This PDF file contains the front matter associated with SPIE Proceedings Volume 7715, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial directions, respectively) has restricted the applications in bio-medical research. However, over the last 10 years various techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF) microscopy, are compared to the properties of the two high resolution techniques. Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined system provides the biologist with an unprecedented view of the sub-cellular organization of life.

Digital holographic microscopy (DHM) provides label-free quantitative phase contrast with low demands on sample preparation. Nevertheless, for DHM measurements on fixed cells the mounting medium has to be considered while the phase contrast of living cells may be influenced by the used buffer solution. To quantify these effects, the maximum cell caused phase contrast and the visibility of the nucleoli were analyzed. A second aim of the study was to identify subcellular components in DHM phase contrast images. Therefore, comparative investigations using bright field imaging, DHM and fluorescence microscopy with 4',6- Diamidino-2-phenylindol (DAPI) staining were performed. DAPI-staining visualizes cell components containing DNA. The obtained results demonstrate exemplarily for two tumor cell lines that from DHM phase contrast images of fixed cells in phosphate buffer saline (PBS) cell thickness values are obtained which are comparable to living cells. Furthermore, it is shown that in many cases nucleus components can be identified only by DHM phase contrast.

Retrieval of the amplitude and phase of electromagnetic waves made digital holographic microscopy (DHM) capable of revealing morphological details at ultrahigh resolution in the order of a few nanometers only and precisely measuring the refractive index across a sample (e.g. cell or neuron). In short,DHM added a new dimension to optical imaging,whic h explains why it is such an excellent instrument for metrological,but also for biological applications. We believe that DHM is,b y nature,ideally suited for nonlinear microscopy. In this work,w e review the advantages of DHM for nonlinear microscopy and present its application to determination of the axial position of nonlinear nanoparticles capable of second harmonic generation.

We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRi's rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

Optical Coherence Tomography (OCT) has found applications in many fields of medicine and has a large potential for the optical biopsy of tumors. One of the technological challenges impairing faster adoption of OCT is the relative complexity of the optical instrumentation required, which translates into expensive and bulky setups. In this paper we report an implementation of Time-Domain Optical Coherence Tomography based on Plasma Enhanced Chemical Vapor Deposition (PECVD) Silicon Carbide (SiC). The devices, with a footprint of 0.3 cm², are fabricated using rib waveguides defined in a SiC layer. While most of the components needed are known when using this material [1], a fast delay line with sufficient scanning range is a specific requirement of Time Domain (TD)-OCT. In the system reported here this is obtained making use of the thermo-optical effect. Though the current implementation still requires external sources and detectors to be coupled to the planar waveguide circuit, future work will include three-dimensional integration of these components onto the substrate to achieve a fully autonomous and compact OCT chip. With the potential to include the read-out and driving electronics on the same die, the reported approach can yield extremely compact and low-cost TD-OCT systems in the visible, enabling a broad range of new applications, including OCT devices for harsh environment.

Significant motion artifacts limit performance of conventional full-field optical coherence tomography for in vivo imaging. A theoretical and experimental study of those limitations is presented and a new FF-OCT system suppressing most of artifacts due to sample motions is demonstrated using instantaneous phase-shifting with non-polarizing optics and pulsed illumination.

We present a novel frequency-swept light source working at 1060nm that utilizes a tapered amplifier as gain medium. These devices feature significantly higher saturation power than conventional semiconductor optical amplifiers and can thus improve the limited output power of swept sources in this wavelength range. We demonstrate that a tapered amplifier can be integrated into a fiber-based swept source and allows for high-speed FDML operation. The developed light source operates at a sweep rate of 116kHz with an effective average output power in excess of 30mW. With a total sweep range of 70 nm an axial resolution of 15 μm in air (~11μm in tissue) for OCT applications can be achieved.

Pulse oximetry (PO) is a non-invasive medical device used for monitoring of the arterial oxygen saturation (SaO₂) and in particular of haemoglobin oxygenation in blood. Oxygen saturation is commonly used in any setting where the patient blood oxygen saturation is unstable, including Neonatal Intensive Care Unit (NICU). The main factor affecting PO's output data is the presence of voluntary or involuntary motion artifacts or imperfect skin-sensor contact. Various methods have been employed to reject motion artifact but have met with little success. The aim of the present work is to propose a novel measurement procedure for real-time monitoring and validation of the oxygen saturation data as measured in standard pulse oxymeter. The procedure should be able to individuate and reject erroneous saturation data due to incorrect transducer-skin contact or motion artifact. In the case of short sequences of rejected SpO₂ data (time duration< 8s), we report on an algorithm able to substitute the sequence of rejected data with the "most-probable" (rescued) SpO₂ data. In total we have analyzed 14 patient for a total of 310 hr, 43 min and 15s, equivalent to a total number of samples of 1118595. For our study, we were interested to download heart rate measured with the ECG (HRECG), the heart rate as measured by the pulse oximeter (HR_SAT) and the SpO₂ value. In order to remove the erroneous SpO₂ values reported in the rough data in coincidence of motion artifact (top, right), we have implemented a specific algorithm which provides at the output a new sequence of SpO₂ data (validated SpO₂ data). With the aim to "rescue" SpO₂ value rejected by the previously presented algorithm, we have implemented an algorithm able to provide the "most-probable" SpO₂ values in the case of single rejected values or in the case of short sequences of invalidated data (< 8 s). From these data it is possible to observe how in the 6.8% of the observation time the SpO₂ data measured by the pulse oximeter are not validated by the use of our method (corresponding to a total time of 16 hr, 8min and 40s). The use of the proposed algorithm aiming to "rescue" data from short sequences of rejected data (< 8s) allows to increase the validated data of the 2.5%t(equivalent to 8hr, 40 min and 16s), allowing a percent of usable data of the 95.7%. Once implemented in clinic, it could be used to identify the period of the day in which the percent of rejected data increase or correlate this data to clinical procedure in order to intensify clinicians and nurses attention.

The use of standard instrumentation for the assessment of the respiration rate as of flow is an important goal in medicine. Spirometers, textile-based capacitive sensors or photopletismography are standard contact instrumentations used for such aim; the main drawback in the use of such instrumentations is the necessity to have a direct contact of the instrument with the patient. In this paper, we present an optical no-contact method for monitoring of both the respiration rate and flow. This method is based on the measurement of external chest wall movement by a laser Doppler vibrometer. The measurement procedure has already been demonstrated to be extremely well performing for what concern the monitoring of the cardiac activity. The proposed method can be operated at a distance of 1.5 m, on different point of the patient thoracic and abdominal area. We have monitored respiration rate and flow on 8 patients with a spirometer and simultaneously with the proposed noncontact measurement procedure. Bland-Altman analysis of the respiration rate measured with both instruments demonstrate a mean error on the determination of the respiration rate of < 1% and of the < 4% for the instantaneous flow. We also report a study on the optimal position on the thoracic area based on quality of the signal measured on the same population of subject.

This paper combines multispectral imaging and simple image processing techniques for the non-destructive identification of Thai rice breeds. Especially, we exploit only two fluorescent wavelengths in a 500-580 nm wavelength band and utilize simple image thresholding, blob filtering, and blob analysis techniques in order to identify 8 different Thai rice breeds. Other key features include no waste produced and fast identification time. In our experimental study, UVC light is used as our exciting light, a liquid crystal tunable optical filter is used for our wavelength selection, and a camera with 644×488 active pixels is used to capture desired wavelength images. Milled rice grains from 8 different Thai rice breeds having similar size and shape are tested. There is also one glutinous rice breed in our experiment. Our experimental result shows that by suitably applying image thresholding, blob filtering, and blob analysis to fluorescent images, all Thai rice breeds can be effectively identified.

We demonstrate that a speckle pattern in the spatially coherent laser field transmitted by a diffuser forms a multitude of three-dimensional bottle-shaped micro-traps. These multiple traps serve as a means for an effective trapping of large number of air-born absorbing particles. Confinement of up to a few thousand particles in air with a single beam has been achieved. The ability to capture light-absorbing particles suspended in gases by optical means opens up rich and diverse practical opportunities, including development of photonic shielding/fencing for environmental protection in nanotechnology industry and new methods of touch-free air transport of particles and small containers, which may hold dangerous substances, or viruses and living cells.

Aggregation peculiarities of red blood cells (RBCs) in autologous plasma are studied using double trap optical tweezers technique. The positions of RBCs are controlled with submicrometer accuracy by two optical traps formed by strongly focused laser beams (λ=1064 nm). Quantitative measurements of interaction forces between RBCs in pair aggregates are performed. Depending on the RBCs aggregation force, four different end-points of disaggregation induced by optical trap movement are revealed. Analysis of experimental force dependence on the distance between two RBCs during disaggregation is in a good agreement with the model of ring-shaped interaction surfaces of RBCs in pair aggregate. Aggregation velocities measured are shown to be strongly different for healthy and pathologic (System Lupus Erythematosis - SLE) blood samples.

Surface plasmon resonance (SPR) biosensors have been widely used for dynamical analysis of molecular affinity, bacterium screening, and drug discovery due to its advantages of label-free detection, dynamic interaction analysis, small sample volume, and ultra sensitivity (feasibility of single molecular detection). Recently, SPR biosensing for cell imaging known as SPR microscopy (SPRM) has attracted great attention due to the characteristics of SPR biosensors. However, it is well known that the trends of sensitivity and spatial resolution are opposite to each other: Surface plasmon waves (SPWs) with shorter wavelength which provides higher spatial resolution has less sensitivity. It is known that the spatial resolution of SPRM is limited by the propagation length of surface plasmon wave (SPW) along the metaldielectric interface. SPW excited by 632.8 nm light has the propagation length of 3 um. This length becomes longer when a longer wavelength is selected. While most of SPR biosensors are built with 632.8 nm or longer wavelength for high sensitivity, using 532nm light to excite SPWs is desired for submicron resolution since the propagation length is around 150 nm. Different from current phase interrogation methods, the proposed phase interrogation method is highly sensitive and suitable for CCD imaging. Although it is generally believed that SPWs with wavelength 532nm has poor sensitivity, the experimental result showed that the setup can reach the sensitivity lower than 2×10^-6 RIU when sucrose is used as the test sample.

Current high-content screening (HCS) techniques involve the analysis of cellular assays using high-resolution imaging combined with sophisticated algorithms for automated image analysis. Commercially available platforms are invariably highly specialised and expensive. Here we present a novel assay utilising changes in fluorescence lifetime in the vicinity of a rough Au film. A mammary carcinoma cell line was created expressing EGFP in the membrane, and cells were plated onto multi-well slides covered with a 30 nm Au film. FLIM images show a large reduction in lifetime for membrane-bound GFP in close proximity to the Au surface. Addition of a suitable ligand leads to internalization of the GFP with a corresponding increase in lifetime. The degree of internalization can be very quickly and easily checked using standard lifetime analysis techniques, with no need for image analysis. We demonstrate the method by comparing the efficacies of two small molecule inhibitors interfering with the internalization process.

We have investigated a series of mono-, bis- and trisvinyl-pyridinium TP derivatives (TP-Py) which exhibit interesting properties : good water solubility, reduced size, high photostability, large two-photon absorption cross-sections (δ up to 700 GM), red emission (λ_em=660-680nm). Most importantly, TP-Py happen to be poorly fluorescent in water whereas their fluorescence is strongly enhanced when binding to various forms of DNA. We showed that this property originates in the spontaneous immobilisation of the dye inside the double-stranded DNA helix. Due to moderate fluorescence quantum yield efficiencies (η), the brightness of TP-Py appears however to be improvable (ηδ=19). For this purpose, a new generation of specifically engineered TP derivatives have been designed and extensively characterized. We showed that switching from a classical TP core to a more electron-rich trinaphtyl (TN) core enables to increase the molecular brightness although reducing drastically the dye-DNA specific interactions.

In the current paper we present preliminary data demonstrating therapeutic efficiency of local laser hyperthermia of mouse tumors with gold nanoparticles. Measuring the tumor temperatures both superficial and inner by means of standard NIR-thermograph and original acoustic thermometer correspondingly we show that the gold nanoparticles increase thermal sensitivity of tumor tissue. Transmission electron microscopy and histopathology of the tumor tissue indicated that the mechanism of apoptotic death of tumor cells is triggered following the laser treatment. 5 days after the treatment tumor growth inhibition was 104 %.

We review our experimental studies on near infrared laser-activated gold nanoparticles in the direct welding of connective tissues. In particular, we discuss the use of gold nanorods excited by diode laser radiation at 810 nm to mediate functional photothermal effects and weld eye's lens capsules and arteries. The preparation of biopolymeric matrices including gold nanorods is described as well, together with preliminary tests for their application in the closure of wounds in vessels and tendons. Finally we mention future perspectives on the use of these nanoparticles for applications in the therapy of cancer.

We present here research work on two optical biosensors which have been developed within two separate European projects (6th and 7th EU Framework Programmes). The biosensors are based on the idea of a disposable biochip, integrating photonics and microfluidics, optically interrogated by a multichannel interrogation platform. The objective is to develop versatile tools, suitable for performing screening tests at Point of Care or for example, at schools or in the field. The two projects explore different options in terms of optical design and different materials. While SABIO used Si₃N₄/SiO₂ ring resonators structures, P3SENS aims at the use of photonic crystal devices based on polymers, potentially a much more economical option. We discuss both approaches to show how they enable high sensitivity and multiple channel detection. The medium term objective is to develop a new detection system that has low cost and is portable but at the same time offering high sensitivity, selectivity and multiparametric detection from a sample containing various components (e.g. blood, serum, saliva, etc.). Most biological sensing devices already present on the market suffer from limitations in multichannel operation capability (either the detection of multiple analytes indicating a given pathology or the simultaneous detection of multiple pathologies). In other words, the number of different analytes that can be detected on a single chip is very limited. This limitation is a main issue addressed by the two projects. The excessive cost per test of conventional bio sensing devices is a second issue that is addressed.

In this work we report on the fabrication of functionalized PSiMc scaffolds that can be used to enhance the detection of MMP-8. Matrix metalloproteinases (MMPs) are the major enzymes that degrade extracellular matrix (ECM) proteins and play a key role in diverse physiological and pathological processes. We are interested in detecting the collagenase-type MMP-8 that is an inflammatory marker in gingival fluid for predicting tooth movement during orthodontic treatment. As presence of an increasing amount of MMP-8 in saliva is directly related with the tooth movement during orthodontic treatment, monitoring continuously the MMP-8 variation is primordial. Porous silicon microcavity (PSiMc) structures were prepared as multilayered stacks of low and high refractive indices and with layer thicknesses in the order of visible light wavelength. Then the PSi surface was functionalized with human antibodies. Both functionalization and MMP-8 infiltration were monitored by specular reflectometry. PSiMc is characterized by a narrow resonance peak in the optical spectrum that is very sensitive to a small change in the refractive index, such as that obtained when a molecule is attached to the large internal surface of porous silicon. The pore dimensions of the used PSiMc structures were evaluated by atomic force microscopy (AFM) and scanning electron microscope (SEM).

Continuous-wave diffuse reflectance or Near Infrared Spectroscopy (NIRS) offers the possibility to perform a preliminary screening of tissue for ischemia or other tissue anomalies. A tool for intracavity NIRS measurements during laparoscopic surgery, developed within the framework of the FP7-IP ARAKNES (Array of Robots Augmenting the KiNematics of Endoluminal Surgery) project, is described. It consists of a probe, that is located on the tip of an appropriately shaped laparoscopic manipulator and then applied to the tissue. Such a probe employs an array of incoherent semiconductor light sources (LEDs) frequency-multiplexed on a single detector using a lock-in technique. The resulting overall tool structure is simple and compact, and allows efficient coupling of the emitted light towards the tissue. The tool has high responsivity and enables fast and accurate measurements. A dataset gathered from in-vivo tissue is presented. The performance both indicates direct applicability of the tool to significant surgical issues (ischemia detection), and clearly indicates the possibility of further miniaturizing the probe head towards catheterized approaches.

Monocyte-derived macrophages play a key role in lipid metabolism in vessel wall tissues. Macrophages can take up lipids by various mechanisms. As phagocytes, macrophages are important for the decomposition of lipid plaques within arterial walls that contribute to arteriosclerosis. Of special interest are uptake dynamics and intra-cellular fate of different individual types of lipids as, for example, fatty acids, triglycerides or free and esterified cholesterol. Here we utilize Raman microscopy to image the metabolism of such lipids and follow subsequent storage or degradation patterns. The combination of optical microscopy with Raman spectroscopy allows visualization at the diffraction limit of the employed laser light and biochemical characterization through the associated spectral information. Relatively long measuring times, due to the weakness of Raman scattering can be overcome by non-linear effects such as coherent anti-Stokes Raman scattering (CARS). With this contribution we introduce first results to monitor the incorporation of lipid components into individual cells employing Raman and CARS microscopy.

Within this contribution, we demonstrate a combination of microarrays, microfluidics and SERS to enable a sequence specific detection of DNA. In this combination, the microarray allows for the immobilisation of DNA sequences as well as the removal of unbound DNA, microfluidics permit the automation of the process and SERS provides a highly sensitive detection by means of an interaction between an analyte molecule and the enhanced electromagnetic field in the proximity of metallic nanostructured surfaces such as spherical nanoparticles. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.

One of the major public health hazards is colon cancer. There is a great necessity to develop new methods for early detection of cancer. If colon cancer is detected and treated early, cure rate of more than 90% can be achieved. In this study we used FTIR microscopy (MSP), which has shown a good potential in the last 20 years in the fields of medical diagnostic and early detection of abnormal tissues. Large database of FTIR microscopic spectra was acquired from 230 human colonic biopsies. Five different subgroups were included in our database, normal and cancer tissues as well as three stages of benign colonic polyps, namely, mild, moderate and severe polyps which are precursors of carcinoma. In this study we applied advanced mathematical and statistical techniques including principal component analysis (PCA) and linear discriminant analysis (LDA), on human colonic FTIR spectra in order to differentiate among the mentioned subgroups' tissues. Good classification accuracy between normal, polyps and cancer groups was achieved with approximately 85% success rate. Our results showed that there is a great potential of developing FTIR-micro spectroscopy as a simple, reagent-free viable tool for early detection of colon cancer in particular the early stages of premalignancy among the benign colonic polyps.

Despite of the recent year's important advances in breast cancer biology, there is a continuous demand for new microscopic studies able to provide complementary information on cell shape that is an essential feature of the tumour cells affecting their proliferation and spreading. Understanding the relationship between cell shape and cellular function is important for regulation of cell phenotype modification in particular during cancerogenesis. Utilizing a multitechnique approach, we have investigated the morphological differences of normal human mammalian epithelial cells (HMEC) and cancerous breast epithelial cells (MCF7) cells, both mammalian epithelial cells, but from the same cell type, allowing us to compare them. The goal of our investigation was to combine information on morphological properties of these cells provided by imaging techniques like atomic force microscopy (AFM), brightfield microscopy with in-depth images of microtubules via the multiphoton microscopy (MPM). Cells morphology studies for both cells' types were first carried out using the contact mode AFM which has gained great potential for studying biological systems. Brightfield optical imaging was operated in correlation with the AFM measurements. Topography analyses were performed for living cells as well as fixed ones for both MCF7 and HMEC 184 A1 cells. Living cancerous cells are much softer than normal ones, smaller in shape, and more difficult to manipulate. Photonic responses of fixed cells have been then evaluated by the multiphoton microscopy. Due to light's good penetration depth (IR excitation) in biological samples, MPM has already proved to be a reliable and powerful tool in medical and biological deep tissue imaging. Moreover, MPM provides useful three-dimensional information on the structural and optical properties of the specimen due to its intrinsic optical sectioning resolution. Combination of these microscopic techniques allows us to correlate external cell morphology, with in-depth images provided by the non-linear optical response of microtubules. Understanding cytoskeletal perturbations and particularly, organization of the microtubules can help us to comprehend biological processes in cancer.

Conventional imaging systems used today in surgical settings rely on contrast enhancement based on color and intensity and they are not sensitive to morphology changes at the microscopic level. Elastic light scattering spectroscopy has been shown to distinguish ultra-structural changes in tissue. Therefore, it could provide this intrinsic contrast being enormously useful in guiding complex surgical interventions. Scatter parameters associated with epithelial proliferation, necrosis and fibrosis in pancreatic tumors were previously estimated in a quantitative manner. Subtle variations were encountered across the distinct diagnostic categories. This work proposes an automated methodology to correlate these variations with their corresponding tumor morphologies. A new approach based on the aggregation of the predictions of K-nearest neighbors (kNN) algorithm and Artificial Neural Networks (ANNs) has been developed. The major benefit obtained from the combination of the distinct classifiers is a significant increase in the number of pixel localizations whose corresponding tissue type is reliably assured. Pseudo-color diagnosis images are provided showing a strong correlation with sample segmentations performed by a veterinary pathologist.

A series of surface plasmonic fibre devices were fabricated using multiple coatings deposited on a lapped section of a single mode fibre and post-fabrication UV laser irradiation processing with a phase mask, producing a surface relief grating structure. These devices showed high spectral sensitivity in the aqueous index regime ranging up to 4000 nm/RIU for wavelength and 800 dB/RIU for intensity. The devices were then coated with human thrombin binding aptamer. Several concentrations of thrombin in buffer solution were made, ranging from 1nM to 1μM. All the concentrations were detectable by the devices demonstrating that sub-nM concentrations may be monitored.

The potential impact of optical fiber sensors embedded into medical textiles for the continuous monitoring of the patient during Magnetic Resonance Imaging (MRI) is now proved. We report how two pure optical technologies can successfully sense textile elongation between, 0% and 3%, while maintaining the stretching properties of the textile substrates for a good comfort of the patient. Investigating influence of different patients' morphology as well as textile integration issues to let free all vitals organs for medical staff actions, the OFSETH harness allows a continuous measurement of respiration movements. For example, anaesthesia for MRI examination uses the same drugs as for any surgical procedure. Even if spontaneous respiration can be preserved most of the time, spontaneous respiration is constantly at risk of being impaired by anaesthetic drugs or by upper airway obstruction. Monitoring of the breathing activity is needed to assess adequate ventilation or to detect specific obstruction patterns. Moreover artefacts due to physiological motions induce a blooming effect on the MRI result. The use of synchronisation devices allows reducing these effects. Positioned at certain strategic places according to the investigated organ, the presented sensors could constitute an efficient and adapted solution for respiratory synchronisation of the MRI acquisition.

The increased concern, emerged in the last few years, on food products safety has stimulated the research on new techniques for traceability of raw food materials. DNA analysis is one of the most powerful tools for the certification of food quality, and it is presently performed through the polymerase chain reaction technique. Photonic crystal fibers, due to the presence of an array of air holes running along their length, can be exploited for performing DNA recognition by derivatizing hole surfaces and checking hybridization of complementary nucledotide chains in the sample. In this paper the application of a suspended core photonic crystal fiber in the recognition of DNA sequences is discussed. The fiber is characterized in terms of electromagnetic properties by means of a full-vector modal solver based on the finite element method. Then, the performances of the fiber in the recognition of mall synthetic oligonucleotides are discussed, together with a test of the possibility to extend this recognition to samples of DNA of applicative interest, such as olive leaves.

A tightly focused ultrafast pulsed laser beam is guided into the volume of the photosensitive material and induces nonlinear photomodification. By translating the sample, the position of the focus is changed relatively, thus point-by-point complex 3D structures can be written inside the bulk. In this report, we present a Laser Two-Photon Polymerization (LTPP) setup for three-dimensional micro/nanostructuring for applications in photonics, microoptics, micromechanics, microfluidics and biomedicine. This system enables fabrication of functional devices over a large area (up to several cm in lateral size) with reproducible sub-micrometer resolution (up to 200 nm). In our experiments a Yb:KGW active media laser oscillator (75 fs, 200 kW, 515 nm frequency doubled, 80 MHz) was used as an irradiation source. The sample was mounted on XYZ wide range linear motor driven positioning stages having 10 nm positioning resolution. These stages enable an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support a linear scanning speed of up to 300 mm/s. Control of all the equipment was automated via custom made computer software "3D-Poli" specially designed for LTPP applications. The model of the structure can be imported as CAD file, this enables rapid and flexible structuring out of various photopolymers like ORMOCERs, ORMOSILs, acrylates and PEGDAs which are commonly used in conventional UV mask, nanoimprint and μ-stereolithographies. In this paper, we demonstrate polymeric microstructures fabricated over a large area on glass, plastic and metal substrates. This opens a way to produce functional devices like photonic crystals, microlenses, micromechanic and microfluidic components and artificial scaffolds as templates for cell growth. Additionally, results of primary myogenic stem cells expanding on microfabricated polymeric scaffolds are provided. Cell proliferation tests show the material and structure to be biocompatible for the biomedical practice.

For sensing with lab-on-chip systems, the use of highly integrated photonic components is a key ingredient for high sensitivity and short response time. Here, we present the efficient integration of organic semiconductor distributed feedback lasers based on Alq3:DCM and deep ultraviolet induced waveguides into a Poly(methyl methacrylate) (PMMA) substrate. The optimized coupling of laser light from the organic semiconductor lasers into the waveguides is discussed. On-chip coupling of laser light at 645 nm to waveguides is demonstrated. Utilizing mass production technologies and simple processes with only a few different materials paves the way for low-cost all-organic chips. In particular, a specific sensing concept is introduced: deep ultraviolet induced waveguides and nanostructured phospholipid gratings, which are bottom-up assembled using Dip-Pen Nanolithography, are combined to form a grating coupler with a grating period of 700 nm. Light of different wavelength is decoupled under different angles by the grating coupler to demonstrate its functional capability. Biosensing with this device is discussed based on a model system.

Based on the optical methods, it could be able to identify the concentrations of the hemoglobin fractions in a few seconds. An array of Light Emitting Diodes "LEDs" with different wavelengths and a photo detector were used to detect the transmitted or reflected light intensities. A system of equations was built according to the modified Lambert-Beer Law to be solved as an inverse problem. Typically practical inverse problems are all ill-posed. Even well-posed problems may be unstable or ill-conditioned when they digitalized. Also, applications in tissue spectroscopy and optical imaging result in an ill-posed problems. The difficulties involved in solving ill-posed systems are known, where large changes in the solution result from small perturbations in the right hand members of the system that can be caused by measurement tolerance or noise. A good numerical method to solve it may be beneficial in the applications to the optimization problems including linear programming and nonlinear programming. To account for the sensitivity to noise, a regularization method is usually applied to solve this sort of ill-posed problem, where a suitable regularized parameter is used to depress the bias in the computed solution by a better balance of approximation error and propagated data error. The Method has better computational efficiency and accuracy even for the highly ill-conditioned linear equations with a large disturbance on the given data. Comparing the results with that obtained from a direct solution of the system of equations, we prove well-posedness, stability and convergence of the method. The estimated hemoglobin fractions were highly correlated to the reference laboratory measurements.

We report preliminary clinical results of autofluorescence imaging of malignant and benign skin lesions, using pulsed 355 nm laser excitation with synchronized detection. The novel synchronized detection system allows high signal-tonoise ratio to be achieved in the resulting autofluorescence signal, which may in turn produce high contrast images that improve diagnosis, even in the presence of ambient room light. The synchronized set-up utilizes a compact, diode pumped, pulsed UV laser at 355 nm which is coupled to a CCD camera and a liquid crystal tunable filter. The excitation and image capture is sampled at 5 kHz and the resulting autofluorescence is captured with the liquid crystal filter cycling through seven wavelengths between 420 nm and 580 nm. The clinical study targets pigmented skin lesions and evaluates the prospects of using autofluorescence as a possible means in differentiating malignant and benign skin tumors. Up to now, sixteen patients have participated in the clinical study. The autofluorescence images, averaged over the exposure time of one second, will be presented along with histopathological results. Initial survey of the images show good contrast and diagnostic results show promising agreement based on the histopathological results.

Photodynamic antimicrobial therapy (PAT) may become a useful clinical tool to treat microbial infections, overcoming microbial resistance that is a major problem nowadays. The aim of our work was to verify the damage caused by photosensitization over a Escherichia col) via atomic force microscopy (AFM), looking for structural changes that might occur in cells after PAT. Cells culture were grown until a stationary phase to reach a concentration of approximately 108 cells/mL allowing the production of extracellular slime in a biofilm-like structure. The cells including the extracellular matrix were put in a slide and its structure was observed using AFM; subsequently a water solution of methylene blue at 60μM was applied over the cells and a pre-irradiation time of 3 minutes was waited and followed by illumination with a diode laser (λ=660nm, power 40mW, 3min, fluence 180J/cm², beam diameter 0.04cm²). The same cells were observed and the images stored. A second set of experiments was performed with a smaller number of cells/area and without extracellular slime, using the parameters abovementioned. The results showed alterations on cellular scaffold markedly dependent on the number of cells and the presence of extracellular slime. The slime is targeted by the photosensitizer, and after irradiation a destruction of the matrix was observed; when fewer cells were evaluated the destruction is much more evident. The images suggested rupture of the cellular membrane and cellular fragments were observed. Our findings indicate that AFM seems is a useful tool to investigate parameters linked with photodestruction of microorganisms.

Protoporphyrin IX (PpIX) displays high tumour-selective uptake following oral administration of 5-aminolaevulinic acid (ALA), a fact that is being exploited for the fluorescence-guided resection (FGR) and photodynamic therapy (PDT) of human brain malignancies. A clinical procedure for interstitial PDT (iPDT) has been established including pre-treatment planning, optical fiber insertion under stereotactic guidance and therapeutic irradiation at non-thermal fluence rates. We have previously reported on median survival in the range of 15 months and the existence of some intriguing long-term survivors (>5 years) following iPDT. Such successful treatments rely on for example sufficient light, PpIX and oxygen levels. We have investigated the absolute PpIX concentration as well as the PDT-induced photobleaching kinetics in brain tissues. Tissue samples acquired during FGR contained PpIX concentrations up to 28 μM. This observation implies that ALA-induced PpIX levels are sufficient for inducing PDT effects in viable tumour tissue upon therapeutic irradiation. However, regions of pre-existing necrosis were characterised by significantly lower photosensitiser levels. Fluorescence spectroscopy was implemented in parallel to iPDT with the aim to employ PpIX photobleaching as a tool for realtime treatment supervision and early treatment prognosis.

Following previous studies where we developed some high performance porphyrin derivatives for photodynamic therapy demonstrating their activity in different cell lines, we now extend our attention to CRL1472 bladder cancer line. In this work the phototoxicity of several diaryl and tetraarylporphyrins with different structures were evaluated with different incubation times. The phototoxicity observed was not directly related to the concentration of photosensitizer inside cells. Uptake studies demonstrate that the brominated derivative 2 which despite the most efficient photosensitizer presents a poor tendency to enter into cells.

Low-level laser therapy (LLLT) is commonly used to accelerate wound healing. Besides, the technique of imaging the light distribution inside biological tissues permits us to understand several effects about light-tissue interaction. The purpose of this study was to determine the relative attenuation coefficient of the light intensity in healthy and burned skin rats during cutaneous repair following LLLT or not. Two burns about 6mm in diameter were cryogenerated using liquid N2 on the back of 15 rats. Lesion L was irradiated by a He-Ne laser (λ= 632.8nm) and fluence 1.0J/cm²; Lesion C was control and received sham irradiation. A healthy skin area (H) was also analyzed. The lesions were irradiated at days 3, 7, 10 and 14 post-burning. The animals were euthanized at days 3, 10 and 31 and skin samples were carefully removed and placed between two microscope slides, spaced by z= 1mm. A laser beam irradiated the sandwiched tissue from epidermis to dermis. A CCD camera was placed orthogonal to the beam path and it photographed the distribution of the scattered light. The light decay occurred according to the Beer's Law. Significance was accepted at p <0.01 by using t-Student test. Our results show that the light decay along any direction was close to an exponential. Burned skin samples presented decay significantly faster than healthy skin samples. Besides, attenuation coefficient changed during burning healing comparing treated and control lesions. These findings suggest that the relative attenuation coefficient is a suitable parameter to optimize LLLT during wound healing.

Development is reported of a small-sized ultraviolet (UV) radiometer designed for measurements of energy characteristics of UV radiation - energy illuminance and energy exposure. Main characteristics are considered of nZnSe(O, Te)/Ni Schottky structures created for the developed UV radiometer and used as UV sensors. Characteristics are presented of optical glass filters and interference light filters designed for separation of different biologically relevant UV spectral regions.

The reduction of pathogenic microorganisms in supragingival plaque is one of the principal factors in caries prevention and control. A large number of microorganisms have been reported to be inactivated in vitro by photodynamic therapy (PDT). The purpose of this study was to develop a rat model to investigate the effects of PDT on bacterial reduction in induced dental caries. Twenty four rats were orally inoculated with Streptococcus mutans cells (ATCC 25175) for three consecutive days. The animals were fed with a cariogenic diet and water with 10% of sucrose ad libitum, during all experimental period. Caries lesion formation was confirmed by Optical Coherence Tomography (OCT) 5 days after the beginning of the experiment. Then, the animals were randomly divided into two groups: Control Group: twelve animals were untreated by either light or photosensitizer; and PDT Group: twelve animals were treated with 100μM of methylene blue for 5min and irradiated by a Light Emitting Diode (LED) at λ = 640±30nm, fluence of 172J/cm², output power of 240mW, and exposure time of 3min. Microbiological samples were collected before, immediately after, 3, 7 and 10 days after treatment and the number of total microaerophiles was counted. OCT images showed areas of enamel demineralization on rat molars. Microbiological analysis showed a significant bacterial reduction after PDT. Furthermore, the number of total microaerophiles in PDT group remained lower than control group until 10 days posttreatment. These findings suggest that PDT could be an alternative approach to reduce bacteria in dental caries.

In this work we made teeth ablation experiments using a laser microprocessing set-up coupled with an amplified femtosecond laser system with 200 fs pulse duration. The experiments were performed at different laser fluences, from 0.21 J/cm² up to 64 J/cm². Structures with computer controlled geometries were created as single round craters, large square craters shapes, and parallel lines. Irradiation was made in single-pulse, multi-pulse, and quasi-continuous (2 kHz) mode at different laser scanning speeds. Characterization of ablated structures was made by optical microscopy and scanning electron microscopy. Ablation areas images show crystalline and regular structures. There are not evidences of material burning under 64 J/cm². Generated structures are reproducible, dependent on dental quality. Enamel ablation threshold under 3 J/cm² was measured. Dimensions of the ablated structures are of tens of micrometers, depending on beam fluence, focusing optics, and material hardness. When a 10x microscope objective was used, craters with about 5-7 micrometers were obtained. Better resolution of the structures can be obtained down to about 1 micron, however more difficult is to observe and work with such ablated structures. Femtosecond laser ablation demonstrates to be a promising method for teeth treatment due to its advantages: ablation precision and no collateral damages.

Endodontic therapy consists in cleaning and shaping the root canal system, removing organic debris and sealing the intra-canal space with permanent filling materials. The purpose of this study was to evaluate various root canal fillings in order to detect material defects, the marginal adaptation at the root canal walls and to assess the quality of the apical sealing. 21 extracted single-root canal human teeth were selected for this study. We instrumented all roots using NiTi rotary instruments. All canals were enlarged with a 6% taper size 30 GT instrument, 0,5 mm from the anatomical apex. The root canals were irrigated with 5% sodium hypochlorite, followed by 17% ethylenediaminetetraacetic acid (EDTA). After the instrumentation was completed, the root canals were obturated using a thermoplasticizable polymer of polyesters. In order to assess the defects inside the filling material and the marginal fit to the root canal walls, the conebeam micro-computed tomography (CBμCT) was used first. After the CBμCT investigation, time domain optical coherence tomography working in en face mode (TDefOCT) was employed to evaluate the previous samples. The TDefOCT system was working at 1300 nm and was doubled by a confocal channel at 970 nm. The results obtained by CBμCT revealed no visible defects inside the root-canal fillings and at the interfaces with the root-canal walls. TDefOCT investigations permit to visualize a more complex stratificated structure at the interface filling material/dental hard tissue and in the apical region.

In this work, our attention was drawn towards developing affinity-based electrical biosensors, using a MESFET (Metal Semiconductor Field Effect Transistor). Semiconductor (SC) surfaces must be prepared before the incubations with biomolecules. The peptides route was adapted to exceed and bypass the limits revealed by other types of surface modification due to the unwanted unspecific interactions. As these peptides reveal specific recognition of materials, then controlled functionalization can be achieved. Peptides were produced by phage display technology using a library of M13 bacteriophage. After several rounds of bio-panning, the phages presenting affinities for GaAs SC were isolated; the DNA of these specific phages were sequenced, and the peptide with the highest affinity was synthesized and biotinylated. To explore the possibility of electrical detection, the MESFET fabricated with the GaAs SC were used to detect the streptavidin via the biotinylated peptide in the presence of the bovine Serum Albumin. After each surface modification step, the IDS (current between the drain and the source) of the transistor was measured and a decrease in the intensity was detected. Furthermore, fluorescent microscopy was used in order to prove the specificity of this peptide and the specific localisation of biomolecules. In conclusion, the feasibility of producing an electrical biosensor using a MESFET has been demonstrated. Controlled placement, specific localization and detection of biomolecules on a MESFET transistor were achieved without covering the drain and the source. This method of functionalization and detection can be of great utility for biosensing application opening a new way for developing bioFETs (Biomolecular Field-Effect Transistor).

In most industrial states a huge amount of newly hatched male layer chickens are usually killed immediately after hatching by maceration or gassing. The reason for killing most of the male chickens of egg producing races is their slow growth rate compared to races specialized on meat production. When the egg has been laid, the egg contains already a small disc of cells on the surface of the yolk known as the blastoderm. This region is about 4 - 5 mm in diameter and contains the information whether the chick becomes male or female and hence allows sexing of the chicks by spectroscopy and other methods in the unincubated state. Different imaging methods like sonography, 3D-X-ray micro computed tomography and magnetic resonance imaging were used for localization of the blastoderm until now, but found to be impractical for different reasons. Optical coherence tomography (OCT) enables micrometer-scale, subsurface imaging of biological tissue and could therefore be a suitable technique for an accurate localization. The intention of this study is to prove if OCT can be an appropriate approach for the precise in ovo localization.

A single-mode biconical tapered optical fiber (BTOF) sensor was utilized for sensing the variation of refractive index (RI) with concentration of D-glucose in double distilled deionized water and measuring of RI of amino acids (AAs) in carbohydrate solutions. This method showed a rewarding ability in understanding the basis of biomolecular interactions in biological systems. The BTOF is fabricated by heat pulling method, utilizing a CO₂ laser. The detection limit of the BTOF was 50 ppb for the D-glucose concentration ranging from 0 to 80 ppm, and RI detection limit corresponding to these concentrations in the range at 1.3333 to 1.3404 was 5.4×10^-6 as a refractometer sensor. The response of the BTOF shows that the different kinds of interactions of various groups of AAs such as L-alanine, L-leucine, and L-cystein with D-glucose, sucrose and water molecules depend on functional groups in AAs such as OH, SH;CH₂;NH₃⁺ ,COO^-. These results can be interpreted in terms of solute-solute and solute-solvent interactions and structure making/breaking ability of solutes in the given solution.

We demonstrate a simple refractive index sensor (RI) sensing system based on a biconical tapered optical fiber (BTOF), which is fabricated by heat pulling method, utilizing a CO₂ laser. In this work we explore the application of these sensors for the detection of label free single stranded DNA (ssDNA) in real time. During the experiment, the target ssDNA did not need to be labeled with a fluorescent tag, which is expensive and complicated. The change in output optical transmission of the tapered fiber was recorded for Poly-L-Lysine (PLL) coating, ssDNA probe immobilization and hybridization. The result indicated that due to the hybridization with the complementary target ssDNA on the tapered surface, the RI of surrounding medium changes which leads to changes in the characteristics of the tapered region and change in the output power of the sensor.

The newest approach in the saturation fluorimetry of photosynthetic organisms by the example of phytoplankton was developed. The theoretical model and the inverse problem of the saturation fluorimetry are discussed. The results of evaluation of molecular photophysical parameters of alga Chlorella pyrenoidosa under various stress factors, such as presence of DCMU and Cu2+ ions are presented. The correlation between theese parameters and the parameters obtained using Fluorescence Induction and Relaxation technique is discussed.

In this studies we used classical and laser methods for cyanobacteria diagnostics: Fluorescence Induction and Relaxation and Non-Linear Laser Fluorimetry in order to obtain the whole set of photophysical parameters of cyanobacteria. Different photophysical processes that take place in photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC6803 were studied with mentioned above fluorescent methods and sets of photophysical parameters were determined. The results allow us to suggest a model of photo adaptation processes under excess irradiance (depending on light intensity and spectrum).

A method for analysis of skin capillary-refill processes in real time by means of reflection photoplethysmography (PPG) contact probe operating in the blue (438nm ± 30 nm) and infrared (938 nm ± 20 nm) regions of spectrum is proposed. The corresponding prototype hardware and software for measurements have been developed and tested in laboratory. Realtime measurements of finger capillary refill kinetics by this technology have been taken and analyzed. Results demonstrated that both AC and DC components of the blue PPG biosignal are sensitive to capillary occlusion and refill.

The biofilm formed by Candida albicans is the mainly cause of infections associated to medical devices such as catheters. Studies have shown that photodynamic antimicrobial therapy (PAT) has lethal effect on C. albicans, and it is based on photosensitizer (PS) in the presence of low intensity light to generate reactive oxygen species in biological systems. The aim of this study was to analyze in real time, by Optical Coherence Tomography (OCT), the alterations in C. albicans biofilm in vitro during PAT using methylene blue (MB) as a PS and red light. An OCT system with working at 930nm was used, sequential images of 2000×512 pixels were generated at the frame rate of 2.5frames/sec. The dimension of the analyzed sample was 6000μm wide by 1170μm of depth corrected by refraction index of 1.35. We recorded 1min. before and after the irradiation with LED for PAT, generating 8min. of video. For biofilm formation, discs were made from elastomeric silicone catheters. The PS was dissolved in PBS solution, and a final concentration of 1mM MB was applied on biofilm, followed by a red LED irradiation (λ=630nm±20nm) during 6min. We performed a curve of survival fraction versus time of irradiation and it was reduced by 100% following 6min. of irradiation. OCT was performed for measurement of biofilm thickness of 110μm when biofilm was formed. During irradiation, the variation of biofilm thickness was ~70μm. We conclude that OCT system is able to show real time optical changes provided by PAT in yeasts organized in biofilm.

Monitoring of the vessel capillary density and blood oxygenation spatial pattern in epithelium is important for shock detection and it's preventing interventions. The challenge is the complex epithelium structure, absorption and scattering of light in the multiple tissue layers. In this paper we describe results of estimating the vessel capillary density pattern in tissue phantoms collecting spatially resolved reflectance spectra. We designed tissue phantom which mimics optical properties of epithelium including microvasculature. Grids grooved in the phantom plates mimicking the blood capillary network containing freshly prepared oxy-hemoglobin solution. The preliminary results shows that the new method can reasonably extracts minor spatial deviations of oxygenation and local volume blood fraction - parameters, directly related to the local vessel density patterns.

The human eye moves continuously even while it appears to be at rest. The involuntary eye movements causing this motion are called fixational eye movements. Ocular Microtremor (OMT) is the smallest (150 - 2500nm amplitude) and fastest (~ 80Hz) of these eye movements. OMT has been proven to provide useful clinical information regarding depth of consciousness and neurological disorders. Most quantitative clinical investigations of OMT have been carried out using an eye-contacting piezoelectric probe. However, this measurement procedure suffers from a number of disadvantages which limit the potential of the technique in the clinical environment. The need for eye contact requires the eye to be anaesthetised and not all subjects can tolerate the procedure. A promising alternative to the piezoelectric technique is speckle metrology. A speckle correlation instrument for measuring OMT was first described by Al-Kalbani et al. The approach presented in this paper is a non contact measurement technique implementing laser speckle correlation and using a highly light sensitive video camera operating at 500Hz. The OMT measurement technique in this paper was investigated using a human subject and an eye movement simulator. Using this system, measurement of speckle on the eye takes only a few minutes, no eye drops are necessary and no discomfort is caused to the subject. The paper describes the preliminary results of capturing speckle from the simulator and from the human eye in-vivo at eye safe laser powers. The effects of tear flow, biospeckle and speckle shifting by larger eye movements on the displacement information carried by the speckle are also discussed.

Starch is one of the major constituents of our everyday diet and it forms granules. Starch granules are basically consisting of amylose and amylopectin molecules and they are among the brightest nature-made second harmonic generation (SHG) converters. In this study, we take advantage of that and we perform polarization sensitive SHG (PSHG) imaging of starch granules. We fit the SHG signal variation of each pixel of the PSHG images into a biophysical model and we extract molecular information below the experimental resolution limit. Specifically, by assuming that the SHG source molecule is a helix with cylindrical symmetry along its long axis, the model extracts the helical pitch angle of the SHG source and the orientation of its supporting filament for every pixel of the image. Pixel by pixel fitting consequently creates new images which their contrast is based on the values of the fitting to the theoretical model. Then we chose a region of interest in the image and we create pixels' values histograms. We applied the above in wheat starch granules and we found a highly peaked pixels' histogram of helical pitch angles at θe = 36.2°. This pitch angle corresponds to the strand of the parallel double helical structure, called amylopectin (as measured by small angle X-ray scattering). Thus, using an optical technique we extracted the helical pitch angle of amylopectin in starch. This angle value can be used as a quantitative biomarker capable of characterizing the quality of starch based structures and products.

Polarization sensitive second harmonic generation (PSHG) imaging can provide useful information which is unreachable by intensity SHG imaging. Specifically, it can provide geometrical characteristics of the SHG source molecular architecture. The information is obtained by rotating the excitation linear polarization and by fitting the SHG intensity variation to a cylindrical symmetry biophysical model. As a result, the ratios of the non-vanishing χ² tensor elements, responsible for the SHG conversion, are retrieved. In the end, by assuming a SHG source with dominant hyperpolarizability, its molecular orientation can be estimated. Here, we developed and used this approach to retrieve submicron structural information from cultured neurons and to provide estimation on the effective orientation of the molecular SHG source in axons. For that purpose, the PSHG images of axons were fitted pixel by pixel using an algorithm based on the above mentioned model. A coefficient of determination of r2>90% was chosen as a filtering mechanism. For a selected region of interest we then retrieved the pixels' values histogram of the harmonophores' effective orientations, θe. The distribution was centred at θe=34.93°, with σ=7.62°. These angle values correspond to the geometrical characteristics of the tubulin heterodimmers forming the microtubules. Modifications on tubulin dimers may alter θe, σ thus the PSHG optical technique suggests novel quantitative biomarkers able to characterize neurons' plasticity as well as disease progression.

Imaging endoscopes have enabled the development of minimally invasive procedures in a wide range of medical applications. Flexible endoscopes have additional advantages over rigid endoscopes. Remarkably, they have an enhanced capability of being guided through the internal conduits of the human body. The development of imaging endoscopes based on coherent optical fiber bundles have made high resolution fiber endoscopy possible. In the last years, multicore fibers have been proposed as an alternative to coherent fiber bundles for high resolution applications. Both types of structures entail several limiting factors. Among them, the most critical one is the optical crosstalk that takes place between the parallel contiguous fibers of the device, which provokes a worsening in the contrast of the images. Therefore it imposes a limit to the quality of the endoscopic system that must be avoided. In this work, we present a theoretical model for the study of optical coupling in multicore fibers, which is based on an electromagnetic optics approach. This model is applied to the analysis of crosstalk within fiber imaging endoscopes. It includes the effect of core non-homogeneities and bendings. The essential equations of the model will be shown. These equations provide us with a theoretical basis that is subsequently applied to fiber endoscopes design. Therefore, we present a robust method for the adjustment of opto-geometrical parameters of the fiber in order to fulfil the quality requirements for a certain application. The key role of core diameter variations in the quality of the image will be specially highlighted.

This paper describes the development of an early detection method for probing pathological tissue variations. The method could be used for classifying various tissue alteration namely tumors tissue or skin disorders. The used approach is based on light scattering and absorption spectroscopy. Spectral content of the scattered light provides diagnostic information about the tissue contents. The importance of this method is using a safe light that has less power than the used in the imaging methods that will enable the frequent examination of tissue, while the exiting modalities have drawbacks like ionization, high cost, time-consuming, and agents' usage. A modality for mapping the oxygen saturation distribution in tissues noninvasively is new in this area of research, since this study focuses on the oxygen molecule in the tissue which supposed to be homogenously distributed through the tissues. Cancers may cause greater vascularization and greater oxygen consumption than in normal tissue. Therefore, oxygen existence and homogeneity will be alternated depending on the tissue state. In the proposed system, the signal was extracted after illuminating the tissue by light emitting diodes (LED's) that emits light in two wavelengths, red (660 nm) and infrared (880 nm). The absorption in these wavelengths is mainly due to oxyhemoglobin (HbO₂) and deoxyhemoglobin (Hb) while other blood and tissue contents nearly have low effect on the signal. The backscattered signal which is received by a photodiodes array (128 PDs) was measured and processed using LabVIEW. Photoplethysmogram (PPG) signals have been measured at different locations. These signals will be used to differentiate between the normal and the pathological tissues. Variations in hemoglobin concentration and blood perfusion will also be used as an important indication feature for this purpose.

A well established navigation method is one of the key conditions for successful brain surgery: It should be accurate, safe and online operable. Recent research shows that Optical Coherence Tomography is a potential solution for this application by providing a high resolution and small probe dimension. In this study a fiber Spectral-Domain OCT system with a super luminescent diode with the center wavelength of 840 nm providing 13.6 μm axial resolution was used. A single mode fiber (Ø 125 μm) was employed as the detecting probe. The information acquired by OCT was reconstructed into grayscale images by vertically aligning several A-scans from the same trajectory with different depth, i.e. forward scanning. For scans of typical white matter, the images showed a higher reflection of light intensity with lower penetration depth as well as a steeper attenuation rate compared to the scans typical for grey matter. Since the axial resolution of this OCT system is very high, some microstructures lying on the striatum, hippocampus and thalamic nucleus were visible in these images. The research explored the potential of OCT to be integrated into a stereotactic surgical robot as a multi-modal navigation method.

An "in vitro" study of Raman spectra from oral human tissues is reported in order to the develop a diagnostic method suitable for "in vivo" oral pathology follow-up. The investigated pathology is Pemphigus Vulgaris (PV) for which new techniques for guiding and monitoring therapy would be particularly useful. Raman spectra were obtained in the wavenumber regions from 1000 to 1800 cm^-1 and 2700 to 3200 cm^-1 from tissues from patients at different stages of pathology (active PV, under therapy and in PV remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage arise in 1150-1250 cm^-1 (amide III) and 1420-1450 cm^-1 (CH₃ deformation) regions and around 1650 cm^-1 (amide I) and 2930 cm^-1 (CH₃ symmetric stretch). A wavelet deconvolution procedure was applied to the spectra for better discriminating among the three different stages of illness and a linear regression analysis was used to fully exploit the content of information of Raman spectra.

Early detection of coronary atherosclerosis is an unmet clinical challenge. The detection system has to be highly sensitive and possess high spacial resolution, in order to provide precise information of the vulnerable plaque location and size. Recently molecular fluorescence probes have been identified as efficient inflammation biomarkers for the inflammation process within vulnerable plaques¹ and being used in the proposed application to detect inflamed lesions in the blood vessel wall. The general principle of the proposed solution is based on a sensor whose head is guided by an intravascular catheter to the region of interest (coronary artery). When the sensor illuminates an activated fluorescent probe, located in inflamed areas of vulnerable plaques, the fluorescence is excited and light is emitted with a slightly shifted spectrum. The emitted light is being collected by the same sensor head, guided through the optical fiber and finally detected by photo-detectors. In this way, by detecting emitted fluorescence one can obtain information about the location of vulnerable plaques. The localization resolution is critically depending on the spot size of the illuminating light beam. Moreover, for a high signal to noise ratio in the detection electronics, as much fluorescent light as possible has to be collected from the plaque location. It has been already demonstrated that using single-mode fibers in combination with graded index fibers, a Gaussian beam, with adjustable waist position and diameter can be formed, representing the fundamental limit of achievable spot size². However, when using single mode fibers in this application, the collection efficiency would be very low due to the small core diameter of this fiber and thus signal to noise ratio would be strongly reduced. In this work, we present a solution to this challenge, combining both principles. A single mode fiber in combination with a graded index fiber is used for illumination purposes, while the fluorescence light is collected by the same fiber, but employing the cladding/coating total reflection to form a multimode fiber for the backwards propagating light. Thus, a narrow spot size can be obtained allowing high resolution images, with high signal to noise ratio due to the multimodal collection scheme. We show preliminary results of spot size and beam diameter measurements from the sensor head and discuss the implication for the improvement of the current catheter-based detection systems.

Doppler Optical Coherence Tomography (DOCT) is a modern technique used for accurate measurements of blood flow in the superficial layers of human skin, retina or other tissues and their phantoms. In this work, we considered the effect of both static and dynamic superficial layer of the scattering medium on the measured velocity of a flow located beneath this layer. In the case of static layer a tissue phantom consisting of a plain glass capillary (inner size 0.3 × 3 mm) embedded into a slab of Intralipid solution mimicking human skin was designed. Flow velocity profiles were measured at different embedding depths and Intralipid concentrations. The obtained results show a decrease in the measured peak velocity value of the flow in the embedded capillary with increasing the embedding depth and/or concentration of the Intralipid solution in the static layer. A dynamic superficial layer was considered in the case with two plain glass capillaries (inner size 0.2 × 2 mm) attached together. Flow rate of the lower capillary was fixed to 100 ml/h, while the parameters of the upper flow were varied (concentration from 1 % to 4 % and flow rate from 0 to 200 ml/h). The results obtained with the above parameters do not show significant distortions in the measured flow velocity profile, only false velocity peaks arising at the rear flow boundaries.

Optical coherence tomography (OCT) is a technique, capable of high resolution and non-invasive 3D imaging in vivo by detection of backscattered light from cellular and sub cellular structures. Due to visualization of micrometer sized tissue constituents and high penetration depths of up to 2 mm, it is already well established in medical fields like ophthalmology and dermatology. Laser scanning confocal microscopy (LSCM), on the contrary, gives further information on structural tissue components stained with suitable dyes. In combination, these two methods yield three dimensional and high resolution data providing geometrical and structural details of tissue. In this study, we present simultaneous OCT and LSCM image acquisition resulting in a lateral resolution of better than 6.2 μm for OCT and 0.8 μm for LSCM, respectively. The axial resolution of the OCT amounts to 8 μm. Two laser lines, 488 nm and 561 nm, are combined to provide fluorescence excitation of green and red dyes. By using a long working distance objective, it is possible to perform experiments on bulky specimens like isolated organs or animal models in vivo. First studies indicate the ability to identify strains of elastic fibers within lung tissue in combination with the three dimensional morphology of the lung.

Clustering of receptor and signalling molecules (such as CD95 or CD40) within ceramide-enriched membrane domains results in a very high density of these proteins facilitating activation of associated enzymes, the exclusion of inhibitory molecules and/or the recruitment of further signalling molecules to transmit the signal into the cells. However, at present the mechanisms of receptor clustering and the exact distribution of proteins within the ceramide-enriched domains are unknown. Therefore, we generated digital images from anti-CD95 stimulated JYcells that were stained with FITC-coupled anti-ceramide and Cy3-labelled anti-CD95 antibodies. We developed image processing methods to determine the spatial distribution of proteins in ceramide-enriched membrane domains and visualized them by volume rendering and surface models. After image preprocessing with appropriate filters for contrast enhancement, noise reduction and logarithmic scaling, 3D models were generated using adapted volume and surface reconstruction. To detect the colocalization of CD95 and ceramide molecules we developed several different methods rasterizing 3D data of each channel into cells and counting intensity values above a specified colour threshold value. The colocalization voxel was set either by normalized product of totals (product intensity) or depending on binarization. In addition, a cross-covariance function to quantify the colocalization was determined and embedded as a 3D object. These computerized techniques allowed for a quantitative analysis of the spatial arrangement of proteins in ceramide-rich domains of living cells.

We studied scars and wounds depths and surfaces thanks to our interferometric fringes projector 3D scanner1, 2. Color information of a wound indicates its deterioration level. That's why the visual color restitution, as realistic as possible, is a highly important parameter. Firstly our acquired 3D pictures were color mapped with an image recorded by a RGB camera. The results were not efficient enough. In order to improve our technique and provide more precise information, we add a spectral characterization to the set-up. Before adding the spectral information and a realistic color mapping to the 3D measurements, we evaluate the performances of colorimetric measurements. The tests have been made on mice with scars on their back.

We present a novel implantable multi-wavelength reflectance sensor for the measurement of blood pressure with pulse transit time (PTT). Continuous long-term monitoring of blood pressure and arterial oxygen saturation is vital for medical diagnostics and the ensuing therapy of cardiovascular diseases. Conventional cuff-based blood pressure monitors do not provide continuous data and put severe constraints on the patients' daily lives. An implantable sensor would eliminate such problems. The new biocompatible sensor is placed subcutaneously on blood perfused tissue. The PTT is calculated by photoplethysmograms and the ECG-signal, that is recorded with intracorporal electrodes. In addition, the sensor detects the arterial oxygen saturation. An ensuing spectralphotometric analysis of the light intensity changes delivers data on the concentration of dysfunctional hemoglobin derivatives. Experimental measurements showed a clear correlation between the estimated PTT and the systolic blood pressure reference. These initial results demonstrate the potential of the sensor as part of an fully implantable sensor system for the longterm-monitoring of cardiovascular parameters.

Photodynamic Therapy (PDT) is a therapeutic technique widely used in dermatology to treat several skin pathologies. It is based in topical or systemic delivery of photosensitizing drugs followed by irradiation with visible light. The subsequent photochemical reactions generate reactive oxygen species which are considered the principal cytotoxic agents to induce cell necrosis. In this work we present a PDT model that tries to predict the photodynamic effect on the skin with a topically administered photosensitizer. The time dependent inhomogeneous distribution of the photoactive compound protoporphyrin IX (PpIX) is calculated after obtaining its precursor distribution (Methyl aminolevulinate, MAL) which depends on the drug permeability, diffusion properties of the skin, incubation time and conversion efficiency of MAL to PpIX. Once the optical energy is obtained by means of the Beer Lambert law, a photochemical model is employed to estimate the concentration of the different molecular compounds taking into account the electronic transitions between molecular levels and particles concentrations. The results obtained allow us to know the evolution of the cytotoxic agent in order to estimate the necrotic area adjusting parameters such as the optical power, the photosensitizer concentration, the incubation and exposition time or the diffusivity and permeability of the tissue.

Based on experimental analyses of colon and rectal tissues by THz spectroscopy and THz imaging, we show it is possible to distinguish between healthy and cancerous zones. Plots of the absorption coefficient and the index of refraction of the healthy and cancer affected tissues as well as 2-D transmission THz images will be presented. The experimental results will be discussed and the conditions for the tissues discrimination will be established.

Optical coherence tomography (OCT) is a technique of choice for micrometer-scale resolution imaging of biological specimens [1,2]. Full-field optical coherence tomography (FF-OCT) was introduced a few years ago as an alternative method to conventional OCT. FF-OCT is based on an interference microscope with a camera as an array detector combined with a low coherence illumination source for parallel acquisition of en-face oriented tomographic images [3- 5]. FF-OCT is a technique of choice for noninvasive three-dimensional imaging of ex vivo biological tissues with ultrahigh spatial resolution (~ 1 μm) [6,7]. FF-OCT is based on phase-shifting interferometry: several interferometric images are acquired with an image sensor, a phase-shift being introduced between each of these frames by using, for example, the displacement of the reference mirror. The amplitude of the interference signal, i.e. the fringe envelope, is calculated by combination of these frames [8-11]. Recent developments in OCT technology have been carried out in order to exploit the spectroscopic response of the imaged sample. This technique, referred to as spectroscopic OCT, detects and processes the interferometric signal to provide spatially-resolved spectroscopic information. It can be used to enhance image contrast, permitting better differentiation of tissues through their spectroscopic properties and providing additional information on the sample composition [12-14]. An alternative method to take advantage of the spectroscopic response of the sample is to image at several distinct wavelengths. This can be achieved by using several detectors [15,16] or several illumination sources. Spectroscopic imaging with FF-OCT, using several detectors, is demonstrated in this paper.

Biomedical imaging by means of nonlinear Raman microspectroscopy offers novel approaches towards individualized decisions on therapy routines as diagnosis is possible on the chemical composition of tissue and cells even during the pre-clinical phase of a disease. As Raman-based microspectroscopy allows for non-invasive, contact-free and label-free investigation of living tissue not disturbed by the presence of water, it contains high potential for biomedical diagnostics. Our experimental approach towards CARS-based in vitro tissue characterization is discussed. The focus lies on the joint use of linear Raman microspectroscopy and CARS microscopy. While linear Raman microspectroscopy is used to obtain the information on critical Raman marker bands at selected spatial positions within the sample, CARS microscopy is focussing on fast image generation using the previously defined Raman marker bands. Complementarily, secondharmonic generation imaging was applied to the samples. Furthermore, we present innovative concepts for CARS-image analysis.

We discuss the combination of a CARS-imaging system with microfluidics. Such system is a versatile tool to quantify the relative contributions of resonant and non-resonant scattering at the CARS frequency. We will show that the twochannel microfluidic chip employed in combination with deuterated isotopomers as an internal standard allows for fast and quantitative detection of organic molecules by CARS microscopy. The experimental design enables the simultaneous measurement of both the chemically relevant Raman-resonant signal and the non-Raman-resonant background.

The multi-spectral imaging technique has been used for distant mapping of in-vivo skin chromophores by analyzing spectral data at each reflected image pixel and constructing 2-D maps of the relative concentrations of oxy-/deoxyhemoglobin and melanin. Instead of using a broad visible-NIR spectral range, this study focuses on narrowed spectral band 500-700 nm, so speeding-up the signal processing procedure. Regression analysis confirmed that superposition of three Gaussians is optimal analytic approximation for the oxy-hemoglobin absorption tabular spectrum in this spectral band, while superposition of two Gaussians fits well for deoxy-hemoglobin absorption and exponential function - for melanin absorption. The proposed approach was clinically tested for three types of in-vivo skin provocations - ultraviolet irradiance, chemical reaction with vinegar essence and finger arterial occlusion. Spectral range 500-700 nm provided better sensitivity to oxy-hemoglobin changes and higher response stability to melanin than two reduced ranges 500-600 nm and 530-620 nm.

This paper presents the experimental analysis of different pathologically modified tissues, using the ultra-short laser polarimetry. Any changes produced in the polarization state of a light beam passing through a sample that posses a polarization inhomogeneous structure, will depend on both the interfaces and the internal structures of the respective sample. The ultra-short laser polarimetry, as a new polarization imagery technique, is able to provide more in-depth increased resolution images of the samples.

Spatially-spectrally-resolved reflectance measurements allow in vivo measuring the optical coefficients of absorption and scattering within the cortical tissue. This method, if applied to neural tissue during enhanced activity, could allow a straightforward monitoring of the blood oxygen saturation changes occurring in the brain cortex during hemodynamic responses. Furthermore, it may provide valuable information on possible absorption and scattering changes occurring during stimulation. The feasibility of such measurements was investigated by carrying a preliminary numerical study using a Monte-Carlo light propagation routine. Experimental parameters such as the geometry of the optical probe, baseline cortex optical coefficients retrieved from the literature and anatomical characteristics of the rat barrel cortex were used as an input for the simulations. The sensitivity of the probe to the local variations of optical coefficients was investigated with this numerical approach. Additionally, the influence of the barrel cortex dimensions and the probe positioning relatively to the activated region were studied for instrumental optimization purpose. It was found that typical variations of optical coefficients can be detected if the activated region of barrel cortex has a volume of typically 1 mm3 or larger. The decay of the probe sensitivity to changes was studied as a function of the depth of the activated region. The results showed that the best sensitivity is achieved by placing the light injection fiber of the optical probe aligned onto the center of the cylindrical barrel.

Plasmonic nanostructures exhibit a strong field enhancement due to the excitation of localized and propagating surface plasmon polaritons. The use of these effects yields in a wide range of analytical applications. For instance, the strong electromagnetic field enhancement may be used to dramatically increase fluorescence, Raman cross sections (surface enhanced Raman spectroscopy - SERS) or IR absorption. Since the requirements to a powerful technique are both a fingerprint specificity and high sensitivity, the SERS method is a powerful tool for a variety of analytical applications in (bio)chemical and biological analysis. Because the reproducibility of established SERS arrays (e. g. roughened metal electrodes and aggregated metal nanoparticle) across a large measuring area is rather low, we have established an e-beam (electron beam lithography) based fabrication process yielding in regularly patterned gold nanorhomb arrays. The anisotropic optical response of the SERS array is characterized. Furthermore, the SERS arrays are investigated with respect to the second part of their electromagnetic enhancement, resulting in design and fabrication criteria of potential SERS arrays.

Due to its enormous signal intensity and high fingerprint sensitivity, surface enhanced Raman spectroscopy (SERS) is a powerful technique in chemical and biological applications. Our goal is the employment of the SERS technique for (bio)analytical devices. A main feature in order to achieve a versatile applicability is to ensure a reproducible and homogeneous signal across large measuring areas. Electron beam lithography is an adequate approach to assemble such reproducible arrays. Within this contribution, the fabrication process of regular patterned nanostructures based on electron beam lithography and argon ion beam etching is described. Using the exposure of crossed gratings of lines within a resist layer, gold nanorhomb and nanorectangle arrays are produced on a quartz wafer. The patterns are periodically arranged with pitches of 200 to 400 nm and exhibit sharp edges with corner radii of less than 10 nm. The pattern dimensions in combination with the small edge radii yield a high electromagnetic field enhancement caused by plasmonic excitation. The SERS arrays were characterized by means of SEM and were tested with respect to their SERS response, especially with regard to their reproducibility.

We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

The broad application of surface-enhanced Raman spectroscopy (SERS) is greatly hampered by the lack of reliable and reproducible substrates; usually the activity of a given substrate has to be determined by time-consuming experiments such as calibration studies or ultramicroscopy. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably characterizable with a technique that does not interfere with the subsequent measurements. Here, we introduce an innovative approach to produce low cost and large scale reproducible substrates for SERS applications, which allows an easy and economical production of micropatterned SERS active surfaces based on an enzyme induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities and exhibits a relationship between electrical conductivity and resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale. Furthermore, the presented substrate shows a high reproducibility and is appropriate for various applications.

Toxic effects of nanoparticles can be analyzed with alveolar macrophages in vitro. To quantify exposure of cells to particles we analyzed the sedimentation of nanoparticle agglomerates in cell culture medium (MEM) by means of phase contrast microscopy. Particles were suspended by brief ultrasonication in MEM and pipetted into a glass bottom culture dish on the stage of a Nikon-Biostation under cell culture condition. Successive images were captured from the lowermost optical plane and were converted into binary images. The number of agglomerates (N) as well as the particle-covered area (A) were determined by image analyses. Typically, N increased to a maximum value before it partially decayed due to overlapping and/or optical interference of particles, and finally became constant. In contrast, A increased in a monophasic manner. By means of mathematical modeling we identified the endpoint of sedimentation of particle agglomerates, which is an important though a largely neglected event in most cell culture experiments. This endpoint could be calculated from an approximated model function. As the method can be employed in the presence of cells, a parallel evaluation of particle sedimentation and particle uptake appears possible.

Conventional laser light sources for the treatment of a hard tissue in dental (Er:YAG laser, Er,Cr:YSGG laser and CO₂ laser etc.) are good for removal of caries. However these lasers cannot achieve to give a selective treatment effect for caries without a side effect for normal tissue. The objective of this study is to develop the less-invasive treatment technique of carious dentin by selective absorption effect using the laser with a wavelength of 6.02 μm which corresponds to an absorption peak of organic matters called amide 1 band. Mid-infrared nanosecond pulsed laser by difference-frequency generation was used for the experiment of selective treatment. A tunable wavelength range, pulse width and repetition rate is from 5.5 to 10 μm, 5 ns and Hz, respectively. The laser with a wavelength of 6.02 μm and predetermined energy parameters was irradiated to the plate of carious dentin model which is made by soaking in lactic acid solution. After laser irradiation, the surface and cross-sectional surface of samples were observed by a scanning electron microscope (SEM). Average power density about 15 W/cm² realized to excavate a demineralized region (carious dentin model) selectively in a SEM observation. In the same energy condition, serious side effect was not observed on the surface of normal dentin. A wavelength of 6.02 μm realizes a selective excavation of carious dentin. Using 6.02 μm is a novel and promising technique toward to next-generation dental treatment procedure for realizing MI.

Nanoparticles of titanium dioxide (TiO₂) and zinc oxide (ZnO) are used in sunscreens as protective compounds against UV radiation. We investigate these particles from the viewpoint of nanosafety (penetration into skin in vivo, production of free radicals when UV-irradiated) as well as UV protection. We show that: a) even after multiple applications, the particles remain within stratum corneum (uppermost skin layer); b) the optimal sizes are 62 nm and 45 nm, respectively for TiO₂ and ZnO particles for 310-nm light and, correspondingly, 122 and 140 nm - for 400-nm radiation; c) in general, small particles (25 nm in diameter) are more photoactive than the larger ones (400 nm in diameter); however, on the background if porcine skin in vitro this difference is not seen and is substantially surpassed by skin contribution into production of free radicals.

Digital Holographic Microscopy (DHM) has been used to investigate the spontaneous cell membrane fluctuations (CMF) of the Red Blood Cell. DHM as an interferometric technique is able to accurately provide the wavefront deformation induced by a transparent specimen, including living cells in a transmission configuration. From a numerical reconstruction of a single hologram, quantitative phase contrast images are obtained. The local phase shift is proportional to the specimen thickness with accuracy of 5-10 nm. As a non invasive full field technique DHM is particularly well suited to assess and study membrane fluctuations of a large number of cells simultaneously. In our analysis we show that CMF amplitudes are unhomogenously distributed on the cellular surface and seem to correlate with the biconcave equilibrium shape of erythrocytes. A mean fluctuation amplitude of 47 nm is measured in a group of 198 erythrocytes.

The effective irradiance is a useful measure to compare performances of different broadband light sources and to more precisely predict the outcome of a topical photodynamic therapy. The effective irradiance (or effective fluence rate) and the exposition time of the optical radiation usually determine the light dose. The effective irradiance (E_eff) takes into account the spectral irradiance of the source as well as the action spectrum, where the wavelength dependence of both optical diffusion through tissue and photosensitizer are considered. In practice there are no standard action spectra for the currently used photosensitizers. As a consequence, measured values of effective irradiance using different action spectra can not be compared. In order to solve this problem, the basis of the calibration theory developed for the broadband ultraviolet radiometry can be applied, where an experimental radiometer is compared with a standard radiometer. Here is presented a simple set of linear relations in the form E_eff = k . E, where E is the source irradiance and k a real positive value, here denoted as a characteristic of the radiometer, as valuable tools for correction of effective irradiances measured according to different action spectra. As a result, for two effective radiometers with different characteristics k₁ and k₂, measured values are Eeff and Qeff respectively, and it is easily shown that the value E_eff = Q_eff • k₁/k₂ .

Biosensors are finding numerous applications in clinical diagnosis, drug discovery, biotechnology, environmental monitoring and etc. Hemoglobin (Hb), a natural heme containing protein, exhibits enzymatic activity towards hydrogen peroxide, which is possible to improve by altering the heme orientation and/or changing the microenvironment in the vicinity of the heme sites. It was shown that hypericin (HY), a naphthodianthrone from Hipericum perforatum and a potent photosensitizer, interacts with Hb and causes conformational changes of the protein. These results were gained both in dark and under visible light exposure by absorption and fluorescence spectroscopy. It was shown that photodynamic influence of HY leads to Hb absorption decrease at Soret band, depending on HY concentration and irradiation doze. Excitation of Hb/HY complexes leads to reduction of some emission peaks, correlating with the concentration of HY, incubation and irradiation time. The incubation and irradiation of complexes leads to an increase in electrophoretic mobility of Hb and its peroxidase activity. Under the HY influence Hb properties as a hydrogen peroxide detector could be improved and an effective determination of peroxide formation could be achieved. This makes Hb an attractive 'recognition' element for construction of third-generation biosensors.

The investigation of materials suitable for enzyme immobilization in biosensing applications has a widespread interest. There are many studies on physico-chemical properties of these materials at macroscopic level but few studies have been devoted to examine and correlate these properties at microscopic level. FT-IR spectroscopy with Micro-Attenuated Total Reflection (Micro-ATR) approach can be extremely useful for understanding a variety of aspects of materials which can be used for optimising immobilization procedures. Moreover, this experimental approach is particularly simple to use (no sample preparation is required) and minimally invasive. Using a Perkin Elmer Spectrum One FT-IR spectrometer equipped with a mercury-cadmium-telluride detector and a micro-ATR element we investigated different materials used for immobilization procedures with various enzymes widely used for biosensing in environmental and clinical applications. In particular, composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus layers of tethered chitosan of different molecular weight have been examined. Also silica gel matrices without and with glucose oxidase have been investigated. Spectra have been analysed and the contribution of principal functional groups has been evidenced.