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The feasibility of air-coupled ultrasound transducers to detect laser-induced ultrasound from artificial blood vessels embedded in an optically scattering phantom is demonstrated. These air-coupled transducers allow new applications in biomedical photoacoustic imaging where contact with tissue is not preferred. One promising application of such transducers is the addition of photoacoustic imaging to the regular x-ray mammographic screening procedure.

Laser Doppler perfusion imaging (LDPI) has been proven to be a useful tool in predicting the burn wound outcome in an early stage. A major disadvantage of scanning beam LDPI devices is their slow scanning speed, leading to patient discomfort and imaging artifacts. We have developed the Twente Optical Perfusion Camera (TOPCam), a whole field laser Doppler perfusion imager based on a CMOS imaging array, which is two orders of magnitude faster than scanning beam LDPI systems. In this paper the first clinical results of the TOPCam in the setting of a burn centre are presented. The paper shows perfusion images of burns of various degrees. While our system encounters problems caused by blisters, tissue necrosis, surface reflection and curvature in a manner similar to scanning beam imagers, it poses a clear advantage in terms of procedure time. Image quality in terms of dynamic range and resolution appears to be sufficient for burn diagnosis. Hence, we made important steps in overcoming the limitations of LDPI in burn diagnosis imposed by the measurement speed., (Copyright 2009 Elsevier Ltd and ISBI. All rights reserved.)

Perfusion measurements using conventional laser Doppler techniques are affected by the variations in tissue optical properties. Differences in absorption and scattering will induce different path lengths and consequently will alter the probability that a Doppler shift will occur. In this study, the fraction of Doppler shifted photons and the Doppler broadening of a dynamic medium, are measured with a phase modulated low coherence Mach-Zehnder interferometer. Path length-resolved dynamic light scattering measurements are performed in various media having a constant concentration of dynamic particles inside a static matrix with different scattering properties and the results are compared with a conventional laser Doppler technique, with a simple model and with Monte Carlo simulations. We demonstrate that, for larger optical path lengths, the scattering coefficient of the static matrix in which the moving particles are embedded have a small to minimal effect on the measured fraction of Doppler shifted photons and on the measured average Doppler frequency of the Doppler shifted light. This approach has potential applications in measuring perfusion independent of the influence of optical properties in the static tissue matrix.

Photoacoustic imaging is a hybrid imaging modality based on the detection of acoustic waves generated by the absorption of short laser pulses in biological tissue. It combines the advantages of excellent contrast achieved in optical techniques with the high resolution of ultrasound imaging. In this article we present a review of the work done at the University of Twente to image tumor angiogenesis in vivo using this technique. We start with a description and the technical details of the different photoacoustic systems developed in our laboratory, with their validation on phantoms. We then discuss small-animal studies with results of serial imaging of angiogenesis over a 10-day period at the site of tumor induction in a rat. Further, we present clinical results using a photoacoustic mammoscope of breast cancer imaging based on angiogenesis-driven optical absorption contrast.

Advances in optical array sensor technology allow for the real time acquisition of dynamic laser speckle patterns generated by tissue perfusion, which, in principle,allows for real time laser Doppler perfusion imaging(LDPI). Exploitation of these developments is enhanced with the introduction of faster algorithms to transform photo currents into perfusion estimates using the first moment of the power spectrum. A time domain (TD)algorithm is presented for determining the first-order spectral moment. Experiments are performed to compare this algorithm with the widely used Fast Fourier Transform(FFT). This study shows that the TD-algorithm is twice as fast as the FFT-algorithm without loss of accuracy.Compared to FFT, the TD-algorithm is efficient in terms of processor time, memory usage and data transport.

When a diffuse object is illuminated with coherent laser light, the backscattered light will form an interference pattern on the detector. This pattern of bright and dark areas is called a speckle pattern. When there is movement in the object, the speckle pattern will change over time. Laser speckle contrast techniques use this change in speckle pattern to visualize tissue perfusion. We present and review the contribution of laser speckle contrast techniques to the field of perfusion visualization and discuss the development of the techniques.

We present the Twente Optical Perfusion Camera (TOPCam), a novel laser Doppler Perfusion Imager based on CMOS technology. The tissue under investigation is illuminated and the resulting dynamic speckle pattern is recorded with a high speed CMOS camera. Based on an overall analysis of the signal-to-noise ratio of CMOS cameras, we have selected the camera which best fits our requirements. We applied a pixel-by-pixel noise correction to minimize the influence of noise in the perfusion images. We can achieve a frame rate of 0.2 fps for a perfusion image of 128x128 pixels (imaged tissue area of 7x7 cm2) if the data is analyzed online. If the analysis of the data is performed offline, we can achieve a frame rate of 26 fps for a duration of 3.9 seconds. By reducing the imaging size to 128x16 pixels, this frame rate can be achieved for up to half a minute. We show the fast imaging capabilities of the system in order of increasing perfusion frame rate. First the increase of skin perfusion after application of capsicum cream, and the perfusion during an occlusion-reperfusion procedure at the fastest frame rate allowed with online analysis is shown. With the highest frame rate allowed with offline analysis, the skin perfusion revealing the heart beat and the perfusion during an occlusion-reperfusion procedure is presented. Hence we have achieved video rate laser Doppler perfusion imaging.

Laser Doppler flowmetry is a non-invasive method of measuring microcirculatory blood flow in tissue. In this review the technique is discussed in detail. The theoretical and experimental developments to improve the technique are reviewed. The limitations of the method are elaborated upon, and the research done so far to overcome these limitations is critically assessed.

Aim: Spinal cord stimulation (SCS) has been shown effective as a therapy for different chronic painful conditions, but the effectiveness of this treatment for pain as a result of peripheral diabetic neuropathy is not well established. The primary objectives of this study were to evaluate the effect and safety of SCS for treatment of pain and the effects on microcirculatory blood flow in the affected areas in patients with refractory peripheral diabetic neuropathy., Method: The study was designed as a prospective, open-label study. Data were collected during screening, at implant and at regular intervals, after initiation of therapy. Eleven diabetic patients with chronic pain in their lower limbs and no response to conventional treatment were studied. The SCS electrode was implanted in the thoracic epidural space. Neuropathic pain relief was assessed by Visual Analogue Scale (VAS) and microcirculatory skin perfusion was measured with Laser Doppler flowmetry., Results: Nine subjects had significant pain relief with the percutaneous electrical stimulator. Average pain score for all nine patients was 77 at baseline and 34 at 6 months after implantation. At the end of the study, eight of nine patients continued to experience significant pain relief and have been able to significantly reduce their pain medication. For six of them, the stimulator was the sole treatment for their neuropathic pain. No significant changes in microcirculatory perfusion were recorded., Conclusion: Spinal cord stimulation offers an effective and safe therapy for chronic diabetic neuropathic pain.

A real-time photoacoustic imaging system is designed and built. This system is based on a commercially available ultrasound imaging system. It can achieve a frame rate of 8 frames/sec. Vasculature in the hand of a human volunteer is imaged, and the resulting photoacoustic image is combined with the ultrasound image. The real-time photo acoustic imaging system with a hybrid ultrasound probe is demonstrated by imaging the branching of subcutaneous blood vessels in the hand.

Background and Objective: To optimize laser therapy of port-wine stains (PWSs), information about the vasculature as well as lesion depth is valuable. In this study we investigated the use of photoacoustic imaging (PAI) to obtain this information., Study Design/materials and Methods: PAI uses pulsed light to generate ultrasound upon absorption of short light pulses by blood. In this study we used PAI to image vasculature in PWSs in three human volunteers. Two-dimensional imaging (scan direction vs. depth) was carried out by scanning a double-ring photoacoustic sensor over the tissue surface., Results: In the photoacoustic images we observed an increased photoacoustic signal intensity at the locations of the PWS that is associated with increased vascularization. From the obtained images we measured the thickness of the vascular layer and estimated lesion depth. In some cases single vessels could be observed at the position of the PWS whereas in other cases the PWS appeared as a region with large photoacoustic signal intensity., Conclusions: PAI has the potential to reveal information about the lesion depth as well as thickness of the vascular layer., ((c) 2008 Wiley-Liss, Inc.)

The influence of tissue optical properties on laser Doppler perfusion imaging (LDPI) is not well understood. We address this problem by quantifying the dependence of the signal response to tissue optical properties based on speckles or coherence areas and on photon statistics. We investigate the effect in vivo, showing the amplitude of photocurrent fluctuations in normal skin and port-wine stain with a range of beam diameters, and its relation to the speckle size variation difference between these two tissues. For the case of a low concentration of moving particles moving within a static turbid medium, a model is described and applied to predict the influence of speckles on the overall and depth sensitivity of LDPI, for a range of scattering levels and absorption levels. The results show that the speckle related effects on overall and depth sensitivity are large and that the depth sensitivity is highly likely to be misinterpreted without taking the speckle phenomenon into account.

The performance of a graded index multimode fiber optic low coherence Mach-Zehnder interferometer with phase modulation is analyzed. Investigated aspects were its ability to measure path length distributions and to perform path length resolved Doppler measurements of multiple scattered photons in a turbid suspension of particles undergoing Brownian and translational motion. The path length resolution of this instrument is compared with a system using single mode fibers for illumination and detection. The optical path lengths are determined from the zero order moment of the phase modulation peak in the power spectrum. The weighted first moment, which is equal to the average Doppler shift, shows a linear response for different mean flow velocities within the physiological range.

We report the first path-length-resolved perfusion measurements on human skin measured with a phase-modulated low-coherence Mach-Zehnder interferometer with spatially separated fibers for illumination and detection. Optical path lengths of Doppler shifted and unshifted light and path-length-dependent Doppler broadening of multiply scattered light from skin are measured from the Doppler broadened interference peaks appearing in the power spectrum. Perfusion and its variations during occlusion are measured in real time for a given optical path length, and the results are compared with the perfusion signal obtained with a conventional laser Doppler perfusion monitor.

We show a novel technique to distinguish between Doppler shifted and unshifted light in multiple scattering experiments on mixed static and dynamic media. With a phase modulated low coherence Mach- Zehnder interferometer, optical path lengths of shifted and unshifted light and path length dependent Doppler broadening are measured in a two-layer tissue phantom, with a superficial static layer of different thickness covering a semi-infinite dynamic medium having identical optical properties. No Doppler broadening is observed until a certain optical path length depending on the thickness of the superficial static layer. From the minimum optical path length corresponding to the Doppler-shifted light the thickness of the static layer that overlies the dynamic layer can be estimated. Validation of the experimentally determined thickness of the static layer is done with the Doppler Monte Carlo technique. This approach has potential applications in discriminating between statically and dynamically scattered light in the perfusion signal and in determining superficial burn depths.

Near-infrared photoacoustic images of regions-of-interest in 4 of the 5 cases of patients with symptomatic breasts reveal higher intensity regions which we attribute to vascular distribution associated with cancer. Of the 2 cases presented here, one is especially significant where benign indicators dominate in conventional radiological images, while photoacoustic images reveal vascular features suggestive of malignancy, which is corroborated by histopathology. The results show that photoacoustic imaging may have potential in visualizing certain breast cancers based on intrinsic optical absorption contrast. A future role for the approach could be in supplementing conventional breast imaging to assist detection and/or diagnosis.

A theoretical model is presented and experimentally validated that allows the prediction of the effect of speckles on the depth sensitivity of laser Doppler perfusion imaging. It is shown that the influence of speckles on depth sensitivity is large. In particular the sensitivity to particle motion in superficial layers is strongly beam diameter dependent: decreasing the beam diameter on the tissue surface increases the sensitivity to superficial motion to a much stronger extent than sensitivity to motion at a larger depth. This can be explained through the effect of beam diameter changes on the fractional coherence areas generated by photons with different penetration depths in the tissue.

Cooling the scalp during administration of chemotherapy can prevent hair loss. It reduces both skin blood flow and hair follicle temperature, thus affecting drug supply and drug effect in the hair follicle. The extent to which these mechanisms contribute to the hair preservative effect of scalp cooling remains unknown. The purpose of this study was to establish a relationship between local scalp skin temperature and cutaneous blood flow during scalp cooling. We measured skin temperature and cutaneous perfusion during a cooling and re-warming experiment. Experiments on a single subject showed that the measurements were reproducible and that the response was identical for the two positions that were measured. Inter-subject variability was investigated on nine subjects. We found that for the first 10 degrees C of cooling, perfusion of the scalp skin decreases to below 40%. Perfusion can be further reduced to below 30% by a few degrees more cooling, but a plateau is reached after that. We found that a generally accepted relation in thermal physiology between temperature and perfusion (i.e. Q(10) relation) does not describe the data well, but we found an alternative relation that describes the average behavior significantly better.

We show experimental validation of a novel technique to measure optical path length distributions and path length resolved Doppler broadening in turbid media for different reduced scattering coefficients and anisotropies. The technique involves a phase modulated low coherence Mach-Zehnder interferometer, with separate fibers for illumination and detection. Water suspensions of Polystyrene microspheres with high scattering and low absorption levels are used as calibrated scattering phantoms. The path length dependent diffusion broadening or Doppler broadening of scattered light is shown to agree with Diffusive Wave Spectroscopy within 5%. The optical path lengths are determined experimentally from the zero order moment of the phase modulation peak around the modulation frequency in the power spectrum and the results are validated with Monte Carlo simulations.

We present a model for predicting the spatial intensity correlation function of dynamic speckle patterns formed by light backscattered from turbid suspensions, and an experimental validation of these predictions. The spatial correlation varies remarkably with multiple scattering. The provided computational scheme is a step towards correctly interpreting signals obtained from instruments based on the measurement of dynamic speckle patterns in the far field.

Laser Doppler flowmetry (LDF) is a non invasive method enabling the monitoring of microvascular blood flow, a very important marker of tissue health. This article gives an overview on the concept of LDF for microvascular perfusion monitoring and imaging. It first describes the theoretical background of the technique. Then, the benefits of LDF signal processing are shown through clinical examples: use of time-frequency representations and wavelets. Afterwards, the paper introduces novel approaches of velocity components. For that purpose, a work providing the determination of the velocities relative contribution in physiologically relevant units (mm/s) is presented. Imaging perfusion is also reviewed through methods based on laser speckle. The most prominent disadvantage of the latter devices being the time needed to produce a perfusion image, solutions are proposed in the last part of the paper.

We present a method to determine the speed of sound in tissue using a double-ring photoacoustic sensor working in reflection mode. This method uses the cross-correlation between the laser-induced ultrasound waves detected by two concentric ring shaped sensors, while a priori information about the depth-position of the photoacoustic source is not required. We demonstrate the concept by estimating the speed of sound in water as a function of temperature. Comparison of the estimated speed with values reported in literature shows an average systematic error of 0.1% and a standard deviation of 0.1%. Furthermore, we demonstrate that the method can be applied to layered media. The method has application in the correction of photoacoustic and ultrasound images afflicted by local speed variations in tissue. Additionally, the concept shows promise in monitoring temperature changes which are reflected in speed of sound changes in tissue.

In optical Doppler measurements, the path length of the light is unknown. To facilitate quantitative measurements, we develop a phase-modulated Mach-Zehnder interferometer with separate fibers for illumination and detection. With this setup, path-length-resolved dynamic light scattering measurements of multiple scattered light in static and dynamic turbid media are performed. Optical path length distributions spanning a range from 0 to 11 mm are measured from the area under the phase modulation peak around the modulation frequency in the power spectrum. 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. Validation of the estimated path length distributions is done by measuring their deformation for increasing absorption and comparing these observations with predictions based on Lambert-Beer's law.

An integrated optoelectronic probe with small dimensions, for direct-contact laser Doppler blood flow monitoring has been realized. A vertical cavity surface emitting laser (VCSEL), and a chip with photodetectors and all necessary electronics are integrated in a miniature probe head connected to a laptop computer. The computer sound processor is utilized for acquisition and digital signal processing of the incoming Doppler signal. In this paper, the design of the laser Doppler perfusion monitor is described and its performance is evaluated. We demonstrate our perfusion monitor to be less sensitive to subject motion than a commercial fiber-optic device. For medium and high perfusion levels, the performance of our integrated probe is comparable to the fiberoptic flowmeter containing a normal edge-emitting laser diode. For very low perfusion levels, the signal-to-noise ratio of the fiber-optic device is higher. This difference can mainly be attributed to the shorter coherence length of the VCSEL compared with the edge-emitting laser diode.

Background and Objectives: In intravenous access to veins there is a risk of puncturing venous valves or blocking of the catheter by the valves. Therefore, we have investigated whether and how photoacoustic imaging (PAI), which visualizes the lumen of blood vessels, can be used to detect these valves., Study Design/materials and Methods: Venous valves in superficial veins on the dorsal side of the hand of human volunteers were located by palpation and visual inspection. Next, this location was imaged using PAI., Results: In 16 of 21 human volunteers venous valves that were found by palpation could be observed by PAI as local discontinuities in the imaged vessel. From these images, four characteristic features by which venous valves can be recognized in photoacoustic images were identified., Conclusions: PAI has the potential to be applied in the detection of venous valves., ((c) 2006 Wiley-Liss, Inc.)

Photoacoustic imaging is a hybrid imaging modality that is based on the detection of acoustic waves generated by absorption of pulsed light by tissue chromophores such as hemoglobin in blood. For this technique, usually large and costly Q-switched Nd:YAG lasers are used. These lasers provide a pulse energy of at least several milliJoules. In search of alternative light sources, we investigated the use of a small and compact pulsed laser diode to image blood vessels. We proved that a pulsed laser diode can be applied for imaging blood vessels in vivo.

We report on the quantitative influence of speckles in laser Doppler perfusion imaging. The influence of speckles on the signal amplitude and on the Doppler spectrum is demonstrated experimentally for particle suspensions with different scattering levels and various beam widths. It is shown that the type of tissue affects the instrumental response through the effect of lateral light diffusion on the number of speckles involved in the detection process. These effects are largest for narrow beams.

We present PAM, the Photoacoustic Mammoscope developed at the University of Twente, intended for initial retrospective clinical studies on subjects with breast tumours. A parallel plate geometry has been adopted and the breast will be gently compressed between a glass plate and a flat ultrasound detector matrix. Pulsed light (5 ns) from an Nd:YAG laser will impinge the breast through the glass plate in regions of interest; an appropriate number of the 590 elements of the detector matrix will be activated in succession to record photoacoustic signals. Three-dimensional image reconstruction employs a delay-and-sum beamforming algorithm. We discuss various instrumental aspects and the proposed imaging protocol. Performance studies of the ultrasound detector are presented in terms of sensitivity, frequency response and resolution. Details of the patient-instrument interface are provided. Finally some imaging results on well-characterized breast tissue phantoms with embedded tumour simulating inserts are shown.

We present photoacoustic images of tumor neovascularization obtained over a 10-day period after subcutaneous inoculation of pancreatic tumor cells in a rat. The images were obtained from ultrasound generated by absorption in hemoglobin of short laser pulses at a wavelength of 1064 nm. The ultrasound signals were measured in reflection mode using a single scanning piezodetector, and images were reconstructed with a weighted delay-and-sum algorithm. Three-dimensional data visualize the development and quantify the extent of individual blood vessels around the growing tumor, blood concentration changes inside the tumor and growth in depth of the neovascularized region.

The standardisation of manoeuvres to perform clinically discriminative microvascular flow reserve tests is still poorly developed, as well as the response analysis. The aim of this study was to establish a reproducible analysis method for the post-occlusive reactive hyperaemia (PORH) test measured using laser Doppler perfusion monitoring (LDPM). LDPM data were measured from the PORH response of 24 Fontaine class II-III peripheral atherosclerotic/arterial obstructive disease (PAOD) patients and 30 healthy subjects. The PORH response was recorded from the dorsum of the foot after 3 min of arterial occlusion at the thigh. The resulting tracings were analysed by describing their morphology through five defined parameters: resting flux (RF), time to RF level (tRF), maximum flux (MF) during reactive hyperaemia, time to maximum flux (tMF), and time to half recovery (tHR). While the time parameters were discriminative between patients and controls, flux parameters were not. The time to resting flux (tRF) led to the most discriminative model that correctly predicted 88.5% of the cases. Hence, we concluded that obtaining t(RF) with the presented procedures provides an optimal model to quantify the patient's microvascular condition from the PORH response.

A photoacoustic double-ring sensor, featuring a narrow angular aperture, is developed for laser-induced photoacoustic imaging of blood vessels. An integrated optical fiber enables reflection-mode detection of ultrasonic waves. By using the cross-correlation between the signals detected by the two rings, the angular aperture of the sensor is reduced by a factor of 1.9, from 1.5 to 0.8 deg. Consequently, photoacoustic images could be obtained in a manner analogous to the ultrasound B-scan mode. Next, the cross section of artificial blood vessels is visualized by reconstruction of the absorbed energy distribution. Finally, in vivo imaging and the subsequent reconstruction of the absorbed energy distribution is demonstrated for superficial blood vessels in the human wrist., (Copyright 2004 Society of Photo-Optical Instrumentation Engineers.)

We present a laboratory version of a photoacoustic mammoscope, based on a parallel plate geometry. The instrument is built around a flat high-density ultrasound detector matrix. The light source is a Q-switched Nd:YAG laser with a pulse duration of 5 ns. To test the instrument, a novel photoacoustic phantom is developed using poly(vinyl alcohol) gel, prepared by a simple procedure that imparts optical scattering suggestive of breast tissue to it without the requirement for extraneous scattering particles. Tumor simulating poly(vinyl alcohol) gel spheres appropriately dyed at the time of preparation are characterized for optical absorption coefficients. These are then embedded in the phantom to serve as tumors with absorption contrasts ranging from 2 to 7, with respect to the background. Photoacoustic studies in transmission mode are performed, by acquiring the laser-induced ultrasound signals from regions of interest in the phantom. Image reconstruction is based on a delay-and-sum beamforming algorithm. The results of these studies provide an insight into the capabilities of the prototype. Various recommendations that will guide the evolving of our laboratory prototype into a clinical version are also discussed., (Copyright 2004 Society of Photo-Optical Instrumentation Engineers.)

A double-ring sensor was applied in photoacoustic tomographic imaging of artificial blood vessels as well as blood vessels in a rabbit ear. The peak-to-peak time (tau(pp)) of the laser (1064 nm) induced pressure transient was used to estimate the axial vessel diameter. Comparison with the actual vessel diameter showed that the diameter could be approximated by 2ctau(pp), with c the speed of sound in blood. Using this relation, the lateral diameter could also precisely be determined. In vivo imaging and monitoring of changes in vessel diameters was feasible. Finally, acoustic time traces were recorded while flushing a vessel in the rabbit ear with saline, which proved that the main contribution to the laser-induced pressure transient is caused by blood inside the vessel and that the vessel wall gives only a minor contribution.

The effects of fiber coupling and fiber length on photocurrent fluctuations are studied when the light of a laser diode transmitted to and from a dynamic turbid medium by a step-index multimode fiber is studied. When the laser light is coupled asymmetrically, filling only the higher-order modes, the photocurrent fluctuations are suppressed significantly when fiber lengths of as much as 16 m are added between the laser and the medium. Addition of as much as 16 m of detection fiber, or any fiber in the case of symmetric light coupling, leads to much less or no suppression of the photocurrent fluctuations.

Background and Objectives: The ability to correctly visualize the architectural arrangement of microvasculature is valuable to many diverse fields in medicine. In this study, we applied photoacoustics (PA) to obtain high-resolution images of submillimeter blood vessels., Study Design/materials and Methods: Short laser pulses are used to generate ultrasound from superficial blood vessels in several animal models. From these ultrasound waves the interior of blood vessels can be reconstructed., Results: We present results from a novel approach based on the PA principle that allows specific in vivo visualization of dermal blood vessels without the use of contrast agents or ionizing radiation., Conclusions: We show PA images of externalized blood vessels and demonstrate in vivo PA imaging of vasculature through layers of skin varying in thickness., ((c) 2004 Wiley-Liss, Inc.)

This paper describes the simultaneous use of two, noninvasive, near-infrared techniques near-infrared spectroscopy (NIRS) and a continuous wave NIR laser Doppler flow system (LDF) to measure changes in the blood oxygenation, blood concentration and blood flow velocity in the brain. A piglet was used as animal model. A controlled change in the arterial CO2 pressure (PaCO2) was applied for achieving changes in the listed cerebrovascular parameters. The time courses of blood concentration parameters (NIRS) and RMS blood flow velocity (LDF) were found to correspond closely with those of carotid blood flow and arterial carbon dioxide pressure (PaCO2). This result shows the additional value of LDF when combined with NIRS, preferably in one instrument. Development of pulsed LDF for regional blood flow measurement is indicated.

The visualization of the brain vascular system could be of great importance for studying its functionality and for diagnosing possible disorders. In this paper we report the use of photoacoustics for imaging brain perfusion on Albino rats in vivo and post mortem. The measurements on the animals were direct on the skin surface. The blood perfusion on skull cartilage was imaged and 2D slices were constructed by using a beamforming algorithm. From the images representation the Interactive Data Language (IDL, Research System Inc.) was used. We also investigated the possibility of using the Evans Blue dye as a substitute of blood for imaging brain structures in vitro. The breakdown of the dye under pulsed laser irradiation was studied and the energy under which this effect occurs was calculated for the wavelength of 532 nm.

To investigate the feasibility of instrument-independent perfusion units for laser Doppler flowmetry, a comparison was performed of two commercial fiberoptic laser Doppler perfusion monitors measuring the same flux situation for two different types of probes. In vivo measurements were performed on the cortex of pig's kidney, with an ultrasonic arterial flow meter as reference. The flow was mainly varied by internal arterial constriction using a balloon catheter. For each probe, instruments are compared in terms of the ratio of laser Doppler flux and arterial flow. For a given probe, the flux-to-flow ratios of the two instruments show a linear mutual relationship for a wide variety of arterial flows and laser Doppler fluxes. In vitro measurements were performed on an aqueous suspension of polystyrene microspheres. For the probe with interfiber distance 500 microm the ratio of the in vivo fluxes appears to agree within 16% to the value found in vitro, while for the 250-microm probe a difference of 28% was found. For a wide range of fluxes, the in vivo flux values of one instrument can be translated into flux values for the other instrument, in spite of the instrumental differences. This enables the user to render experimental results independent of the specific instrument, thus facilitating multi-center studies.

Materials for solid photoacoustic breast phantoms, based on poly(vinyl alcohol) hydrogels, are presented. Phantoms intended for use in photoacoustics must possess both optical and acoustic properties of tissue. To realize the optical properties of tissue, one approach was to optimize the number of freezing and thawing cycles of aqueous poly(vinyl alcohol) solutions, a procedure which increases the turbidity of the gel while rigidifying it. The second approach concentrated on forming a clear matrix of the rigid poly(vinyl alcohol) gel without any scattering, so that appropriate amounts of optical scatterers could be added at the time of formation, to tune the optical properties as per requirement. The relevant optical and acoustic properties of such samples were measured to be close to the average properties of human breast tissue. Tumour simulating gel samples of suitable absorption coefficient were created by adding appropriate quantities of dye at the time of formation; the samples were then cut into spheres. A breast phantom embedded with such 'tumours' was developed for studying the applicability of photoacoustics in mammography.

Microcirculatory blood flow can be measured using a laser Doppler flowmetry (LDF) probe. However, the readings are affected by the tissue's optical properties (absorption and scattering coefficients, mu(a) and mu(s)) and probe geometry. In this study the influence of optical properties [mu(a)in(0.053,0.23) mm-1,mu(s)in(14.7,45.7) mm-1] on LDF perfusion and LDF sampling depth was evaluated for different fiber separations. In vitro measurements were made on a sophisticated tissue phantom with known optical properties that mimicked blood flow at different depths. Monte Carlo simulations were carried out to extend the geometry of the tissue phantom. A good correlation between measured and simulated data was found. The simulations showed that, for fixed flow at a discrete depth, the influence of mu(s) or mu(a) on LDF perfusion increased with an increase in flow depth and decreased with an increase in fiber separation. For a homogeneous flow distribution, however, the perfusion varied 40% due to variations in the optical properties, almost independent of the fiber separation (0.23-1.61 mm). Therefore, the effect in real tissue is likely to vary due to the unknown heterogeneous blood flow distribution. Further, the LDF sampling depth increased with a decrease in mu(s) or mu(a) and an increase in fiber separation. For fiber separation of 0.46 mm, the e-1 sampling depth ranged from 0.21 to 0.39 mm.

We utilized a complimentary metal oxide semiconductor video camera for fast flow imaging with the laser Doppler technique. A single sensor is used for both observation of the area of interest and measurements of the interference signal caused by dynamic light scattering from moving particles inside scattering objects. In particular, we demonstrate the possibility of imaging the distribution of the moving red blood cell concentration. This is a first step toward laser Doppler imaging without scanning parts, leading to a much faster imaging procedure than with existing mechanical laser Doppler perfusion imagers.

We have developed a blood velocimeter based on the principle of self-mixing in a semiconductor laser diode through an optical fiber. The intensity of the light is modulated by feedback from moving scattering particles that contain the Doppler-shift frequency. Upon feedback the characteristics of the laser diode change. The threshold current decreases, and an instable region may become present above the new threshold. The amplitude of the Doppler signal turns out to be related to the difference in intensity between situations with and without feedback. This amplitude is highest just above feedback. The suppression of reflection from the glass-fiber facets is of paramount importance in the obtaining of a higher signal-to-noise ratio. Using an optical stabilization of the feedback, we optimized the performance of the laser-fiber system and the Doppler modulation depth and clarified its behavior with a suitable physical model. We also investigated the effect of the finite coherence length of the laser. We tested the efficiency of the self-mixing velocimeter in vivo with the optical glass fiber inserted in the artery with endoscopic catheters, both in upstream and in downstream blood flow conditions. For the latter we used a special side-reflecting device solution for the fiber facet to allow downstream measurements.

We report first results of measurements by low-coherence Doppler interferometry of the path-length distribution of photons undergoing multiple scattering in a highly turbid medium. We use a Mach-Zehnder interferometer with multimode graded-index fibers and a superluminescent diode as the light source. The path-length distribution is obtained by recording of the heterodyne fluctuations that arise from the Brownian motion of particles in an Intralipid suspension as a function of the optical path length. The experimental path-length distribution is in good agreement with predictions of Monte Carlo simulations. In the heterodyne spectrum, an increase of the mean Doppler frequency with path length is observed.

Laser Doppler perfusion imaging (LDPI), is a further development in laser Doppler flowmetry (LDF). Its advantage is that it enables assessment of microvascular blood flow in a predefined skin area rather than, as for LDF, in one place. In many ways this method seems to be more promising than LDF in the assessment of burn wounds. However, several methodological issues that are inherent in the LDPI technique, and are relevant for the assessment of burn depth, must be clarified. These include the effect of scanning distance, curvature of the tissue, thickness of topical wound dressings, and pathophysiological effects of skin colour, blisters, and wound fluids. Furthermore, we soon realised that to examine the perfusion image generated by LDPI adequately the process of analysis was appreciably improved by the simultaneous use of digital photography. In the present investigation we used both in vitro and in vivo models and also examined burned patients, and found that the listed factors all significantly affected the LDPI output signal. However, if these factors are known to the examiner, most of them can be adjusted for. If the technique is further improved by minimizing such effects and by reducing the practical difficulties of applying it to a burned patient in the burns unit, the technique may find uses in everyday clinical decision-making.

Intra-arterial measurements of the velocity and the average flow of red-blood cells were investigated by means of a fiber-coupled laser Doppler velocimeter based on the self-mixing effect. The velocity of the red cells was calculated from the frequency of the signal that occurs when light, scattered back from a moving object in front of a fiber into a laser-diode cavity, interferes with the laser cavity's proper mode. These fluctuations occur at the Doppler frequency. The signal was obtained from the photodiode that is present in the laser diode's housing. Temperature control and stabilization of the diode cavity were introduced to reduce the light-intensity fluctuation that is due to mode hopping of the diode. The velocimeter was calibrated with a rotating disk covered with white paper (nonlinearity of 2.6% for velocities up to 0.4 m/s) and tested in vitro as a fluid velocimeter. The velocimeter was used in in vivo tests on the iliac artery of a 35-kg pig and on the arteria pulmonaris of a healthy calf. The optical fiber was placed in the iliac artery by a basket catheter 4 cm proximal to the bifurcation of the femoral artery. The average arterial blood flow velocity of the red cells were measured upstream and downstream. A special cleaving procedure for the fiber tip in downstream measurement is reported. Blood-velocity measurement is compared with values generated by an ultrasound flowmeter, and a difference of less than 9% is found.

An experimental investigation was performed into the effect of simple shear on the light-scattering properties of undiluted human blood. Undiluted human blood was enclosed between two glass plates with an adjustable separation between 30 and 120 microns and with one plate moving parallel to the other. For various shear rates and layer thicknesses, the angular light distribution and the collimated transmission were measured for 633-nm light. For shear rates above 150 s-1, the transmission results directly yielded a total attenuation coefficient of 120 mm-1. At lower shear rates the total attenuation followed an irregular pattern. From the angular intensity distributions, the anisotropy for single scattering was deduced by inverse Monte Carlo simulations. A continuous increase of the average cosine g with the shear rate was observed, with g in the range 0.95-0.975.

Doppler Monte Carlo (DMC) simulations of the transport of light through turbid media, e.g., tissue, can be used to predict or to interpret measurements of the blood perfusion of tissue by laser-Doppler perfusion flowmetry. We describe the physical and mathematical background of Doppler Monte Carlo calculations, and present some comparisons with measurements, performed with experimental flow models, built to mimic skin tissue characteristics and for the calibration of perfusion instruments,which are important goals of the Biomed-I concerted action. The measurements deal with coherence effects.