Technological innovations of the 20^th century provided medicine and surgery with new tools, among which virtual reality and robotics belong to the most revolutionary ones. Our work aims at setting up new techniques for detection, 3D delineation and 4D time follow-up of small abdominal lesions from standard mecial images (CT scsan, MRI). It also aims at developing innovative systems making tumor resection or treatment easier with the use of augmented reality and robotized systems, increasing gesture precision. It also permits a realtime great distance connection between practitioners so they can share a same 3D reconstructed patient and interact on a same patient, virtually before the intervention and for real during the surgical procedure thanks to a telesurgical robot. In preclinical studies, our first results obtained from a micro-CT scanner show that these technologies provide an efficient and precise 3D modeling of anatomical and pathological structures of rats and mice. In clinical studies, our first results show the possibility to improve the therapeutic choice thanks to a better detection and and representation of the patient before performing the surgical gesture. They also show the efficiency of augmented reality that provides virtual transparency of the patient in real time during the operative procedure. In the near future, through the exploitation of these systems, surgeons will program and check on the virtual patient clone an optimal procedure without errors, which will be replayed on the real patient by the robot under surgeon control. This medical dream is today about to become reality.

In the field of dentistry, effectiveness of USPL irradiation is researched because USPL has less thermal side effect to dental hard tissue. In this paper, we observed morphological change and optical change of dental hard tissue irradiated by USPL for discussing the safety and effectiveness of USPL irradiation to dental hard tissues. Irradiated samples were crown enamel and root dentin of bovine teeth. Lasers were Ti:sapphire laser, which had pulse duration (P_d)of 130 fsec and pulse repetition rate (f) of 1kHz and wavelength (l) of 800nm, free electron laser (FEL), which had P_d of 15 μsec and f of 10Hz and wavelength of 9.6μm, and Er:YAG laser, which had P_d of 250 μsec and f of 10Hz and wavelength of 2.94μm. After laser irradiation, the sample surfaces and cross sections were examined with SEM and EDX. The optical change of samples was observed using FTIR. In SEM, the samples irradiated by USPL had sharp and accurate ablation with no crack and no carbonization. But, in FEL and Er:YAG laser, the samples has rough ablation with crack and carbonization. It was cleared that the P/Ca ratio of samples irradiated by USPL had same value as non-irradiated samples. There was no change in the IR absorption spectrum between samples irradiated by USPL and non-irradiated sample. But, they of samples irradiated by FEL and Er:YAG laser, however, had difference value as non-irradiated samples. These results showed that USPL might be effective to ablate dental hard tissue without thermal damage.

In this paper we present an original approach to laser welding of ocular media. Attention is focused on laser welding of the cornea and lens capsule. The process is based on the interaction of near infrared diode laser radiation (at 810 nm) with tissue that was previously stained with an Indocyanine Green solution in sterile water. The topical application of the chromophore makes possible a selective heating of the tissue, which results in a homogenous welding effect with low thermal damage to the surrounding tissue. Experimental tests were performed ex vivo on both capsule and cornea, and in vivo (rabbits) only on the cornea, in order to characterize the process as a whole. Spectrophotometric, biomechanical, and thermal measurements were carried out in order to study the laser-tissue interaction, while morphological, histological and auto-florescence microscopy analyses made during a follow-up study provided information on the healing process in welded rabbit corneas. The welding procedure was set up according to the type of tissue, with the staining procedure and irradiation conditions being optimized in each case. Our test indicated that: 1) laser welding of corneal wounds, which is a non contact technique performed at low continuous wave laser power (12 W/cm²), can be proposed as a support to or substitute for the standard suturing technique in cataract surgery and in penetrating keratoplasty (in corneal transplants); 2) laser welding of the lens capsule requires a "contact irradiating technique" in order to be efficiently performed, since the tissue is in underwater conditions, with single spot pulses of about 100 J/cm² fluence and pulse duration around 100 ms. In the latter case, laser welding was proposed as a tool for assisting closure of the lens capsule after the lens refilling procedure (Phaco-ersatz), or for repairing capsular breaks induced by accidental traumas or produced intraoperatively.

Introduction: One of the most appreciated features of the sperm is its motility, which depends on a big energy consumption despite differences among species. Laser acts direct or indirectly on mitochondria increasing ATP production. Material and method: By means of a Computer Aided Sperm Analysis (CASA) we have studied the effects of a 655 nm continuous wave diode laser irradiation at different power outputs with a dose of 3.3418 J on sperm motility. After an eosine-nigrosine stain to establish its quality, the second fraction of fresh beagle dog sperm was divided into 5 groups, 1 control and four to be irradiated respectively with an average output power of 6.84 mW, 15.43 mW, 33.05 mW and 49.66 mW. At times 0 and 45 minutes from irradiation pictures were taken and analysed with the Sperm class Analyzer SCA2002 programme. The motility parameters of 4987 spermatozoa studied were: curvilinear velocity (VCL), progressive velocity (VSL), straightness (STR), wobble (WOB), average path velocity (VAP), linearity (LIN), mean amplitude of lateral head displacement (ALHmed), beat cross frequency (BCF) and the total motility (MT). At time 15 minutes after irradiation a hypoosmotic swelling test (HOST) was done. Results: Several motility parameters that affect the overall motile sperm subpopulation structure have been changed by different output powers of a 655 nm diode laser irradiation, and prevents the decrease of the sperm motility properties along time.

During the last couple of years new imaging techniques using femtosecond lasers (fs-lasers) in the near infrared spectral range evolved for a variety of in vitro applications. We wanted to know, whether fs-lasers have a non-invasive imaging potential for in vivo applications for human skin. So far, little is known about possible risks of this irradiation type. To estimate the risk of irradiation damage in human skin we used a "biological dosimeter" in this investigation. We irradiated fresh human skin samples with both an fs-laser and a solar simulator (UV-source) for comparison. DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immuno-fluorescence image analysis. Various fs-irradiation regimes were evaluated differing in laser power and step width of horizontal irradiation scans. When using 15 mW or 30 mW fs-laser power combined with horizontal irradiation scans applied every 5 mm in depth around the epidermal-dermal junction no induction of CPDs was found. However, induction of CPDs could be seen using 60 mW laser power and 5 μm step width. Narrowing the step width to 1 mm and using increasing laser power (up to 35 mW) from the surface of the skin to the epidermis led to CPD formation, too. Quantitative comparison of CPD production at various laser regimes with CPD production using a solar simulator was done. We could show that the number of CPDs formed by the 60 mW laser irradiation regime is comparable to an UV-irradiation giving 0.6 MED (minimal erythemal dose). The smaller step width laser irradiation regime (1 μm step width and up to 35 mW) was comparable to a UV-irradiation regime resulting in 0.45 MED.

There is increasing interest in photoacoustic techniques for non-invasive soft tissue investigation, this in part is due to the successful in vivo trials and investigation using biomedical soft tissue phantoms. The work presented in this paper focuses on the construction and calibration of a semiconductor laser system for the generation of high power optical pulses required for photoacoustic signal generation in biomedical phantoms. Such energy levels are achieved using combination of commercial semiconductor laser sources. Results presented in this paper show that this combination process is effective and an efficient means to obtain photoacoustic signals in biomedical samples.

For many medical laser applications, a particular beam shape is required. The output beam of a laser can be approximated by a Gaussian, higher-order Gaussian, annular or a flat-top (uniform) distribution. Here, we investigate, analytically and experimentally, the effects of laser beam shapes on the depths of penetration in treatments of any types of vascular malformation. In order to do this, the physical and optical parameters of the skin must be known and measured correctly. Using the Monte-Carlo method for seven layers of skin, a software predicting the beam propagation and intensity distribution inside of tissue has been developed in our centre. In this paper, a 15 watts copper vapour laser producing (511nm and 578 nm) for treatments of patients having PWS (Port Wine Stains) of different sizes is employed. The output beam of this laser was Gaussian. We have designed a beam homogenizer converting a Gaussian beam into flat-top distribution. Therefore, the effects of the laser irradiance beam shape (before and after beam shaping) on the depth of penetration have been investigated before people's treatments. Initially, two laser beams having Gaussian output distribution of the same power are considered. The diameter of one beam is 5mm and the other one is 10 mm. The intensity distribution of these beam inside of similar tissues are predicted and it is concluded that for deep but small size PWS the Gaussian beam having smaller beam diameter is more suitable than the larger spot size. Then, the beam intensity distribution inside of the same tissue (similar parameters) for two flat-top beams of the same power but different diameters (one is 5mm and the other is 10 mm) is calculated. It can be seen that the flat top beam of bigger spot-size has smaller penetration depth but it illuminates a larger area uniformly (suitable for large but not deep area). The depth of penetration of flat-top beam with smaller spot size is deeper but it illuminates a smaller area uniformly and is suitable for treatments of patients with small but deep PWS. Using a low power laser, we have been able to image the PWS area and roughly determine the depths of malformations. Finally, depends on the size, depth and shapes of the area the required beam shape can be chosen. A clinical protocol is developed for 25 patients and it is shown an extreme improvement in related medical procedures.

In this work, we numerically show that a continuum of light, generated by a femtosecond laser and a microstrured fiber (MF) can be used as tunable source for STED microscopy. The envisaged setup is composed by a microscope on which such a continuum is inserted. The spectral excitation and STED bands selection is performed by a monochromator based on an acousto-optic tunable filter (AOTF). Pulse temporal shapes fluctuations and intensity fluctuations effects are investigated. Those fluctuations are consistent with the experimental measurements. Temporal variations have little influence on the STED efficiency, whereas intensity fluctuations have much more effects.

Membrane dynamics of human glioblastoma cells were investigated using the intercalating fluorescence marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). In particular its generalized polarization (GP), which describes a spectral shift depending on the phase of membrane lipids, was used as a measure of membrane stiffness, whereas its fluorescence lifetime τ and its rotational diffusion time t_r were used to characterize membrane fluidity. Upon excitation with linearly polarized pulsed laser light the parallel and perpendicular components of fluorescence from the sample were measured simultaneously using an imaging device with polarization sensitivity. So far, membrane dynamics depended on temperature and cell age as well as the on intracellular amount of cholesterol. In addition, the plasma membrane (assessed by illumination with an evanescent electromagnetic wave) appeared to be stiffer than intracellular membranes (assessed by epiillumination of the cells).

Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.

Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.

Fluorescence fluctuation spectroscopy is applied to study molecules, passing through a small observation volume, usually subjected to diffusive or convective motion in liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or imbedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres), DNA-chips, and dye-stained histidine molecules anchored on silanized glass surfaces.

The introduction of naked DNA or other membrane impermeable substances into a cell (transfection) is a ubiquitous problem in cell biology. This problem is particularly challenging when it is desired to load membrane impermeable substances into specific cells, as most transfection technologies (such as liposomal transfection) are based on treating a global population of cells. The technique of optical transfection, using a focused laser to open a small transient hole in the membrane of a biological cell (photoporation) to load membrane impermeable DNA into it, allows individual cells to be targeted for transfection, while leaving neighbouring cells unaffected. Unlike other techniques used to perform single cell transfection, such as microinjection, optical transfection can be performed in an entirely closed system, thereby maintaining sterility of the sample during treatment. Here, we are investigating the introduction and subsequent expression of foreign DNA into living mammalian cells by laser-assisted photoporation with a femtosecond-pulsed titanium sapphire laser at 800 nm, in cells that are adherent.

We present a method for aberration correction in a confocal microscope that successfully combines both a spatial light modulator and a deformable membrane mirror. An active locking technique is used that benefits from the fast update rate of the deformable membrane mirror and the large effective stroke of the spatial light modulator. Concentrating on defocus, we were able to track 'best focus' over a distance of 80 μm with a lock RMS precision of 57 nm. In principle, this technique can be applied to any Zernike mode or aberration that can be accurately reproduced on the deformable membrane mirror.

3-D optical fluorescence microscopy is an efficient tool for volumic investigation of biological samples. Nevertheless the image acquired by this way is altered by the properties of the microscope, according to its Point Spread Function (PSF). The aim of deconvolution algorithms is the reassignment of defocused information. This method provides an improvement in data quality and the possibility to compare specimens acquired using different systems. But deconvolution requires making a compromise between the precision of the result and the stability of the process, since this stability is directly related to the noise level of the data. This noise can be of different types, mainly electronic noise due to the sensors but we also include in the term "noise" the variation of fluorescence during the acquisition. Numerous deconvolution algorithms exist, giving variable results according to specimen characteristics. For the cases where deconvolution is not enough to obtain usable data, we developed some pre-process treatments. These tools can be used separately or consecutively depending on the application needs and specimen requirements.

Orthopedics and neurosciences are fields of medicine where the analysis of objective movement parameters is extremely important for clinical diagnosis. Moreover, as there are significant differences between static and dynamic parameters, there is a strong need of analyzing the anatomical structures under functional conditions. In clinical gait analysis the benefits of kinematical methods are undoubted. In this paper we present a 4D (3D + time) measurement system capable of automatic location of selected anatomical structures by locating and tracing the structures' position and orientation in time. The presented system is designed to help a general practitioner in diagnosing selected lower limbs' dysfunctions (e.g. knee injuries) and also determine if a patient should be directed for further examination (e.g. x-ray or MRI). The measurement system components are hardware and software. For the hardware part we adapt the laser triangulation method. In this way we can evaluate functional and dynamic movements in a contact-free, non-invasive way, without the use of potentially harmful radiation. Furthermore, opposite to marker-based video-tracking systems, no preparation time is required. The software part consists of a data acquisition module, an image processing and point clouds (point cloud, set of points described by coordinates (x, y, z)) calculation module, a preliminary processing module, a feature-searching module and an external biomechanical module. The paper briefly presents the modules mentioned above with the focus on the feature-searching module. Also we present some measurement and analysis results. These include: parameters maps, landmarks trajectories in time sequence and animation of a simplified model of lower limbs.

We combined a homebuilt multiphoton microscope and a prism-CCD based spectrograph to develop a spectral imaging system capable of imaging deep into live tissues. The spectral images originate from the two-photon autofluorescence of the tissue and second harmonic signal from the collagen fibers. A highly penetrating near-infrared light is used to excite the endogenous fluorophores via multiphoton excitation enabling us to produce high quality images deep into the tissue. We were able to produce 100-channel (330 nm to 600 nm) autofluorescence spectral images of live skin tissues in less than 2 minutes for each xy-section. The spectral images rendered in RGB (real) colors showed green hair shafts, blue cells, and purple collagen. Analysis on the optical signal degradation with increasing depth of the collagen second-harmonic signal showed 1) exponential decay behavior of the intensity and 2) linear broadening of the spectrum. This spectral imaging system is a promising tool for both in biological applications and biomedical applications such as optical biopsy.

Franz Joebsis first used near infrared spectroscopy (NIRS) as a tool for the in vivo monitoring of tissue oxygenation. Today, NIRS instruments are more and more used in clinical environments since these systems are now easy to use, sensitive, robust, give rapid analysis and have multiple measuring points. In the present work, optic fibre probes were used as optical head of a CW-NIR instrument adapted for in vivo NIRS measurements in the brain of rodents. This prototype was designed for non-invasive analysis of the two main forms of haemoglobin: oxy-haemoglobin (HbO₂) and deoxy-haemoglobin (Hb), chromophores present in biological tissues. In the present experiments it was applied to measure non- invasively HbO₂ and Hb levels in the rat brain; that are markers of the degree of tissue oxygenation, thus providing an index of blood levels and therefore of brain metabolism. In addition, the same animals set for central NIRS studies, were also surgically prepared for electrophysiological monitoring of cell firing in discrete brain areas. These are raphe dorsalis nucleus, locus coeruleus, ventral tegmental area that are defined as main serotoninergic, noradrenergic and dopaminergic cell containing regions of the CNS and therefore involved in the major cerebral syndromes. Then, following a control recording period, exogenous oxygen (O2, 0.1bar, 2min) or carbon dioxide (CO2 0.1bar, 20min) was inflated orally. The data gathered indicate an original relationship between NIRS analysis of brain metabolism and electrical changes in three major nuclei of CNS involved in neurophysiologic and pathologic activities.

As we know Quantum mechanics is a mathematical theory that can describe the behavior of objects that are at microscopic level. Time-resolved autofluorescence spectrometer monitors events that occur during the lifetime of the excited state. This time ranges from a few picoseconds to hundreds of nanoseconds. That is an extremely important advance as it allows environmental parameters to be monitored in a spatially defined manner in the specimen under study. This technique is based on the application of Quantum Mechanics. This principle is applied in our project as we are trying to use different fluorescence spectra to detect biological molecules commonly found in cancerous colorectal tissue and thereby differentiate the cancerous and non-cancerous colorectal polyps more accurately and specifically. In this paper, we use Fluorescence Lifetime Spectrometer (Edinburgh Instruments FL920) to measure decay time of autofluorescence of colorectal cancerous and normal tissue sample. All specimens are from Department of Colorectal Surgery, Singapore General Hospital. The tissues are placed in the time-resolved autofluorescence instrument, which records and calculates the decay time of the autofluorescence in the tissue sample at the excitation and emission wavelengths pre-determined from a conventional spectrometer. By studying the decay time,τ, etc. for cancerous and normal tissue, we aim to present time-resolved autofluorescence as a feasible technique for earlier detection of malignant colorectal tissues. By using this concept, we try to contribute an algorithm even an application tool for real time early diagnosis of colorectal cancer for clinical services.

Optical Coherence Tomography is an emerging technique for biomedical diagnostic help. This is a non-invasive, high resolution, non-destructive mean for some optical biopsy. Since a few years new developments have been undergone in the field of OCT trying to functionalize OCT measurements. One of them is Spectroscopic OCT where simultaneous access to depth resolution as well as spectral features depth resolved in the media are obtained. These spectroscopic OCT system are mainly based on post processing of classical OCT signals what is time consuming and which add numerical noise. We propose an 'all optical' system for real-time direct display of depth-frequency analysis of media.

Digital Holographic Microscopy (DHM) provides three-dimensional (3D) images with a high vertical accuracy in the nanometer range and a diffracted limited transverse resolution. This paper focuses on 3 different tomographic applications based on DHM. First, we show that DHM can be combined with time gating: a series of holograms is acquired at different depths by varying the reference path length, providing after reconstruction images of slices at different depths in the specimen thanks to the short coherence length of the light source. Studies on enucleated porcine eyes will be presented. Secondly, we present a tomography based on the addition of several reconstructed wavefronts measured with DHM at different wavelengths. Each wavefront phase is individually adjusted to be equal in a given plane of interest, resulting in a constructive addition of complex waves in the selected plane and destructive addition in the others. Varying the plane of interest enables the scan of the object in depth. Thirdly, DHM is applied to perform optical diffraction tomography of a pollen grain: transmission phase images are acquired for different orientations of the rotating sample, then the 3D refractive index spatial distribution is computed by inverse radon transform. The presented works will exemplify the versatility of DHM, but above all its capability of providing quantitative tomographic data of biological specimen in a quick, reliable and non-invasive way.

This paper reports a three-dimensional (3D) analysis of shift-invariant pattern recognition applied to holographic images reconstructed digitally from holographic microscopes. It is shown that the sequential application of a 2D filter to plane-by-plane reconstructions of an optical field is exactly equivalent to the application of a more general filter with a 3D impulse response. We show that any 3D filter with arbitrary impulse response can be implemented in this way. The process is illustrated (in 3D) by filtering a holographic image of different sized glass spheres suspended in water.

A modular digital holographic microscopy system for integration in commercial microscopes has been developed. The reconstruction of the digitally captured holograms is performed by application of a spatial phase shifting non diffractive reconstruction method. As a consequence of the applied algorithms the reconstructed holographic images contain not the disturbing terms "twin image" and "zero order". In combination with microscope lenses the system's lateral resolution is improved up to the diffraction limit. Digital holographic focus adjustment with constant imaging scale allows multi focus imaging of object planes and subsequent focus correction from only a single captured hologram. Results of investigations on technical specimen characterize the lateral and axial resolution of the system. The applicability of the system is demonstrated by results obtained from drug stimulated cellular morphology changes and apoptosis monitoring of living pancreas cells.

We have developed a digital holographic microscope (DHM), in a transmission mode, adapted to the quantitative study of cellular dynamics. Living cells are optically probed by measuring the phase shift they produce on the transmitted wave front. The high temporal stability of the phase signal, equivalent to λ/1800, and the low acquisition time (down to 20 μs) enables to monitor cellular dynamics processes. An experimental procedure allowing to calculate both the intracellular refractive index and the cellular thickness (morphometry) from the measured phase shift is presented.

A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. In general, two different screening procedures (1) High Throughput Screening (HTS) and (2) High Content Screening (HCS) are distinguished, where in the first case a rapid measurement of large sample numbers, and in the second case a high information content from a single sample is desired. In particular, a HCS system for the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) has been established and integrated into an existing HTS-system.

Due to their extraordinary photophysical properties CdSe/ZnS core/shell nanocrystals (quantum dots) are excellent luminescence dyes for fluorescence resonance energy transfer (FRET) systems. By using a supramolecular lanthanide complex with central terbium cation as energy donor, we show that commercially available biocompatible biotinilated quantum dots are excellent energy acceptors in a time-resolved FRET fluoroimmunoassay (FRET-FIA) using streptavidin-biotin binding as biological recognition process. The efficient energy transfer is demonstrated by quantum dot emission sensitization and a thousandfold increase of the nanocrystal luminescence decay time. A Foerster Radius of 90 Å and a picomolar detection limit were achieved in quantum dot borate buffer. Regarding biological applications the influence of bovine serum albumin (BSA) and sodium azide (a frequently used preservative) to the luminescence behaviour of our FRET-system is reported.

Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Forster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu³⁺-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Forster-radius, estimated from the absorption and emission bands, was ca. 77Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.

In the present work, we report the synthesis of ZnO nanoparticles by adopting femtosecond laser ablation of ZnO targets in pure ethanol, and in a solution of Tetramethylrhodamine B isothiocyante. An ultrafast intensified CCD device was used to study the plasma formation in the liquid environment. Dye molecules were grafted onto the ZnO nanoparticles by adding the dye solution to the ablated ZnO-ethanol solution. From the optical studies of the ZnO nanoparticles by photoluminescence spectroscopy, we observed energy transfer from the ZnO to the attached dye molecules. Strongly facetted nanohybrid particles were observed with an average size of 15 nm, by HRTEM measurements. Finally, we show that the in situ ablation in the dye solution allows to increase the efficiency of the energy transfer from the ZnO nanoparticle to the grafted dye molecules.

Two distributive tactile sensing systems are presented, based on fibre Bragg grating sensors. The first is a onedimensional metal strip with an array of 4 sensors, which is capable of detecting the magnitude and position of a contacting load. This system is compared experimentally with a similar system using resistive strain gauges. The second is a two-dimensional steel plate with 9 sensors which is able to distinguish the position and shape of a contacting load. This system is compared with a similar system using 16 infrared displacement sensors. Each system uses neural networks to process the sensor data to give information concerning the type of contact.

Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and biotechnical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. For routine measurements by fluorescence techniques the existence of an improved quality assurance is one of the basic needs. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values only. Typical requirements onto fluorescence reference materials or standards deal with the verification of the instrument performance as well as the improvement of the data comparability. Especially for biomedical applications fluorescence labels are used for the detection of proteins. In particular these labels consist of nano crystalline materials like CdS and CdSe. The field of Non-Cadmium containing materials is under investigation. In order to evaluate whether glass based materials can be used as standards it is necessary to calculate absolute values like absorption/excitation cross sections or relative quantum yields. This can be done using different quantities of dopands in glass, glass ceramics or crystals. The investigated materials are based on different types of glass, silicate, phosphate and boron glass, which play a dominant role for the absorption and emission mechanism. Additional to the so-called elementary fluorescence properties induced by raw earth elements the formation of defects lead to higher cross sections additionally. The main investigations deal with wavelength accuracy and lifetime of doped glasses, glass ceramics and crystalline samples. Moreover intensity patterns, homogeneity aspects and photo stability will be discussed.

Using vertical-cavity surface-emitting lasers (VCSELs) as light sources in optical traps offers various advantages compared to the common approaches. In particular, these are small dimensions, a circularly symmetric output beam, and the simple fabrication of two-dimensional laser arrays. We investigate the application of VCSELs in a standard tweezers setup, where trapping forces of up to 4.4 pN are achieved with 15 μm polystyrene particles and a transverse multi-mode VCSEL. The latter has improved trapping characteristics compared to a single-mode device. By introducing a small-spaced array of three VCSELs in the setup, non-mechanical movement with average velocities of up to 3 μm/s is demonstrated with 10 μm particles. Furthermore, the novel concept of the integrated optical trap is presented. By integrating a microlens directly on the VCSEL output facet, two-dimensional optical trapping is achieved in a small-sized system without any external optics. Elevation and trapping of 10 μm polystyrene particles is demonstrated at optical output powers of about 5 mW. In order to improve the beam quality of the lasers, the inverted surface relief technique is applied, which eliminates a previously observed offset between laser center and trapped particle.

We show that speckles play an important role in laser Doppler perfusion imaging. The influence of speckles on the signal amplitude and the Doppler spectrum is demonstrated experimentally on particle suspensions with different scattering levels and varying beam width. Polystyrene microsphere suspensions with known optical properties are used to make scattering samples. A theoretical model is explained to calculate the speckle size from the back scattered intensity distributions. The coherence area is calculated with Monte Carlo simulations on different scattering samples and experiments are performed to validate our theoretical model. The experimental results are in good agreement with our theoretical predictions.

The present work aims at comparing simulations of photon transport phenomena in biological multi-layered tissues by means of two methods: a finite element code and a Monte Carlo procedure. We apply these codes to model time-dependent light propagation in multi-layered media. The physical situation refers to the case of a narrow incident laser pulse of 1 ps duration acting upon the surface of the media. Time-resolved spectra are reported for different geometries and optical properties for high media. Here, codes are applied to simulate data acquired from measurements on tissues in extremities. The results of the study provide information to calibrate the finite element characteristics for future experiences including fluorescence events in multi-layered complex systems.

We study the fluctuations of light multiply scattered by particles under Brownian motion in a fluid. We focus on the behavior of the time correlation function of the field in the non-diffusive regime, in both transmission and reflection. In transmission through optically thin systems, an extended Diffusing-Wave Spectroscopy (DWS) model based on the Radiative Transfer Equation (RTE) is described, which predicts substantial deviations from the standard DWS theory. For backscattered light, experiments using unpolarized light show a clear dependence on the anisotropy factor g. This behavior is not described by the standard DWS theory. A good agreement with the data is obtained using the RTE model, and an approximate model in which the path-length distribution of the standard DWS is corrected by a prefactor which depends on the level of anisotropy. These results should have broad applications in diffuse-light biomedical imaging, and in the field of soft-materials and biomaterials analysis.

The study presented demonstrates the ability of the self-mixing interferometry configuration with a laser diode to measure flow through scattering media. A flow model has been used based on a 1.5mm-diameter tube covered with layers of scattering media. The laser intensity power spectrum has been obtained when changing the flow value and the thickness of the layer placed on top of the tube. Different data processing algorithms have been compared including first moment, normalized first moment, RMS, and Lorentzian and exponential curve fitting parameters. It was found that an exponential curve in semi-logarithm scale well describe the spectrum and can be best used when monitoring flow under layers of up to 1.1mm in thickness.

Hip and knee prostheses are complex products with high quality standards. The aim is to reproduce the behavior of a natural joint. This can be achieved by using a hard and a soft component. The research project "Optical Geometry Acquisition of Medical Implants and Prostheses", short "OptiGIP", explores methods for an automated quality assurance included in the production chain of these components. The approach is divided in two sections, a three dimensional and a two dimensional measurement technique. First a stripe projection method is used to produce a three dimensional model of the component. This model can be used to verify the geometry of the component. Furthermore it enables a new examination method, the Model-Based RSA, which is used to explore the effect of loosening. A late loosening of the components within the bones is the most important quality criterion for a successful implantation. The second part of described measurement method aims at a reduction of an early loosening of the prosthesis. Even very small scratches on the prosthesis's surface can have an impact on an early loosening because of friction between the soft and hard surface. Scratches produce bigger particles of the soft component than an undamaged surface would do. Recent research activities show that these bigger particles have an influence on an early loosening mechanism. The two-dimensional measurement checks the quality of the surface of the hard component of the implant. The approach is to use an extended dark field method. The prosthesis is illuminated from various angles producing a sequence of images. These images are filtered to distinguish between reflections from scratches and direct reflections. A combination of the filtered images shows the scratches.

We report the development of non-invasive, path length resolved Doppler measurements of the multiply scattered light in turbid media, for different absorptions using phase modulated Mach-Zehnder low coherence interferometer, with separate fibers for illumination and detection. A Doppler broadened phase modulation interference peak is observed that shows an increase in the average Doppler shift with optical path length, independent of absorption. The estimated path length distributions indicate suppression and narrowing for increasing absorption and can be related by Lambert-Beer's law.

We demonstrate a novel quantum dot based probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This probe may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically-degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Peptide cleavage results in release of AuNPs and restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 hours of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker.

We devised a multimodal planar imaging system for in vivo mouse imaging, employing four modalities: optical imaging, green and red fluorescence reflectance imaging, radionuclide imaging and X-ray radiography. We are testing separately, and then in a combined way, each detection mode, via in vivo mouse imaging, with the final purpose of identifying small implanted tumor masses, of providing early tumor detection and following metastatic dissemination. We describe the multimodal system and summarize its main performance, as assessed in our research work in the various stages of the development, in fluorescence and radionuclide tests on healthy or tumor bearing mice. For gamma-ray detection we used a semiconductor pixel detector (Medipix1 or Medipix2) that works in single photon counting. Laser-induced fluorescence reflectance imaging was performed in vivo using a pulsed light source to excite the fluorescence emission of injected hematoporphyrin (HP) compound, a CCD camera, a low pass filter and a commercial image analysis system. The bimodal system was used for the acquisition of combined images of the tumor area (fluorescence: animal top view; radionuclide: bottom view). It was shown that the tumor area can be imaged in a few minutes, with a few millimeter resolution (1 mm pinhole diameter), radioactively (^99mTc radiotracer), and with the fluorescence system and that, in one case, only one of the two modalities is able to recognize the tumor. A phantom study for thyroid imaging with ¹²⁵I source embedded in a simulated tissue indicated a spatial resolution of 1.25 mm FWHM with a 1 mm pinhole.

The aim of this work was to study synthetic polycation effects on erythrocyte agglutination mediated by anti-glycophorin using image digital analysis. Polycations are oligomers or polymers of natural or synthetic origin, which bear a great number of positive charges at pH 7.4. Several of these polycations are nowadays used in clinic for human and veterinary purposes. New applications of polycations to the development of new drug delivery systems are investigated, in order to promote the drug absorption through the gastro-intestinal and blood brain barriers. However, up to now, there are no clear relationships between macromolecular features of polycations (molecular weight, mean charge density, charge repartition, etc.) and their interactions with blood elements (which bear superficial negative charges). The interaction on the red blood cell membrane with synthetic polycations having well-controlled macromolecular features and functionalized with pendent polyethylene glycol segments was investigated. The alterations over stationary and dynamic viscoelastic properties of erythrocyte membranes were analyzed through laser diffractometry. Image digital analysis was used to study erythrocyte agglutination mediated by anti-glycophorin. Results show different reactivities of the polycations on the erythrocyte membrane. These findings could provide more information about the mechanisms of polycation interaction on erythrocyte membranes. We consider that this work could provide useful tools to understand and improve the haemocompatibility of polycations and enlarge their potential in clinic.

An analytical theory has been proposed to describe multiple dynamic light scattering (MDLS) in a multi-layered turbid medium with optical and dynamical heterogeneities. To examine the validity of this theory, Monte Carlo method is used for simulating the photon correlation diffusion in the medium. The simulation results are then compared with that of the analytical prediction. A comprehensive investigation has been carried out, including cases of one finite layer, one semi-infinite layer, two finite layers, and three layers with the last layer being semi-infinite. In simulations optical parameters are varied in a large range: the absorption and the reduced scattering coefficients are taken from 0.1 to 0.7 cm^-1 and 2.0 to 20.0 cm^-1, respectively. The Monte Carlo results, in most cases, are in an excellent agreement with that of the analytical theory, demonstrating the effectiveness of the analytical theory for multi-layered MDLS.

In this work the mutagenic effect on Escherichia coli strains induced by UV radiation emitted by a XeCl laser (λ = 308 nm) has been analysed as a function of the exposure dose and compared with the effect induced by 254 nm radiation emitted by a conventional germicidal lamp. E. coli strains, wild-type (recA+) and mutant (recA1, defective in DNA damage repair systems), plated on LB agar, supplemented with rifampicin when requested, were irradiated by means of a germicidal lamp in the dose range 0 - 9 mJ/cm². Similar strains were exposed to 308 nm pulsed laser radiation (τ = 20 ns FWHM; max. pulse energy: 100 mJ) in the dose range 0-1.0 x 10⁴ mJ/cm². The discrepancy between the results obtained with the lamp and the laser on the mutation frequency, suggested that the biological response to the two radiation sources involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV 308 nm induced mutagenesis in the recA-defective strain at an extent considerably higher than in the recA-proficient strain.

Manipulation of material by optical means represents an emerging field with numerous applications. Especially in biology and medicine, the flexible and powerful potential of laser utilization holds great promises. For many applications, the resolution of the induced effects is essential. Besides focusing of the beam by various means, the use of sub-wavelengths nanoantenna could overcome this problem. The optical absorption of certain nanostructures is based on plasmon effects. We present studies of the use of metal (homogeneous gold or gold/silver core/shell systems) nanoparticles as antennas that convert the incident laser light into irreversible destructive effects. Based on the established field of DNA-conjugated nanoparticles, we investigated the sequence-specific attachment of DNA-nanoparticle complexes onto DNA with complementary sequences, in the state of double-stranded either isolated or metaphase chromosomal DNA. Important points were the adjustment of the absorption properties of the nanoparticles by control of their material composition (e.g., by addition of a silver layer to a gold core) and diameter. Another group of experiments studied chromosome-conjugated particles before and after laser treatment, in order to reveal the lateral extension of damages as well as the underlying mechanism.

Cytogenetical study of lymphocytes using the light microscopy could reveal a large amount of chromosomal abnormalities, which determine corresponding hereditary disorders. However, geneticists sometimes observe the cases where the same chromosomal rearrangements seen in light microscope cause quite different phenotype (from normal to abnormal) in relatives. The aim of the study was to explain the mechanisms of the different phenotype appearance in family members carrying the same reciprocal translocations. It was carried out the standard chromosome analysis in 12 families, where some relatives had reciprocal translocations. Chromosomes were differentially stained using G-method. The samples were analysed in optical microscope (x1000). Using OMIM gene map, UCSC Genome Browser, eGenome Release v2.3 and Unigene databases it was revealed transposons and transposon derivates in chromosome regions involved in translocations. We suppose that the variability of clinical manifestations in translocation-bearing patient is caused by the influence of the transposons, such as Hsmar2, Alu-elements or some others. We propose the following mechanisms of transposone action in these patients. The first may lie on recombination between the 2 specific DNA-transposon containing sites on different chromosomes resulting in balanced reciprocal translocation with no significant influence on the most genes' activity in corresponding regions. The weakening of transposase repression, which may follow in gametes, increases the transposase activity, and hereby, the probability of transposon dislocation. Dislocation can change the activity of groups of genes, because transposons often carry the regulatory sequences. This can induce multiply innate disorders in the progeny of the phenotypically healthy parents, carrying the translocation. According to the second mechanism, the reciprocal translocation is caused by recombination between 2 Alu repeats. These repeats can undergo reverse transcription, and a DNA-product, formed during this process, can paste in a new chromosome region in gametes. As the Alu repeats contain the CpG-islands, they can change the gene activity resulting in a disorder. The understanding of the cases of such genetical disorders might help to predict the appearance of the progeny with pathological karyotype, making the light microscopy more informative in diagnostic of the diseases.

The non-invasive methods of treatments have been studying for the improvement of quality of life (QOL) of patients undergoing treatment. A photodynamic therapy (PDT) is one of the non-invasive treatments. PDT is the method of treatment using interactions of a laser and a photosensitizer. PDT has few risks for patients. Furthermore, PDT enables function preservation of a disease part. PDT has been used for early cancer till now, but in late years it is applied for age-related macular degeneration (AMD). AMD is one of the causes of vision loss in older people. However, PDT for AMD does not produce the best improvement in visual acuity. The skin photosensivity by an absorption characteristic of a photosensitizer is avoided. We examined new PDT using combination of an ultra-short pulsed laser and indocyanine green (ICG).

The orientation and attachment of neuronal cells were controlled by submicron-scale topographical patterns. The surface structure is realized with a laser beam and photo-responsive azobenzene polymer thin films. A surface relief grating (SRG) can be produced by self-organization of molecules under the action of light. The cells are attached onto the SRG and preferentially grown along the groove direction. The use of polymer thin films is good candidate for cellular engineering applications.

The intrinsic quality of an image is related to the concepts of spatial resolution, noise and contrast. A common method to measure these parameters is by using Edge Response Function measurements with a black mask. We investigated these parameters using an experimental apparatus mainly composed by a an argon pumped Ti:Sa laser working in femtosecond regime and a time-correlated single photon-counting system. The investigated samples were suspensions of Intralipid 10% with distilled water in which a black mask was inserted and bidimensional scanning were performed at different depths ( z = L/2, L/4, 3L/4). The experimental data were analyzed in order to get information on the above-mentioned image quality parameters. A comparison with similar results obtained with a streak camera has been done and for spatial resolution a comparison with random walk predictions has been performed. The results of this study can be particularly useful in identifying the best working conditions and in improving the performance of image reconstruction algorithms since the clinical prototypes of optical mammographers nowadays under pre-clinical investigation adopt time-correlated single photon-counting technique.

In this paper, we present the conception of a holographic combiner for an augmented reality Head Mounted Display (HMD) dedicated to surgical applications. The recording of this holographic component has been performed at the Laboratoire des Systemes Photoniques (LSP) in Strasbourg, France. We present in this paper two different approaches for the recording of such a component: one using plane waves, and the other using spherical waves. The setup linked to the first approach has been developed and built, so that measurments of the diffraction efficiency can be shown. For the other way of recording the holographic combiner, we have performed numerical simulations to find the best recording setup to fit our specifications.