We studied the ability to detect small absorbing objects embedded in a highly scattering medium. Absorbing spheres of varying size, from 0.8 mm to 6.8 mm radius, submerged in a solution of highly scattering, low absorbing liquid: skim milk, were studied in a trans-illumination geometry. Groups of more than one sphere and a single circular disk, with radius identical to that of one of the spheres, were also studied. Single linear raster scans in the plane of the sphere, with the spheres centered between the source and detector, were made. Data was taken in the frequency-domain, yielding profiles of the objects in each of the three measurable quantities: dc intensity, phase, and modulation. The diffraction pattern form the sphere differed from that of the disk, demonstrating a volume effect associated with photon diffusion. The diffraction pattern of multiple spheres differed from that of single spheres.

We have experimentally investigated the meaning of the effective optical absorption [mu(a)((eff))] and the reduced scattering [mu(s)?((eff))] coefficients measured on the surfaces of two-layered turbid media, using the diffusion equation for homogeneous, semi-infinite media. We performed frequency-domain spectroscopy in a reflectance geometry, using source-detector distances in the range 1.5-4.5 cm. We measured 100 samples, each made of one layer (thickness in the range 0.08-1.6 cm) on top of one semi-infinite block. The optical properties of the samples were similar to those of soft tissues in the near infrared. We found that the measured effective optical coefficients are representative of the underlying block if the superficial layer is less than ~0.4 cm thick, whereas they are representative of the superficial layer if it is more than ~1.3 cm thick.

In recent years researchers have made significant progress in understanding the physics of fluorescence in highly scattering materials such as tissues in the near-infrared. We have quantitatively verified a model which describes fluorescence in ideal (homogeneous and infinite) tissue-like media. Given the quantitative accuracy of this model, one can use measurements of the fluorescence of a tissue with a homogeneous distribution of fluorophore to obtain the quantum yield, lifetime of the probe, and the absorption and scattering coefficients of the tissue at the fluorescent wavelength. We demonstrate that this can be done with a simple measurement of the photon density as a function of source-detector separation at the excitation and emission wavelengths. To verify our approach we present the lifetime, quantum yield of the fluorescent probe (rhodamine B), and the absorption and scattering coefficients of the medium at the emission peak wavelength that are obtained by a fit of the model to experimental measurements.

Tissue glucose levels affect the refractive index of the extracellular fluid. The difference in refractive index between the extracellular fluid and the cellular components plays a role in determining the reduced scattering coefficient (micro(s)') of tissue. Hence a physical correlation may exist between the reduced scattering coefficient and glucose concentration. We have designed and constructed a frequency-domain near-infrared tissue spectrometer capable of measuring the reduced scattering coefficient of tissue with enough precision to detect changes in glucose levels in the physiological and pathological range.

Light injected at a point on a surface of a scattering medium is emitted at the surface after traveling a quasisemicircular path deep into the medium. This phenomenon can be exploited to detect objects immersed in the medium from time-resolved measurements of light intensity at the surface. Our experiments on model systems demonstrate that absorbing objects, surrounded by bone and other scattering material, can be detected. The technique yields surface images of absorbing objects submerged in a scattering medium. Images of the same phantoms inside the cavity of a skull can be obtained by the same technique.

Our research is aimed at the development of a frequency-domain instrument for conducting non-invasive, real-time, near-infrared, optical tomography of tissue in vivo. Our goal is to reconstruct a spatial map of the optical properties of a strongly scattering medium in a semi-infinite-geometry sampling configuration. Specifically, we focus our attention on the absorption coefficient ((mu) a ) and the reduced scattering coefficient ((mu) s ') of the medium. We have developed a frequency-domain measurement protocol (which we call precalibrated), which permits one to recover the values of (mu) a and (mu) s ' of a uniform tissue-like phantom from a measurement at a single source-detector separation and a single modulation frequency. It requires a preliminary reference measurement on a calibration sample of known optical properties before the measurement on the investigated sample. This approach is in principle rigorous only in macroscopically homogeneous media. We have verified that the equations valid for uniform media can still be applied to yield qualitative information on the optical nature of the inhomogeneity if the effect of macroscopic inhomogeneities on the measured phase and intensity is not too large. In vitro measurements on turbid media containing scattering and absorbing homogeneities, with optical properties very similar to the background medium, gave encouraging results. We plan to implement this measurement protocol in a multisource, multidetector instrument for optical tomography.

Near-IR optical tomography is thwarted by the highly scattering nature of light propagation in tissue. We propose a weighted back-projection method to produce a spatial map of an optical parameter which characterized the investigated medium. We have studied the problem of the choice of the back-projection weight function for the absorption coefficient ((mu) a ) and for the reduced scattering coefficient ((mu) s ') of tissuelike phantoms. Working in frequency-domain optical imaging, we have initially approached the problem of quantifying the effect caused by a small absorbing defect embedded in the medium on the measured DC intensity, AC amplitude, and phase. The collection of DC, AC, and phase changes during a 1 mm step raster scan of the absorbing defect provides information on the photon path distributions and, in general, on the probed spatial region when DC, AC, and phase are, respectively, the measured parameters. We report experimentally determined weight functions for (mu) a and (mu) s '. They indicate that absorption and scattering maps can significantly differ in terms of resolution.

In the near-infrared spectral region (700-900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the DC fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable DC fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in rhodamine concentration on the order of 1% using the DC fluorescence intensity. This DC fluorescence sensitivity is characterized experimentally at two concentrations (55 and 360 nM) and over a range of source-detector separations. Other aspects of the sensitivity are discussed over a large range of concentrations using a fluorescence photon migration model.

High frequency, intensity-modulated light waves are attenuated and phase-shifted by the absorption and scattering properties of highly scattering media, such as tissue. The simultaneous measurement of the average light intensity, modulation amplitude, and phase-shift at a fixed distance from a sinusoidally modulated light source, permits a quantitative determination of the absolute values of the absorption and scattering coefficients from a frequency-domain scan. Our studies have established the range of modulation frequencies that give the highest sensitivity to changes of the optical parameters in model systems. We have measured the optical absorption spectra of dyes suspended in highly scattering media. These spectra match those found in non-scattering media. This frequency-domain approach provides a simple method to perform quantitative spectroscopy in highly scattering media.