnormalities inside tissues to the camera of the smartphones. Different simulation methods have been developed in the past, by solving light diffusion equations, or running Monte Carlo simulations. Several algorithms exist for the latter, including MCML and the recently developed MCX. For imaging purpose, the observable parameter of interest is the reflectance profile of a tissue under some specific pattern of illumination and optical setup. Extensions of the MCX algorithm to simulate this observable under these conditions were developed. These extensions were validated against MCML and diffusion theory for the simple case of contact measurements, and reflectance profiles under colposcopy imaging geometry were also simulated. To validate this model, the diffuse reflectance profiles of tissue phantoms were measured with a spectrometer under several illumination and optical settings for various homogeneous tissues phantoms. The measured reflectance profiles showed a non-trivial deviation across the spectrum. Measurements of an added absorber experiment on a series of phantoms showed that absorption of dye scales linearly when fit to both MCX and diffusion models. More work is needed to integrate a pupil into the experiment.

Today around 275000 women a year in the world keep dying from the cancer of uterine cervix due to the difficulty to meet the logistic requirements of an organized screening in the developing world. Polarimetric imaging is a new promising technique with a tremendous potential for applications in biomedical diagnostics: it is sensitive to slight morphological changes in tissues, can provide wide field images for the screening and requires light sources such as a LED for example. This work intends to characterize the polarimetric response of the uterine cervix in its healthy and pathological states. An extensive series of ex-vivo measurements is in progress the Kremlin Bicêtre hospital near Paris using an imaging multispectral Mueller polarimeter in backscattering configuration. The goal of this study is to evaluate the performances of polarimetric imaging technique in terms of sensitivity and specificity for the detection of healthy epithelia (Healthy Squamous epithelium and Malpighian Metaplasia) with respect to the diagnosis provided by pathologists from histology slides as the “gold standard”. We show that, at λ=550nm, performances as high as 62% sensitivity and 64% specificity are achieved by optimizing a simple threshold on the scalar retardance values.

The paper presents the evaluation of photodynamic therapy (PDT) procedures with an application of a microsystem. Two cell lines were used in the experiments, i.e. human lung carcinoma - A549 and normal human fetal lung fibroblast MRC5. Mono-, coculture and mixed cultures were performed in a microsystem at the same time. The microsystem consisted of a concentration gradient generator (CGG) which generates different concentrations of a photosensitizer, and a set of microchambers for cells. The microchambers were linked by microchannels of various length in order to allow cells migration and in this way cocultures were created. Transparent materials were used for the chip manufacture, i.e. glass and poly(dimethylsiloxane). A high power LED was used to test photodynamic therapy effectiveness in the microsystem.

Collagen hydrogels are natural biomaterials that comprise 3D networks of high water content and have viscoelastic properties and biocompatibility similar to native tissues. Consequently, these materials play an important role in tissue engineering and regenerative medicine for quite some time. Second harmonic generation (SHG) and two-photon fluorescence (TPF) contrasts transpire as valuable label-free spectroscopic probes for analysis of these biomaterials and this presentation will report the structural, mechanical and physicochemical parameters leading to the observed optical SHG and TPF effects in synthesized 3D collagen hydrogels. We will present results regarding understanding the dependency of collagen fiber formation on ion types, new results regarding strengthening of these biomaterials with a nontoxic chemical cross-linker genipin and polarization selection of collagen fibers’ orientations.