Differential Pathlength Research Articles

Multispectral reflectance imaging of brain activation in rodents: methodological study of the differential path length estimations and first in vivo recordings in the rat olfactory bulb

Dynamic maps of relative changes in blood volume and oxygenation following brain activation are obtained using multispectral reflectance imaging. The technique relies on optical absorption modifications linked to hemodynamic changes. The relative variation of hemodynamic parameters can be quantified using the modified Beer-Lambert Law if changes in reflected light intensities are recorded at two wavelengths or more and the differential path length (DP) is known. The DP is the mean path length in tissues of backscattered photons and varies with wavelength. It is usually estimated using Monte Carlo simulations in simplified semi-infinite homogeneous geometries. Here we consider the use of multilayered models of the somatosensory cortex (SsC) and olfactory bulb (OB), which are common physiological models of brain activation. Simulations demonstrate that specific DP estimation is required for SsC and OB, specifically for wavelengths above 600 nm. They validate the hypothesis of a constant path length during activation and show the need for specific DP if imaging is performed in a thinned-skull preparation. The first multispectral reflectance imaging data recorded in vivo during OB activation are presented, and the influence of DP on the hemodynamic parameters and the pattern of oxymetric changes in the activated OB are discussed.

Non-invasive measurement of the microvascular properties of non-dysplastic and dysplastic oral leukoplakias by use of optical spectroscopy

Differential Path-length Spectroscopy (DPS) was used to non-invasively determine the optical properties of oral leukoplakias in vivo. DPS yields information on microvascular parameters such as the mucosal blood content, the microvascular blood oxygenation and the average micro-vessel diameter as well as on tissue morphological parameters such as the scattering slope and scattering amplitude. DPS measurements were made on non-dysplastic and dysplastic oral leukoplakias using a novel fiber-optic probe, and were correlated to the histological outcome of biopsies taken from the same location. Our data show borderline significant increases in mucosal blood content in dysplastic lesions compared to non-dysplastic lesions, with no changes in microvascular oxygen saturation and light scattering signatures. These results suggest that dysplastic and non-dysplastic leukoplakias may be discriminated non-invasively in vivo through differences in their microvascular properties, if they can be reproducibly quantified in the presence of a variable thickness keratin layer that optically shields the mucosal layer.

Changes in Bladder Wall Blood Oxygen Saturation in the Overactive Obstructed Bladder

Several studies suggest that hypoxia of the bladder wall contributes to bladder dysfunction but the exact relation between bladder function and blood oxygen saturation, a surrogate marker for hypoxia, is not known. We determined bladder wall blood oxygen saturation in vivo in an animal model of bladder outlet obstruction to establish the exact relation between blood oxygen saturation and bladder function. In 8 sham operated and 8 urethrally obstructed guinea pigs we measured blood oxygen saturation of the bladder wall by differential path length spectroscopy before surgery and 8 weeks postoperatively. Urodynamic investigations performed during the whole 8-week period provided data on bladder function. Before surgery and 8 weeks after sham surgery blood oxygen saturation in the bladder wall was between 88% and 95% during filling. It decreased during voiding and returned to greater than 90% within 30 seconds. Eight weeks after obstruction saturation was significantly lower than in the sham operated group during filling and voiding. The decrease was positively related to bladder pressure during filling and voiding, and was more pronounced when overactivity was present. Local bladder contractions occurred without a measurable increase in bladder pressure but were associated with a decrease in saturation. A normal bladder maintains a high oxygen saturation level during filling. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder contractions without a measurable increase in bladder pressure were associated with a decrease in blood saturation.

Application of differential pathlength spectroscopy in head and neck optical diagnostics

Application of differential pathlength spectroscopy in head and neck optical diagnostics

Effects of assuming constant optical scattering on haemoglobin concentration measurements using NIRS during a Valsalva manoeuvre.

Resolving for changes in concentration of tissue chromophores in the human adult brain with near-infrared spectroscopy has generally been based on the assumption that optical scattering and pathlength remain constant. We have used a novel hybrid optical spectrometer that combines multi-distance frequency and broadband systems to investigate the changes in scattering and pathlength during a Valsalva manoeuvre in 8 adult volunteers. Results show a significant increase in the reduced scattering coefficient of 17% at 790nm and 850nm in 4 volunteers during the peak of the Valsalva. However, these scattering changes do not appear to significantly affect the differential pathlength factor and the tissue haemoglobin concentration measurements.

Non-invasive measurement of photosensitiser concentration using fluorescence differential path-length spectroscopy: validation for different liposomal formulations of m-THPC: Foscan, Foslip and Fospeg

As previously described, Fluorescence differential path length spectroscopy (FPDS) can determine chromophore concentrations non-invasively after injection with m-THPC (Foscan) in the rat liver [1]. Our first aim is to validate FDPS for two other, liposomal formulations of m-THPC; pegylated liposomes (Fospeg) and conventionel liposomes (Foslip), and compare them to Foscan. As a proof of principle we use the highly vascularised, optically homogenous liver of the rat [1] Validation of the FDPS-measurements was done by chemical extraction of the same liver [2]. Our second aim is to validate FDPS measurements of the tongue, which is optically less homogenous, but clinically more relevant. After successful validation in both liver and tongue tissue, the pharmacokinetic-profile in other tissue types could be assessed by FDPS alone. Therefore, FDPS can lower the need for labour-intensive chemical extraction. Fifty-four male Wistar rats were intravenously injected with one of the three formulations of m-THPC; eighteen rats for each formulation. All rats were injected with 0.15 mg kg m-THPC. FDPS measurements were performed on liver, palate, tongue, spleen and kidney 2, 4, 8, 24, 48, and 96 h after m-THPC administration. For validation of our FDPS measurements, liver and tongue were harvested for chemical extraction [2]. Concentration estimates in liver and tongue measured by FDPS are here compared with chemical extraction. At the HNODS-meeting we will present the results of our first step in the validation of FDPS; the correlation of FDPS measurements with chemical extraction for the three different formulations in the liver.

Differential Pathlength Spectroscopy for diagnosis of head and neck cancer

The development of optical techniques for non-invasive diagnosis of cancer is an ongoing challenge to biomedical optics. For oral cancer biopsy has a low specificity because of a thick keratin layer that often covers potential malignancies. We investigated the possibility to distinguishing potentially malignant visible lesions from benign ones to reduce the number of unnecessary biopsies. Major challenge here is to measure through the thick keratin layer that often covers the biologically active layers. Differential Pathlength Spectroscopy is a fibre optic measurement technique that samples tissue in a predetermined sampling volume. We constructed a probe for sampling up to 1mm deep into the tissue. By analysing the white light spectrum with a mathematical model developed earlier we could determine scattering parameters, blood volume, micro vascular saturation and the average blood vessel diameter in the sample volume just below the probe. A total of 110 measurements were performed in 21 patients on 75 suspicious lesions that were biopsied after measurement and 35 clinically normal locations that were not biopsied. Lesions were classified as normal, hyperplasia, inflammation, dysplasia or cancer. With this data we built a logistic regression model that predicts the need for a biopsy (if classification equals dysplasia or cancer) on the basis of the spectra. The model was trained and evaluated using the leave-one-out-approach. The results showed a very good combination of relative sensitivity and specificity with an area under the curve of 0.943. When removing the obvious lesions from the dataset (i.e. the unbiopsied normals and half the samples classified as cancer that were clinically unmistakable, the area under the ROC curve even improved to 0.951. In practice this comes down to reducing the number of biopsies to 50% without reducing the overall sensitivity of the diagnostic procedure, or reducing the number of biopsies by 90% at a loss of 5% relative sensitivity. We believe Differential Pathlength Spectroscopy is a very promising technique that may help to reduce the costs of healthcare without compromising the quality.

Multiplexed fluorescence lifetime measurements by frequency-sweeping Fourier spectroscopy

We report simultaneous measurements of fluorescence lifetimes at multiple excitation wavelengths with a Fourier transform frequency domain fluorescence lifetime spectrometer. The spectrometer uses a Michelson interferometer with its differential optical path length scanning at a 22,000 Hz scan rate. The scan speed of the optical delay varies linearly during each scan and creates interference modulations that sweep from -150 to 150 MHz in 45.5 micros. The frequency-sweeping modulation allows nanosecond fluorescence lifetime measurements within 45.5 micros. Because the interference modulation frequency is wavelength dependent, under the Fourier multiplexing principle, the spectrometer can perform lifetime measurements on multiple excitation wavelengths simultaneously.

Optical Spectroscopy to Guide Photodynamic Therapy of Head and Neck Tumors

In contrast to other interstitial applications of photodynamic therapy (PDT), optical guidance or monitoring in the head and neck is at a very early stage of development. The present paper reviews the use of optical approaches, in particular optical spectroscopy, that have been used or have the potential to guide the application of PDT. When considering the usefulness of these methods, it is important to consider the volume over which these measurements are acquired, the influence of differences in and changes to the background optical properties, the implications for these effects on the measured parameters and the difficulty of incorporating these types of measurements in clinical practice in head and neck PDT. To illustrate these considerations, we present an application of a recently developed technique, which we term fluorescence differential path length spectroscopy for monitoring meta-tetra(hydroxyphenyl)-chlorin or Foscan-PDT of interstitial head and neck cancer.

Assessment of ultrasound modulation of near infrared light on the quantification of scattering coefficient

To assess the effect of ultrasound modulation of near infrared (NIR) light on the quantification of scattering coefficient in tissue-mimicking biological phantoms. A unique method to estimate the phase of the modulated NIR light making use of only time averaged intensity measurements using a charge coupled device camera is used in this investigation. These experimental measurements from tissue-mimicking biological phantoms are used to estimate the differential pathlength, in turn leading to estimation of optical scattering coefficient. A Monte-Carlo model based numerical estimation of phase in lieu of ultrasound modulation is performed to verify the experimental results. The results indicate that the ultrasound modulation of NIR light enhances the effective scattering coefficient. The observed effective scattering coefficient enhancement in tissue-mimicking viscoelastic phantoms increases with increasing ultrasound drive voltage. The same trend is noticed as the ultrasound modulation frequency approaches the natural vibration frequency of the phantom material. The contrast enhancement is less for the stiffer (larger storage modulus) tissue, mimicking tumor necrotic core, compared to the normal tissue. The ultrasound modulation of the insonified region leads to an increase in the effective number of scattering events experienced by NIR light, increasing the measured phase, causing the enhancement in the effective scattering coefficient. The ultrasound modulation of NIR light could provide better estimation of scattering coefficient. The observed local enhancement of the effective scattering coefficient, in the ultrasound focal region, is validated using both experimental measurements and Monte-Carlo simulations.

Differential Pathlength Spectroscopy for the Quantitation of Optical Properties of Gold Nanoparticles

An accurate estimation of optical absorption coefficient (microabs) and scattering coefficient (microsca) is important in characterizing nanoparticles for identifying or optimizing applications such as photothermal therapy and photoacoustic imaging. In this exciting period where several fascinating methods have been unveiled for the synthesis of various nanoparticles, the field is still lacking in the availability of efficient characterization methods. We introduce an accurate and simple methodology to optically characterize nanoparticles which could fill the gap. This is based on differential pathlength spectroscopy (DPS), a dual optical fiber approach, originally developed to detect cancer endoscopically by measuring the optical properties of tissue in small interrogation volumes. We expand its use to nanoparticles in a method that allows us to resolve the effects of microabs and microsca in the extinction coefficient of low concentration samples. We outline the measurement protocol using the DPS system and describe the analysis of the data taking additional inputs from electron microscopy and discrete dipole approximation (DDA) simulations. The DPS signal from the sample is first translated into the backscattering coefficient using a calibration constant. Further, the backscattering coefficient is converted via the simulated scattering phase function into the scattering coefficient. With this knowledge and extinction coefficient measured using a conventional photospectrometer, the absorption coefficient is calculated. We prove the validity of the method using spherical and rod-shaped gold nanoparticles, comparing the results with outputs from DDA simulations. We also briefly touch upon the dilemma of the choice of the appropriate dielectric function for gold at the nanoscale.

Fiber modulators and fiber amplifiers for LISA

We present the sideband phase characteristics of a fiber-coupled integrated electro-optical modulator (EOM) at a modulation frequency of 2 GHz for Fourier frequencies from 0.1 mHz to 1 Hz. The upper phase noise limit was almost an order of magnitude better than required for LISA. The EOM's phase dependencies on temperature and transmitted optical power were measured and found to be uncritical. Additionally we have investigated three optical amplifiers emitting 1 W. Their differential phase noise and optical pathlength noise as one contribution to differential phase noise were measured. The measured differential phase noise was within the requirement. The dependencies of differential phase noise on pump power were measured and requirements for the operation of the amplifier on the LISA satellite derived.

Differential pathlength spectroscopy for diagnosis of head and neck cancer

Differential pathlength spectroscopy for diagnosis of head and neck cancer

A broadband continuous-wave multichannel near-infrared system for measuring regional cerebral blood flow and oxygen consumption in newborn piglets

Near-infrared spectroscopy (NIRS) is a promising technique for assessing brain function in newborns, particularly due to its portability and sensitivity to cerebral hemodynamics and oxygenation. Methods for measuring cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) have been developed based on broadband continuous-wave NIRS. However, broadband NIRS apparatus typically have only one detection channel, which limits their applicability to measuring regional CBF and CMRO(2). In this study, a relatively simple multiplexing approach based on electronically controlled mechanical shutters is proposed to expand the detection capabilities from one to eight channels. The tradeoff is an increase in the sampling interval; however, this has negligible effects on CBF measurements for intervals less than or equal to 1 s. The ability of the system to detect focal brain injury was demonstrated in piglets by injecting endothelin-1 (ET-1) into the cerebral cortex. For validation, CBF was independently measured by computed tomography (CT) perfusion. The average reduction in CBF from the source-detector pair that interrogated the injured region was 51%+/-9%, which was in good agreement with the CBF reduction measured by CT perfusion (55%+/-5%). No significant changes in regional CMRO(2) were observed. The average regional differential pathlength prior to ET-1 injection was 8.4+/-0.2 cm (range of 7.1-9.6 cm) and did not significantly change after the injury.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; approximately 0.2 mm(3) and approximately 10(2) mm(3), respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Monitoring ALA‐induced PpIX Photodynamic Therapy in the Rat Esophagus Using Fluorescence and Reflectance Spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence of 54 J cm(-2). Forty-eight hours after PDT the esophagus was excised and histologically examined for PDT-induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two-phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two-phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb + Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert–Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation ( R = 0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb + Mb] was 0.9–1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700–965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF] = 2.7–2.8) in a living heart tissue.

Assessment of Near-Infrared Path Length in Fibrous Phantom and Muscle Tissue

The first derivative of the pseudo-absorption spectrum of a water-loaded cotton wool (water-CW) phantom, which mimics muscle tissues, was used to determine the light path length in the near-infrared (NIR) region. The light path length increased as the density of the turbid medium decreased. It is independent of both water content in the range of 75-85% (by weight) and the diffuse reflecting reference used to determine the pseudo-absorbance. The path length determination procedure was verified by measurements of diffuse reflectance in chicken breast tissue for which the path length of 1.8 mm (differential path length factor, DPF = 2.1) was found to be similar to the path length of NIR light of 1.5-2.2 mm (DPF = 1.8-2.6) in a water-CW phantom of density similar to chicken breast. We conclude that the NIR light path length can serve as a characteristic of muscle tissue density.

Quantitative fluorescence spectroscopy in turbid media using fluorescence differential path length spectroscopy

We have developed a new technique, fluorescence differential path length spectroscopy (FDPS), that enables the quantitative investigation of fluorophores in turbid media. FDPS measurements are made with the same probe geometry as differential path length spectroscopy (DPS) measurements. Phantom measurements are performed for two fiber diameters (400 microm and 800 microm) and for a wide range of optical properties (mu(s)': 0 to 10 mm(-1); mu(a): 0 to 2 mm(-1)) to investigate the influence of the optical properties on the measured differential fluorescence signal. The differential fluorescence signal varies by a factor of 1.4 and 2.2 over the biologically relevant scattering range (0.5 to 5 mm(-1)) for a given fluorophore concentration for 400 microm and 800 microm fibers, respectively. The differential fluorescence signal is attenuated due to absorption at the excitation wavelength following Lambert-Beer's law with a path length equal to the differential path length.

Empirical model description of photon path length for differential path length spectroscopy: combined effect of scattering and absorption

Differential path length spectroscopy (DPS) is a method of reflectance spectroscopy that utilizes a specialized fiber geometry to make the photon path length (tau) insensitive to variations in tissue optical properties over a wide range of absorption (mu(a)) and total scattering (mu(s)) coefficients, which are common within the ultraviolet/visible (UV/VIS) wavelength region. This study extends the description of tau to larger mu(a) and smaller mu(s) values, optical properties that are representative of the near-infrared region (NIR), a region where the DPS path length may be dependent on both coefficients. This study presents a novel empirical relationship between tau and the combined effect of both mu(a) (range: 0.1-12 mm(-1)) and mu(s) (range: 1.5-42 mm(-1)), anisotropy of 0.8, and is applicable to DPS probes containing a wide range of fiber diameters (range: 100-1000 microm). The results indicate that the simple empirical formula, including only one fitted parameter, is capable of accurately predicting tau over a wide range (r=0.985; range: 80-940 microm) and predictions are not biased versus mu(a) or mu(s). This novel relationship is applicable to analysis of DPS measurements of tissue in both the UV/VIS and NIR wavelength regions and may provide information about the wavelength-specific tissue volume optically sampled during measurement.