U. A. Gamm

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 (RSF) and the fiber diameter (dfib) 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 RSF and dimensionless scattering ( μs′dfib). For μs′dfib > 10 we find that RSF is insensitive to p(θ). A solid optical phantom is constructed with μs′ ≈ 220 mm−1 and is used to convert RSF 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 dfib ([0.2 – 1] mm).

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.

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 (dfiber), 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 (dfiber ∈ [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.

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 (μ′s) 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.

Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle

Abstract. We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μa, reduced scattering coefficient, μs′, phase function parameter, γ, and intrinsic fluorescence, Qμa,xf, across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμa,xf.

Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo

Abstract. Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48]  h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

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.

Microscopic analysis of the localization of two chlorin-based photosensitizers in OSC19 tumors in the mouse oral cavity

The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT.

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 (μs = 0-7 mm (-1)). We also introduce a small modification to the model to account for the case of μs = 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.

Measurements of tissue scattering properties using multi-diameter single fiber reflectance spectroscopy: Experimental validation

MDSFR spectroscopy is a method that allows the quantification of µ’s and the phase function parameter γ We are presenting an experimental validation of this method based on phantoms containing polystyrene spheres and show preliminary in vivo data.