KEYWORDS: Endoscopes, In vivo imaging, Calibration, Visualization, High speed imaging, Image resolution, Video, 3D image processing, Biomedical optics, Semiconductor lasers
We report the design of a novel laser line-triangulation laryngoscope for the quantitative visualization of the three-dimensional movements of human vocal folds during phonation. This is the first successful in vivo recording of the three-dimensional movements of human vocal folds in absolute values. Triangulation images of the vocal folds are recorded at the rate of 4000 fps with a resolution of 256×256 pixels. A special image-processing algorithm is developed to precisely follow the subpixel movements of the laser line image. Vibration profiles in both horizontal and vertical directions are calibrated and measured in absolute SI units with a resolution of ±50 µm. We also present a movie showing the vocal folds dynamics in vertical cross section.
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.
We utilized a CMOS (Complementary Metal Oxide Semiconductor) video-camera for fast flow imaging with use of 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. Particularly we demonstrate the possibility to image the distribution of the moving read blood cell concentration. This is a first step towards laser Doppler imaging without scanning parts, leading to a much faster imaging procedure compared to existing mechanical laser Doppler perfusion imagers.
This paper reviews the development and use of laser Doppler perfusion monitors and imagers. Despite their great success and almost universal applicability in microcirculation research, they have had great difficulty in converting to widespread clinical application. The enormous interest in microvascular blood perfusion coupled with the 'ease of use' of the technique has led to 2000+ publications citing its use. However, useful results can only be achieved with an understanding of the basic principles of the instrumentation and its application in the various clinical disciplines. The basic technical background is explored and definitions of blood perfusion and laser Doppler perfusion are established. The calibration method is then described together with potential routes to standardisation. A guide to the limitations in application of the technique gives the user a clear indication of what can be achieved in new studies as well as possible inadequacy in some published investigations. Finally some clinical applications have found acceptability and these will be explored.
In laser Doppler flowmetry (LDF) deep perfusion measurements can be realized by using a large separation between the fibers used for illumination and detection. In order to achieve a sufficient signal-to-noise ratio, the power of the laser light can be increased, but only to the limit indicated by the safety regulations. In this paper, pulsed laser Doppler flowmetry (pLDF) is presented as a manner to increase the SNR without exceeding the safety limits. The method is based on the principle that light is needed only when the signal is being sampled. The setup is presented, and we will show results that indicate that equivalent results are obtained for a pulsed and continuous wave setup (cwLDF), however with a much smaller tissue exposure. Furthermore, the limits encountered in realizing a pulsed system will be discussed.
A double-ring photoacoustic sensor to image and monitor blood content in tissue has been developed. This sensor has a very small opening angle. Using this sensor we are able to image artifical blood vessels, as well as vessels in a rabbit ear. Furthermore, the feasibility of in vivo imaging is demonstrated with a photoacoustic reconstruction of the joining of two palmar veins a few centimeter proximal to the wrist in a human arm.
A novel photoacoustic breast phantom was developed using poly(vinyl alcohol) gel prepared by a simple technique that imparts optical scattering to the gel without the neccessity for scattering particles. Tumour simulating gel samples of suitable absorption coefficient were also prepared using a second technique, by adding appropriate quantities of dye at the time of formation; the samples were then cut into spheres. The optical absorption coefficient of the spheres was chosen as between 4 - 7 times that of breast tissue. A breast phantom with a thickness of 60 mm, embedded with such 'tumours' was developed for studying the applicability of photoacoustics in mammography.
Time resolved photoacoustics, in a transmission mode, was used to image the inhomogeneities. Light excitation was from a liquid-light guide coupled Nd:YAG laser at 1064 nm, with a 5 ns pulse duration. The guide was mechanically scanned across the surface of the phantom, with the time-of-flight signals recorded using a PVDF based detector array. A modified delay and sum beamforming algorithm was used to reconstruct the photoacoustic sources. Results of these experiments are discussed.
To localize and monitor the blood content in tissue we developed very sensitive photoacoustical detectors. In these detectors a PVdF-layer has been used as piezo-electric material and also fibers for the illumination of the sample are integrated. The resolution is about 20 im in depth and about 50-100 im laterally. The wavelengths ofthe laser light were 532and 1064 nm. With these colors we can measure at different depths in tissue. We will report measurements on real tissue: vessels in chicken breast, in the human arm, and in test animals at various positions.
It is possible to delineate the contribution of surface layers from the bulk optical properties of the medium by using selective adjacent distances of the detection fibers in an optical probe with small source-detectors distances. Using an optical probe with small source-detector separation, the measurement at r < 0.8 mm carries more information about the surface layers, the measurement at greater r > 1.4 mm is dominated by optical properties of deeper layers and is less sensitive to differences in surface optical properties.
We report 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 light source. The path length distribution is obtained by recording the heterodyne fluctuations arising due to 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 the path length is observed. The path length resolution of the setup was directly evaluated by replacing the turbid medium with randomly moving scatterers by a mirror attached to a harmonically oscillating piezo-element. The maximum (peak-to-peak) mirror displacement was 10% of the optical wavelength. We observed a narrow and strong (signal/noise ratio ~300) interference peak with the full width at the half maximum ~50 microns equal to the coherence length of the superluminescent diode. However, additional weaker satellite peaks are also observed, which may be caused by the intermodal dispersion in our multimode fibers. We demonstrate that our setup allows achieving high path length resolution for biological tissues where the width of the path length distribution is several millimeters.
Using very sensitive photoacoustical detectors we localized and monitored the blood content in tissue. In these detectors a PVdF-layer has been used as piezo-electric material and also fibers for the illumination of the sample are integrated. The resolution is about 20micrometers in depth and about 50-100micrometers laterally. The wavelengths of the laser light were 532 and 1064 nm. With these colors we can measure at different depths in tissue. The measurements concerned blood perfusion in real tissue: vessels in chicken breast, in test animals at various positions and in the human arm.
Integrated probes have been developed containing the essential optoelectronic components necessary for monitoring tissue perfusion using the laser Doppler principle. The device contains a VCSEL for illumination, and a chip with photodetectors, amplifiers for signal enhancement, and digital circuitry for external probe control. VCSEL and detector chip are mounted on a common ceramic platform. A Peltier element may be included for temperature stabilisation, or thermal cycling for physiological purposes. Two special chips have been developed: one containing an array of five detectors, at various distances from the laser, which will allow for some degree of depth discrimination, and a single detector chip. In this paper the probe designs are presented and some results of in vivo measurements are shown.
Measurements have been carried out using a pulsed laser-Doppler setup. The main advantage of pulsing a laser-diode is that much higher peak powers can be used, allowing a larger source-detector separation, resulting in a larger penetration depth. The method enables e.g. monitoring of cerebral perfusion as well as monitoring perfusion through organs (e.g. kidney).
Photo-acoustics (PA) is a technique, which can be used as the basis for non-invasive medical tomography. It is based on the absorption contrast between the biological target and the surrounding tissue and as a result of that does not suffer from strong light scattering. This fact makes PA suitable for imaging of the microvasculature in skin (532nm) or of deeper structures by using infrared light. In this paper, we present images of a vasculature cast, which was obtained by a Wistar rat and measurements on the absorption coefficient of Evans Blue.
We will present an overview of some new optical diagnostic techniques that have emerged in the past few years. Among those: *Photon Transillumination of Tissue. Pulsed or modulated laser light is scattered and/or absorbed in tissue. The position of scattering layers and discrete absorbers will influence the pattern of light emerging from inside. Image analysis will reveal those positions. *Photoacoustics of Tissue. Not the emerging light but ultrasound waves, produced by adiabatic heating (approximately 1 K) of blood cells by absorption of laser photons, are detected at the surface. The relatively low sound velocity allows to perform time-of-flight measurements, thus enabling to obtain depth-sensitive information. *Laser Doppler Monitoring and Imaging. On one hand the traditional monitoring technique is being upgraded in a large-scale European Standardization project. On the other, new imaging techniques are emerging. Also, laser-Doppler using self-mixing techniques renders flow information from inside arteries.
We suggest a method for quantitative determination of the total fraction of Doppler-shifted photons in light scattered by an object containing both moving and static scatterers. The method is based on two approaches: time-varying speckle and laser Doppler were we consider the speckle fluctuations as the primary phenomenon with the Doppler effect being the main cause of these fluctuations. A validation of the method and one of its practical applications is demonstrated. The total fraction of Doppler-shifted photons in light backscattered from human skin has been measured in vivo.
To localize and monitor the blood content in tissue we developed a very sensitive photo-acoustical detector. PVDF has been used as piezo-electric material. In this detector also fibers for the illumination of the sample are integrated. Resolution is about 20 (m in depth and about 50-100 m laterally). We use 532 nm light. We will show how photoacoustics can be used for measuring the thickness of tissue above bone. We will also report measurements on tissue phantoms: e.g. a vessel delta from the epigastric artery branching of a Wistar rat, filled with an artificial blood-resembling absorber. The measurements have been carried out on phantoms containing vessels at several depths. Signal processing was enhanced by Fourier processing of the data.
Aiming at development of tools for accurate non-invasive diagnosis, we investigate the accuracy of experimental and numerical approaches for monitoring of deep structures, such as blood vessels, together with determination of their optical parameters. Among numerical techniques developed for determination of structure and optical parameters of biotissue basing on measurement data, Monte Carlo simulations is the best tool, but requires a considerable computation time to preserve accuracy when one deals with large source detector separations. In contrast, diffusion approximation is fast but suffers from a difficulty of estimation of accuracy. We implemented an intermediate approach, deriving and solving frequency-domain-specific equations basing on radiative transfer theory. We conducted measurements and corresponding simulations using multilayered tissue-simulating sample and estimate accuracy novel techniques can achieve. We obtained an accuracy of 0.002 mm-1 in measurements of the reduced scattering coefficient.
An integrated probe has been developed containing the essential optoelectronic components necessary for monitoring tissue perfusion using the laser Doppler principle. The device includes a VCSEL for illumination, and a chip containing photodetectors, analog amplifiers for signal enhancement, and digital circuitry for external probe control. VCSEL and detector chip are mounted on a common ceramic platform, which also includes an integrated temperature sensor. A Peltier element may be included for temperature stabilization, or thermal cycling for physiological purposes. Two special chips have been developed: one containing an array of five detectors, at various distances from the laser, which will allow for some degree of depth discrimination, and a single detector chip. In this paper the multidetector chip and the probe design are presented.
In this work we are going to discuss a role of speckles in laser-Doppler. Despite the speckle approach and laser Doppler were developed separately the both are used to describe the phenomena of the intensity oscillations on the photodetector caused by dynamic light scattering. Actually, those techniques are used when an information about the velocity of moving object must be obtained. Recently it was noticed by David Briers that finally both techniques give the same quantitative answer and might be integrated in one approach having advantages of both. By this work we tried to extend the speckle approach to the work on laser Doppler. We will show our own way in which the both approaches could be integrated in one. The contents of the presentation: first a short introduction into the subject will be done and main goals of the work will be pointed out. Then we will overview shortly two interferometric methods, laser-Doppler and time-varying speckle, trying to find what are the similarities and distinctions between of those. Next, our theoretical framework on integrating of two techniques in one will be proposed. We will show you some experimental validation of our approach and a possible practical application of the one. And finally, conclusion and summary.
Development of efficient analytical and numerical techniques for the determination of optical parameters of biotissues on the base of measured data is a crucial part of successful implementation of non- invasive diagnostic techniques in clinical conditions. Widely used approximations, like diffusion approximation (DA), were shown to fail in most real-life circumstances due to simplifications of modeling or neglect of various involve phenomena, like boundary effects, tissue inhomogeneity, skin roughness and deviations of optical properties of skin in time due to physiological effects. In this work we compare experimental results with results of numerical simulations. For our measurements, we used both spatial-resolved and frequency-domain techniques. To describe propagation of photons we numerically solved the radiatve transfer equation (RTE). We found that the Monte-Carlo method (MC) is too time-consuming for large source- detector separations. We achieved flexibility in preparation of experimental medium with tissue simulating sample containing several homogeneous layers. Our objective is the investigation of accuracy in determining unknown structures and optical coefficients from measured data, based on the realistic model of the tissue described in the RTE.
An experimental facility has been developed that allows for the determination of the scattering cross section and the scattering phase function of thin layers of undiluted blood. The layer is formed between to flat glass plates. By rotating one of the plates, a simple shear can be imposed onto the blood. Experiments have been performed with 633 nm light on layers with a thickness between 20 and 60 micrometer. It was shown that for shear rates in the range 150 - 500 s-1, the scattering coefficient has a constant value of 115 mm-1. Also, the scattering anisotropy for single scattering g ranges from 0.95 at lower shear rates, to 0.975 at higher shear rates. The value of g increases with the shear, both in a direction parallel to and perpendicular to the shear direction.
In this work we compare experimental results with results of numerical simulations. For our measurements, we used both spatial-resolved and frequency-domain techniques. To describe propagation of photons we solved the rigorous radiative transfer equation (RTE). We found that the Monte- Carlo method (MC) is too time-consuming for large source- detector separations. We achieved flexibility in preparation of experimental medium with tissue-simulating sample containing of several homogeneous layers. Our objective is the investigation of accuracy in determining unknown structures and optical coefficients from measured data, based on the realistic model of the tissue described in the RTE. We have shown that, by comparing the use of the RTE to the diffusion approximation or MC, we achieve better accuracy or universality in source-detector distances.
To localize and monitor the blood content in tissue we developed a very sensitive double-ring photo-acoustical detector. PvdF has been used as piezo-electric material. In this detector also a fiber for illumination of the sample is integrated. This detector has the advantage that it is very sensitive in the forward direction. A ratio of FWHM to depth of 1:70 can be obtained with this detector.
Our goal is the development of a photo-acoustic instrument for 3D imaging of the microvascular structure in tissue, in real time. A photo-acoustic multi-element detector has been designed, which measures in reflection mode. The light source is a pulsed laser with a wavelength of 532nm and the active piezo-material is PVdF. Using a disk detector we have achieved to reconstruction 3D images with a depth and lateral resolution of 10-20 micrometers and 200 micrometers respectively. With the new probe we expect to reduce the measuring time and to sped up the signal and image processing.
In the present paper, recent experimental advances obtained with a laser Doppler self-mixing velocimeter are reported. The self-mixing effect in a semiconductor laser is used to realize the velocimeter. The velocity is calculated measuring the frequency peak of the frequency spectrum of the intensity signal generated by the laser diode when modulated by feedback light coming from the moving scattering particles. A special optical fiber version of this velocimeter to be used specifically for intra-arterial blood velocity measurement has been realized and a solution for reducing temperature influence on the semiconductor performances is proposed. The results of the in vivo tests carried out with the proposed sensor are presented.
We report here first results of measurements of the pathlength distribution of scattered photons by low- coherence Doppler interferometry. Laser Doppler Flowmetry (LDF) is used to non-invasively monitor the blood micro circulation in biological tissue. On the other hand, the LDF response is also affected by the optical properties of the tissue itself, which complicates the problem of the exact evaluation of the blood perfusion. In a scattering medium like skin photons travel along different paths of variable length. The longer the pathlength, the higher the change for the photon to be scattered by a blood cell. An aqueous suspension of Intralipid is used to mimic the most important properties of the skin. Using a free beam Michelson interferometer we measure the AC component of the intensity of the pattern formed by interfering the scattered light with a coherent component from the reference channel. In each measurement the mirror in the reference channel is kept fixed while the AC component arises due to the Doppler effect in light scattered by the micro-particles experiencing Brownian motion. The pathlength distribution is extracted from the dependence of the detected signal on the reference mirror position.
A theoretical framework is proposed for the description of the photodetector signal generated by Doppler-induced speckle fluctuations. The theory allows for predicting the power of the photo current fluctuations. It is valid for a detector of arbitrary size. The input data required for application of the theory are the angular distribution of the detected light, the fraction of Doppler shifted photons and the active detector size. The theory is based on the time domain approach to the statistics of dynamic speckle patterns on the photodetector. An experiment has been carried out to validate some aspects of our theory. The consequences of the speckle dynamics for the various modes of laser Doppler Flowmetry are discussed. As one of a practical application of the theory the fraction of Doppler- shifted photons in light back scattered from human skin has been measured in vivo. .
We describe a non-invasive method for the determination of optical parameters of highly scattering media, such as biological tissue. An advantage of this method is that it does not rely on diffusion theory, thus it is applicable to strongly absorbing media and at small source-detector separations. Monte Carlo simulations and phantom measurements are used to illustrate the achievable accuracy of the system. The method was applied to non-invasive in- vivo tracking of haemoglobin concentration in biological tissue. The results correlated well to clinically determined Hb concentrations.
A method for the absolute determination of the fraction of Doppler-shifted photons in light scattered by mixture of moving and stationary scatterers is proposed. It based on the theoretical approach developed in our group to predict the signal power fluctuations of an integrated Doppler- induced speckle pattern on the photodetector. A benefit of using a multimode step-index optical fiber as a scattering sample-to-detector interface is considered. An experimental validation of the method has been performed and experimentally obtained data are in a good agreement with the theory. As one of a practical application of the method the fraction of Doppler-shifted photons in light backscattered from human skin has been measured and the results are reported.
We developed Photoacoustic Tissue Scanning to tomografically image optically absorbing structures in tissue. With 532 nm light depths down to 6-9 mm were reached. As the samples we used capillaries with blood, human hairs or a variable dilution of Evans blue, embedded in real tissue (chicken breast) or a 10 % solution of Intralipid-10%. Small PVdF piezoelectric hydrophones were used for detection, in scanning mode for imaging purposes. The depth resolution is about 10 ?m, and the lateral resolution is limited by the diameter of the detector (200 ?m in our case).
An improved version of our laser Doppler tester has been developed. The tester contains a tissue phantom made in the form of a thin film. Films with a thickness in the range 20 - 90 micrometer are stacked to reach a total thickness of several millimeters. Motion is realized by rotating discs with a thickness of 20 - 22 micrometer, separated by a static layer of 95 micrometer thickness. The total number of movable layers is 13. The tester contains four phantom stacks, each mimicking a different type of tissue. Since each tissue consists of a stack of thin layers separated by a matching medium, the consequence of a possible mismatch on the photon penetration depth is investigated with the Monte Carlo simulation technique. This leads to a range of refractive indices in which the matching medium must be taken to keep the effect of mismatch below an acceptable level. It appears that for a tissue structure composed of layers with a thickness of 30 micrometer, the refractive index of the matching material should be between 1.50 and 1.54 for a refractive index of the matrix material of the phantom equal to 1.52. Within this range, the effect of mismatch on the probed depth and the total reflection is limited to 2%.
KEYWORDS: Glucose, Raman spectroscopy, Signal to noise ratio, Luminescence, Spectroscopy, In vivo imaging, Raman scattering, Blood, Light scattering, Error analysis
We have investigated the possibilities of applying Raman spectroscopy for the in-vivo determination of blood glucose levels. To this end we measured Raman spectra of glucose dissolved in pure water and in the presence of other analytes such as glycogen and proteins. Secondly, we determined the fluorescence of blood serum for different excitation wavelengths. Since all measurements were done in an absolute way, we were able to predict if the Raman signal level of glucose was high enough to permit the in-vivo determination of the physiological glucose levels in blood.
We study the influence of analytes on the scattering phase function of tissue mimicking phantom materials. These analytes change the scattering of the sample by a small fraction. We developed a system for measuring the scattering phase function of turbid media and used it to study the influence of analytes on the scattering phase function and the scattering cross section. Measurement of the scattering phase function of complex turbid media, such as human tissue mimicking phantoms, provides a means of increasing the accuracy of calculations of light scattering by such media. Instead of an assumed - generally simplified - phase function, one can use a more realistic phase function in e.g., Monte Carlo simulations. It also provides information on the scattering and absorption properties of the medium. The influence of analyte concentration is derived from changes in scattering, making it a relative measurement. This has the advantage that there is no need to determine the scattering itself. This paper discusses the experimental set-up and test measurements on monodisperse polystyrene latex suspension in comparison to Monte Carlo simulations, the possibility of extracting the scattering phase function. We also present measurements of the influence of an analyte on the scattering properties of biological tissue in vitro. The change in scattering coefficient found from these measurements is in good agreement with theoretically predicted values.
Photoacoustics was used to image optically absorbing structures, like hairs and blood vessels in tissue samples. With 532 nm light depths down to 6 - 9 mm were reached. As the samples we used a 10% solution of Intralipid-10% or real tissue (chicken breast), containing capillaries with blood or a variable dilution of Evans blue. Small PVdF piezoelectric hydrophones were used for detection, in scanning mode for imaging purposes. The depth resolution is about 10 micrometers . The lateral resolution is limited by the diameter of the detector (200 micrometers in our case). This study was undertaken to develop Photoacoustic Tomography of tissue for the localization of blood vessels, e.g. for the detection of blood concentrations (angiogenesis) around tumors.
A theory is presented that allows for the prediction of the power of the photocurrent fluctuations in laser Doppler blood flowmetry (LDBFM). The method is complementary to the Monte Carlo simulation method which is only capable of predicting the shape of the Doppler spectrum. The input data required for application of the theory are the angular distribution of the detected photons and the relative amount of Doppler shifted photons. The consequences of the speckle dynamics for the various modes of LDBFM are discussed. The speckle behavior is particularly important in those cases where the photodetector is directly exposed to the light scattered back from the tissue, as found in a certain setup of laser Doppler imaging (LDI). In that case, the optical properties of the tissue govern the size of the speckles. Hence, the type of tissue affects the instrumental response through the speckles in an independent way. In this paper, this effect has been quantified for a number of typical tissues. The speckle effect not only influences the overall response of the instrument in a strong manner, but also its sensitivity to motion at various depths. The speckle related variation of the instrumental response may be suppressed when a sufficiently wide beam is used.
A new optical tissue phantom is presented, which consists of a polymer film containing scatterers and absorbers. With this tissue phantom, tissue models can be constructed having optical properties and layered structures similar to those in living tissue. Optical properties can be specified for each layer, with a resolution of 20 micrometers. With this tissue phantom, a new laser Doppler testing principle is developed using a repetitive construction of static and moving layers. In this paper, the consequences of the usage of uniform motion in thin layers are discussed, as compared with the more random motion of blood in tissue. This issue is studied by means of Monte Carlo simulations. Measurements are shown performed with a laser Doppler imager. In particular, the normalization of the flux signal is discussed. It turns out, that normalization with the DC-level of the backscattered light gives better images than normalization with DC2. Also, results of depth sensitivity measurements are shown.
Photoacoustic (PA) signals were used to detect and image optically absorbing structures in highly scattering media. Tissue phantoms were constructed from 10 μm diameter carbon fibres and nylon vessels containing blood or a variable dilution of Evens blue that were situated within a I% dilution oflntralipid A PVdF piezoelectric hydrophone with a bandwidth of 70 MHZ was used to detect the PA signals. A Q-switched frequency doubled Nd: Y AG laser provided 1 O ns pulses at 1 O Hz repetition rate and 532 nm wavelength. The sensitivity of the PA technique was demonstrated by the photoacoustic detection of single red blood cells that were situated on a glass plate in a PBS buffer. PA images of the samples were constructed from scanned array detection of the acoustic signals. The imaging algorithm is based on scanning synthetic delay-and-sum focus forming. The transducer directivity was simulated, verified and used to determine the weight factors of the algorithm. Threads and capillaries with various diameters situated at various depths have been imaged. The lateral resolution is limited by the diameter (200 μm) of the transducer. In the experimental images is shown that the depth resolution can be less than 10 μm. The accuracy of the source location reconstruction depends both on the method of signal processing and the PA signal duration. The PA images of larger blood vessels in phantom tissue show only the front and back side of these vessels due to the relatively low optical penetration depth in whole blood, acoustic interference and acoustic reflection at the walls of the vessels. The photoacoustic image of optically absorbing characters located at a depth of 1.8 mm and illuminated through 2 mm Intralipid dilution is presented. The results of this study are promising for the development ofphotoacoustic tomography. Keywords: Photoacoustics, imaging, phantom tissue, tomography, blood, piezoelectric detection.
A new optical tissue phantom is presented, which consists of a polymer film containing scatterers and absorbers. With this tissue phantom, tissue models can be constructed having the optical properties and layered structure similar to those in living tissue. Optical properties can be specified for each layer, with a resolution of 20 micrometers . With this tissue phantom, a new laser Doppler testing principle is developed using a repetitive construction of static and moving layers. Monte Carlo simulations show that the requirements to the optical matching quality provided by the medium between the layers are not very restrictive. Two applications of the new tester are shown. For a single laser Doppler blood flow monitor, the depth sensitivity characteristics of two fiber optic probes are measured. Furthermore, an attempt is made to compare the outputs of two different laser Doppler instruments measuring on the same tissue phantom, with the same flow situation. Such a comparison may be important when new instruments are used for a specific medical application.
In laser doppler blood flowmetry (LDBFM), the flow is derived from the first moment of the power spectrum of the photocurrent. This quantity depends on both the spectral width and the modulation depth of the signal. This modulation depth depends on the amount of speckles on the detector surface. The speckle size is determined by the wavelength, the local wave form and the angular distribution of the incoming light.In theoretical treatments of laser speckle, the distance between the light source and the illuminated screen or detector is often taken much larger than the light source dimensions. In direct contact LDBFM where the detector is placed directly on or close to the skin, such assumptions do not hold, making analytic solutions of the problem impossible. The issue is studied by means of a model experiment. A scattering medium is illuminated by tow beams originating from the same laser, one of which is shifted in frequency. With a detector close to the surface of the medium, beats are observed at the difference frequencies. From the modulation depth of these beats, it is possible to derive the speckle size. The main purpose of this paper is to present this conceptually simple experiment. The first experimental results are shown. Although still of limited quality, they show the potential of the method to study the effects of scattering anisotropy, mean free path length, the detector size and its distance to the tissue.
Pulsed photoacoustic (PA) signals may be used for the detection and imaging of blood vessels in tissue. A relatively strong absorption by red blood cells and low absorption by the surrounding tissue, combined with a reasonable penetration depth of the light is found at a wavelength of ca. 577 nm. Experiments were performed with a pulsed frequency doubled Nd:YAG laser which delivered 10 ns pulses at 532 nm wavelength. Ten percent dilutions of India ink and 50% suspensions of red blood cells in PBS were used as optical absorbers. Blood vessels were simulated by hollow nylon fibers with an inner diameter of ca. 250 micrometer through which these suspensions flow. The optical scattering of the surrounding tissue was simulated by a 12% dilution of Intralipid-10% to get a solution with a reduced scattering coefficient of 1.8 mm-1. The PA signals were detected with a hydrophone that contained four wide band piezoelectric transducers made of 9 micrometer thick PVdF film with an effective diameter of 200 micrometers. Laser pulses with energies up to 8 microjoules were delivered to the sample by a 50 or 100 micrometer core diameter glass fiber. Pulsed optical heating of red blood cells up to 30 - 35 degrees for more than 12,000 times did not affect the photoacoustic response of the cells. If a single fiber is used to illuminate the sample, then even at a depth of 1 mm the PA signals show that the volume that is effectively illuminated is laterally restricted to a diameter of ca. 1 mm. Vessels with blood or ink dilutions were detected up to a depth of more than 1 mm in the scattering medium. Monte- Carlo (MC) simulations were used to simulate the spatial distribution of light absorption in phantom tissue. From this distribution the PA response of blood vessels was simulated. A delay-and-sum beam forming algorithm was developed for 3-D near field configurations and applied to a PA image reconstruction program. The images based on MC simulations as well as experimental data show that the side of larger vessels that is facing the illuminating fiber can be located with a resolution that depends on the configuration and varies between 0.1 and 1 time the inner vessel diameter. This shows the principle and the feasibility of three dimensional photoacoustic dermal tissue imaging.
Time-resolved laser tomography is a promising technique for noninvasive laser examination of structure and condition of biotissues. The results of Monte-Carlo simulations discussed in this paper are related with time-resolved laser Doppler tomography of blood transport in the tissues. In this technique the temporal variations of the optical signal carry information about blood transport. By simulating laser light propagation in a human skin model an opportunity for in-depth by selecting the photons with short, medium, and long transit times. As an example a possibility for detection of a small blood vessel embedded in dermis is considered.
An approach for accelerated simulating of the Doppler frequency spectra histogram (DFSH) of a signal from the laser Doppler flowmeter (LDF) using a Monte-Carlo method is suggested. An extra averaging over permutations of the moving and stationary particles along the photon trajectories enables noticeably less trajectories to be simulated in order to obtain an estimate of the DFSH with a pre-set accuracy as compared with the traditional method. The convergence of the DFSH by this 'fast' method to that by the traditional method is demonstrated for different experimental conditions and the computational time advantage of the fast method is evaluated.
Recently a blood velocimeter was developed, based on the principle of self-mixing in a semiconductor laser. This means that the intensity of the light is modulated by feedback from moving scattering particles, which contains the Doppler shift frequency. Upon feedback the characteristics of the laser diode will change. The threshold current will decrease and an instable region may become present just above the new threshold. It turns out that the amplitude of the Doppler signal is related to the difference in intensity between the situations with and without feedback. This amplitude is highest, but also most unstable, just above feedback. The suppression of reflection from the glass fiber facets is of paramount importance. Using an optical stabilization of the feedback, we are able to optimize the performance of the laser-fiber system and the Doppler modulation depth, and to clarify the behavior with a suitable physical model. The velocimeter has been used in vivo with the glass fiber inserted in normal catheters, but in upstream and in downstream situations. For the latter, the fiber facet in the liquid has been provided with a special side-reflecting device.
When performing direct-contact laser-Doppler flowmetry on experimental flow models, the power spectra of the detector signal can be obtained by homodyne or by heterodyne detection. Especially with uniformly moving probe particles coherence effects are observed, leading to changes in the width of the power spectrum. Due to destructive interference effects at the detector surface, in the measured homodyne spectra the contribution of the relatively high Doppler frequencies is suppressed compared with that of lower frequencies. This results in spectra which are narrower than expected theoretically. This effect allows us to investigate the importance of the relative amount of coherent areas at the surface of the detector.
Photoacoustic (PA) techniques may be used for imaging of absorbing structures within a light scattering medium. By use of an array of detectors, the macroscopic structure of the absorbers in the medium may be determined, based on differences in light absorption. A physical explanation of the spherical PA profile is presented, from which the signals generated by other source geometries can be derived. The potentialities of PA imaging of blood perfused tissue have been investigated. Experiments were performed with a pulsed frequency-doubled Nd:YAG laser which delivered 10 ns pulses at 532 nm wavelength. Dilutions of India ink, dyed epoxy strands and dyed polystyrene spheres acted as PA sources. Characteristic source dimensions varied between ca. 10 and 250 micrometer. The PA signals were detected with wide band piezoelectric transducers made of 9 and 28 micrometer thick PVdF film with mm and sub-mm lateral dimensions. Detection distances were between 1 and 50 mm. Nonlinear effects have been observed for higher levels of absorbed energy. Dilutions of Liposyn were used as optically scattering media. The calculations suggest the applicability of the method. For imaging of dermal blood vessels up to a depth of 1 mm, piezoelectric signals in the (mu) V range may be expected. The experimental PA signals contain significant frequency components up to 75 MHz, depending on the source characteristics. In principle from such signals the detection distance can be determined with micrometer resolution. The detectors show a forward directivity caused by acoustic interference on the detector surface which depends on the lateral dimensions and the acoustic pulse shape and pulse duration. Optical attenuation coefficients of the Liposyn dilutions have been determined photoacoustically. A spatial resolution of ca. 5-50 micrometer was achieved in the reconstruction of the PA source locations.
In pulse oximetry, the red and infrared intensity fluctuations are often assumed to have the same form. However, we observed strong phase delays between these signals during measurements on the forehead of some of our subjects when no pressure was applied onto the probe, which practically disappeared when pressure was applied on the probe. The signals obtained at different distances from the light source were transformed into fluctuations in the absorption and reduced scattering coefficient by means of results from Monte Carlo simulations. The changes in the reduced scattering coefficient appeared to be inverted when no pressure was applied onto the probe. Although the calculated relation between the red and infrared fluctuations in the absorption coefficient was sometimes free of hysteresis, the ratio between the fluctuations still depend on pressure on the probe, and on the chosen optical properties of the medium.
Laser Doppler flowmetry (LDF) is a method that can be used for measuring blood flow changes in the microcirculation. We have contributed to the development of a new device for LDF, based on digital signal processing. A method for correcting the disregarding of frequency components was developed, by approximating the noise-free Doppler spectrum with an exponential shape. The frequency components from 40 kHz to 50 kHz can be used to correct for white noise. We introduced variable resistors for the case common mode components from both detectors have different magnitudes. However, after adjustment we found that noise may still be present. We have observed, that cutting off at 150 Hz suppresses many noise contributions and still provides sufficient Doppler information. For the transfer of a moment calculated from 150 Hz - 20 kHz into 0 - infinity Hz, the correction method mentioned above can be applied.
A tissue-optical model is presented in which changes in the blood volume fraction, fv, and tissue saturation, SO2, are calculated from non-invasively measured intensity changes at two wavelengths during ischemia. Measurements were performed during occlusion and during muscle contraction at the human forearm with a sensor containing two LEDs, (lambda) equals 660 nm and (lambda) equals 940 nm, and photodiodes at 7.0 mm, 9.5 mm, and 20 mm from the LEDs. We used diffusion theory for a homogeneous semi-infinite medium to obtain registrations of (Delta) fv and (Delta) SO2 from measured changes in the photon fraction, (Delta) I, during the experiment for each detector separately. As expected, fv stays nearly constant during occlusion, whereas SO2 decreases, for each detector. During muscle contraction we observed that the intensity changes at each detector are much smaller than during occlusion. As expected, both fv and SO2 decrease at the beginning of the contraction period, but increase before the end of the contraction period. (Delta) SO2 depends more strongly than (Delta) fv on the assumed myoglobin concentration, the scattering coefficients, the blood volume fraction and the saturation at the beginning of the session. The success of the homogeneous model in the occlusion experiment is probably caused by simultaneous deoxygenation in the muscle and in the skin. However, during muscle contraction the changes in SO2 and fv were different at each detector. The failure of the homogeneous model in that case may be explained by the deoxygenation which is expected to be larger in muscle tissue than in skin tissue.
When performing direct-contact laser-Doppler flowmetry on experimental flow models, the power spectra of the detector signal can be obtained by homodyne or by heterodyne detection. Especially with uniformly moving probe particles coherence effects are observed, leading to changes in the width of the power spectrum. Due to destructive interference effects at the detector surface, in the measured homodyne spectra the contribution of relatively high Doppler frequencies is suppressed compared with that of lower frequencies. This results in spectra which are narrower than expected theoretically. This effect allows us to investigate the importance of the relative amount of coherence areas at the surface of the detector.
In order to study the behavior of laser-Doppler based tissue blood perfusion meters an experimental flow model has been developed consisting of a set of layers with dispersed scatterers and/or absorbing material which are moveable with respect to each other and to the laser-Doppler probe. As the material for the layers gelatin was used, and for the scatterers polystyrene spheres were chosen. Light (from a diode laser) scattering in the sample was measured in reflection using a photodiode array. The intensity and the Doppler spectrum were recorded as a function of the source-detector distance and the angle of laser light incidence. For comparison a number of Monte Carlo simulations of the dynamic light scattering in the sample were performed. The simulations included data regarding the Doppler spectrum, the number of scatter events, paths lengths, positions and angles of emergence and penetration depths. It is seen that in the heterodyne detection case good agreement between measurements and simulations is obtained, while in the homodyne the simulations have to be downscaled in frequency (factor 3). This may be caused by coherence effects due to the finite aperture of the detector. In the simulations the averaged Doppler frequency and averaged absolute Doppler frequency turn out to be quadratic and linear dependent on the numerical aperture. This effect was verified with an independent calculation.
In order to investigate the applicability of Monte-Carlo simulations for (Doppler) light scattering in tissue, two upscaled experimental models were constructed. The models consisted of thin layers, either water or gelatin, with scatterers, which can be moved relative to each other. Measurements and simulations of the scattered intensity and the Doppler frequency moments are in rather good agreement.
In reflectance pulse oximetry the ratio R/IR between the red and infrared intensity fluctuations, as measured at the skin surface, is used to estimate the arterial oxygen saturation. This ratio is influenced by light propagation in tissue, as measurements at several distances between light sources and detectors simultaneously show that R/IR depends on this distance. In the present study the influence of the estimated tissue properties on R/IR and its distance dependence are investigated by means of Monte Carlo simulations, a method to vary the optical properties without the need for a new Monte Carlo simulation. A three wavelength model has been introduced, because of secondary emission of the red LED. The influence of water absorption has been taken into account. The simulation results depend on the chosen optical properties. Results of R/IR for SaO2 equals 98% with properties from in vivo experiments agree much better with the measured values than the predictions based on in vitro data available in literature. The results show that the condensed Monte Carlo simulation is a valuable tool to gain insight in the principles of reflectance pulse oximetry: The model, assuming a homogeneous distribution of pulsations, is able to describe the experimental results for pulse sizes, R/IR and its distance dependence very well.
A velocimeter, consisting of a semiconductor laser, coupled to a glass fiber, to be inserted in the flow, and applying self-mixing as the detection technique, is described. A special application is the measurement of blood velocity in veins and arteries. Technical aspects, including flow profile calculations and measurements, and in-vivo and in-vitro velocity measurements are described and discussed.
A novel velocimeter for the measurement of blood velocity in veins and arteries is described. It consists of a semiconductor laser, coupled to a glass fiber, to be inserted in the flow, and applying self-mixing as the detection technique. Theoretical aspects and in-vivo and in-vitro measurements are described and discussed.
The validity of the similarity parameter ∑'s ≡ ∑s(1 - g), the reduced scattering coefficient, where g is the average cosine of the scattering phase function is investigated. Attenuation coefficients α and diffusion patterns are obtained from solutions of the transport equation for isotropic scattering and Rayleigh-Gans scattering, applied to infinite media. Similarity is studied for the attenuation coefficient α, as well as for the Kubelka-Munk absorption and backscattering coefficients in the positive and negative directions, and for predictions of the internal reflection at interfaces. Similarity between solutions of the Boltzmann equation for highly forward scattering and isotropic scattering (g = 0) exist only when ∑a << ∑s(l - g). However, because similarity between results, both with g > 0.9, is independent of the value of the absorption coefficient, it is advantageous to simulate highly forward scattering media like biological tissues with g > 0.9, e.g., by Monte Carlo simulations, instead of using isotropic scattering or diffusion theory. Monte Carlo simulations on slabs confirm the deviations from the diffusion approximation and show the behavior near boundaries. Application of similarity may save calculation time in Monte Carlo simulations, because simulation with a lower value for g will increase the mean free path.
A laser Doppler velocimeter has been developed to measure blood flow velocity in vivo. It consists of a semiconductor laser coupled to a fiber. Laser light is guided into a blood vessel and backscattered light (by red blood cells) is guided back into the laser. The backscattered Doppler shifted light produces an intensity modulation of the laser (self-mixing effect). The beat-frequency of the intensity modulation is related to the Doppler shift of the backscattered light. A model is presented to calculate modulation signals, and results of measurements in vitro and in vivo are shown.
Laser Doppler velocimetry provides a method for non invasive measurements of the perfusion of tissue. Therefore the tissue is illuminated with a monochromatic light source and back scattered light from the tissue is collected at a detector at an adjacent site. Some of the back scattered photons have had interaction with moving red blood cells and are frequency shifted. Due to interference of frequency shifted and non-frequency shifted photons the intensity at the detector fluctuates. These fluctuations provide the information from which a rate for the perfusion can be derived. In this paper we present perfusion measurements and Monte Carlo (MC) simulations on both a scale model and human skin tissue. The Monte Carlo results are used to quantify the size and position of the probe volume. Three different ways are presented to vary the size and position of the probe volume.
Laser Doppler velocimetry provides a method for non invasive measurements of the
perfusion of tissue. Therefore the tissue is illuminated with a monochromatic light
source and back scattered light from the tissue is collected at a detector at an
adjacent site. Some of the back scattered photons have had interaction with moving
red blood cells and are frequency shifted. Due to interference of frequency shifted
and non-frequency shifted photons, the intensity at the detector fluctuates. These
fluctuations provide the information from which a rate for the perfusion can be
derived. ' Recently, the possibility of performing depth dependent measurements on
the skin by3 4vrying the position an size of the sample volume has been
investigated. In this paper we demonstrate that there are at least three
different ways to vary the position and size of the probe volume:
1) Using a different wavelength of the laser.
2) Varying the distance from the laser to the detector.
3) Varying the angle of penetration of the laser light into the tissue.
The method of Raman microspectrosopy can be used to determine position-defined
absolute water content in young and old human lenses from the ratio between the
Raman intensity at 3390 cm and 2935 cm1, as described in previous studies1'2.
Especially when using lens slices accurate positioning is possible and an
excellent spatial resolution can be obtained.
The aim of the present study is to investigate position-defined variations in
absolute water content in human lenses of varying age.
The Phase Mismatching CARS technique uses the nonresonant signal of the glass cuvette walls to coherently compensate the nonresonant signal of the sample yielding a method of measuring background free CARS spectra. The technique is especially suitable under (pre) resonant nditions where many molecular Raman vibrationsNend to have depolarization ratios p close to the nonresonant depolarization ratio p In comparison with the Polarization Sensitive CARS technique the Phase Mismatching technique has a higher net suppression ratio because of the lower loss of vibration resonant signal. The combination of the Phase mismatching and Polazation Sensitive CARS technique yields the possibility of direct measurements of XIjkl components and resolving overlapping bands with different depolarization ratios without an interfering nonresonant background. 1.
A sensitive confocal Raman microspectrometer (CRM) has been developed in our
laboratory enabling the study of single living cells and chromosomes [1,2].
Characteristics of the CR14 are; efficient signal collection (by a high power
microscope objective), confocal detection achieved by positioning a small pinhole in
the image plane of the microscope objective, a high signal throughput of the
spectrograph (including a Chevron type dielectric band pass filter set for laser
1 ight suppression , with a 80-90% transmission of Raman signal [3] ) and virtually
photon-noise limited signal detection by a liquid nitrogen cooled CCD-camera
(quantum efficiency 40% at 700 nm). Laser light of 660 nm from a DCM-operated dye
laser is used for excitation. It prevents degradation of the samples in the focused
beam, a phenomenon observed when using the 514.5 nm line of an argon-ion laser. The
degradation in that case is ascribed to as yet unidentified photo-chemical processes
since the possibilities of excessive heating of the samples and multiphoton
absorption could be ruled out [51 . When using 660 nm laser light Raman spectra can
be obtained even of single fixed metaphase chromosomes in air without any signs of
damage [6] . Radial and axial spatial resolution of the CRM in the standard
configuration are 0.45 m and 1.3 un respectively [4]. This enables the recording of
Raman spectra of specific cellular or chromosomal regions in situ.
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