Towards the Optical Detection of Field Cancerization in the Buccal Mucosa of Patients with Lung Cancer.

INTRODUCTION: An increase in detection of early-stage asymptomatic lung tumors could increase the overall survival rate of lung cancer patients. A new approach to cancer (pre-)screening focusses on detecting field cancerization instead of the tumor itself. The objective of this study was to investigate the use of optical spectroscopy to detect field cancerization in the buccal mucosa of lung cancer patients. METHODS: Optical buccal mucosa measurements were performed in lung cancer patients and controls using multidiameter single-fiber reflectance spectroscopy. We analyzed whether the measured optical parameters could distinguish lung cancer patients from controls. RESULTS: Twenty-three lung cancer patients, 24 chronic obstructive pulmonary disease (COPD) control patients, and 36 non-COPD controls were included. The majority of tumors were non-small-cell lung carcinomas (96%) and classified as stage I (48%). The tissue scattering properties µs' and γ at 800 nm and the tissue bilirubin concentration were all near-significantly different (P=.072, 0.058, and 0.060, respectively) between the lung cancer and COPD group. µs' at 800 nm had a sensitivity of 74% and a specificity of 63%. The microvascular blood oxygen saturation of the lung cancer patients was also higher than the COPD patients (78% vs. 62%, P=.002), this is probably a consequence of the systemic effect of COPD. CONCLUSIONS: We have demonstrated that µs' at 800 nm is increased in the buccal mucosa of patients with lung cancer compared to controls with COPD. This might be an indication of field cancerization in the oral cavity of patients with lung cancer.

Correction for tissue optical properties enables quantitative skin fluorescence measurements using multi-diameter single fiber reflectance spectroscopy.

BACKGROUND AND OBJECTIVE: Fluorescence measurements in the skin are very much affected by absorption and scattering but existing methods to correct for this are not applicable to superficial skin measurements. STUDY DESIGN/MATERIALS AND METHODS: The first use of multiple-diameter single fiber reflectance (MDSFR) and single fiber fluorescence (SFF) spectroscopy in human skin was investigated. MDSFR spectroscopy allows a quantification of the full optical properties in superficial skin (µa, µs' and γ), which can next be used to retrieve the corrected - intrinsic - fluorescence of a fluorophore Qµa,x(f). Our goal was to investigate the importance of such correction for individual patients. We studied this in 22 patients undergoing photodynamic therapy (PDT) for actinic keratosis. RESULTS: The magnitude of correction of fluorescence was around 4 (for both autofluorescence and protoporphyrin IX). Moreover, it was variable between patients, but also within patients over the course of fractionated aminolevulinic acid PDT (range 2.7-7.5). Patients also varied in the amount of protoporphyrin IX synthesis, photobleaching percentages and resynthesis (>100× difference between the lowest and highest PpIX synthesis). The autofluorescence was lower in actinic keratosis than contralateral normal skin (0.0032 versus 0.0052; P<0.0005). CONCLUSIONS: Our results clearly demonstrate the importance of correcting the measured fluorescence for optical properties, because these vary considerably between individual patients and also during PDT. Protoporphyrin IX synthesis and photobleaching kinetics allow monitoring clinical PDT which facilitates individual-based PDT dosing and improvement of clinical treatment protocols. Furthermore, the skin autofluorescence can be relevant for diagnostic use in the skin, but it may also be interesting because of its association with several internal diseases.

Extraction of intrinsic fluorescence from single fiber fluorescence measurements on a turbid medium: experimental validation.

The detailed mechanisms associated with the influence of scattering and absorption properties on the fluorescence intensity sampled by a single optical fiber have recently been elucidated based on Monte Carlo simulated data. Here we develop an experimental single fiber fluorescence (SFF) spectroscopy setup and validate the Monte Carlo data and semi-empirical model equation that describes the SFF signal as a function of scattering. We present a calibration procedure that corrects the SFF signal for all system-related, wavelength dependent transmission efficiencies to yield an absolute value of intrinsic fluorescence. The validity of the Monte Carlo data and semi-empirical model is demonstrated using a set of fluorescent phantoms with varying concentrations of Intralipid to vary the scattering properties, yielding a wide range of reduced scattering coefficients (µ's = 0-7 mm (-1)). We also introduce a small modification to the model to account for the case of µ's = 0 mm (-1) and show its relation to the experimental, simulated and theoretically calculated value of SFF intensity in the absence of scattering. Finally, we show that our method is also accurate in the presence of absorbers by performing measurements on phantoms containing red blood cells and correcting for their absorption properties.

In vivo quantification of the scattering properties of tissue using multi-diameter single fiber reflectance spectroscopy.

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (µs') and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g2)/(1-g1) and g1 and g2 the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of µs'(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of µs'(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.

Use of a coherent fiber bundle for multi-diameter single fiber reflectance spectroscopy.

Multi-diameter single fiber reflectance (MDSFR) spectroscopy enables quantitative measurement of tissue optical properties, including the reduced scattering coefficient and the phase function parameter γ. However, the accuracy and speed of the procedure are currently limited by the need for co-localized measurements using multiple fiber optic probes with different fiber diameters. This study demonstrates the use of a coherent fiber bundle acting as a single fiber with a variable diameter for the purposes of MDSFR spectroscopy. Using Intralipid optical phantoms with reduced scattering coefficients between 0.24 and 3 mm(-1), we find that the spectral reflectance and effective path lengths measured by the fiber bundle (NA = 0.40) are equivalent to those measured by single solid-core fibers (NA = 0.22) for fiber diameters between 0.4 and 1.0 mm (r ≥ 0.997). This one-to-one correlation may hold for a 0.2 mm fiber diameter as well (r = 0.816); however, the experimental system used in this study suffers from a low signal-to-noise for small dimensionless reduced scattering coefficients due to spurious back reflections within the experimental system. Based on these results, the coherent fiber bundle is suitable for use as a variable-diameter fiber in clinical MDSFR quantification of tissue optical properties.

Quantification of the reduced scattering coefficient and phase-function-dependent parameter γ of turbid media using multidiameter single fiber reflectance spectroscopy: experimental validation.

Multidiameter single fiber reflectance (MDSFR) spectroscopy is a method that allows the quantification of µs' and the phase-function-dependent parameter γ of a turbid medium by utilizing multiple fibers with different diameters. We have previously introduced the theory behind MDSFR and its limitations, and here we present an experimental validation of this method based on phantoms containing a fractal distribution of polystyrene spheres both in the absence and presence of the absorber Evans Blue.

Scattering phase function spectrum makes reflectance spectrum measured from Intralipid phantoms and tissue sensitive to the device detection geometry.

Reflectance spectra measured in Intralipid (IL) close to the source are sensitive to wavelength-dependent changes in reduced scattering coefficient ([Formula: see text]) and scattering phase function (PF). Experiments and simulations were performed using device designs with either single or separate optical fibers for delivery and collection of light in varying concentrations of IL. Spectral reflectance is not consistently linear with varying IL concentration, with PF-dependent effects observed for single fiber devices with diameters smaller than ten transport lengths and for separate source-detector devices that collected light at less than half of a transport length from the source. Similar effects are thought to be seen in tissue, limiting the ability to quantitatively compare spectra from different devices without compensation.

Optical detection of preneoplastic lesions of the central airways.

Current routine diagnosis of premalignant lesions of the central airways is hampered due to a limited sensitivity (white light bronchoscopy) and resolution (computer tomography (CT), positron emission tomography (PET)) of currently used techniques. To improve the detection of these subtle mucosal abnormalities, novel optical imaging bronchoscopic techniques have been developed over the past decade. In this review we highlight the technological developments in the field of endoscopic imaging, and describe their advantages and disadvantages in clinical use.

Semi-empirical model of the effect of scattering on single fiber fluorescence intensity measured on a turbid medium.

Quantitative determination of fluorophore content from fluorescence measurements in turbid media, such as tissue, is complicated by the influence of scattering properties on the collected signal. This study utilizes a Monte Carlo model to characterize the relationship between the fluorescence intensity collected by a single fiber optic probe (F(SF)) and the scattering properties. Simulations investigate a wide range of biologically relevant scattering properties specified independently at excitation (λ(x)) and emission (λ(m)) wavelengths, including reduced scattering coefficients in the range µ'(s)(λ(x)) ∈ [0.1 - 8]mm(-1) and µ'(s)(λ(m)) ∈ [0.25 - 1] × µ'(s)(λ(x)). Investigated scattering phase functions (P(θ)) include both Henyey-Greenstein and Modified Henyey-Greenstein forms, and a wide range of fiber diameters (d(f) ∈ [0.2 - 1.0] mm) was simulated. A semi-empirical model is developed to estimate the collected F(SF) as the product of an effective sampling volume, and the effective excitation fluence and the effective escape probability within the effective sampling volume. The model accurately estimates F(SF) intensities (r=0.999) over the investigated range of µ'(s)(λ(x)) and µ'(s)(λ(m)), is insensitive to the form of the P(θ), and provides novel insight into a dimensionless relationship linking F(SF) measured by different d(f).

Measurement of tissue scattering properties using multi-diameter single fiber reflectance spectroscopy: in silico sensitivity analysis.

Multiple diameter single fiber reflectance (MDSFR) measurements of turbid media can be used to determine the reduced scattering coefficient (µ'(s)) and a parameter that characterizes the phase function (γ). The MDSFR method utilizes a semi-empirical model that expresses the collected single fiber reflectance intensity as a function of fiber diameter (d(fiber)), µ'(s), and γ. This study investigated the sensitivity of the MDSFR estimates of µ'(s) and γ to the choice of fiber diameters and spectral information incorporated into the fitting procedure. The fit algorithm was tested using Monte Carlo simulations of single fiber reflectance intensities that investigated biologically relevant ranges of scattering properties (µ'(s) ∈ [0.4 - 4]mm(-1)) and phase functions (γ ∈ [1.4 - 1.9]) and for multiple fiber diameters (d(fiber) ∈ [0.2 - 1.5] mm). MDSFR analysis yielded accurate estimates of µ'(s) and γ over the wide range of scattering combinations; parameter accuracy was shown to be sensitive to the range of fiber diameters included in the analysis, but not to the number of intermediate fibers. Moreover, accurate parameter estimates were obtained without a priori knowledge about the spectral shape of γ. Observations were used to develop heuristic guidelines for the design of clinically applicable MDSFR probes.

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.

Measurement of the reduced scattering coefficient of turbid media using single fiber reflectance spectroscopy: fiber diameter and phase function dependence.

This paper presents a relationship between the intensity collected by a single fiber reflectance device (R(SF)) and the fiber diameter (d(fib)) and the reduced scattering coefficient ( µs') and phase function (p(θ)) of a turbid medium. Monte Carlo simulations are used to identify and model a relationship between R(SF) and dimensionless scattering ( µs'dfib). For µs'dfib > 10 we find that R(SF) is insensitive to p(θ). A solid optical phantom is constructed with µs' ≈ 220 mm-1 and is used to convert R(SF) of any turbid medium to an absolute scale. This calibrated technique provides accurate estimates of µs' over a wide range ([0.05 - 8] mm(-1)) for a range of d(fib) ([0.2 - 1] mm).

Monte Carlo analysis of single fiber reflectance spectroscopy: photon path length and sampling depth.

Single fiber reflectance spectroscopy is a method to noninvasively quantitate tissue absorption and scattering properties. This study utilizes a Monte Carlo (MC) model to investigate the effect that optical properties have on the propagation of photons that are collected during the single fiber reflectance measurement. MC model estimates of the single fiber photon path length (L(SF)) show excellent agreement with experimental measurements and predictions of a mathematical model over a wide range of optical properties and fiber diameters. Simulation results show that L(SF) is unaffected by changes in anisotropy (g epsilon [0.8, 0.9, 0.95]), but is sensitive to changes in phase function (Henyey-Greenstein versus modified Henyey-Greenstein). A 20% decrease in L(SF) was observed for the modified Henyey-Greenstein compared with the Henyey-Greenstein phase function; an effect that is independent of optical properties and fiber diameter and is approximated with a simple linear offset. The MC model also returns depth-resolved absorption profiles that are used to estimate the mean sampling depth (Z(SF)) of the single fiber reflectance measurement. Simulated data are used to define a novel mathematical expression for Z(SF) that is expressed in terms of optical properties, fiber diameter and L(SF). The model of sampling depth indicates that the single fiber reflectance measurement is dominated by shallow scattering events, even for large fibers; a result that suggests that the utility of single fiber reflectance measurements of tissue in vivo will be in the quantification of the optical properties of superficial tissues.

Empirical model of the photon path length for a single fiber reflectance spectroscopy device.

A reflectance spectroscopic device that utilizes a single fiber for both light delivery and collection has advantages over classical multi-fiber probes. This study presents a novel empirical relationship between the single fiber path length and the combined effect of both the absorption coefficient, mua (range: 0.1-6 mm-1), and the reduced scattering coefficient, micro's (range: 0.3 - 10 mm-1), for different anisotropy values (0.75 and 0.92), and is applicable to probes containing a wide range of fiber diameters (range: 200-2000 microm). The results indicate that the model is capable of accurately predicting the single fiber path length over a wide range (r = 0.995; range: 180-3940 microm) and predictions do not show bias as a function of either microa or micro's .

Controlling the optical path length in turbid media using differential path-length spectroscopy: fiber diameter dependence.

We have characterized the path length for the differential path-length spectroscopy (DPS) fiber optic geometry for a wide range of optical properties and for fiber diameters ranging from 200 microm to 1000 microm. Phantom measurements show that the path length is nearly constant for scattering coefficients in the range 5 mm(-1)< micros <50 mm(-1) for all fiber diameters and that the path length is proportional to the fiber diameter. The path length decreases with increasing absorption for all fiber diameters, and this effect is more pronounced for larger fiber diameters. An empirical model is formulated that relates the DPS path length to total absorption for all fiber diameters simultaneously.

Non-invasive measurement of the morphology and physiology of oral mucosa by use of optical spectroscopy.

Differential path-length spectroscopy (DPS) was used to non-invasively determine the superficial optical properties of oral mucosa in vivo. DPS yields information on physiological parameters such as the mucosal blood content, the microvascular blood oxygenation and the average micro-vessel diameter as well as on morphological parameters such as the scattering slope and scattering amplitude. DPS measurements were made on normal and cancerous oral mucosa using a novel fiber-optic probe, and were correlated to the histological outcome of punch biopsies taken from the same location. Our data shows that the mucosa of oral squamous cell carcinoma is characterised by a significant decrease in microvascular oxygenation and increase in mucosal blood content compared to normal oral mucosa as well as a significant decrease in scattering amplitude and increase in scattering slope.

HIF1a expression in bronchial biopsies correlates with tumor microvascular saturation determined using optical spectroscopy.

Tumor hypoxia is generally considered to be related to aggressive behaviour of a tumor. As in lung cancer direct determination of oxygenation is difficult, hypoxia-related proteins have been studied. A number of studies on these proteins show different results and the usefulness of these protein expressions remains questionable. In this article, we relate one of these hypoxia-related proteins (hypoxia-inducible factor, HIF1a) to a direct in vivo spectroscopic measurement of tumor blood saturation performed during bronchoscopy. Seventeen samples from malignancies and non-malignant tissues were studied. Microvascular saturation levels in the no malignancy group equalled 87+/-11.5% (range 71-100%) and in the malignant group 43+/-21% (range 6-63%). This difference was statistically significant (p<0.0002). There was a significant difference in the spectroscopically determined saturations between the biopsies with negative expression of HIF1a and the biopsies with positive expression of HIF1a (p<0.005). From these data, it can be concluded that HIF1a expression is related to a low microvascular blood saturation as determined in vivo by optical spectroscopy. This study may lead to a better acceptance of the usage of different techniques to establish hypoxia in order to study the effect of hypoxia on therapeutic interventions and prognosis of lung cancer.

Monitoring PDT by means of superficial reflectance spectroscopy.

Monitoring of relevant parameters during photodynamic therapy (PDT) and correlating these with treatment response is necessary to guarantee optimal and reproducible treatment outcome. In this paper we study the correlation between changes in the local tissue optical properties (absorption and scattering coefficients) during ALA-PDT and changes in PpIX fluorescence. The optical properties are measured extremely superficially by employing a single fiber for the delivery and collection of white light to and from the tissue. The measured reflectance spectrum is modeled in terms of four relevant parameters: blood saturation, relative blood volume fraction, scattering intensity and wavelength dependence of the scattering. All these parameters, except the relative blood volume fraction, are shown to correlate with the rate of photobleaching of PpIX, which in turn has previously been shown to correlate with the response of tissues to PDT. These results yield valuable insight in the behavior of these parameters during PDT and their suitability to predict PDT-response for other photosensitizers for which monitoring through photobleaching is not possible.