This PDF file contains the front matter associated with SPIE Proceedings Volume 7000, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing

We use patterned 3D multi-spot illumination to perform neuronal multi-site stimulation in rat brain tissue. Using a spatial light modulator, we holograpically project 3D light fields for multi-site two-photon photolysis of caged neurotransmitters to generate synaptic inputs to a neuron. Controlled photostimulation of multiple synapses from various locations in the dendritic tree provides a way to analyze how neurons integrate multiple inputs. Our holographic projection setup is incorporated into a two-photon 3D imaging microscope for visualization and for accurate positioning of specific uncaging sites along the neuron's dendritic tree. We show two-photon images and the neuron's response to holographic photostimulation of synapses along dendrites.

Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

This research proposes a new imaging technique for near real time multispectral acquisition using CCD RGB cameras of the so called "Degree Of Polarization" (DOP) in polarimetry for future clinical investigation. The aim of exploiting the DOP as the contrast element is to demonstrate that the elliptical DOP provides more information characterizing complex medium than the more traditional linear and circular ones. The system considers an incoherent input white light beam and opportunely calibrated nematic crystals (LCVR), so no mechanical tools are necessary. The particular features of the system indicate it to be the perfect candidate for a new imaging system considering in-vivo (as well as ex-vivo) non invasive superficial diagnostic for medical application as dermatologic diagnostics, since no type of sample preparation is necessary, i.e. tissue biopsy, radiation or contrast agent injection. Thus the biomedical application of this method suggests a simple, direct, fast and also easily exploitable future employment, as a desirable mean for clinical investigation but also for digital recognition in biometrics. Further new elements to improve the model of light scattering and matter-light interaction will be acquired, in particular considering a very complete characterization of the system response using latex microspheres suspension to simulate turbid media with different concentration.

Quantitative digital holographic multi-focus phase imaging enables label-free minimally invasive live cell analysis by high resolution detection of sample induced optical path length changes. However, a drawback of many experimental arrangements for the analysis of living cells with digital holography is the requirement for a separate reference wave which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, a self interference digital holographic microscopy (DHM) approach was explored which only requires a single object illumination wave. Due to the Michelson interferometer design of the proposed experimental setup two wave fronts with an almost identical curvature are superimposed. This results in a simplified evaluation of the digital holograms. The applicability of the proposed self interference principle is illustrated by results from a technical specimen and living single cells. Furthermore, adherent cancer cells have been analyzed for morphology changes in perfusion chambers due to flow and the refractive index of suspended cells was determined. In summary, the method prospects to be a versatile tool for quantitative phase imaging as simplification is important for the establishment of these methods in live cell analysis.

Optical tweezers are a versatile technique to manipulate living biological specimen in a contact-less way. The interaction with living cells can be performed, for example, through direct manipulation of cell organelles or by movement of an internalized particle within the cytoplasm. However, the risk of damage that the trapping beam may induce in the biological sample due to the energy deposition has to be considered. This optically induced damage or photodamage depends mainly on the wavelength of the trapping beam, the exposure time and the biological specimen that is investigated. In this work, we explore a method to analyse the photo damage in living cells in a multimodal biophotonic workstation that is based on combining a holographic optical tweezers (HOT) microscope with a self-interference digital holographic microscopy (DHM) module. A time-dependent investigation shows that no observable changes in the cell morphology are induced at room conditions with the used laser power of the trapping beam during periods of time < 20 min of laser application. In addition, results from investigations of the photodamage increasing the working temperature to 37°C demonstrate that the optical tweezers beam can provoke severe but reversible morphology changes in the cell.

Quantitative Phase Imaging techniques including DHM have been applied recently in the field of cell imaging to monitor and quantify non-invasively dynamic cellular processes modifying cell morphology and/or content . Concretely, the DHM phase signal is highly sensitive to cell thickness and intracellular integral RI variations associated with transmembrane water movements. As net water flow across the cell membrane leads at the same time to changes in cell thickness and intracellular RI, the interpretation of phase signal variations remains difficult. To overcome this drawback, we have developed a Dual-wavelength Digital Holographic Microscopy (DHM) setup allowing to separately measure, with a single CCD camera acquisition, thickness and integral RI of living cells. The method is based on the use of an absorbing dye that enhances the refractive index dispersion of the extracellular medium. Practically, two significantly different phase signals can be obtained when measuring at two appropriate wavelengths. From the two phase measurements, both cell RI and thickness can be univocally determined.

The spectroscopic and photophysical properties of ruthenium polypyridyl polypeptide conjugates of the type [Ru(bpy)₂PIC-Argn]ⁿ⁺²⁺, where bpy is 2,2-bipyridyl (bpy), PIC is 2-(4-carboxyphenyl)imidazo[4,5- f][1,10]phenanthroline and PIC-Argn is this ligand peptide bonded to polyarginine where n is 5 or 8, is described. The resonance Raman spectroscopy of the peptide conjugated complex and parent are strongly pH dependent and demonstrate a switch of lowest energy charge transfer transition between bpy and pic ligands as s function of pH. The pKa of the imidazole ring on the complex is obtained from resonance Raman spectroscopy as 7.8 ± 0.2. The luminescence lifetime of the complex is strongly oxygen dependent and a Stern-Volmer plot of O₂ quenching for [Ru(bpy)2(PIC-Arg8)]¹⁰⁺ yielded a KSV value of 2300 ± 420 M^-1 which was independent of pH over the range 2 to 11. The complexes, because of their large Stokes shifts can, uniquely, be used under identical conditions of probe concentration and excitation wavelength for resonance Raman and luminescence cellular imaging. Cellular imaging was conducted using SP2 myeloma cells which confirmed that the [Ru(bpy)2(PIC-Arg8)]¹⁰⁺ is readily taken up by mammalian cells although the parent and pentarginine analogues are not membrane permeable. Preliminary examples of multi-parameter imaging using these probes were presented. Resonance Raman maps of [Ru(bpy)₂(PIC-Arg8)]¹⁰⁺ within living myeloma cells showed on the basis of spectral discrimination, attributed to pH, three distinct regions of the cell could be identified, ascribed to the nucleus, the cytoplasm and the membranes. Luminescence lifetime imaging showed quite large variations in the probe lifetime within the living cell which was tentatively ascribed to variation in O₂ concentration about the cell. Preliminary estimates of O₂ concentration were made and it was found that the membranes, both inner and outer are the most O₂ rich regions of the cell. Overall, we propose that such peptide labeled luminescent metal are potentially a valuable addition to cellular imaging by providing tools for multiplexed analysis of the cell environment.

We have recently demonstrated a particularly economic approach to analyze large arrays of microring resonator (MRR) sensor elements coupled to a single bus waveguide. The sensor elements can be individually functionalized to specifically promote the accumulation of target molecules. The binding of target molecules to the surface of a particular MRR results in an increase of its resonance wavelengths which can be measured with high accuracy. In order to measure the response of the individual MRR from an array to external stimuli, we employ a special frequency modulation scheme in which each MRR is independently modulated and phase sensitive lock-in detection is used to filter the respective frequency component from the superimposed complex transmission spectrum of the bus waveguide. We fabricated test arrays comprising up to 12 MRR coupled to a single bus waveguide. A silicon nitride based material system was chosen to realize the devices. Each element of an array is equipped with a platinum heater electrode for thermo-optical modulation. A tunable laser system was used for optical characterization and a clear readout of the individual MRR resonance frequencies was possible by employing the modulation scheme above. Furthermore, we demonstrated a bulk refractive index sensitivity of 190 nm/RIU for a frequency modulated MRR. With our first results, we point out the large potential for multiplexed label-free detection of diverse bio molecular compounds. Due to the miniaturization of the multisensor arrays the realization of portable sensor systems will be feasible.

In this work, we investigate the usability of layered polymer - inorganic composite waveguides for label-free sensing of surface bound bioreactions in an aqueous environment. The waveguide structure consists of a nanoimprint fabricated polymeric inverted rib waveguide with a sputtered Ta₂O₅ thin film on top. The interaction of the optical field with the surface is increased as a consequence of the mode profile localization near the surface, when high-index coating is deposited on a low-index waveguide. Young interferometer configuration with reference and sensors waveguide arms was utilized in sensor chips. Light from a laser source was end-fire coupled into the chips and interference pattern produced by the outcoupled light was investigated. External μ-fluidic pump was used to produce the analyte flow. Ambient refractive index change was characterized by applying DI-water with varying glucose concentration on waveguides. With the waveguide length of 1 cm a detection limit in the order of 10^-7 - 10^-6 refractive index unit (RIU) was achieved. Specific binding reactions on the surface were investigated with C - reactive protein (CRP) antibodies and antigens.

One of the early predictors of cardiovascular diseases, with growing interest, is the arterial stiffness which is typically evaluated through the velocity and morphology of the arterial pressure wave. In each cardiac cycle the heart generates a pressure wave which propagates through the arterial tree. Along its path, the pressure wave interacts with the arterial walls and, consequently, the morphology of a local arterial pressure wave can be assessed by the arterial distention movement. Due to its superficiality, proximity of the heart and high probability of atherosclerosis development, the carotid artery has particular interest to be monitored. In this work, the development of a non-invasive fibre Bragg grating (FBG) probe for the acquisition of the arterial distention wave is presented. Comparing to traditional methods, optical FBG based sensors can offer many advantages, namely, compactness, immunity to electromagnetic interference, high sensitivity, low noise and immunity to light source intensity due to its codification in the wavelength domain. The arterial movements induce strain on a uniform FBG, with the arterial distention pattern. The carotid pulse wave was successful accessed in young human carotid artery, with an acquisition rate of 950 Hz, allowing a clear distinction of the carotid pulse identification points.

Assessment of glycaemia in diabetes is crucially important for prevention of both, acute and long term complications. Continuous glucose monitoring (CGM) is certainly the most appropriate way for optimizing the glycaemic control, since it prevents or delays the progression of complications associated with hypo- or hyperglycaemic events, reducing morbidity, mortality, and overall costs in health care systems. In this paper we describe the concept and first in vitro results of a minimally invasive, chip-based NIR-Sensor for continuous glucose monitoring. The sensor concept is based on difference infrared absorption spectroscopy, which was evaluated within laboratory measurements of D+-Glucose dissolved in water. The laboratory measurements revealed a linear relationship between glucose concentration and the integrated difference spectroscopy signal with a coefficient of determination of 99.6% in the concentration range of 0- 500 mg/dL. Suitable wavelength bands were identified in which the correlation is preserved and commercial light sources are available for realisation of a spectrometer-less, integrated NIR-sensor. In the designed sensor the component area (non-disposable) is separated from the detection area (disposable, low-cost). The disposable part of the sensor is fluidically connected to a micro-dialyses needle, accessing glucose subcutaneously via the ISF (interstitial fluid) or intravascularly. The non-disposable part contains all the optical elements, like LED´s and photo-detectors. The in- and out-coupling of the optical signal is achieved across the plane of the chip by using total internal reflection on mirrors integrated into the fluidic chip. The glucose is continuously measured by considering the difference signals of light at the corresponding wavelengths, as a function of time or in defined intervals if the light sources are modulated. The in-vitro measurements show an absolute error of about 5 mg/dL with a relative error of 5% for glucose concentrations larger than 50 mg/dL and about 12 % in the hypoglycemic range (<50 mg /dL).

Photoplethysmography (PPG) is an optical method of blood pulsation recording and has been extensively studied for decades. Recently PPG is widely used in the medical equipment for patient monitoring and in laboratories for research and physiological studies. In spite of the technological progress in the field of medical equipment, there are no generally accepted standards for clinical PPG measurements up to date. One of the most important factors affecting PPG waveform is the contact pressure between tissue and PPG probe. The aim of the current study was to develop and evaluate a system for software-assisted PPG signal acquisition from the unloaded artery. Novel PPG waveform derived Optimal Pressure Parameter (OPP) has been proposed as the reliable indicator of unloaded artery condition. We affirm that PPG measurements provided in balanced transmural arterial pressure conditions might serve as a reference for the unification of contact manner optical plethysmography methods. It is a step forward towards the standardization of the PPG methodology, and showed that the maximal value of the OPP, obtained in the particular experimental trial, indicates the optimal PPG probe contact pressure at that moment. Our developed system has been validated in the experimental series and showed the possibility of determining the correct PPG contact pressure value with high repeatability. It is concluded that this system can provide the necessary feedback to perform reliable PPG signal acquisition from the unloaded conduit artery.

Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

Utilising the versatility of holographic optical tweezers and high speed video analysis, we present a scheme for the analysis of the rotational properties of multiple rod-shaped bacteria directly from video microscopy data. The bacterial body and flagella rotation frequency of Bacillus subtilis are determined by temporally resolved monitoring of the position of the bacterial body. In contrast to established methods, the video-based approach can be extended to the simultaneous analysis of several bacteria within the field of view. Monitoring multiple bacteria simultaneously will allow resolving the role of hydrodynamic interactions of multiple flagella motors on their mutual dynamics.

Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

We report on the results of using self-developed combined laser system consisting of a femtosecond laser scalpel (Cr:Forsterite seed oscillator and a regenerative amplifier, 620 nm, 100 fs, 10 Hz) and optical tweezers (cw laser, 1064 nm) for performing noncontact laser-mediated polar body (PB) and trophectoderm (TE) biopsy of early mammalian embryos. To perform PB biopsy the femtosecond laser scalpel was initially used to drill an opening in the zona pellucida, and then the PB was extracted out of the zygote with the optical tweezers. Unlike PB biopsy, TE biopsy allows diagnosing maternally-derived as well as paternally-derived defects. Moreover, as multiple TE cells can be taken from the embryo, more reliable diagnosis can be done. TE biopsy was performed by applying laser pulses to dissect the desired amount of TE cells that had just left the zona pellucida during the hatching. Optical tweezers were then used to trap and move the dissected TE cells in a prescribed way. Laser power in optical tweezers and energy of femtosecond laser pulses were thoroughly optimized to prevent cell damage and obtain high viability rates. In conclusion, the proposed techniques of laser-based embryo biopsy enable accurate, contamination-free, simple and quick microprocessing of living cells.

In monitoring the quality of drinking water with respect to the presence of hazardous bacteria there is a strong need for on-line sensors that allow quick identification of bacterium species at low cost. In this respect, the combination of photonics and microfluidics is promising for lab-on-a-chip sensing of these contaminants. Photonic crystal slabs have proven to form a versatile platform for controlling the flow of light and creating resonant cavities on a wavelength scale. The goal of our research is to use photonic crystal cavities for optical trapping of microorganisms in water, exploiting the enhanced evanescent field of the cavity mode. We optimize the H0, H1 and L3 cavities for optical trapping of bacteria in water, by reducing out-of-plane losses and taking into account the trapping-induced resonance shift and the in-plane coupling with photonic crystal waveguides. The cavities are fabricated on silicon-on-insulator material, using e-beam lithography and dry etching. A fluidic channel is created on top of the photonic crystal using dry film resist techniques. Transmission measurements show clear resonances for the cavities in water. In the present state of our research, we demonstrate optical trapping of 1 μm diameter polystyrene beads for the three cavities, with estimated trapping forces on the order of 0.7 pN.

In recent years, microfluidic devices have become important tools for cell analysis in biology and medicine. They enable fast and inexpensive analysis with reduced consumption of analytes. However, for optical detection involving FACS (fluorescence-activated cell sorting), sample preparation by attaching an antibody-labeled fluorochrome to the cell is required. Cell tagging by fluorochromes is a mature technology but might affect cell viability and function. In this paper we present a novel concept for marker-free detection and first realization steps. We show the integration of a microfluidic chip and an electrically pumped GaAs-based oxide-confined VECSEL (vertical-extended- cavity surface-emitting laser). Particles in the microchannel flow through the laser resonator and induce a change of the cavity resonance, thus allowing sensitive detection to trigger a subsequent sorting process.

We studied fluctuations of an optically trapped bead connected to a single DNA molecule anchored between the bead and a cover glass or between two optically trapped beads. Power spectral densities of the bead position for different extensions of the molecule were compared with the power spectral density of the position fluctuations of the same bead without the molecule attached. Experiments showed that the fluctuations of the DNA molecule extended up to 80% by a force of 3 pN include the colored noise contribution with spectral dependence 1/fα with α~ 0.75.

Penetration of nanoparticles into tooth tissues is of significant interest in solving problems related to reduction of tooth sensitivity, enamel strengthening and restoration and cosmetic bleaching. In this work we demonstrate two-photonexcited autofluorescence and second-harmonic generation microscopy for visualization of penetration of TiO₂ and ZnO nanoparticles into tooth tissues.

Early detection and typing of tumors is pressing matter in clinical research with important impacts for prognosis and successful treatment. Currently, staining is the golden standard in histopathology but requires surgical removal of tissue. In order to avoid resection of non-diseased tissue a non-invasive real-time imaging method is required which can be applied ideally intrasurgically. In this proceeding a combination of second harmonic generation (SHG), two photon excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) imaging has been employed to investigate tissue sections of head and neck carcinomas focussing on laryngeal carcinoma. Primary laryngeal and other head and neck carcinomas consist to 99% of squamous cell carcinoma. By fusing the various imaging methods it is possible to measure the thickness of the epithelial cell layer as a marker for dysplastic or cancerous tissue degradation and to differentiate keratinizing and nonkeratininzing squamous cell carcinomas (SCC). As nonkeratinizing SCCs of the oropharynx correlate with a human papillomavirus (HPV) infection as a subentity of head and neck cancer, and HPV related tumors are associated with a better clinical prognosis, the differentiation between keratinizing and non-keratinizing forms of SCCs is of high diagnostic value. TPEF is capable of displaying cell nuclei, therefore, morphologic information as cell density, cell to cytoplasm ratio, size and shape of cell nuclei can be obtained. SHG - on the other hand - selectively reveals the collagen matrix of the connective tissue, which is useful for determination of tumor-islets boundaries within epithelial tissue - a prerequisite for precise resection. Finally CARS in the CH-stretching region visualizes the lipid content of the tissue, which can be correlated with the dysplastic grade of the tissue.

Optical microspectroscopic tools reveal great potential for dermatologic diagnostics in the clinical day-to-day routine. To enhance the diagnostic value of individual nonlinear optical imaging modalities such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) or two-photon excited fluorescence (TPF), the approach of multimodal imaging has recently been developed. Here, we present an application of nonlinear optical multimodal imaging with Raman-scattering microscopy to study sizable human-tissue cross-sections. The samples investigated contain both healthy tissue and various skin tumors. This contribution details the rich information content, which can be obtained from the multimodal approach: While CARS microscopy, which - in contrast to spontaneous Raman-scattering microscopy - is not hampered by single-photon excited fluorescence, is used to monitor the lipid and protein distribution in the samples, SHG imaging selectively highlights the distribution of collagen structures within the tissue. This is due to the fact, that SHG is only generated in structures which lack inversion geometry. Finally, TPF reveals the distribution of autofluorophores in tissue. The combination of these techniques, i.e. multimodal imaging, allows for recording chemical images of large area samples and is - as this contribution will highlight - of high clinically diagnostic value.

Viscoelastic and spectroscopic properties of single RBC are probed using dual beam optical tweezers and Raman techniques, respectively. Complex response function of cell was measured by means of one and two particles passive microrheology at different stretching states yielding local and overall mechanical properties of exactly the same human erythrocyte. The frequency dependent response function (measured up to 10 kHz) was corrected for the presence of the traps and spectral distribution of complex stiffness over controlled range of cell deformation is calculated and discussed. The presence of non-thermal sources of membrane motions is also explored based on comparison of passive and active microrheology experiments. In order to get insight into structural changes of RBC due to deformation, Raman spectra of single cell were recorded. Evolution of Raman bands with cell deformation was analyzed using sensitive 2D correlation method. The combination of force and Raman spectroscopy is promising and potentially very powerful method to establish essential linkages between structure, mechanical properties and functions of living cells.

It has been described that benign MCF10A breast cancer cell line suffers phenotypic changes toward malignancy when are cultured in sparse conditions. Using Raman spectroscopy with an InVia Raman microscope (Renishaw) with a backscattered configuration, we have studied the metabolic changes of confluent and sparse MCF10A cell cultures. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the metabolic composition of breast cancer cells. The results indicated that Raman spectroscopy together with multivariate analysis is a useful technique to distinguish metabolic changes in malignant transformation. The identification of new metabolites, implementing the catalogue on the characterization of the different phenotypes associated to cell malignancy using Raman spectroscopy is under study.

We have designed and developed an optical fiber-probe for spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multidimensional approach. Concerning fluorescence spectroscopy, the excitation is provided by two laser diodes, one emitting in the UV (378 nm) and the other emitting in the visible (445 nm). These two lasers are used to selectively excite fluorescence from NADH and FAD, which are among the brightest endogenous fluorophores in human tissues. For Raman and NIR spectroscopy, the excitation is provided by a third laser diode with 785 nm excitation wavelength. Laser light is delivered to the tissue through the central optical fiber of a fiber bundle. The surrounding 48 fibers of the bundle are used for collecting fluorescence and Raman and for delivering light to the spectrograph. Fluorescence and Raman spectra are acquired on a cooled CCD camera. The instrument has been tested on fresh human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin, finding an optimal correlation with the subsequent histological exam. In some cases our examination was not in agreement with the clinical observation, but it was with the histological exam, demonstrating that the system can potentially contribute to improve clinical diagnostic capabilities and hence reduce the number of unnecessary biopsies.

Retinal nervous tissue sustains a substantial damage during the autoimmune inflammatory processes characteristic for Multiple Sclerosis (MS). The damage can be characterized non-surgically by Raman Spectroscopy, a non-invasive optical imaging technology. We used non-resonant near-infrared Raman spectrosocopy to create a spectral library of eight pivotal biomolecules known to be involved in neuroinflammation: Nicotinamide Adenine Dinucliotide (NADH), Flavin Adenine Nucleotide (FAD), Lactate, Cytochrome C, Glutamate, N-Acetyl- Aspartate (NAA), Phosphotidylcholine, with Advanced Glycolization End Products (AGEs) analyzed as a reference. Principal Component Analysis (PCA) of 50 spectra taken of murine retinal tissue culture undergoing an inflammatory response and healthy controls was used in order to characterize the molecular makeup of the inflammation. The loading plots revealed a heavy influence of peaks related to Glutamate, NADH, and Phosphotidylcholine to inflammation-related spectral changes. Partial Least Squares - Discriminant analysis (PLS-DA) was performed to create a multivariate classifier for the spectral diagnosis of neuroinflammed tissue and yielded a diagnostic sensitivity of 100% and specificity of 100%. We demonstrate then the effectiveness of combining Raman spectroscopy with PCA and PLS-DA statistical techniques to detect and monitor neuroinflamation in retina. With this technique Glutamate, NAA and NADH are detected in retina tissue as signs for neuroinflammation.

Living cells and single molecules as DNA experiences numerous mechanical events, necessitating single molecule force spectroscopy techniques to provide insight into cellular mechanics as a whole system. This paper shows results on Raman spectroscopy of a single red blood cell which is gradually stretched using optically trapped beads attached to the cell. The applied force is intended to simulate step-by-step deformation experienced by cells in normal conditions (induced by blood flow) as they squeeze through microvasculature. To further improve the sensitivity of the experiments and facilitate their interpretation, 2D correlation and principal component analysis techniques were applied. The purpose of this work is to help unravel direct relationship between mechanical deformation of RBC and chemical changes occurring in the cell structure on molecular level. We also obtained Raman spectra from single DNA molecules in their natural aqueous environment as a first step to establish a direct relationship between DNA's extension and structure in the low force, entropic regime.

Raman spectroscopy has increasing importance in a wide field of applications: particularly in real time monitoring of chemical processes, testing of foodstuffs, identification of ingredients in unknown material mixtures etc. Many materials of interest have resonance wavelength close to the excitation wavelength. Resonant Raman spectroscopy can be used to advantage in these cases. The disadvantage of this technology is the presence of a strong fluorescence background in the Raman spectrum. A combination of the mechanism of resonant Raman spectroscopy with shifted excitation Raman difference spectroscopy can be used to suppress the fluorescence background. The applicability of inexpensive green lasers for this purpose and their tunability by temperature and current is investigated in this paper. The setup consists of two pigtailed lasers at a wavelength of 532 nm with a small wavelength difference switched by a fiber switch with a frequency up to 50 Hz. Every switching pulse triggers an optical spectrometer to measure the backscattered light. A resonant Raman spectrum with a minimized fluorescence background is obtained by subtraction of the two different spectra. The specific wavelengths of the two lasers were set by thermal tuning. The Raman spectra of Isopropanol and Carbon Tetrachloride have been measured in order to verify the setup.

Optical coherence tomography (OCT) in the 1060nm range is interesting for in vivo imaging of the human posterior eye segment (retina, choroid, sclera), as it permits a long penetration depth. Complementary to structural images, polarization-sensitive OCT (PS-OCT) images visualize birefringent, polarization-maintaining or depolarizing areas within the sample. This information can be used to distinguish retinal layers and structures with different polarization properties. High imaging speed is crucial for imaging ocular structures in vivo in order to minimize motion artifacts while acquiring sufficiently large datasets. Here, we demonstrate PS-OCT imaging at 350 kHz A-scan rate using a two-channel PS-OCT system in conjunction with a Fourier domain mode-locked laser. The light source spectrum spans up to 100nm around the water absorption minimum at 1060 nm. By modulating the laser pump current, we can optimize the spectrum and achieve a depth resolution of 9 μm in air (6.5 μm in tissue). We acquired retinal images in vivo with high resolution and deep penetration into choroid and sclera, and features like the depolarizing RPE or an increasing phase retardation at the chorio-scleral interface are clearly visualized.

Glaucoma is a disease of the optic nerve that is usually associated with an increased internal pressure of the eye and can lead to a decreased vision and eventually blindness. It is the second leading cause of blindness worldwide with more than 80 million people affected and approximately 6 million blind. The standard clinical treatment for glaucoma, after unsuccessful administration of eyedrops and other treatments, is performing incisional surgery. However, due to post-surgical complications like scarring and wound healing, this conventional method has a global success rate of only about 60%. In comparison, as femtosecond laser surgery may be performed in volume and is a priori less invasive and less susceptible of causing scarring, glaucoma laser surgery could be a novel technique to supplement the conventional glaucoma surgery. We have been working on the development of a new tool for glaucoma treatment that uses an optimized femtosecond laser source centered at 1.65 μm wavelength for making the surgery and an imaging system based on optical coherence tomography (OCT) for guiding the laser surgery. In this proceeding, we present the results obtained so far on the development and utilization of Fourier-domain OCT imaging system working at 1.3 μm center wavelength for guiding the laser incision. Cross-sectional OCT image of pathological human cornea showing the Schlemm's canal, where the surgery is intended to be done, is presented. By coupling OCT imaging system with the laser incision system, we also demonstrate real-time imaging of femtosecond laser incision of cornea.

The diagnosis of dental caries at an early stage enables the implementation of conservative treatments based on dental preservation. Several diagnostic methods have been developed, like visual-tactile and radiographic are the most commons but are limited for this application. The Optical Coherence Tomography is a technique that provides information of optical properties of enamel, which may change due to the decay process. The objective of this study was to evaluate the ability of OCT to detect different stages of demineralization of tooth enamel during the development of artificial caries lesions, taking as a reference standard for comparison sectional microhardness testing. Different stages of caries lesions were simulated using the pH cycling model suggested Feathestone and modified by Argenta. The samples were exposed to 0 (control group), 5, 10, 15, 20 and 25 days at a daily regimen of three hours demineralization followed by remineralization during 20 hours. It was used an OCT system with at 930nm. Sectional images were generated in all lesion region. The results obtained from the OCT technique presented similar behavior to microhardness, except for the group 25 days, due to inability to perform indentations reading in areas of more intense demineralization. A linear relationship was observed between the OCT and microhardness techniques for detection of demineralization in enamel. This relationship will allow the use of OCT technique in quantitative assessment of mineral loss and for the evaluation of incipient caries lesions.

Optical coherence tomography (OCT) is a non-invasive three-dimensional imaging system that is capable of producing high resolution in-vivo images. OCT is approved for use in clinical trials in Japan, USA and Europe. For OCT to be used effectively in a clinical diagnosis, a method of standardisation is required to assess the performance across different systems. This standardisation can be implemented using highly accurate and reproducible artefacts for calibration at both installation and throughout the lifetime of a system. Femtosecond lasers can write highly reproducible and highly localised micro-structured calibration artefacts within a transparent media. We report on the fabrication of high quality OCT calibration artefacts in fused silica using a femtosecond laser. The calibration artefacts were written in fused silica due to its high purity and ability to withstand high energy femtosecond pulses. An Amplitude Systemes s-Pulse Yb:YAG femtosecond laser with an operating wavelength of 1026 nm was used to inscribe three dimensional patterns within the highly optically transmissive substrate. Four unique artefacts have been designed to measure a wide variety of parameters, including the points spread function (PSF), modulation transfer function (MTF), sensitivity, distortion and resolution - key parameters which define the performance of the OCT. The calibration artefacts have been characterised using an optical microscope and tested on a swept source OCT. The results demonstrate that the femtosecond laser inscribed artefacts have the potential of quantitatively and qualitatively validating the performance of any OCT system.

In this paper we present a model of interference microscope image formation, which allows for analysis on unified basis of imaging different types of samples with different interference microscope modalities. The applicability of the proposed model to analysis of the coherence effects, arising in confocal and full-field interference microscopes, when imaging in depth of a layered sample, is shown. The effect of sample transversal structure on the coherence signal is also analyzed, showing that in this case the interference microscope can not be characterized by a single effective aperture and both illuminating and imaging apertures are important.

An optical imaging system has been developed which uses measurements of diffusely reflected near-infrared light to produce maps of changes in blood flow and oxygenation occurring within the cerebral cortex. Optical sources and detectors are coupled to the head via an array of optical fibers, on a probe held in contact with the scalp, and data is collected at a rate of 10 Hz. A clinical electroencephalography (EEG) system has been integrated with the optical system to enable simultaneous observation of electrical and hemodynamic activity in the cortex of neurologically compromised newborn infants diagnosed with seizures. Studies have made a potentially critically important discovery of previously unknown transient hemodynamic events in infants treated with anticonvulsant medication. We observed repeated episodes of small increases in cortical oxyhemoglobin concentration followed by a profound decrease in 3 of 4 infants studied, each with cerebral injury who presented with neonatal seizures. This was not accompanied by clinical or EEG seizure activity and was not present in nineteen matched controls. The underlying cause of these changes is currently unknown. We tentatively suggest that our results may be associated with a phenomenon known as cortical spreading depolarization, not previously observed in the infant brain.

Wide field multispectral imaging of light backscattered by brain tissues provides maps of hemodynamics changes (total blood volume and oxygenation) following activation. This technique relies on the fit of the reflectance images obtain at two or more wavelengths using a modified Beer-Lambert law^1,2. It has been successfully applied to study the activation of several sensory cortices in the anesthetized rodent using visible light^1-5. We have carried out recently the first multispectral imaging in the olfactory bulb⁶ (OB) of anesthetized rats. However, the optimization of wavelengths choice has not been discussed in terms of cross talk and uniqueness of the estimated parameters (blood volume and saturation maps) although this point was shown to be crucial for similar studies in Diffuse Optical Imaging in humans7-10. We have studied theoretically and experimentally the optimal sets of wavelength for multispectral imaging of rodent brain activation in the visible. Sets of optimal wavelengths have been identified and validated in vivo for multispectral imaging of the OB of rats following odor stimulus. We studied the influence of the wavelengths sets on the magnitude and time courses of the oxy- and deoxyhemoglobin concentration variations as well as on the spatial extent of activated brain areas following stimulation. Beyond the estimation of hemodynamic parameters from multispectral reflectance data, we observed repeatedly and for all wavelengths a decrease of light reflectance. For wavelengths longer than 590 nm, these observations differ from those observed in the somatosensory and barrel cortex and question the basis of the reflectance changes during activation in the OB. To solve this issue, Monte Carlo simulations (MCS) have been carried out to assess the relative contribution of absorption, scattering and anisotropy changes to the intrinsic optical imaging signals in somatosensory cortex (SsC) and OB model.

Optical imaging of turbid media is a challenging problem mainly due to the scattering process that reduces image contrast and degrades spatial resolution. The development of fluorescent probes has recently improved the noninvasive optical technique. In this paper, we are interested in the time gating fluorescence signals. The diffusion approximation is used in order to describe the light propagation of a laser pulse in a turbid media that mimics breast like biological tissue. A numerical model based on a finite element method is proposed. Fluorescence time dependent numerical simulations are performed in order to compute time-gated intensities resulting from line scans across partially absorbing and scattering slab configurations. Optical properties of embedded objects are chosen to be the same as optical properties of breast tumor. Tacking into account two hidden objects, we investigate the lateral resolution aimed by fluorescence modality, and we also compared the results to thus obtained by photon propagation. Different widths of the time gate are computed and it is demonstrated that both lateral localization of one inclusion, and resolution of two inclusions, are enhanced when the time-gate width (▵t) is decreased. The overall computations confirm that fluorescent time-gating technique is very sensitive to local variations in optical properties that are due to breast-like tumors in turbid media.

We have applied an interstitial radiance-based technique based on a spectro-angular mapping approach to the identification and angular localization of 250-nm and 5-nm Au nanoparticle-based inclusions and non-scattering (water only) inclusions in the Intralipid-1% liquid phantom. A combination of the point radiance spectroscopy and white light spectroscopy was used to measure angular resolved light distribution in 450-900 nm spectral range in Intralipid-1% with and without localized inclusions. Characteristic spectro-angular snapshots of the liquid phantom alone and with the localized inclusions were obtained. For liquid phantoms without inclusions, the snapshots demonstrate wavelength dependent light distribution inside the turbid medium. For liquid phantoms with gold inclusions, the approach allows to isolate the spectroscopic signatures of the inclusions from the background, identify locations of the inclusions in the angular domain and show how a presence of water in the inclusion affects spectral identification and angular localization of the target. For liquid phantoms with water-based inclusions, an ability of the inclusion to enhance photon density above reference values and angular dependent signatures were demonstrated. The technique is seen as a potential tool in prostate treatment and diagnostics with gold nanoparticles.

The anomalous behavior of the expansion coefficient of water near 4 °C was studied in a thin photoacoustic cell. We show that the thermal expansion of water vanishes at temperatures lower than 3 ºC when the optical path is shortened below 0.1 mm. We explain this behavior in terms of less favorable hydrogen bonding near the surface of water, which becomes relevant when the fraction of molecules near the surface contributes appreciable to the observed photoacoustic signal. The photoacoustic spectra of water and oleic acid in a thin photoacoustic cell matches the optical spectra from 720 nm to 2200 nm. The high sensitivity of the front-face photoacoustic cell allows for spectroscopy of weakly absorbing media but surface effects may have to be taken into account.

In the recent past, there have been enormous efforts to understand effect of drugs on human body. Prior to understand the effect of drugs on human body most of the experiments are carried out on cells or model organisms. Here we present our study on the effect of chemotherapeutic drugs on cancer cells and the acetaminophen (APAP) induced hepatotoxicity in mouse model. Histone deacetylase inhibitors (HDIs) have attracted attention as potential drug molecules for the treatment of cancer. These are the chemotherapeutic drugs which have indirect mechanistic action against cancer cells via acting against histone deacetylases (HDAC). It has been known that different HDAC enzymes are over-expressed in various types of cancers for example; HDAC1 is over expressed in prostate, gastric and breast carcinomas. Therefore, in order to optimise chemotherapy, it is important to determine the efficacy of various classes of HDAC inhibitor drugs against variety of over-expressed HDAC enzymes. In the present study, FTIR microspectroscopy has been employed to predict the acetylation and propionylation brought in by HDIs. The liver plays an important role in cellular metabolism and is highly susceptible to drug toxicity. APAP which is an analgesic and antipyretic drug is extensively used for therapeutic purposes and has become the most common cause of acute liver failure (ALF). In the current study, we have focused to understand APAP induced hepatotoxicity using FTIR microspectroscopy. In the IR spectrum the bands corresponding to glycogen, ester group and were found to be suitable markers to predict liver injury at early time point (0.5hr) due to APAP both in tissue and serum in comparison to standard biochemical assays. Our studies show the potential of FTIR spectroscopy as a rapid, sensitive and non invasive detection technique for future clinical diagnosis.

Glioblastoma multiforme represents a highly lethal brain tumor. A tumor model has been developed based on the U-251 MG cell line from a human explant. The tumor model simulates different malignancies by controlled expression of the tumor suppressor proteins PTEN and TP53 within the cell lines derived from the wild type. The cells from each different malignant cell line are grown on slides, followed by a paraformaldehyde fixation. UV / VIS and IR spectra are recorded in the cell nuclei. For the differentiation of the cell lines a principal component analysis (PCA) is performed. The PCA demonstrates a good separation of the tumor model cell lines both with UV / VIS spectroscopy and with IR spectroscopy.

In this paper, the design of a metamaterial-based sensor, operating in the mid-infrared frequency range, is proposed. The sensor consists of a planar array of complementary circular inclusions. The resonant frequencies of the sensor are designed to coincide with the proteins and lipids spectral characteristics, in order to detect the presence of cancer tissues, by absorption measurements. This sensor can be also used for the recognition of different benign tumours in a highly accurate and sensitive way. A new analytical circuit model has been developed, useful to describe its resonant behavior. The sensing device is, then, optimized to obtain high selectivity performances and has been tested through proper full-wave simulations. The structure can be used as a biological sensor with possible applications in medical diagnostics.

In this work, a combination of Direct Laser Writing (DLW), PoliDiMethylSiloxane (PDMS) soft lithography and UV lithography was used to create cm- scale microstructured polymer scaolds for cell culture experiments out of dierent biocompatible materials: novel hybrid organic-inorganic SZ2080, PDMS elastomer, biodegradable PEG- DA-258 and SU-8. Rabbit muscle-derived stem cells were seeded on the fabricated dierent periodicity scaolds to evaluate if the relief surface had any eect on cell proliferation. An array of microlenses was fabricated using DLW out of SZ2080 and replicated in PDMS and PEG-DA-258, showing good potential applicability of the used techniques in many other elds like micro- and nano- uidics, photonics, and MicroElectroMechanical Systems (MEMS). The synergetic employment of three dierent fabrication techniques allowed to produce desired objects with low cost, high throughput and precision as well as use materials that are dicult to process by other means (PDMS and PEG-DA-258). DLW is a relatively slow fabrication method, since the object has to be written point-by-point. By applying PDMS soft lithography, we were enabled to replicate laser-fabricated scaolds for stem cell growth and micro-optical elements for lab-on-a-chip applications with high speed, low cost and good reproducible quality.

Photodynamic therapy (PDT) is an emerging technique for the treatment of cancerous and non-cancerous conditions. Gold nanoparticles (GNPs) possess unique physical and chemical properties which allow them to act as multifunctional agents in nanomedicine. GNP- photosensitizer conjugates have attracted increasing attention in drug delivery for photodynamic cancer therapy. In the present investigation, we prepared covalent conjugates of the photosensitizer Toluidine Blue O (TBO) and thiol protected GNPs. The suitability of TBO- GNPs conjugates for in vitro PDT was assayed using the SW480 Human colon adenocarcinoma cell line. Our results suggest that gold nanoparticle conjugates are an excellent vehicle for delivery of photosensitizer agents in the photodynamic therapy of cultured tumour cells.

In this paper we have studied effect of a hyperosmotic optical clearing agent (OCA), such as polyethylene glycol, on the fluorescence intensity from a target located in subcutaneous area in the model experiments. As a fluorescence agent the nanocomposite including gold nanorods with hematophorphyrin was used. The remitted fluorescent signal traveling to the tissue surface was monitored over time as the tissue was treated with the OCA. The detected fluorescent signal increased as the scattering in tissue samples was substantially reduced. The study has shown how OCA can be used to improve the detected signal at localization of subcutaneous target tissue at the photothermal or photodynamic therapy. Immersion clearing of skin can be also useful for improvement of laser exposure efficiency due to the increasing of light penetration depth.

Due to the great number of applications of Low-Level-Laser-Therapy (LLLT) in Central Nervous System (CNS), the study of light penetration through skull and distribution in the brain becomes extremely important. The aim is to analyze the possibility of precise illumination of deep regions of the rat brain, measure the penetration and distribution of red (λ = 660 nm) and Near Infra-Red (NIR) (λ = 808 nm) diode laser light and compare optical properties of brain structures. The head of the animal (Rattus Novergicus) was epilated and divided by a sagittal cut, 2.3 mm away from mid plane. This section of rat's head was illuminated with red and NIR lasers in points above three anatomical structures: hippocampus, cerebellum and frontal cortex. A high resolution camera, perpendicularly positioned, was used to obtain images of the brain structures. Profiles of scattered intensities in the laser direction were obtained from the images. There is a peak in the scattered light profile corresponding to the skin layer. The bone layer gives rise to a valley in the profile indicating low scattering coefficient, or frontal scattering. Another peak in the region related to the brain is an indication of high scattering coefficient (μs) for this tissue. This work corroborates the use of transcranial LLLT in studies with rats which are subjected to models of CNS diseases. The outcomes of this study point to the possibility of transcranial LLLT in humans for a large number of diseases.

Tissue-equivalent phantom is becoming widespread as a substitute in the biological field to verify optical theories, test measuring systems and study the tissue performances for varying boundary conditions, sample size and shape at a quantitative level. Compared with phantoms made with Intralipid solution, ink and other liquid substances, phantom in solid state is stable over time, reproducible, easy to handle and has been testified to be a suitable optical simulator in the visible and near-infrared region. We present accurate determination of the complex refractive index (RI) of a solid tissueequivalent phantom using extended derivative total reflection method (EDTRM). Scattering phantoms in solid state were measured for p-polarized and s-polarized incident light respectively. The reflectance curves of the sample as a function of incident angle were recorded. The real part of RI is directly determined by derivative of the reflectance curve, and the imaginary part is obtained from nonlinear fitting based on the Fresnel equation and Nelder-Mead simplex method. The EDTRM method is applicable for RI measurement of high scattering media such as biotissue, solid tissue-equivalent phantom and bulk material. The obtained RI information can be used in the study of tissue optics and biomedical field.

Understanding the behaviour of light propagation in biological materials is essential for biomedical engineering and its applications. Among the key optical properties of biological media is the angular distribution of the scattered light, characterized by the average cosine of the scattering angle, called the scattering anisotropy coefficient (g). The value of g can be determined by experimentally irradiating the material with a laser beam and making angular-scattering measurements in a goniometer. In this work, an experimental technique was used to determine g by means of goniometric measurements of the laser light scattered off two different dental-resin composites (classified as nano and hybrid). To assess the accuracy of the experimental method, a Mie theory-based computational model was used. Independent measurements were used to determine some of the required input parameters for computation of the theoretical model. The g values estimated with the computational method (nano-filled: 0.9399; hybrid: 0.8975) and the values calculated with the experimental method presented (nano-filled: 0.98297 ± 0.00021; hybrid: 0.95429 ± 0.00014) agreed well for both dental resins, with slightly higher experimental values. The higher experimental values may indicate that the scattering particle causes more narrow-angle scattering than does a perfect sphere of equal volume, assuming that with more spherical scattering particles the scattering anisotropy coefficient increases. Since g represents the angular distribution of the scattered light, values provided by both the experimental and the computational methods show a strongly forward-directed scattering in the dental resins studied, more pronounced in the nano-filled composite than in the hybrid composite.

Knowledge of the optical properties of biological structures is useful for clinical applications, especially when dealing with incoming biomaterials engineered to improve the benefits for the patient. One ceramic material currently used in restorative dentistry is yttrium cation-doped tetragonal zirconia polycrystal (3Y-TZP) because of its good mechanical properties. However, its optical properties have not been thoroughly studied. Many methods for the determination of optical parameters from biological media make the assumption that scattered light is isotropically distributed over all angles. Nevertheless, real biological materials may have an angular dependence on light scattering, which may affect the optical behaviour of the materials. Therefore, the recovery of the degree of anisotropy in the scattering angular distribution is important. The phase function that represents the scattering angular distribution is usually characterized by the anisotropy coefficient g, which equals the average cosine of the scattering angle. In this work, we measured angularscattering distributions for two zirconia ceramic samples, pre-sintered and sintered, with similar thicknesses (0.48 mm and 0.50 mm, respectively) and also for a human dentine sample (0.41 mm in thickness). The samples were irradiated with a He-Ne laser beam (λ = 632.8 nm) and the angular-scattering distributions were measured using a rotating goniometer. The g values yielded were: -0.7970 ± 0.0016 for pre-sintered zirconia, -0.2074 ± 0.0024 for sintered zirconia and 0.0620 ± 0.0010 for dentine. The results show that zirconia sintering results in optical behaviour more similar to those of dentine tissue, in terms of scattering anisotropy.

The laser Doppler flowmetry allows the non-invasive assessment of the skin perfusion in real-time, being an attractive technique to study the human microcirculation in clinical settings. Low-frequency oscillations in the laser Doppler blood flow signal from the skin have been related to the endothelial, endothelial-metabolic, neurogenic and myogenic mechanisms of microvascular flow control, in the range 0.005-0.0095 Hz, 0.0095-0.021 Hz, 0.021-0.052 Hz and 0.052- 0.145 Hz respectively. The mean Amplitude (A) of the periodic fluctuations in the laser Doppler blood flow signal, in each frequency range, derived from the respective wavelet-transformed coefficients, has been used to assess the function and dysfunctions of each mechanism of flow control. Known sources of flow signal variances include spatial and temporal variability, diminishing the discriminatory capability of the technique. Here a new time domain method of analysis is proposed, based on the Time of Correlation (TC) of flow fluctuations between two adjacent sites. Registers of blood flow from two adjacent regions, for skin temperature at 32 ⁰C (basal) and thermally stimulated (42 ⁰C) of volar forearms from 20 healthy volunteers were collected and analyzed. The results obtained revealed high time of correlation between two adjacent regions when thermally stimulated, for signals in the endothelial, endothelial-metabolic, neurogenic and myogenic frequency ranges. Experimental data also indicate lower variability for TC when compared to A, when thermally stimulated, suggesting a new promising parameter for assessment of the microvascular flow control.

Micro-Raman technique can be particularly useful to investigate the chemical changes induced in structure, protein, nucleic acid, lipid, and carbohydrate contents of cells. The aim of this work is to inspect the possibility to employ Raman microspectroscopy to detect biochemical modifications in human mammary epithelial cells after exposure to different Xray doses. The samples consisted of cells cultured on polylysine-coated glass coverslips. After the exposition, control and treated cells were washed in phosphate-buffered saline (PBS) and then fixed in paraformaldehyde 3.7%. They were examined using a confocal micro-Raman system equipped with a He-Ne laser (λ = 632.8 nm; power on the sample= 3.5mW). Differences in the intensity ratio of specific Raman vibrational markers commonly assigned to phenylalanine and tyrosine amino acids (at 1000, 1030, 1618 cm^-1), DNA bases (787, 1090, 1305 cm^-1), and amide III (1237, and 1265 cm^-1) with respect a reference peak (the one of lipids at 1450 cm^-1) were evidenced between control and exposed cells. These differences may be indicative of damage in exposed cells as the fragmentation of individual amino acids and DNA bases, crosslink effects in molecular structure of DNA and protein conformational change that especially tend to "unwind" the protein due to the breaking of hydrogen bonds between peptide chains.

We report the development of the Three-dimensional Real-time Uninvasive Imaging and Evaluation (TRUImagE) system based on digital holographic microscopy to study the morphological changes in cells undergoing photodynamic therapyinduced cell death. The optical system, based on the Michelson interferometer and configured in transmission mode, and the sample holder incorporating a stage incubator have been developed for monitoring various tumorigenic cell samples without the use of markers. Off-axis digital holograms were recorded with a CCD sensor and numerically reconstructed to provide quantitative phase imaging and 3D morphology of the cells in real time. The system was used to continuously monitor and study, at different time points, the changes in cells after incubation with the photosensitizer followed by activation by the appropriate light dose. Results obtained from the TRUImagE system and biochemical assays will be given.

Nonlinear optical imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or second-harmonic generation (SHG) show great potential for in-vivo investigations of tissue. While the microspectroscopic imaging tools are established, automized data evaluation, i.e. image pattern recognition and automized image classification, of nonlinear optical images still bares great possibilities for future developments towards an objective clinical diagnosis. This contribution details the capability of nonlinear microscopy for both 3D visualization of human tissues and automated discrimination between healthy and diseased patterns using ex-vivo human skin samples. By means of CARS image alignment we show how to obtain a quasi-3D model of a skin biopsy, which allows us to trace the tissue structure in different projections. Furthermore, the potential of automated pattern and organization recognition to distinguish between healthy and keloidal skin tissue is discussed. A first classification algorithm employs the intrinsic geometrical features of collagen, which can be efficiently visualized by SHG microscopy. The shape of the collagen pattern allows conclusions about the physiological state of the skin, as the typical wavy collagen structure of healthy skin is disturbed e.g. in keloid formation. Based on the different collagen patterns a quantitative score characterizing the collagen waviness - and hence reflecting the physiological state of the tissue - is obtained. Further, two additional scoring methods for collagen organization, respectively based on a statistical analysis of the mutual organization of fibers and on FFT, are presented.

Analysis of breath and gases emanated from skin can be used for early and non-invasive diagnosis of various kinds of diseases. Two portable, compact, photoacoustic spectroscopy based trace gas sensors were developed for the detection of methane emanated from skin and ammonia emanated from oral cavity. The light sources were distributed feedback diode lasers emitting at the absorption lines of ammonia and methane, at 1.53 μm and 1.65 μm, respectively. Photoacoustic method ensures high selectivity, therefore cross-sensitivity was negligible even with large amount of water vapor and carbon dioxide in the gas sample. In case of ammonia a preconcentration unit was used to achieve lower minimum detectable concentration. Gas sample from the oral cavity was drawn through a glass tube to the preconcentration unit that chemically bonded ammonia and released it when heated. The minimum detectable concentration of ammonia was 10 ppb for 15 s gas sampling time (gas sample of 250 cm³). For methane minimum detectable concentration of 0.25 ppm was found with 12 s integration time, and it was proved to be adequate for the detection of methane emanated from human skin and from mice. Instruments measuring methane and ammonia are currently installed at two medical research laboratories at University of Szeged and tested as instruments for non-invasive clinical trials. The aim of the measurements is to determine correlations between diseases or metabolic processes and emanated gases.

A simple, dual wavelength, multiple-angle, light scattering system has been developed for detecting cryptosporidium suspended in water. Cryptosporidium is a coccidial protozoan parasite causing cryptosporidiosis; a diarrheal disease of varying severity. The parasite is transmitted by ingestion of contaminated water, particularly drinking-water, but also accidental ingestion of bathing-water, including swimming pools. It is therefore important to be able to detect these parasites quickly, so that remedial action can be taken to reduce the risk of infection. The proposed system combines multiple-angle scattering detection of a single and two wavelengths, to collect relative wavelength angle-resolved scattering phase functions from tested suspension, and multivariate data analysis techniques to obtain characterizing information of samples under investigation. The system was designed to be simple, portable and inexpensive. It employs two diode lasers (violet InGaN-based and red AlGaInP-based) as light sources and silicon photodiodes as detectors and optical components, all of which are readily available. The measured scattering patterns using the dual wavelength system showed that the relative wavelength angle-resolved scattering pattern of cryptosporidium oocysts was significantly different from other particles (e.g. polystyrene latex sphere, E.coli). The single wavelength set up was applied for cryptosporidium oocysts'size and relative refractive index measurement and differential measurement of the concentration of cryptosporidium oocysts suspended in water and mixed polystyrene latex sphere suspension. The measurement results showed good agreement with the control reference values. These results indicate that the proposed method could potentially be applied to online detection in a water quality control system.

Laser based techniques offer non invasive means of imaging and optical signal acquisition in the biomedical field. Laser Doppler flowmetry and laser speckle imaging are important laser based methods in current research and have been explored for the analysis of blood flow. Doppler flow meters as well as laser speckle imagers use tissue backscattered light to non-invasively assess the blood flow rate. While because of large spatial variability and the temporal heterogeneity in tissue microvasculature, the measured blood flow rate is expressed in relative units in laser Doppler, laser speckle methods offers a whole field imaging resulting in absolute measurements of flow velocity. Measurement of and analysis of blood flow is vital in evaluating normal as well as differently diseased conditions of the human body. Several parameters related to flow along with flow velocity are important in characterizing tissues based on blood flow. Complexity of the flow is one of such important parameters which could be explored by looking at the fractality of the acquired Doppler signals / speckle images. In this paper, we are comparing the results of blood flow complexities assessed through fractal dimensions of Doppler signals and speckle images acquired from different parts of the body. The method adopted is expected to serve as a helping tool in characterizing normal and malignant tissues with associated variation in blood flow complexities based on the values of obtained fractal dimensions in such cases.

The hand-foot-syndrome presents a severe dermal side-effect of chemotherapeutic cancer treatment. The cause of this side-effect is the elimination of systemically administered chemotherapeutics with the sweat. Transported to the skin surface, the drugs subsequently penetrate into the skin in the manner of topically applied substances. Upon accumulation of the chemotherapeutics in the skin the drugs destroy cells and tissue - in the same way as they are supposed to act in cancer cells. Aiming at the development of strategies to illuminate the molecular mechanism underlying the handfoot- syndrome (and, in a second step, strategies to prevent this severe side-effect), it might be important to evaluate the concentration and distribution of chemotherapeutics and antioxidants in the human skin. The latter can be estimated by the carotenoid concentration, as carotenoids serve as marker substances for the dermal antioxidative status.Following the objectives outlined above, this contribution presents a spectroscopic study aiming at the detection and quantification of carotenoids and selected chemotherapeutics in human skin. To this end, spontaneous Raman scattering and coherent anti-Stokes Raman scattering (CARS) microspectroscopy are combined with two-photon excited fluorescence. While the latter technique is Please verify that (1) all pages are present, (2) all figures are correct, (3) all fonts and special characters are correct, and (4) all text and figures fit within the red margin lines shown on this review document. Complete formatting information is available at http://SPIE.org/manuscripts Return to your MySPIE To Do List at http://myspie.org and approve or disapprove this submission. Your manuscript will not be published without this approval.restricted to the detection of fluorescent chemotherapeutics, e.g., doxorubicin, the vibrational spectroscopic techniques can - in principle - be applied to any type of analyte molecules. Furthermore, we will present the monitoring of doxorubicin uptake during experiments.

In this work an application of gold nanoparticles in in-vitro photothermal cancer cell therapy is demonstrated. Gold nanoparticles with different diameters - 40, 100 and 200 nm are mixed with HeLa cancer cells. After incubation, the nanoparticles are found to be deposited on the cell's membrane or enter into the cells. Pulsed laser radiation at wavelength of 532 nm delivered by Nd:YAG system is used to irradiate the samples. The experiments are performed at fluences in the range from 50 mJ/cm² up to the established safety standard for medical lasers of 100 mJ/cm². The cell viability as a function of the particle dimensions and laser fluence is estimated. The nanoparticles heating and cooling dynamics is traced by a numerical model based on heat diffusion equation combined with Mie theory for calculation of the optical properties of nanoparticles. The particle response to the nanosecond laser heating is investigated experimentally as gold colloids are irradiated at different fluences. The threshold fluences for particle's melting and boiling are defined. We show that at the presented fluence range the particles are decomposed into smaller fragments and even short irradiation time leads to decrease of cell viability.

The development of highly specific markers for fluorescent microscopy has become a very popular research topic. Organic fluorophores have several drawbacks, such as photobleaching and autofluorescence. Therefore increasing interest in inorganic nanoparticles has been observed because of their unseen photostability, chemical robustness and straightforward synthesis. The surface of iron oxide nanoparticles was coated with trialkoxy silanes, which introduced functional groups for possible subsequent coupling reactions. An additional gold layer was added to the surface of the particle to show the enhanced contrast improvement. The nanoparticles were imaged by an optical microscope, in dark field mode, on a glass substrate and inside microorganisms. This proved that the reported method could have great potential as a labelling technique, since it combines the non-photobleaching, photostable nanoparticles with a straightforward and rapid imaging technique.

High rate, full field image acquisition in multiphoton imaging is achievable by parallelization of the excitation and of the detection paths. Via a Diffractive Optical Elements (DOEs) which splits a pulsed laser, and a spatial resolved descanned detection path, a new approach to microscopy has been developed. By exploiting the three operating mode, single beam, 16 beamlets or 64 beamlets, the best experimental conditions can be found by adapting the power per beamlet. This Multiphoton Multispot system (MCube) has been characterized in thick tissue samples, and subsequently used for the first time for Ca²⁺ imaging of acute heart slices. A test sample with fixed mice heart slices with embedded sub-resolution fluorescent beads has been used to test the capability of optical axial resolution up to ~200 microns in depth. Radial and axial resolutions of 0.6 microns and 3 microns have been respectively obtained with a 40X water immersion objective, getting close to the theoretical limit. Then images of heart slices cardiomyocites, loaded with Fluo4-AM have been acquired. The formation of Ca²⁺ waves during electrostimulated beating has been observed, and the possibility of easily acquire full frame images at 15 Hz (16 beamlets) has been demonstrated, towards the in vivo study of time resolved cellular dynamics and arrhythmia trigger mechanisms in particular. A very high speed two-photon Random Access system for in vivo electrophysiological studies, towards the correlation of voltage and calcium signals in arrhythmia phenomena, is now under developing at Light4tech.

We report the development and performance of high resolution spectral domain optical coherence tomography (SD-OCT) system based on 2×2 planar lightwave circuit (PLC) splitter that was designed as a single mode splitter at near infrared and used as the beam splitter for a SD-OCT system. The splitter has been made by coupling SMFs to a planar lightwave circuit (PLC) splitter chip. The PLC splitter chip was fabricated to have a single mode property with 740 nm cutoff wavelength and the SMFs, which have 730 nm cutoff wavelength, were securely connected to the PLC chip through fiber block arrays having lithographically fabricated V grooves. With the implemented PLC splitter, we have obtained a low excess loss of 0.4 dB at 840 nm with wide band coupling property. With the proposed 2×2 PLC splitter and fabricated WDM coupler, SD-OCT images of samples successfully obtained by using combined source with 840 nm and 880 nm SLDs.

In this study, a tuning fork-fiber probe system for a near-field scanning optical microscope (NSOM probe), whose fiber tip was partially dipped into liquid and vibration behavior was mechanically controlled with two nodal-wedges method, was modeled by bending vibration equations, and its resonant characteristics were numerically investigated by solving the system of equations. For the two nodal-wedges method, pin point and knife-edge balance point were newly introduced into a typical NSOM probe, and their contact positions were adjusted along the axis of the fiber probe in order to invoke versatile vibrational modes. From the numerical analysis, it was shown that the resonant frequency and the Q value of the NSOM probe were changed periodically with shifting the contact position of either the knife-edge balance point or the pin point, and that the Q value of the NSOM probe could be controlled by adjusting their contact positions as in air. Additionally, it was found that the Q value was gradually decreased with increasing the dipping depth of the fiber tip and that the Q value could be greatly enhanced at the optimal contact positions of two nodal points.

Optical waveguide biosensors are attracting more and more attentions and presenting great potential applications. Polymer-based optical biosensors are promising for the their unique advantages: low cost, easy fabrication, possibility of functionalization with chemicals for the detection of biological molecules, and flexible operating wavelength in both the infrared communication wavelength band (1310-1550nm) and the visible wavelength region (500-800nm). Operating in the visible wavelength, the optical biosensing can avoid the high optical absorption loss of water solution, which can hardly be done for Si-based optical sensors. In this paper, an optical biosensor utilizing polymer-based athermal optical waveguide microring resonator is presented. The athermal design of the microring resonator can make the resonant wavelength drift with temperature be greatly reduced, and an optical biosensing platform with high thermal stability can be achieved. The simulation results show that the maximal resonant wavelength drift is -0.0085nm when the temperature varies from 20°C to 65°C and the maximal wavelength drift slope is -0.0009nm/K. With the microring resonators fabricated by using a simple UV based soft imprint technique with self-developed UV-curable polymer PSQ-L materials, experimental investigations on the specific surface detection of target molecules have been preliminarily performed. The results shows that the optical biosensors based on the polymer optical microring resonators would have potential applications for label-free surface sensing.

A clinical trial involving multi-spectral imaging of histologically confirmed 8 basaliomas and 30 melanomas was performed. Parametric maps of the melanin index, erythema index and melanoma-nevus differentiation parameter have been constructed and mutually compared. Specific features of basalioma and melanoma images were analyzed and discussed.

A portable multi-spectral photoplethysmography device has been used for studies of 40 subjects. Multi-spectral monitoring was performed by means of a four - wavelengths (465 nm, 530 nm, 630 nm and 870 nm) light emitted diodes (LED) and a single photodiode with multi-channel signal output processing. The proposed methodology and potential clinical applications are discussed.

Two-photon fluorescence (TPEF) microscopy is a powerful tool to image human tissues up to 200 microns depth without any exogenously added probe. TPEF can take advantage of the autofluorescence of molecules intrinsically contained in a biological tissue, as such NADH, elastin, collagen, and flavins. Two-photon microscopy has been already successfully used to image several types of tissues, including skin, muscles, tendons, bladder. Nevertheless, its usefulness in imaging colon tissue has not been deeply investigated yet. In this work we have used combined two-photon excited fluorescence (TPEF), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two-photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of colon. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa, polyp, and colon samples in a good agreement with common routine histology. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or colonoscope to be used in in-vivo imaging applications.

A prototype RGB imaging system for mapping of skin chromophores consists of a commercial RGB CMOS sensor, RGB LEDs ring-light illuminator and orthogonally orientated polarizers for reducing specular reflectance. The system was used for monitoring of vascular malformations (hemagiomas and telangiectasias) therapy.

We propose full-range spectral domain optical coherence tomography equipped with a fiber-based sample scanner, which is used for sample scanning and phase shifting for full-range image at the same time. For a fiber-based sample scanner, since the fiber tip oscillates as a free standing cantilever in general, unintentional phase shift occurs inevitably. The unintentional phase shift was used for eliminating the bothersome complex conjugate ghost image of OCT. In addition, fiber was tilted a few degree to give proper phase shift. In this scheme, moreover, image can be obtained without any physical modification of the scanner. To realize this technique, we constructed the SD-OCT system and fabricated a magnetically actuated single-body lensed fiber scanner due to advantages of simple design, low operating voltage, cost-effectiveness and low insertion loss. The scanner was made of lensed fiber loaded with an iron-based bead and a solenoid which is placed perpendicular to the lensed fiber. When a sinusoidal current is applied into the solenoid, the lensed fiber oscillated due to magnetic force between the iron-based bead and the solenoid. With the suggested full range method, we obtained contrast enhanced full-range SD OCT images of pearl and tooth. This simple and effective method can be applied to any fiber-based scanner and it has great potential as a handheld probe/endoscopic probe in biomedical imaging field.

The waveguide type biosensors for noninvasive glucose detection based on LSPR of silver nanoparticles were fabricated by thermal diffusion in UV-irradiated photo-thermo-refractive (PTR) glasses and by ion-exchange method in sodiumborosilicate glasses in water vapor atmosphere. The optical and structural properties of the obtained nanocomposites were investigated. The D-glucose/D-galactose binding protein (GGBP) was chosen as a sensitive element of biosensor and successable immobilized on top of PTR glass. The change in absorption spectra were judged due to the presence of GGBP on the substrate surface.

Accurate knowledge of the optical properties of turbid media in the light path is important in NIR absorption spectroscopy of biological tissues where multiple scattering complexes the collected light signals due to the non-uniform tissue architecture. Several approaches such as time resolved spectroscopy and spatially resolved spectroscopy have been proposed to measure the bulk optical properties of turbid media. Among them, double integrating sphere (DIS) measurements are recognized as the "golden standard" for in vitro optical properties measurement of turbid media because of its high accuracy and robustness in different conditions. A DIS system is convenient to measure the in vitro optical properties of turbid media like intralipid solutions and biological tissues, since it measures the diffuse reflectance and transmittance simultaneously. However, DIS measurements have been mostly limited to the optical window region (400-1000 nm) or suffered from low signal levels on the detectors due to the absorption by water in the NIR region. In this study, we developed a DIS system for optical property measurement in the 1300-2350 nm region based on a novel wavelength tunable spectroscopic setup which incorporates a high power broadband supercontinuum laser and a high precision monochromator. With this system, optical properties of intralipid solutions were measured in the wavelength region of 1300-2350nm.

Transgenic animals are an essential means for investigating genetic processes in vivo, and depend on efficient delivery techniques to introduce exogenous genetic material into the organism, often at the zygote stage. In this study, we demonstrate an optical approach to microinjection based on a holographic system using a spatial light modulator and a Ti: Sapphire laser. This integrated system is capable of both optical orientation and injection of 60- μm diameter Pomatoceros lamarckii (P.lamarckii) embryos. Individual blastomeres of P. lamarckii embryos were optoinjected with varying sizes of dextran molecules and Propidium iodide using an 800-nm femtosecond laser with controlled dosage. We also show that the technique is able to deliver materials to cells located deep within a welldeveloped embryo. As a visual confirmation of successful optoinjection, the presence of gas bubbles was observed as a function of laser power and exposure time. Small gas bubbles, less than 5-μm in diameter, were found to be tolerated by the irradiated embryo. Furthermore, when switched to the continuous wave mode, the laser could exert optical forces upon the embryo. This facilitated computer-controlled handling and orientation of P. lamarckii embryos without compromising viability. Our multimodal optical platform offers a sterile, non-contact and robust alternative to traditional microinjection. This work is a step towards applications in developmental biology such as cell lineage mapping and formation of transgenic animals using an optical approach.

Retinal image quality is usually analysed through different parameters typical from instrumental optics, i.e, PSF, MTF and wavefront aberrations. Although these parameters are important, they are hard to translate to visual quality parameters since human vision exhibits some tolerance to certain aberrations. This is particularly important in postsurgery eyes, where non-common aberration are induced and their effects on the final image quality is not clear. Natural images usually show a strong dependency between one point and its neighbourhood. This fact helps to the image interpretation and should be considered when determining the final image quality. The aim of this work is to propose an objective index which allows comparing natural images on the retina and, from them, to obtain relevant information abut the visual quality of a particular subject. To this end, we propose a individual eye modelling. The morphological data of the subject's eye are considered and the light propagation through the ocular media is calculated by means of a Fourier-transform-based method. The retinal PSF so obtained is convolved with the natural scene under consideration and the obtained image is compared with the ideal one by using the structural similarity index. The technique is applied on 2 eyes with a multifocal corneal profile (PresbyLasik) and can be used to determine the real extension of the achieved pseudoaccomodation.

Wireless PPG devices were developed and embedded in everyday clothes (bandage, scarf, cycling glove and wrist strap) to monitor cardiovascular state of free-moving persons. The corresponding software for measurements also has been developed and tested in laboratory. Real-time measurements of PPG signals were taken in parallel with a professional ECG reference device, and high correlation was demonstrated.

Influence of low power laser irradiance on healthy skin using diffuse reflectance spectroscopy and multispectral imaging was studied. Changes of diffuse reflectance spectra in spectral range from 500 to 600 nm were observed after 405 nm, 473 nm and 532 nm laser provocation, leading to conclusion that the content of skin hemoglobin has changed. Peaks in spectral absorbance (optical density) curves corresponded to well-known oxy-hemoglobin absorbance peaks at 542 and 577 nm.

This paper is intended to demonstrate a facile and effective method to construct single layer graphene films onto the self-assembled monolayer (SAM) at Au electrodes based surface plasmon resonance (SPR) biochips integrated loop-mediated isothermal amplification (LAMP) for tuberculosis bacillus (TB) detection. It is a novel Au-SAM-graphene nanocomposites and taking advantages of the striking properties of both graphene and Au film, fundamental understanding in hybrid material manipulation and new electrochemical properties can be obtained. The sensitivity of TB detection in the LAMP-based assay for the amplification of the Insertion Sequence 6110 (IS6110) samples was determined by a single-layer graphene/Au thin film and compared with that of a conventional Au/Cr-based SPR chips. The results show that a graphene/Au SPR offers a potentially powerful assay, with a highly sensitive analysis, that may be applicable as an important tool for bio-marker detection.

There are a number of techniques for body composition assessment in clinics and in field-surveys, but in all cases the applied methods have advantages and disadvantages. High precision imaging methods are available, though expensive and non-portable, however, the methods devised for the mass population, often suffer from the lack of precision. Therefore, the development of a safe, mobile, non-invasive, optical method that would be easy to perform, precise and low-cost, but also would offer an accurate assessment of subcutaneous adipose tissue (SAT) both in lean and in obese persons is required. Thereof, the diffuse optical spectroscopy is advantageous over the aforementioned techniques. A prototype device using an optical method for measurement of the SAT thickness in vivo has been developed. The probe contained multiple LEDs (660nm) distributed at various distances from the photo-detector which allow different light penetration depths into the subcutaneous tissue. The differences of the reflected light intensities were used to create a non-linear model, and the computed values were compared with the corresponding thicknesses of SAT, assessed by B-mode ultrasonography. The results show that with the optical system used in this study, accurate results of different SAT thicknesses can be obtained, and imply a further potential for development of multispectral optical system to observe changes of SAT thickness as well as to determine the percentage of total body fat.

The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm^-1 resolution, 30 scans, in the 1800-900 cm^-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm^-1 (amine I) and 1255 cm^-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

We present a new algorithm to process captured images of reflected Placido rings. Up to our knowledge, conventional topographers transform from Cartesian to polar coordinates and vice-versa, thus extrapolating corneal data and introducing noise and image artefacts. Moreover, captured data are processed by the device according to proprietary algorithms and offering a final map of corneal curvature. Corneal topography images consists of concentric rings of approximately elliptical shape. Our proposal consists of considering the information that provides each separate ring. A snake-annealing-like method permits identifying the ring even with discontinuities due to eye-lashes and reflections. By analysing the geometrical parameters of rings (centre, semi-axis and orientation), one can obtain information about small morphological micro-fluctuations and local astigmatisms. These parameters can be obtained with sub-pixel accuracy so the method results of high precision. The method can be easily adapted to work on any topographer, so that it can provide additional information about the cornea at no additional cost.

The rapidly growing application of nanotechnology in medicine has placed new demands on monitoring the clearance rate of various nanoparticles with different shapes, compositions, and conjugations within blood circulation. No clinically relevant method has been developed for rapid and sensitive detection of nanoparticles in blood flow. Our laboratory has developed a laser-based platform with the purpose of in vivo real-time monitoring of nanoparticles using a highly advanced high-speed, multicolor photoacoustic flow cytometry (PAFC). As most nanoparticles have intrinsic absorption, PAFC is an ideal tool for real-time, label-free monitoring of nanoparticle pharmacokinetics. We used four laser wavelengths to verify the concept of in vivo multicolor PAFC, and hypothesize that the potential exists to increase the number of spectral channels. The capability of this platform was demonstrated for detection of magnetic nanobeads and gold nanorods of different sizes and conjugations in blood circulation of animal models. The advantages as well as potential limitations of the new technique are discussed in detail.

Conventional dental lasers have not realized a selective excavation of carious dentin. Objective of this study is to determine the optimal irradiation condition for the selective excavation by using a wavelength around 5.8 μm. A nanosecond pulsed laser with a wavelength of 5.8 μm was obtained by difference-frequency generation technique. The laser delivers 5 ns pulse width at a repetition rate of 10 Hz. 5.8 μm wavelength range, a short wavelength required high excavation energy and a long wavelength required low excavation energy to induce the selective excavation with a low thermal side effect. 5.8 μm wavelength provides a selective excavation technique for minimal intervention.

The Gram-negative, oral bacterium Aggregatibacter actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. The new periodontal disease treatments are emergence in order to prevent infection progression. Antimicrobial photodynamic therapy (a-PDT) can be a useful tool for this purpose. It involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent in the presence of oxygen for eradication of target cells, and appears effective in photoinactivation of microorganisms. The phthalocyanine metal complexes of Pd(II)- (PdPcC) and Al(III)- (AlPc1) were evaluated as photodynamic sensitizers towards a dental pathogen A. actinomycetemcomitans in comparison to the known methylpyridyloxy-substituted Zn(II) phthalocyanine (ZnPcMe). The planktonic and biofilm-cultivated species of A. actinomycetemcomitans were treated. The photophysical results showed intensive and far-red absorbance with high tendency of aggregation for Pd(II)-phthalocyanine. The dark toxicities of both photosensitizers were negligible at concentrations used (< 0.5 log decrease of viable cells). The photodynamic response for planktonic cultured bacteria was full photoinactivation after a-PDT with ZnPcMe. In case of the newly studied complexes, the effect was lower for PdPcC (4 log) as well as for AlPc1 (1.5-2 log). As it is known the bacterial biofilms were more resistant to a-PDT, which was confirmed for A. actinomycetemcomitans biofilms with 3 log reductions of viable cells after treatment with ZnPcMe and approximately 1 log reduction of biofilms after PdPcC and AlPc1. The initial results suggest that a-PDT can be useful for effective inactivation of dental pathogen A. actinomycetemcomitans.

Aim of the study. Assessment by stereomicroscopy of the severity of lesions in osteoporotic bone at both sexes and to correlate micro-and macro-bone fracture due to low bone density values with the disease evolution. Material and method: The study material consists of fragments of bone from the femoral head, vertebral bone, costal and iliac crest biopsy obtained from patients aged over 70 years, female and male, treated in the County Hospital of Timisoara, Department of Orthopedics. For the purpose of studying the samples in stereomicroscopy and trough polarized light it has been used the Olympus Microscope SZ ×7 and an Olympus camera with 2,5 × digital zoom and a 3× optical zoom in the Vest Politechnic Univesity. Results and discussions: Subchondral bone presents osteolysis associated with a osteoporotic bone transformation. Pseudocystic chondrolisis was noted in the osteoarticular cartilage, in addition with areas of hemorrhagic postfractural necrosis. The osteoporotic bone exhibits ischemic necrosis and focal hemorrhagic necrosis adjacent fracture. Microporosity pattern of the bone observed by stereomicroscopy correspond to the spongy bone osteoporosis images. Morphometry of the bone spiculi reveals length of 154.88 and 498.32 μ. In men we found a greater thickness of bone trabeculi compared with bone texture porosity in women. The subchondral bone supports and fulfills an important role in transmitting forces from the overlying articular cartilage inducing the bone resorbtion. The femoral head fracture may be the final event of many accumulated bone microcracks. Conclusions: Bone fragility depends not only of the spongy bone but also of the cortical bone properties. Osteolysis produced by loss of balance in the process of remodeling in favor of bone resorption leads to the thinning of the subchondral bone at both sexes.

Digital Holographic Microscopy (DHM) is a potentially non-invasive new technology which can be applied in many areas from applied imaging science to biomedical optics. DHM is an interferometric technique that gives us a number of important advantages such as the possibility to acquire holograms at high speed, to obtain complete information about amplitude and phase and to use image processing techniques. In this sense, DHM offers rapid 3D imaging with a theoretically higher resolution than OCT (Optical Coherent Tomography). By this technique optical path measurements with sensitivities in the nanometer range of reflective and transparent objects can be obtained. In this work, we use DHM to study the effect of ablation using 4.5 nJ pulses on chicken corneas. For this, a titanium sapphire laser at 800 nm and 76 MHz frequency (Vitesse, Coherent Inc. USA) was focused to its diffraction-limited spot size by a 10x objective of 0.3 numerical aperture. The width of the pulse (170 fs) at the sample was measured by spectral techniques. The average beam power at the sample was 340 mW and all the system was mechanically driven by a XY synchronization unit that controls the speed of the sample movement. The speed of the sample was varied between 1-50 μm/s. The studied chicken corneal tissue was previously processed by Trypan dye in order to visualize the irradiated area. The photodisrupted zone was analyzed by a HDM technique by illuminating it using a laser diode source (λ=683 nm) linearly polarized in a modified Mach-Zehnder with an off-axis geometry configuration. The reflected object wave by the tissue surface (specimen) interferes with the reference wave and a CCD camera records the hologram. As a result, the influence of the speed of photodisruption in the depth of the ablated corneas was analyzed. Therefore, it is possible to analyze thermal and photoirradiated effects on corneal tissues which allow us the possibility to optimize the interaction of intratissue and the intratissue target region of interest.

Objectives: Stereomicroscopy allows a three-dimensional study of the images and of laterality at superior quality in comparison with other methods. Those advantages are given by the large examination fields and the wide work distances. The adding of the clinical and morphological data at the results gathered with stereomicroscopy and the stereo micrometry is useful in order to appreciate the deepness and the widening of the carious process, and the necessity to reconsider the therapeutically strategy. Materials and methods: During 2009-2011 the study material was represented by 10 surgically removed impacted third molars, and by 20 premolars extracted for orthodontic purposes, with closed and macroscopically apparently integer surfaces. 13 premolars with different degrees of carious affectation and periodontal lesions, which were surgically extracted without trauma, were also selected. The in situ measurements at the occlusal site were realized through the utilization of a fluorescent laser device - DIAGNOdent. The basic principles in stereomicroscopy stood at the base of the obliquely and circularly coaxial illumination techniques, one with optical alignment adjustment of the optical microscope and mechanical adjustment for the optimal illumination and micrometry. The Olympus Microscope SZ ×7 and an Olympus camera with 2,5 × digital zoom and a 3× optical zoom has been used to study the samples in stereomicroscopy and through polarized light it. Results: The DiagnoDent measured the following data: out of 43 apparently healthy teeth, 18 presented values between 2 and 13 (D1), 13 showed values between 14 and 24 (D2), 12 measured values over 24 (D3). After the histological examination in stereomicroscopy and in the polarized light: 25 teeth were healthy, 10 presented caries extended in dental enamel and 8 presented dentinal caries. Stereomicroscopy has allowed the morphological study, the color absorption, the appreciation of the lesions' deepness and substance loss that is very useful in grading the progression of the carious lesion. Conclusions: The stereomicroscopic study correlated with clinical and morphological data allowed to appreciate the extent of tissue involved in the carious process, but also the understanding of the enamel, dentine and cement matrix demineralization process, in proximity with the morpho-embryological markings of the human tooth structure.

The aim of this study was to examine cellular and matriceal dynamics within pulp tissue of the teeth with repeated bleaching. Material and method - The study was made on 25 patients aged between 15 and 45, to whom bleaching method of the premolars with indication of extraction in orthodontic purposes was applied. None of the subjects smoked and throughout the investigation no antibiotics had been used. We initiated an intensive oral hygiene program, and we removed the supragingival and subgingival deposits. Oral hygiene and the gingival health were evaluated before every session of bleaching. During each visit the dentition was cleaned professionally and if needed the subjects were reinstucted in proper oral hygiene. After 3 and 5 successive bleachings of the teeth, we removed the dental pulps and we extracted the premolars. The pulpal biopsies were fixed in buffed formaldehyde 10% for 48 hours, then paraffinized, sectioned at 3-5 μ and stained with topographic, H&E and trichrome stained. For the electonomicroscopic study we used the Lehner technique to process the biopsies (n=3) after the reinclusion of the pieces from the paraffine blocks in Epon, postfixated in buffered glutaraldehyde, micro sectioned at 0,5 μ, contrastated with Pb citrate (stained) and examination in transmission electronic microscopy with Philips microscope. Results - At cellular and matriceal level we observed a marked collagen fibrillogenesis in the presence of active fibroblasts, with well developed cellular organites and fibroclastic aspects which suggest matriceal active repair. The microvascular network presents an activated endothelium with turgescent endothelial cells, with intracitoplasmatic resorbtion vacuols, well developed Golgi Complex. Conclusion - We interpreed the cell - matriceal lesions in the context of the acute inflammatory process in the first lesional phase and chronic scleroatrophic process after successive bleaching.

Zn(II)-phthalocyanines with tetra-substitution of D-galactose group on non-peripheral (nGalPc) and peripheral (pGalPc) positions have been studied as photodynamic sensitizers. The both complexes are water-soluble and highly aggregated in water and cell culture medium. The non-peripheral galactose units attached to the phthalocyanine macrocycle (nGalPc) lead to far red shift of absorbance maximum at 703 nm as compared to peripherally substituted pGalPc with maximum at 683 nm. The fluorescence maxima of the studied GalPcs were red shifted (8-14 nm) depending on the used solvent as compared to the absorption maxima. The relatively low fluorescence quantum yields in dimethylsulfoxide (0.06 for nGalPc and 0.21 for pGalPc) were determined. The singlet oxygen generation was determined with lower quantum yield for pGalPc (0.21) as compared to nGalPc (0.38). The lack of dark toxicity towards breast cancer cell line (MCF-7) in wide concentration range (0.125 - 10 μM) was observed. The uptake into the tumor cells and the subcellular localization in MCF-7 cells were determined with higher accumulation for pGalPc, compared to nGalPc. The in vitro photodynamic activity of GalPcs towards breast cancer cells was investigated for different dye concentrations and soft light parameters of 635 nm irradiation. The antitumor activity of nGalPc was superior to the pGalPc-induced cytotoxicity, due to higher generation of singlet oxygen and other reactive oxygen species.

The study presents the PDT with metal phthalocyanines on biofilms grown in root canals of ten representatives of the Gram-positive and the Gram-negative bacterial species and a fungus Candida albicans which cause aqute teeth infections in root canals.. The extracted human single-root teeth infected for 48 h with microorganisms in conditions to form biofilms of the above pathogens were PDT treated. The stage of biofilm formation and PDT effect of the samples of the teeth were determined by the scaning electron microscopy and with standard microbial tests. The PDT treating procedure included 10 min incubation with the respected phthalocyanine and irradiated with 660 nm Diode laser for 10 min. The most strongly antibacterial activity was achieved with zinc(II) phthalocyanine (ZnPc) against Enterococcus faecalis, Staphylococcus aureus and Moraxella catarrhalis. The other Gram-negative bacteria and Candida albicans were 10-100 times more resistant than the Gram-positive species. The Gram-negative Moraxella catarrhalis and Acinetobacter baumannii were more sensitive than the enterobacteria, but eradication of Pseudomonas aeruginosa in biofilm was insignificant. The influence of the stage of biofilm formation and the initial conditions (bacterial density, photosensitizer concentration and energy fluence of radiation) to the obtained level of inactivation of biofilms was investigated. The PDT with ZnPc photosensitizers show a powerful antimicrobial activity against the most frequent pathogens in endodontic infections and this method for inactivation of pathogens may be used with sucsses for treatment of the bacterial biofilms in the root canals.

The aspects of application of the hollow core photonic crystal waveguides for spectroscopic analysis of liquid medium were considered. The possibility of using these structures for analysis of a fruit juice was evaluated. The principles of processing of photonic crystal waveguide transmission spectra, which is sensitive to quality of juice, its composition, and main component concentration, were revealed.

This paper presents the results of experimental study of full field laser speckle imaging due to cortex microcirculation state monitoring for laboratory rats under conditions of stroke and the introduction of agents. Three groups of experimental animals from five animals in each group were studied. The behavior of blood flow, studied by speckle imaging technique, matched the expected physiological response to an impact.

The "specific structures" on the fat cells' membranes in vitro as a result of photodynamic treatment was registered. These structures were identified as cytoplasm/oil microdrops flowed out through the pores in the membranes. The impact of Brilliant Green dissolved in water-ethanol solutions and irradiation by a LED lamp on the quantity and size of "specific structures" on the membranes was investigated. It was demonstrated that optical selective action on fat cells sensitized by Brilliant Green led to the growth of "specific structures" (pores) number during the time interval after light exposure. A high degree of correlation between the optical clearing of fat tissue and quantity of "specific structures" (pores) was found. This result proves our early prediction about mechanism of light-induced fat cells' lipolysis via increased cell membrane porosity.

Raman scattering spectroscopy allows the direct and fast study of molecules by analysis of their vibrational normal modes. However, for certain materials the scattered signal is superimposed by fluorescence, which - if present - overwhelms the intrinsically weak Raman signal by orders of magnitude. An approved method to resolve the instantaneous Raman signal of interest from the delayed fluorescence background is time-correlated single-photon counting (TCSPC). For that, a single-photon detector with fast dynamics is required. The, so-called, superconducting nanowire single-photon detector (SNSPD) is a promising candidate for TCSPC. We have developed an optical instrument using such a SNSPD for the TCSPC method. The detector is made from a 5 nm thick NbN film, patterned by electron-beam lithography in a meander line with a width of 100 nm and a filling-factor of 50 %, covering an active area of 4 × 4 μm². As a proof of concept we have shown that it is possible to resolve low power optical signals (λ between 520 and 630 nm) with a timing jitter of about 35 ps. Based on our experimental results we will discuss perspectives and limits of SNSPD application for spectroscopy.

We use an Anti-Brownian ELectrokinetic (ABEL) trap to probe spectral emission shifts in solution-phase single Peridinin-Chlorophyll-Proteins (PCPs). The ABEL trap allows localization of single biomolecules in solution in a small volume for extended observation without immobilization. The essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in a sub-micron-sized field of view for hundreds of milliseconds to seconds. Peridinin-chlorophyll-protein is a water-soluble antenna protein found in dinoflagellates which uses peridinins (carotenoids) as accessory light harvesting pigments to absorb sunlight in the green region of the spectrum before transferring electronic excitation to chlorophyll. PCP is simpler than many other antenna complexes in that there are only two chlorophyll pigments per monomer which do not form an exciton. We use the ABEL trap to study single PCP monomers in solution for several seconds each. A significant fraction of the molecules show slow spectral shifts (spectral diffusion) relative to the bulk PCP spectrum. This is the first spectral emission measurement conducted in the ABEL trap.

Full-field Optical coherence tomography is an en-face interferometric imaging technology capable of carrying out high resolution cross-sectional imaging of the internal microstructure of an examined specimen in a non-invasive manner. The presented system is based on competitively priced optical components available at the main optical communications band located in the 1550 nm region. It consists of a superluminescent diode and an anti-stokes imaging device. The single mode fibre coupled SLD was connected to a multi-mode fibre inserted into a mode scrambler to obtain spatially incoherent illumination, suitable for OCT wide-field modality in terms of crosstalk suppression and image enhancement. This relatively inexpensive system with moderate resolution of approximately 24um x 12um (axial x lateral) was constructed to perform a 3D cross sectional imaging of a human tooth. To our knowledge this is the first 1550 nm full-field OCT system reported.