This PDF file contains the front matter associated with SPIE-OSA Proceedings Volume 6631, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Commercial imaging systems, such as computed tomography and magnetic resonance imaging, are frequently used powerful tools for observing structures deep within the human body. However, they cannot precisely visualized several-tens micrometer-sized structures for lack of spatial resolution. In this presentation, we propose photoacoustic imaging using multiphoton absorption technique to generate ultrasonic waves as a means of improving depth resolution. Since the multiphoton absorption occurs at only the focus point and the employed infrared pulses deeply penetrate living tissues, it enables us to extract characteristic features of structures embedded in the living tissue. When nanosecond pulses from a 1064-nm Nd:YAG laser were focused on Rhodamine B/chloroform solution (absorption peak: 540 nm), the peak intensity of the generated photoacoustic signal was proportional to the square of the input pulse energy. This result shows that the photoacoustic signals can be induced by the two-photon absorption of infrared nanosecond pulse laser and also can be detected by a commercial low-frequency MHz transducer. Furthermore, in order to evaluate the depth resolution of multiphoton-photoacoustic imaging, we investigated the dependence of photoacoustic signal on depth position using a 1-mm-thick phantom in a water bath. We found that the depth resolution of two-photon photoacoustic imaging (1064 nm) is greater than that of one-photon photoacoustic imaging (532 nm). We conclude that evolving multiphoton-photoacoustic imaging technology renders feasible the investigation of biomedical phenomena at the deep layer in living tissue.

Photoacoustic imaging is a promising new way to generate unprecedented contrast in ultrasound diagnostic imaging. It differs from other medical imaging approaches, in that it provides spatially resolved information about optical absorption of targeted tissue structures. Because the data acquisition process deviates from standard clinical ultrasound, choice of the proper image reconstruction method is crucial for successful application of the technique. In the literature, multiple approaches have been advocated, and the purpose of this paper is to compare four reconstruction techniques. Thereby, we focused on resolution limits, stability, reconstruction speed, and SNR. We generated experimental and simulated data and reconstructed images of the pressure distribution using four different methods: delay-and-sum (DnS), circular backprojection (CBP), generalized 2D Hough transform (HTA), and Fourier transform (FTA). All methods were able to depict the point sources properly. DnS and CBP produce blurred images containing typical superposition artifacts. The HTA provides excellent SNR and allows a good point source separation. The FTA is the fastest and shows the best FWHM. In our study, we found the FTA to show the best overall performance. It allows a very fast and theoretically exact reconstruction. Only a hardware-implemented DnS might be faster and enable real-time imaging. A commercial system may also perform several methods to fully utilize the new contrast mechanism and guarantee optimal resolution and fidelity.

Photoacoustic imaging with line detectors that are longer than the maximum size of an object is able to reveal the three-dimensional (3D) structure of the thermoelastic pressure distribution within the object. The image acquisition and reconstruction is a two step process. In the first step acoustic signals that are measured while the line detector is translated around the object are used to reconstruct a projection of the initial pressure in direction of the line. This is a two-dimensional (2D) image reconstruction problem. In the second step projections taken at multiple line orientations are used to reconstruct the 3D image by inversion of the Radon transform. In this study methods for solving the 2D reconstruction problem are presented. In an experiment using an optical interferometer as acoustic line detector the complete 3D imaging procedure is demonstrated on a phantom.

Besides x-ray imaging, sonography is the most common method for breast cancer screening. The intention of our work is to develop optoacoustical imaging as an add-on to a conventional system. While ultrasound imaging reveals acoustical properties of tissue, optoacoustics generates an image of the distribution of optical absorption. Hence, it can be a valuable addition to sonography, because acoustical properties of different tissues show only a slight variation whereas the optical properties may differ strongly. Additionally, optoacoustics gives access to physiological parameters, like oxygen saturation of blood. For the presented work, we combine a conventional ultrasound system to a 100 Hz laser. The laser system consists of a Nd:YAG-laser at a wavelength of 532 nm with 7 ns pulse duration, coupled to a tunable Optical Parametric Oscillator (OPO) with a tuning rage from 680 nm to 2500 nm. The tunable laser source allows the selection of wavelengths which compromising high spectral information content with high skin transmission. The laser pulse is delivered fiber-optically to the ultrasound transducer and coupled into the acoustical field of view. Homogeneous illumination is crucial in order to achieve unblurred images. Furthermore the maximum allowed pulse intensities in accordance with standards for medical equipment have to be met to achieve a high signal to noise ration. The ultrasound instrument generates the trigger signal which controls the laser pulsing in order to apply ultrasound instrument's imaging procedures without major modifications to generate an optoacoustic image. Detection of the optoacoustic signal as well as of the classical ultrasound signal is carried out by the standard medical ultrasound transducer. The characterization of the system, including quantitative measurements, performed on tissue phantoms, is presented. These phantoms have been specially designed regarding their acoustical as well as their optical properties.

Photoacoustic tomography (PAT) is based on the recording of the acoustic signals excited by illumination of a sample with short laser pulses. The detection of the acoustic signals can be realized either by small (point-like) detectors or by extended integrating detectors. The commonly applied detectors are arrays of small ultrasound transducers or single detectors scanning around the object. A rather new approach is the use of extended integrating detectors for acoustic wave monitoring to avoid the blurring effects of finite aperture sensors in PAT. The present study is focused on the development of integrating line detectors. This is implemented by a combination of a planar waveguide (PWG) and a common path polarization interferometer (CPPI). An arriving acoustic pulse modifies the birefringence of the waveguide material. This leads to a change of phase difference between two orthogonally polarized fundamental waveguide modes, which is converted into a modulation of intensity by an analyzer. The obtained noiseequivalent pressure value is ~1bar without averaging which is rather poor compared to other methods but it can be increased by using polymer waveguide materials with better relative elasto-optic coupling coefficients than polystyrene (C–-19•10^-7 bar^-1). The guiding polystyrene film had a thickness of 1.3 μm and was fabricated with a spin coating method. The bandwidth of the PWG sensor was limited only by the detection electronics to 125 MHz.

Photoacoustic imaging is based on the generation of acoustic waves in a semitransparent sample (e.g. soft tissue) after illumination with short pulses of light or radio waves. The goal is to recover the spatial distribution of absorbed energy density inside the sample from acoustic pressure signals measured outside the sample (photoacoustic inverse problem). If the acoustic pressure outside the illuminated sample is measured with a large-aperture detector, the signal at a certain time is given by an integral of the generated acoustic pressure distribution over an area that is determined by the shape of the detector. For example a planar detector measures the projections of the initial pressure distribution over planes parallel to the detector plane, which is the Radon transform of the initial pressure distribution. Stable and exact three-dimensional imaging with planar integrating detector requires measurements in all directions of space and so the receiver plane has to be rotated to cover the entire detection surface. We have recently presented a simpler set-up for exact imaging which requires only a single rotation axis and therefor the fragmentation of the area detector into line detectors perpendicular to the rotation axis. Using a two-dimensional reconstruction method and applying the inverse two-dimensional Radon transform afterwards gives an exact reconstruction of the three-dimensional sample with this set-up. In order to achieve high resolution, a fiber based Fabry-Perot interferometer is used. It is a single mode fiber with two fiber bragg gratings on both ends of the line detector. Thermal shifts and vibrations are compensated by frequency locking of the laser. The high resolution and the good performance of this integrating line detector has been demonstrated by photoacoustic measurements with line grid samples and phantoms using a model-based time reversal method for image reconstruction. The time reversed pressure field can be calculated directly by retransmitting the measured pressure on the detector positions in a reversed temporal order.

Gold nanorods are seen as possible contrast agents for photoacoustic imaging since they have strong absorption peaks at near-infrared wavelengths. Also they are easy to conjugate with various proteins. If these particles can be conjugated with cancer affinity proteins then these particles can accumulate specifically at a tumor site. By detecting the presence of accumulation of gold nanorods inside the tissue the indirect detection of tumor can be realized. When these particles are irradiated with light pulses of appropriate temporal properties and energy the temperature around these particles can be high enough to induce apoptosis or necrosis in the surrounding cells. In order to use these particles at their full potential we must determine precisely their optical properties. We simulated the optical properties of gold nanorods synthesized by us using the DDSCAT code. The simulated spectra agree qualitatively with the spectra determined using spectrometry and also determined using photoacoustic spectroscopy. Further the values of molar extinction coefficient derived from the simulations were similar to the data measured experimentally by other groups. These results validated qualitatively the model used in the simulations. During simulations we found that the choice of the dielectric function used in simulations plays an important role in the results.

Photoacoustics is a hybrid imaging technique that combines the contrast available to optical imaging with the resolution that is possessed by ultrasound imaging. The technique is based on generating ultrasound from absorbing structures in tissue using pulsed light. In photoacoustic (PA) computerized tomography (CT) imaging, reconstruction of the optical absorption in a subject, is performed for example by filtered backprojection. The backprojection is performed along circular paths in image space instead of along straight lines as in X-ray CT imaging. To achieve this, the speed-of-sound through the subject is usually assumed constant. An unsuitable speed-of-sound can degrade resolution and contrast. We discuss here a method of actually measuring the speed-of- sound distribution using ultrasound transmission through the subject under photoacoustic investigation. This is achieved in a simple approach that does not require any additional ultrasound transmitter. The method uses a passive element (carbon fiber) that is placed in the imager in the path of the illumination which generates ultrasound by the photoacoustic effect and behaves as an ultrasound source. Measuring the time-of-flight of this ultrasound transient by the same detector used for conventional photoacoustics, allows a speed-of-sound image to be reconstructed. This concept is validated on phantoms.

A time-domain fNIRS system was developed for simultaneous acquisition with fMRI. Preliminary results during motor activity indicate good sensitivity and temporal resolution of the system. To our knowledge this is the first time-domain fNIRS and fMRI study on human brain.

We demonstrate the feasibility of time-resolved diffuse reflectance at small source-detector separations using a single-photon avalanche diode (SPAD) operated in time-gated mode. Photon time distributions at an interfiber distance of 0.2 cm were obtained on tissue phantoms with a reduced scattering coefficient of 10 cm^-1, and an absorption coefficient of 0.1 cm^-1, with a dynamic range of 10⁶ and collecting photons at arrival times up to 4 ns. By time-gating the initial photons, carrying information mainly from superficial layers, it is possible to detect longer lived photons that have explored deeper depths even at almost null interfiber distances. The proposed approach should provide higher number of photons at any arrival time, higher contrast, and better spatial resolution as compared to longer interfiber distances.

The estimation of optical properties of highly turbid and opaque biological tissue is a difficult task since conventional purely optical methods rapidly loose sensitivity as the mean photon path length decreases. Photothermal methods, such as pulsed or frequency domain photothermal radiometry (FD-PTR), on the other hand, show remarkable sensitivity in experimental conditions that produce very feeble optical signals. Photothermal Radiometry is primarily sensitive to absorption coefficient yielding considerably higher estimation errors on scattering coefficients. Conversely, purely optical methods such as Local Diffuse Reflectance (LDR) depend mainly on the scattering coefficient and yield much better estimates of this parameter. Therefore, at moderate transport albedos, the combination of photothermal and reflectance methods can improve considerably the sensitivity of detection of tissue optical properties. The authors have recently proposed a novel method that combines FD-PTR with LDR, aimed at improving sensitivity on the determination of both optical properties. Signal analysis was performed by global fitting the experimental data to forward models based on Monte-Carlo simulations. Although this approach is accurate, the associated computational burden often limits its use as a forward model. Therefore, the application of analytical models based on the diffusion approximation offers a faster alternative. In this work, we propose the calculation of the diffuse reflectance and the fluence rate profiles under the δ-P₁ approximation. This approach is known to approximate fluence rate expressions better close to collimated sources and boundaries than the standard diffusion approximation (SDA). We extend this study to the calculation of the diffuse reflectance profiles. The ability of the δ-P₁ based model to provide good estimates of the absorption, scattering and anisotropy coefficients is tested against Monte-Carlo simulations over a wide range of scattering to absorption ratios. Experimental validation of the proposed method is accomplished by a set of measurements on solid absorbing and scattering phantoms.

We discuss a new approach to laser speckle biomedical imaging with the goal to establish a quantitative link between the measured signal and the local dynamic properties of Brownian motion or blood flow. We demonstrate that the presence of a static component in laser speckle imaging signal can significantly complicate the quantitative interpretation of the imaging data. With Monte-Carlo simulations and model experiments we show that the error in the mean particles velocity extracted using traditional approaches can reach several orders of magnitude. With a proper data treatment on the other side the error can be substantially reduced. We suggest a simple data processing scheme that properly accounts for a static component in the scattered light intensity.

This paper describes two setups suitable for interference microscopy for high resolution morphological measurements on living cells in culture medium. The first system incorporated a PZT actuator in the reference path of a Mach Zehnder configuration to facilitate digital phase-stepping interferometry. The second system employed two phase-locked acoustooptic modulators to generate a temporal optical carrier to allow a heterodyne approach to phase demodulation. This setup incorporated a digital CMOS camera with full random pixel access which allowed the heterodyne approach to be implemented as a full-field method without any need for electromechanical scanning. The heterodyne approach offers benefits over the phase-stepping method in terms of measurement resolution and speed, typically offering the equivalent of nanometer resolution for cell height measurements with a bandwidth in the order of 200-300 Hz for 1000 pixels. Results for morphological measurements using both systems on red blood cells and keratinocytes are presented.

Digital Holography in microscope configuration thanks to the numerical reconstruction procedure is a flexible and useful tool for analysis of biological material. We present the investigation of lipid particles growth in in-vitro mouse cell using a Digital Holographic Microscopy (DHM) employed in combination with Lateral Shear Interferometry (LSI). The optical setup is based on a Mach-Zehnder interferometer in transmission geometry. The sample cell is placed in one interferometer arm while the other one is used as a reference beam. By means of the Rayleigh-Sommerfield integral is possible to retrieve the complex object field and then to calculate the amplitude and phase of the laser light transmitted by the sample. Traditional microscopy allows to obtain amplitude contrast image only, DH, instead, enables to calculate the phase map of the complex wave that is simply related to the optical phase difference (OPD) experienced by the light when it is transmitted through the object. In this way it is possible to obtain phase contrast image that is very useful for biological materials that often present low amplitude contrast for quantitative amplitude image. The main difficulty of this technique is to remove the optical aberrations produced by the optical setup components. Several methods have been proposed, such as subtraction of a reference phase map (without sample) or numerical multiplication of a parametric lens. We propose a fast and effective solution of this problem based on LSI. We digitally introduce a lateral shear of one pixel in x and y directions calculating the phase difference ΔØ_x,y, between the actual phase map and its sheared replica in both directions. ΔØ_x,y include a linear term due to defocus aberration and the object phase difference. The linear term can be easily eliminated and sample phase map retrieved by numerical integration. This technique allows to obtain the correct phase contrast image removing optical aberration, avoiding unwrapping problems.

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy (Marquet et al., 2005). Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. We have recently proposed (Rappaz et al., 2005) a new and efficient decoupling procedure allowing to directly obtain separate measurements of the thickness and the integral refractive index of a given living cell. Consequently, it has been possible, for the first time to our knowledge, to accurately measure (with a precision of 0.0003) the mean refractive index of living erythrocytes.. On the other hand, the cellular thickness measurements allow to calculate the volume and shape of erythrocytes. In addition, DHM, thanks to its subwavelength phase shift measurements, was found to yield an efficient tool to assess erythrocyte cell membrane fluctuations (ECMF). Typically, ECMF characterized by an amplitude within the range of 45 nm were observed.

For the planning and documentation of maxillofacial surgery highly resolved tissue information is needed. In our approach, the surface of an object is displayed and measured with pulsed holography. With a single laser pulse (Nd:YAG) of 20 ns the object surface is recorded on a CCD sensor, movement artifacts are systematically avoided. With the knowledge of the recording parameters, the original wave field is synthesized numerically from the holographic interference pattern. The calculated slices are combined into an image stack, representing the digitized real image. This wave field represents the object geometrically correct, but focussed and unfocussed regions overlay. The focussed regions are identified numerically and combined into a height map, the texture information is extracted from the real image simultaneously. Both, height and texture are combined, yielding pixel-precise textured surface models. With this novel method it is possible to capture the surface of moving objects, even 3d motion series are possible. Skin can be detected in the real image, giving the potential application for facial measurements. Compared to our analog holographic topometry, there are still limitations regarding the extent of the imageable field and the axial resolution. The quick display of the reconstructed real image allows a direct appraisal of the object topology. This method is a valuable tool for the surface visualization of living subjects, offering potential for completely new fields of application.

We proposed and developed an optical system for imaging of the surface profiles of biological cells and tissues with nanometer resolution. The system is a low-cost system which can perform the imaging of the surface morphology in a large area and large depth range without any special preparation of the sample. The system consists of two interferometers in which one is in the configuration of a Michelson interferometer and the other is in the configuration of a Mach-Zehnder interferometer. The former is used for scanning of the surface profile of the sample, and the latter is used to compensate the phase shift due to the different traveling ranges of the reference mirror in successive scannings. The phase difference between the interferograms detected in both interferometers is proportional to the surface height of the sample at that point. The system was demonstrated to possess the axial resolution within ±5 nm and its lateral resolution is at the diffraction limit. We used the system for the imaging of various samples including biological cells and tissues. The system was also used for dynamic imaging to observe the morphological change of the surface of biological cells.

We realized new adaptive holographic sensor and interferometer, which allows to visualize high-resolution 3D images of diffuse reflected objects in Continue Hologram Registration Regime- CHRR. The coupled laser wave nonlinear theory was applied for optimization of hologram recording in crystals symmetry 23 and optimized experimental set up. Experimentally demonstrated dynamical holographic image sensors on doped 23 symmetry photosensitive crystals, with resolution 7900-lines/mm at 632 nm and 11641 lines/mm at 440 nm for 15 mW CW HeNe and He-Cd lasers. The results are presented for holographic visualization of Cryogenic and Ultrasonic near field images of Surgical Medical Instrument. Application of CHRR interferometer for hologram registration of moving biological object in "vivo" is illustrated.

Hyperspectral imaging of the retina presents a unique opportunity for direct and quantitative mapping of retinal biochemistry - particularly of the vasculature where blood oximetry is enabled by the strong variation of absorption spectra with oxygenation. This is particularly pertinent both to research and to clinical investigation and diagnosis of retinal diseases such as diabetes, glaucoma and age-related macular degeneration. The optimal exploitation of hyperspectral imaging however, presents a set of challenging problems, including; the poorly characterised and controlled optical environment of structures within the retina to be imaged; the erratic motion of the eye ball; and the compounding effects of the optical sensitivity of the retina and the low numerical aperture of the eye. We have developed two spectral imaging techniques to address these issues. We describe first a system in which a liquid crystal tuneable filter is integrated into the illumination system of a conventional fundus camera to enable time-sequential, random access recording of narrow-band spectral images. Image processing techniques are described to eradicate the artefacts that may be introduced by time-sequential imaging. In addition we describe a unique snapshot spectral imaging technique dubbed IRIS that employs polarising interferometry and Wollaston prism beam splitters to simultaneously replicate and spectrally filter images of the retina into multiple spectral bands onto a single detector array. Results of early clinical trials acquired with these two techniques together with a physical model which enables oximetry map are reported.

The technique of Laser Doppler Perfusion Imaging (LDPI) is widely used for determining cerebral blood flow or skin perfusion in the case of burns. The commonly used Laser Doppler Perfusion Imagers are scanning systems which point by point scan the area under investigation and use a single photo detector to capture the photoelectric current to obtain a perfusion map. In that case the imaging time for a perfusion map of 64 x 64 pixels is around 5 minutes. Disadvantages of a long imaging time for in-vivo imaging are the bigger chance of movement artifacts, reduced comfort for the patient and the inability to follow fast changing perfusion conditions. We present a Laser Doppler Perfusion Imager which makes use of a high speed CMOS-camera. By illuminating the area under investigation and simultaneously taking images at high speed with the camera, it is possible to obtain a perfusion map of the area under investigation in a shorter period of time than with the commonly used Laser Doppler Perfusion Imagers.

This article describes the theoretical development and design of a real-time microcirculation imaging system, an extension from a previously technology developed by our group. The technology utilises polarisation spectroscopy, a technique used in order to selectively gate photons returning from various compartments of human skin tissue, namely from the superficial layers of the epidermis, and the deeper backscattered light from the dermal matrix. A consumer-end digital camcorder captures colour data with three individual CCDs, and a custom designed light source consisting of a 24 LED ring light provides broadband illumination over the 400 nm - 700 nm wavelength region. Theory developed leads to an image processing algorithm, the output of which scales linearly with increasing red blood cell (RBC) concentration. Processed images are displayed online in real-time at a rate of 25 frames s^-1, at a frame size of 256 x 256 pixels, and is limited only by computer RAM memory and processing speed. General demonstrations of the technique in vivo display several advantages over similar technology.

We report the realization of a surface plasmon resonance imaging biosensor potentially capable of dynamically characterizing optical anisotropy by means of polarimetric measurements. Our approach relies on a light beam propagating through a high refractive index glass-prism (Kretschmann-Raether configuration) in order to excite a surface plasmon wave along a metal-dielectric interface. This evanescent wave probes the metal-dielectric vicinities with subnanometer sensitivity, thus resolving optical characteristics of adsorbed biomolecular targets. Fixing wavelength and angle of incidence of the beam enables real-time monitoring of adsorptions and desorptions of targets onto the whole surface of the chip, allowing for example characterization of DNA:DNA interaction kinetics with applications to genetic diagnosis. The polarimetric surface plasmon resonance imaging device uses a pyramid of high index glass and two orthogonal SPR imaging sensor arms. The interface is probed along two orthogonal directions. A signal difference in reflection between the two arms should allow us to resolve local optical anisotropy of the dielectric medium, keeping the parallel and real-time capabilities of the system. Additional information can be obtained by varying the angle of incidence of the light beam or tuning its wavelength. We believe that this type of sensor will be useful for studying collective biomolecular assemblies' conformational changes.

Multiphoton autofluorescence imaging offers minimal-invasive examination of cells without the need of staining and complicated confocal detection systems. Therefore, it is especially interesting for non-invasive clinical diagnostics. To extend this sophisticated technique from superficial regions to deep lying cell layers, internal body parts and specimens difficult of access, the bulky optics need to be reduced in diameter. This is done by tiny GRIN-optics, based on a radial gradient in the reflective index. Of especial interest for multi-photon applications is the newly developed GRIN-lens assembly with increased numerical aperture. High resolution images of plant tissue, hair and cells show the improved image quality,compared to classical GRIN-lenses. The rigid GRIN-endoscopes are already applied in wound healing studies. Here, the GRIN-lenses with diameters smaller than 3 mm enter small skin depressions. They reproduce the focus of a conventional laser scanning tomograph tens of mm apart in the specimen under study. We present first clinical measurements of elastin and SHG of collagen of in-vivo human skin of venous ulcers (ulcer curis).

We have developed a new two-photon system for in vivo flow cytometry, thereby allowing us to simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice, markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB- 435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold, enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to investigate dynamics of circulating cells in cancer and other important diseases.

We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.

To achieve high lateral resolution in microscopy, we exploit the localized electromagnetic field of a Solid Immersion Lens (SIL). The lens is mounted on a cantilever of a Scanning Probe Microscope (SPM) to allow a dynamic scan with constant tip-sample force. This unit can be integrated into a micro fluorescence (Zeiss UMSP) or Raman spectrometer (Renishaw) to allow spectroscopy and spectrally resolved imaging in the near field. Three methods can be applied with a lateral resolution of less than 30 nm: 1) Reflectance-SNOM: the sample is imaged by illuminating the surface through the SIL and detecting the reflected near-field. 2) Photon-tunneling-SNOM: the contrast is generated by the ability of the photons to tunnel through the energy barrier into the substrate. 3) Fluorescence- SNOM: the chromophore is excited and the fluorescence light is collected by the SIL. The collection efficiency for the fluorescence is increased by a factor of 10 due to the high refractive index of the SIL compared to conventional methods.

The highly malignant brain tumor, glioblastoma multiforme, is difficult to totally resect without aid due to its infiltrative way of growing and its morphological similarities to surrounding functioning brain under direct vision in the operating field. The need for an inexpensive and robust real-time visualizing system for resection guiding in neurosurgery has been formulated by research groups all over the world. The main goal is to develop a system that helps the neurosurgeon to make decisions during the surgical procedure. A compact fiber optic system using fluorescence spectroscopy has been developed for guiding neurosurgical resections. The system is based on a high power light emitting diode at 395 nm and a spectrometer. A fiber bundle arrangement is used to guide the excitation light and fluorescence light between the instrument and the tissue target. The system is controlled through a computer interface and software package especially developed for the application. This robust and simple instrument has been evaluated in vivo both on healthy skin but also during a neurosurgical resection procedure. Before surgery the patient received orally a low dose of 5-aminolevulinic acid, converted to the fluorescence tumor marker protoporphyrin IX in the malignant cells. Preliminary results indicate that PpIX fluorescence and brain tissue autofluorescence can be recorded with the help of the developed system intraoperatively during resection of glioblastoma multiforme.

Bladder cancer is widely spread. Moreover, carcinoma in situ can be difficult to diagnose as it may be difficult to see, and become invasive in 50 % of case. Non invasive diagnosis methods like photodynamic or autofluorescence endoscopy allow enhancing sensitivity and specificity. Besides, bladder tumors can be multifocal. Multifocality increases the probability of recurrence and infiltration into bladder muscle. Analysis of spatial distribution of tumors could be used to improve diagnosis. We explore the feasibility to combine fluorescence and spatial information on phantoms. We developed a system allowing the acquisition of consecutive images under white light or UV excitation alternatively and automatically along the video sequence. We also developed an automatic image processing algorithm to build a partial panoramic image from a cystoscopic sequence of images. Fluorescence information is extracted from wavelength bandpass filtered images and superimposed over the cartography. Then, spatial distribution measures of fluorescent spots can be computed. This cartography can be positioned on a 3D generic shape of bladder by selecting some reference points. Our first results on phantoms show that it is possible to obtain cartography with fluorescent spots and extract quantitative information of their spatial distribution on a "wide" field of view basis.

An optical detector suitable for inclusion in tomographic arrangements for non-contact in vivo bioluminescence and fluorescence imaging applications is proposed. It consists of a microlens array (MLA) intended for field-of-view definition, a large-field complementary metal-oxide-semiconductor (CMOS) chip for light detection, a septum mask for cross-talk suppression, and an exchangeable filter to block excitation light. Prototype detector units with sensitive areas of 2.5 cm x 5 cm each were assembled. The CMOS sensor constitutes a 512 x 1024 photodiode matrix at 48 μm pixel pitch. Refractive MLAs with plano-convex lenses of 480 μm in diameter and pitch were selected resulting in a 55 x 105 lens matrix. The CMOS sensor is aligned on the focal plane of the MLA at 2.15mm distance. To separate individual microlens images an opaque multi-bore septum mask of 2.1mm in thickness and bore diameters of 400 μm at 480 μm pitch, aligned with the lens pattern, is placed between MLA and CMOS. Intrinsic spatial detector resolution and sensitivity was evaluated experimentally as a function of detector-object distance. Due to its small overall dimensions such detectors can be favorably packed for tomographic imaging (optical diffusion tomography, ODT) yielding complete 2 π field-of-view coverage. We also present a design study of a device intended to simultaneously image positron labeled substrates (positron emission tomography, PET) and optical molecular probes in small animals such as mice and rats. It consists of a cylindrical allocation of optical detector units which form an inner detector ring while PET detector blocks are mounted in radial extension, those gaining complementary information in a single, intrinsically coregistered experimental data acquisition study. Finally, in a second design study we propose a method for integrated optical and magnetic resonance imaging (MRI) which yields in vivo functional/molecular information that is intrinsically registered with the anatomy of the image object.

Systems using intense pulsed light are being increasingly used in therapy applications where issues related to safety of devices and also of performance are becoming more urgent to address. Mechanisms to address this include a suitable standards framework and also the development and application of appropriate measurement techniques. An approach of using conventional bandpass optical filters and silicon photodetectors has been implemented using an analogue USB data capture interfaces linked to a laptop PC. An initial system with 8 concurrent channels has been upgraded to a separate system sampling up to 16 analogue channels. Sampling takes place at the maximum hardware conversion rate of the USB device. Observations have been made of a range of intense pulsed light systems, including a Lumenis One unit with a range of discrete filters. The system has been of value in determining the basic parameters of output pulse profile and spectral composition. This has in turn been related to aspects of standards development for both device manufacture and allocation of appropriate safety eyewear. Initial assessments of a subset of intense pulsed light systems indicate significant complexities in terms, for example, of variation in spectral content as a function of device output setting.

We use the theory of two dimensional discrete wavelet transforms to derive inversion formulas for the Radon transform of terahertz datasets. These inversion formulas with good localised properties are implemented for the reconstruction of terahertz imaging in the area of interest, with a significant reduction in the required measurements. As a form of optical coherent tomography, terahertz CT complements the current imaging techniques and offers a promising approach for achieving non-invasive inspection of solid materials, with potentially numerous applications in industrial manufacturing and biomedical engineering.

We present a Monte Carlo (MC) program for determining the temporal behavior of radiation in turbid media, in general, and a tissue, in particular. An object-oriented MC program with flexibility for parallel implementation, and for performing stochastic analysis, is described. We determine the temporal probability distribution functions for three tissues: I ) a lipid-based tissue phantom, II ) a healthy dental tissue, and III ) a dental tissue with caries. The expected time of flight for transmitted radiation is calculated. Signal-to-noise ratios (SNR) are then obtained for several temporal thresholds. By restraining the time threshold, a two orders of magnitude increase in SNR is predicted. A multivariate analysis is finally proposed to complement the discrimination process, necessary for trans-illumination interferometry.

An integrating sphere system has been developed to non-invasively study the optical properties of biological tissues over a broad spectral range. Using the integrating sphere as both a diffuse illumination source and a detector provides a technically simple measurement apparatus with numerous advantages. A primary advantage is the reduction of the effect of spatial inhomogeneities on the determination of optical properties, afforded by the increased area of detection through the port-opening of the sphere, which challenges many fibre-based, spatially-resolved measurements. Through a single measurement of total diffuse reflectance, an estimation of the transport albedo of homogeneous, liquid phantoms can be made for those cases where scattering is greater than a determined threshold. Further estimations can be made to describe the absorption environment. The effects of the sphere geometry, particularly port-opening size, on the accuracy of the estimated optical properties will be discussed. These results will be used to modify the design of the integrating sphere as an efficient illuminator and light collector, in order to optimize its use in determining the optical properties of biological tissues.

The aim of this work is the utilization of individual multimode fibers for the purposes of microendoscopy. In the present contribution we discuss the question of image aberration induced by transmission along the fiber proposing a restoration algorithm. Under LP conditions, and for not too large core diameters, the main cause of aberration is intermodal dispersion, but in this work we demonstrate that it may be computed and corrected. We implemented a restoration algorithm based on the separation and equalization of the contribution of each mode, for both step-index and gradedindex fibers. Simulations show that fibers with a diameter of few tens of microns can transmit even quite detailed images, and the proposed algorithm is effective for both the above types of fibers, for different fiber lengths and for a variety of images. Experimental tests were performed by transmitting a Gaussian beam through a graded index silica fiber (diameter 62.5 μm, NA=0.275). After applying the proposed post-processing to the aberrated image exiting from the fiber, we obtained an error of 0.25 &mgr;m on the FWHM of the original Gaussian beam. In conclusion, it appears possible to "capture" an external image and transmit the same through the fiber towards an observer at the other fiber end, after appropriate phase correction.

An experimental technique which allows one to perform pump-probe transient absorption spectroscopy in real-time is an important tool to study irreversible processes. This is particularly interesting in the case of biological samples which easily deteriorate upon exposure to light pulses, with the formation of permanent photoproducts and structural changes. In particular pump-probe spectroscopy can provide fundamental information for the design of optical chromophores. In this work a real-time pump-probe imaging spectroscopy system has been realized and we have explored the possibility to further reduce the number of laser pulses by using a time-gated camera. We believe that the use of a time-gated camera can provide an important step towards the final goal of pump-probe single shot spectroscopy.

For endoscopic application, inexpensive, safe, and extremely flexible hollow infrared optical fibers have been fabricated based on the polycarbonate (PC) capillary with silver and cyclic olefin polymer (COP) as inner coatings. By optimizing the drawing condition of PC capillary from a commercially available polycarbonate tube and inner-coating process, transmission efficiency of hollow PC fibers is shown to be equal to those of glass capillary based ones. Both Er:YAG laser light and green pilot beam were delivered through the endoscope with low losses even when it was sharply bent with a bending radius as small as 1 centimeter. Preliminary experiments were also conducted on possibility of transmitting infrared thermal image by using bundled silver-coated PC hollow fibers.

Pulsed photothermal radiometry (PPTR) can be used for non-invasive depth profiling of skin vascular lesions (e.g., port wine stain birthmarks), aimed towards optimizing laser therapy on an individual patient basis. Optimal configuration of the experimental setup must be found and its performance characterized on samples with well defined structure, before introducing the technique into clinical practice. The aim of our study is to determine how sample structure and width of spectruml acquisition band affect the accuracy of measured depth profiles. We have constructed tissue phantoms composed of multiple layers of agar and of thin absorbing layers between the agar layers. Three phantoms had a single absorber layer at various depths between 100 and 500 μm, and one phantom had two absorber layers. In each sample we induced a non-homogeneous temperature profile with a 585 nm pulsed laser and acquired the resulting radiometric signal with a fast InSb infrared camera. We tested two configurations of the acquisition system, one using the customary 3-5 um spectruml band and one with a custom 4.5 μm cut-on filter. The laser-induced temperature depth profiles were reconstructed from measured PPTR signals using a custom algorithm and compared with sample structure as determined by histology and optical coherent tomography (OCT). PPTR determined temperature profiles correlate well with sample structure in all samples. Determination of the absorbing layer depth shows good repeatability with spatial resolution decreasing with depth. Spectruml filtering improved the accuracy of reconstructed profiles for shallow absorption layers (100-200 μm). PPTR technique enables reliable determination of structure in tissue phantoms with thin absorbing layers. Narrowing of the spectruml acquisition band (to 4.5 - 5.3 μm) improves reconstruction of objects near the surface.

We have developed a novel platform-concept, which allows the construction of an automated upright light-microscope-based slide-scanning device. In order to achieve maximal speed at maximal image quality we have paid special attention to a highly rigid, highly stiff construction. By using novel materials we have been able to achieve vibration-damping characteristics many times better than conventional approaches. We have combined these new concepts with a voice-coil based digitally controlled focus drive, which achieves the speed and stability of a piezo-element, yet allows travels of up to 10 mm. Using scan-modes which avoid stop & go but instead keep the slide moving at a constant speed we have been able to cut scan-times to levels approaching that of multi-objective approaches, yet with much higher flexibility and better image quality.

Biomedical optics and photomedicine applications are challenged by the turbid nature of most biological tissue systems. This nature limits the penetration depth of light into the tissue. Optical clearing improves the penetration depth of light by the application of optical clearing agents which produce an equalization of refractive indices between tissue components and causes a decrease in tissue scattering, and thus increase in optical transmittance. In this paper we examine the effects of optical clearing agents on ex vivo porcine skin using the immersion method. We develop a simple model that can be used to compare different aspects of optical clearing agents such as the rate at which the clearing agents enters the tissue and also the reduction in scattering achieved. We examine the change in the reflected light spectrum over time as the clearing agent enters the skin. This is examined via point probe measurements and also a wide field imaging technique with a consumer-end digital camera. The consumer-end digital camera offers a cheap and simple method for analyzing optical clearing agents over a wider field, overcoming the limitations of single point measurements.

Recently, we have presented a thin optical detector assembly consisting of a microlens array (MLA) coupled to a large area CMOS sensor through a septum mask. The sensor is placed in the physical focal plane of the MLA. Each lens of the MLA forms a small image on the sensor surface, with individual images being separated from each other by the septum mask. The resulting sensor image thus shows a multitude of small sub-images. A low-resolution image can be attained by extracting only those pixels that are located on the optical axis of a microlens, as reported previously. Herein we describe an improved post-processing method to extract images of higher resolution (which can be focused to an arbitrary plane) from a single raw sensor image: Each lens of the MLA results in a mapping from points in object space to corresponding sensor pixels. By tracing back the light paths from sensor pixels through the lenses onto an arbitrary focal plane in object space this mapping can be inverted. Intensities captured on individual sensor pixels can be attributed to virtual pixels on that focal plane using the computed inverse mapping. As a result, from a single acquisition by the detector, images focused to any plane in object space can be calculated. In contrary to the approach of extracting focal point intensities, the spatial resolution is not limited by microlens pitch. We present experimental examples of extracted images at various object plane distances and studies determining the spatial resolution.

A series of nine in-line curvature sensors on a garment are used to monitor the thoracic and abdominal movements of a human during respiration for application to Human Respiratory Plethysmography. These results are used to obtain volumetric tidal changes of the human torso which show agreement with data from a spirometer used simultaneously to recorded the inspired and expired volume at the mouth during both rhythmic and transient breathing patterns. The curvature sensors are based upon long period gratings which are written in a progressive three layered fibre to render them insensitive to refractive index changes. The sensor consists of the long period grating laid upon a carbon fibre ribbon, with this then encapsulated in a low temperature curing silicone rubber. The sensing array is multiplexed and interrogated using a derivative spectroscopy based technique to monitor the response of the LPGs' attenuation bands to curvature. The versatility of this scheme is demonstrated by applying the same garment and sensors to various human body types and sizes. It was also found from statistical analysis of the sensing array data, in conjunction with the measurements taken with a spirometer, that 11 to 12 sensors should be required to obtain an absolute volumetric error of 5%.

The resolution of the images obtained from the eye fundus are limited by the ocular aberrations. As most of the aberrations are due to the eye optics, they do not affect the light intensity measured in the eye iris plane. By illuminating the retina with a laser and collecting the light in a pupil plane conjugate, it is possible to apply the imaging correlography technique. From processing series of pupil plane images, this technique gives information about the retina in the form of the squared modulus of the Fourier transform or, equivalently, the autocorrelation of the diffraction-limited image intensity. Two factors make this technique suitable for retinal imaging: 1) For this technique to work, changes of phase distribution in the retinal plane are necessary between each frame. Small eye movements naturally provide these changes; 2) This method does not provide directly the phase of the Fourier transform. Therefore it is of most use for centro-symmetric objects like the retina's photoreceptor mosaic. Preliminary data have been obtained in vivo showing the feasibility of applying such a technique in the eye. Experimental results are compared against simulation based on retinal scattering model.

Monitoring of bacterial cell numbers is of great importance not only in microbiological industry but also for control of liquids contamination in the food and pharmaceutical industries. Here we describe a novel low-cost and highly efficient technology for bacterial cell monitoring during cultivation process. The technology incorporates previously developed monitoring device and algorithm of its action. The devise analyses light scattered by suspended bacterial cells. Current stage utilizes monochromatic coherent light and detects amplitudes and durations of scattered light impulses, it does not require any labeling of bacterial cell. The system is calibrated using highly purificated bacteria-free water as standard. Liquid medial are diluted and analyzed by the proposed technology to determine presence of bacteria. Detection is done for a range of particle size from 0.1 to 10 μm, and thus particles size distribution is determined. We analyzed a set of different bacterial suspensions and also their changes in quantity and size distribution during cultivation. Based on the obtained results we conclude that proposed technology can be very effective for bacteria monitoring during cultivation process, providing benefits of low simplicity and low cost of analysis with simultaneous high detection precision.

In this work the authors present an optical corneal tomographer that uses two Scheimpflug cameras attached to an innovative illumination system that allows a rotary scanning of the entire cornea. The measurements are made from corneal optical sections obtained by illumination with a collimated beam expanded in a fan by a small cylindrical lens. This lens is provided with motor driven rotation in order to perform automated rotary scan of the whole cornea. The authors expect to achieve a scanning speed that will allow producing complete tomography maps without consideration of eye movements. Two Scheimpflug cameras are used to capture the images of the optical sections. With this system it is possible to obtain 3-D representation of the corneal thickness as well as corneal topography. Maps of the corneal thickness and elevation maps are shown. As Scheimpflug cameras are used, it is expected to obtained data from the lens too.

An in-line holography setup with pinholes is discussed. A set of coherent light cones generated by a pinhole array illuminates the sample. Compared to a single pinhole the effective numerical aperture is increased. The axial resolution is determined by the effective aperture and is increased, too.