Pixel-Based Absorption Correction for Dual-Tracer Fluorescence Imaging of Receptor Binding Potential

Ratiometric approaches to quantifying molecular concentrations have been used for decades in microscopy, but have rarely been exploited in vivo until recently. One dual-tracer approach can utilize an untargeted reference tracer to account for non-specific uptake of a receptor-targeted tracer, and ultimately estimate receptor binding potential quantitatively. However, interpretation of the relative dynamic distribution kinetics is confounded by differences in local tissue absorption at the wavelengths used for each tracer. This study simulated the influence of absorption on fluorescence emission intensity and depth sensitivity at typical near-infrared fluorophore wavelength bands near 700 and 800 nm in mouse skin in order to correct for these tissue optical differences in signal detection. Changes in blood volume [1-3%] and hemoglobin oxygen saturation [0-100%] were demonstrated to introduce substantial distortions to receptor binding estimates (error \u3e 30%), whereas sampled depth was relatively insensitive to wavelength (error \u3c 6%). In response, a pixel-by-pixel normalization of tracer inputs immediately post-injection was found to account for spatial heterogeneities in local absorption properties. Application of the pixel-based normalization method to an in vivo imaging study demonstrated significant improvement, as compared with a reference tissue normalization approach

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

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 (μs′)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 consistentl y 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

Extraction of Intrinsic Fluorescence from Single Fiber Fluorescence Measurements on a Turbid Medium: Experimental Validation

Abstract 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

Biphasic Oxidation of Oxy-Hemoglobin in Bloodstains

Background: In forensic science, age determination of bloodstains can be crucial in reconstructing crimes. Upon exiting the body, bloodstains transit from bright red to dark brown, which is attributed to oxidation of oxy-hemoglobin (HbO2) to methemoglobin (met-Hb) and hemichrome (HC). The fractions of HbO 2, met-Hb and HC in a bloodstain can be used for age determination of bloodstains. In this study, we further analyze the conversion of HbO2 to met-Hb and HC, and determine the effect of temperature and humidity on the conversion rates. Methodology: The fractions of HbO2, met-Hb and HC in a bloodstain, as determined by quantitative analysis of optical reflectance spectra (450–800 nm), were measured as function of age, temperature and humidity. Additionally, Optical Coherence Tomography around 1300 nm was used to confirm quantitative spectral analysis approach. Conclusions: The oxidation rate of HbO2 in bloodstains is biphasic. At first, the oxidation of HbO2 is rapid, but slows down after a few hours. These oxidation rates are strongly temperature dependent. However, the oxidation of HbO2 seems to be independent of humidity, whereas the transition of met-Hb into HC strongly depends on humidity. Knowledge of these decay rates is indispensable for translating laboratory results into forensic practice, and to enable bloodstain age determination on the crime scene

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, mu(a) (range: 0.1 - 6 mm(-1)), and the reduced scattering coefficient, mu(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 mu m). 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 mu m) and predictions do not show bias as a function of either mu(a) or mu(S)

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 ( ) 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 is unaffected by changes in anisotropy (g. [0.8, 0.9, 0.95]), but is sensitive to changes in phase function (Henyey-Greenstein versus modified Henyey-Greenstein). A 20% decrease in 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 ( ) of the single fiber reflectance measurement. Simulated data are used to define a novel mathematical expression for that is expressed in terms of optical properties, fiber diameter and . 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 tissue