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In this study we employ a near-infrared fluorescence lymphatic imaging (NIRFLI) technique to longitudinally image spatial and temporal changes in the lymphatics in mice bearing vascular endothelial growth factor (VEGF)-C overexpressing B16F10 (VEGF-C-B16F10) or mock-transduced B16F10 (mock-B16F10) melanoma tumors. Our NIRFLI data show that ICG-laden lymph accumulates into a VEGF-C-B16F10 tumor compared to mock-B16F10 at 3 days post implantation, presumably due to increased lymphatic vessel permeability. Quantification shows a significantly greater percentage of ICG-perfused area in VEGF-C-B16F10 (7.6 ± 2) as compared to MOCK-B16F10 (1 ± 0.5; p = 0.02), which is also confirmed by quantification of the lymphatic leakage of evans blue dye (optical density at 610nm; VEGF-C-B16F10, 10.5 ± 2; mock-B16F10, 5.1 ± 0.5; p = 0.009); thereafter, lymphatic leakage is visualized only in the peritumoral region. Our imaging data also show that anti-VEGF-C treatment in VEGF-C-B16F10 restores normal lymphatic vessel integrity and reduces dye extravasation. Because NIRFLI technology can be used to non-invasively detect lymphatic changes associated with cancer, it may provide a new diagnostic to assess the lack of lymphatic vessel integrity that promotes lymphovascular invasion and to assess therapies that could arrest invasion through normalization of the lymphatic vasculature., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

In this short communication, we demonstrate for the first time, the use of far red fluorescent gene reporter, iRFP to longitudinally and non-invasively track the in vivo process of lymphatic metastases from an orthotopic site of mammary implantation through lymphatic vessels and to draining lymph nodes. Potentially useful to accelerate cancer drug discovery as an in vivo screening tool to monitor the pharmacological arrest of metastasis, we show that the custom as well as commercial small animal imaging devices have adequate performance to detect the gene reporter in stably expressing metastatic cancer cells.

One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery.

As part of a proof-of-concept study for future delivery of targeted near-infrared fluorescent (NIRF) tracers, we sought to assess the delivery of micrograms of indocyanine green to all the axillary lymph nodes following intraparenchymal breast injections and intradermal arm injections in 20 subjects with advanced breast carcinoma and undergoing complete axillary lymph node dissection. Lymphatic vessels and nodes were assessed in vivo. Ex vivo images demonstrated that 87% of excised lymph nodes, including 81% of tumor-positive lymph nodes, were fluorescent. Future clinical studies using microdose amounts of tumor-targeting NIRF contrast agents may demonstrate improved surgical intervention with reduced morbidity.

Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression due in part to the lack of imaging techniques currently available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal aspect of the left hindpaw for baseline data or directly to the popliteal lymph node (PLN) and until 21 days post-implantation (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were also detected at 21 days p.i. in mice with tumor in the paw and at 11 days p.i. in mice with tumor in the PLN, due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.

Despite the development of a large number of promising candidates, few contrast agents for established medical imaging modalities have successfully been translated over the past decade. The emergence of new imaging contrast agents that employ biomedical optics is further complicated by the relative infancy of the field and the lack of approved imaging devices compared to more established clinical modalities such as nuclear medicine. Herein, we propose a navigational approach (as opposed to a fixed "roadmap") for translation of optical imaging agents that is (i) proposed through consensus by four academic research programs that are part of the cooperative U54 NCI Network for Translational Research, (ii) developed through early experiences for translating optical imaging agents in order to meet distinctly varied needs in cancer diagnostics, and (iii) adaptable to the rapidly changing environment of academic medicine. We describe the pathways by which optical imaging agents are synthesized, qualified, and validated for preclinical testing, and ultimately translated for "first-in-humans" studies using investigational optical imaging devices. By identifying and adopting consensus approaches for seemingly disparate optical imaging modalities and clinical indications, we seek to establish a systematic method for navigating the ever-changing "roadmap" to most efficiently arrive at the destination of clinical adoption and improved outcome and survivorship for cancer patients.

ALFIA (Automated Lymphatic Function Imaging Analysis), an algorithm providing quantitative analysis of investigational near-infrared fluorescence lymphatic images, is described. Images from nine human subjects were analyzed for apparent lymphatic propagation velocities and propulsion periods using manual analysis and ALFIA. While lymphatic propulsion was more easily detected using ALFIA than with manual analysis, statistical analyses indicate no significant difference in the apparent lymphatic velocities although ALFIA tended to calculate longer propulsion periods. With the base ALFIA algorithms validated, further automation can now proceed to provide a clinically relevant analytic tool for quantitatively assessing lymphatic function in humans.

Current treatment of unilateral breast cancer-related lymphedema (BCRL) is only directed to the afflicted arm. Near-infrared fluorescent imaging (NIRF) of arm lymphatic vessel architecture and function in BCRL and control subjects revealed a trend of increased lymphatic abnormalities in both the afflicted and unafflicted arms with increasing time after lymphedema onset. These pilot results show that BCRL may progress to affect the clinically "normal" arm, and suggest that cancer-related lymphedema may become a systemic, rather than local, malady. These findings support further study to understand the etiology of cancer-related lymphedema and lead to better diagnostics and therapeutics directed to the systemic lymphatic system.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

Photoacoustic (PA) imaging is based upon the generation of an ultrasound pulse arising from subsurface tissue absorption due to pulsed laser excitation, and measurement of its surface time-of-arrival. Expensive and bulky pulsed lasers with high peak fluence powers may provide shortcomings for applications of PA imaging in medicine and biology. These limitations may be overcome with the frequency-domain PA measurements, which employ modulated rather than pulsed light to generate the acoustic wave. In this contribution, we model the single modulation frequency based PA pressures on the measurement plane through the diffraction approximation and then employ a convolution approach to reconstruct the sectional image slices. The results demonstrate that the proposed method with appropriate data post-processing is capable of recovering sectional images while suppressing the defocused noise resulting from the other sections.

We demonstrate the ability to non-invasively and quantitatively image lymphatic architecture and contractile function using dynamic near-infrared (NIR) fluorescence imaging with injection of indocyanine green in normal and transgenic mice. Unlike normal mice, which showed well defined lymphatic drainage patterns and orthograde propagation of contraction waves, we observed tortuous and mispatterned lymphatic vessels and persistent retrograde lymph flow in mice with deficiency in Prox1, a transcription factor essential for lymphatic vascular development. NIR fluorescence imaging provides a method for quantifying lymphatic function for future studies designed to discern differences in lymphatic function in murine models of human lymphatic disease.

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.

In this contribution, different measurement noise pre-filtering techniques were developed using frequency-domain fluorescence measurements of homogeneous breast phantoms. We demonstrated that implementing noise pre-filtering, based on modulation depth and measurement error in amplitude, can improve model match between experimental and simulated data under varying experimental conditions (target depths, 1-3 cm and fluorescence optical contrast, 1:0 and 100:1). Noise pre-filtering also improves the qualitative estimation of target(s) location in reconstructed images in deep target(s) when there was fluorescence in the background. Interestingly, decreases in model mismatch did not necessarily correlate with increases in reconstructed target accuracy. In addition, it was observed that pre-filtering measurement noise using different criteria can help differentiate target(s) from artifacts, thus possibly minimizing the false-positive cases in a clinical environment.

We have developed fluorescence enhanced optical tomography based upon fully adaptive finite element method (FEM) using tetrahedral dual-meshing wherein one of the two meshes discretizes the forward variables and the other discretizes the unknown parameters to be estimated. We used the 8-subtetrahedron subdivision scheme to create the nested dualmesh in which each are independently refined. However, two tetrahedrons from the two different meshes pose an intersection problem that needs to be resolved in order to find the common regions that the forward variables (the fluorescent diffuse photon fluence fields) and the parameter estimates (the fluorescent absorption coefficients) can be mutually assigned. Using an efficient intersection algorithm in the nested tetrahedral environments previously developed by the authors, we demonstrate fully adaptive tomography using a posteriori error estimates. Performing the iterative reconstructions using the simulated boundary measurement data, we demonstrate that small fluorescent targets embedded in the breast simulating phantom in point illumination/detection geometry can be resolved at reasonable computational cost.

This study represents a first attempt to assess the detection capability of a fluorescence-enhanced optical imaging system as quantified by the Hotelling observer. The imaging system is simulated by the diffusion approximation of the time-dependent radiative transfer equation, which describes near infra-red (NIR) light propagation through clinically relevant tissue volumes. The random structures in the background are introduced using a lumpy-object model as a representation of anatomical structure as well as non-uniform distribution of disease markers. The systematic errors and noise associated with the actual experimental conditions are incorporated into the simulated boundary measurements to acquire imaging data sets. A large number of imaging data sets are considered in order to perform Hotelling observer studies. We find that the signal-to-noise ratio (SNR) of Hotelling observer (i) decreases as the strength of lumpy perturbations in the background increases, (ii) decreases as the target depth increases, and (iii) increases as excitation light leakage decreases, and reaches a maximum for filter optical density values of 5 or higher.

This article describes a novel non-contact fluorescence optical tomography scheme which utilizes multiple area illumination patterns, to reduce the ill-posedness of the inverse problem involved in recovering interior fluorescence yield distributions in biological tissue from boundary fluorescence measurements. The image reconstruction is posed as an optimization problem which seeks a tissue optical property distribution minimizing, for all illumination patterns simultaneously, a regularized difference between the observed boundary measurements of light distribution, and the boundary measurements predicted from a physical model. Multiple excitation source illumination patterns are described by line and Gaussian sources scanning the simulated tissue phantom surface and by employing diffractive optics-generated patterns. Multiple measurement data sets generated by scanning excitation sources are processed simultaneously to generate the interior fluorescence distribution in tissue by implementing the fluorescence tomography algorithm in a parallel framework suitable for multiprocessor computers. Image reconstructions for single and multiple fluorescent targets (5mm diameter) embedded in a 512ml simulated tissue phantom are demonstrated, with depths of the fluorescent targets from the illumination plane between 1cm to 2cm. We show both qualitative and quantitative improvements of our algorithm over reconstructions from only a single measurement.

We present three-dimensional fluorescence yield tomography of a tissue phantom in a noncontact reflectance imaging setup. The method employs planar illumination with modulated light and frequency domain fluorescence measurements made on the illumination plane. An adaptive finite-element algorithm is used to handle the ill-posed and computationally demanding inverse image reconstruction problem. Tomographic images of fluorescent targets buried at 1-2 cm depths from the illumination surface demonstrate the feasibility of fluorescence tomography from reflectance tomography in clinically relevant tissue volumes.

To date, the assessment of fluorescence-enhanced optical imaging has not been performed owing to (i) the lack of tools necessary for objective assessment of image quality (OAIQ), and (ii) the difficulty to test an untested diagnostic contrast agent in patient studies. Herein, we focus upon the development of a framework for OAIQ which includes a model to simulate both natural tissue heterogeneity as well as heterogeneous distribution of a molecularly targeted fluorophore. Specifically, we use a novel tomographic algorithm previously developed and validated in our laboratory (Roy and Sevick-Muraca, IEEE Trans. Med. Imaging, 2005). Our results show that image generation is (i) unaffected by normal anatomical heterogeneity manifested in endogenous tissue optical properties of absorption and scattering, and (ii) restricted by heterogeneous distribution of fluorophore in the background as the contrast is decreased.

Frequency domain photon migration (FDPM) measurements were employed to accurately quantify optical properties of both the suspending fluid and particles within dense polystyrene suspensions of 143- or 226-nm mean diameter at varying concentrations (5-30% by volume). The measured absorption coefficients varied linearly with particle volume fraction whereas the isotropic scattering coefficients varied nonlinearly in agreement with the prediction that utilizes the hard-sphere structure factor model. These results validate the interference approximation of light scattering to describe light propagation accurately within dense suspensions. Furthermore, owing to the accuracy of FDPM absorption measurements, the imaginary refractive indices for both particles and their suspending fluid were determined and were found to compare favorably with literature values.

Fluorescence frequency-domain photon migration measurements were acquired from tissue phantoms, each containing a fluorescent target, by means of area illumination and area detection on the same surface and for the first time, to our knowledge, compared with predictions computed with a numerical solution to the coupled photon diffusion equations. We accomplished area illumination and area detection using a planar, intensity-modulated excitation light source and a gain-modulated intensified charge-coupled device camera, respectively. A 1-ml vessel containing 1-microm solution of Indocyanine Green in 1% Liposyn was immersed 1 cm deep in each 512-ml tissue phantom. For most tissue phantoms, the background surrounding the 1-ml target was composed of Liposyn solution containing Indocyanine Green or 3,3'-Diethylthiatricarbocyanine Iodide such that the target-to-background ratio of fluorescence yield was > or = 10:1. Measurements of fluorescence modulation amplitude and phase were predicted with a mean error ranging from 10.1% to 13.6% and 0.56 degrees to 1.72 degrees, respectively. These numbers are similar to those obtained by use of single-pixel frequency-domain photon migration techniques and validate the potential use of area illumination and area detection for biomedical imaging of tissues. Results also demonstrate that target-to-background ratios of fluorescence yield and fluorescence lifetime significantly affect target detectability.

The ultimate success of near-infrared optical tomography rests on the precise measurement of light propagation within tissues or random media, the accurate prediction of these measurements from a light propagation model, and an efficient three-dimensional solution of the inverse imaging problem. To date, optical tomography algorithms have focused on frequency-domain photon migration (FDPM) measurements of phase-delay and amplitude attenuation, which are reported relative to the incident light, even though phase-delay and amplitude of incident light are nearly impossible to measure directly. In this contribution, we examine referenced, fluorescence-enhanced frequency-domain photon migration measured at excitation and/or emission wavelengths and report on a measurement strategy to minimize measurement and calibration error for efficient coupling of data to a distorted Born iterative imaging algorithm. We examine three referencing approaches and develop associated inversion algorithms for (1) normalizing detected emission FDPM data to the predicted emission wave arising from a homogeneous medium, (2) referencing detected emission FDPM data to that detected at a reference point, and (3) referencing detected emission FDPM data to detected excitation FDPM data detected at a reference point. Our results show the latter approach to be practical while reducing the nonlinearity of the inverse problem. Finally, in light of our results, we demonstrate the method for eliminating the influence of source strength and instrument functions for effective fluorescence-enhanced optical tomography using FDPM.

We present three-dimensional tomographic images of the absorption coefficient that is due to the presence of a fluorophore reconstructed from frequency domain fluence measurements of a tissue phantom containing a single, fluorescence contrast-enhanced inclusion. We show that such a reconstruction may be improved when the importance of measurement error correlations between relative phase shift and amplitude is assessed and when measurements are preprocessed to reduce the magnitude and the bias of system error.

The development of near-infrared (NIR) optical imaging for biomedical optical imaging is hampered by the computational intensiveness of large-scale three-dimensional (3-D) image reconstruction and the potential lack of endogenous contrast for detection of relevant tissue features. In this contribution the inverse optical imaging problem is formulated in three dimensions in a noncompressive geometry as a simple-bound constrained minimization problem in order to recover the interior fluorescence properties of exogenous contrast agent from frequency-domain photon migration measurements at the boundary. The solution of the forward optical diffusion problem for the frustum shape containing fluorescence inclusions of 10:1 contrast is accomplished by use of the Galerkin finite-element formulation. The inverse approach employs the truncated Newton method with trust region and a modification of automatic reverse differentiation to speed the computation of the optimization problem. The image-reconstruction results confirm that the constrained minimization may offer a more logical approach for the 3-D optical imaging problem than unconstrained optimization.

In the past, nonlinear unconstrained optimization of the optical imaging problem has focused on Newton-Raphson techniques. Besides requiring expensive computation of the Jacobian, the unconstrained minimization with Tikhonov regularization can pose significant storage problems for large-scale reconstructions, involving a large number of unknowns necessary for realization of optical imaging. We formulate the inverse optical imaging problem as both simple-bound constrained and unconstrained minimization problems in order to illustrate the reduction in computational time and storage associated with constrained image reconstructions. The forward simulator of excitation and generated fluorescence, consisting of the Galerkin finite-element formulation, is used in an inverse algorithm to find the spatial distribution of absorption and lifetime that minimizes the difference between predicted and synthetic frequency-domain measurements. The inverse approach employs the truncated Newton method with trust region and a modification of automatic reverse differentiation to speed the computation of the optimization problem. The reconstruction results confirm that the physically based, constrained minimization with efficient optimization schemes may offer a more logical approach to the large-scale optical imaging problem than unconstrained minimization with regularization.

A method is presented to determine fluorescence decay lifetimes within tissuelike scattering media. Fluorescence lifetimes are determined for micromolar concentrations of the dyes 3,3'-Diethylthiatricarbocyanine Iodide and Indocyanine Green by frequency-domain investigations of light propagating in turbid media. Dual-wavelength photon-migration measurements that use intensity-modulated sources at excitation and emission wavelengths of the fluorophores provide optical parameters of the media as well as fluorescence properties of the dyes. The deduction of fluorescence lifetimes requires no calibration with reference fluorophores, and the results are shown to be independent of dye concentration.

Stochastic reconstruction techniques are developed for mapping the interior optical properties of tissues from exterior frequency-domain photon migration measurements at the air-tissue interface. Parameter fields of absorption cross section, fluorescence lifetime, and quantum efficiency are accurately reconstructed from simulated noisy measurements of phase shift and amplitude modulation by use of a recursive, Bayesian, minimum-variance estimator known as the approximate extended Kalman filter. Parameter field updates are followed by data-driven zonation to improve the accuracy, stability, and computational efficiency of the method by moving the system from an underdetermined toward an overdetermined set of equations. These methods were originally developed by Eppstein and Dougherty [Water Resources Res. 32, 3321 (1996)] for applications in geohydrology. Estimates are constrained to within feasible ranges by modeling of parameters as beta-distributed random variables. No arbitrary smoothing, regularization, or interpolation is required. Results are compared with those determined by use of Newton-Raphson-based inversions. The speed and accuracy of these preliminary Bayesian reconstructions suggest the near-future application of this inversion technology to three-dimensional biomedical imaging with frequency-domain photon migration.

From analytical and numerical solutions that predict the scattering of diffuse photon density waves and from experimental measurements of changes in phase shift theta and ac amplitude demodulation M caused by the presence of single and double cylindrical heterogeneities, we show that second- and higher-order perturbations can affect the prediction of the propagation characteristics of diffuse photon density waves. Our experimental results for perfect absorbers in a lossless medium suggest that the performance of fast inverse-imaging algorithms that use first-order Born or Rytov approximations might have inherent limitations compared with inverse solutions that use iterative solutions of a linear perturbation equation or numerical solutions of the diffusion equation.

The feasibility of employing fluorescent contrast agents to perform optical imaging in tissues and other scattering media has been examined through computational studies. Fluorescence lifetime and yield can give crucial information about local metabolite concentrations or environmental conditions within tissues. This information can be employed toward disease detection, diagnosis, and treatment if noninvasively quantitated from reemitted optical signals. However, the problem of inverse image reconstruction of fluorescence yield and lifetime is complicated because of the highly scattering nature of the tissue. Here a light propagation model employing the diffusion equation is used to account for the scattering of both the excitation and fluorescent light. Simulated measurements of frequency-domain parameters of fluorescent modulated ac amplitude and phase lag are used as inputs to an inverse image-reconstruction algorithm, which employs the diffusion model to predict frequency-domain measurements resulting from a modulated input at the phantom periphery. In the inverse image-reconstruction algorithm, a Newton-Raphson technique combined with a Marquardt algorithm is employed to converge on the fluorescent properties within the medium. The successful reconstruction of both the fluorescence yield and lifetime in the case of a heterogeneous fluorophore distribution within a scattering medium has been demonstrated without a priori information or without the necessity of obtaining absence images.

Measurements of nanosecond and subnanosecond fluorescence lifetimes are restricted to dilute, nonscattering systems since excitation and emission photon times of flight significantly affect measured fluorescent decay kinetics. We provide the theoretical rationale for frequency-domain measurements of phase-shift and amplitude demodulation made at excitation and emission wavelengths for direct determination of lifetimes in tissues and other scattering media. We confirm our analytical expressions using standard laser dyes such as 3,3'-diethylthiatricarbocyanine iodide, IR- 125, and IR- 140 in polystyrene suspensions with similar scattering properties as tissues. Our results have significant implication for lifetime-based spectroscopy in tissues and other scattering media.

We investigate the origin of fluorescent or phosphorescent signals reemitted from highly scattering media (such as tissues), using diffusion theory and probability analysis. Results show that the lifetime of a uniformly distributed phosphorescent or fluorescent optical probe will profoundly affect the volume interrogated by noninvasive reflectance measurements. When the lifetime is greater than photon migration times, the origin of the reemitted signal is confined closest to the surface. Our computations suggest that noninvasive measurements of tissue oxygen concentration may not necessarily interrogate deep tissues when systemically administered phosphorescent dyes are used.