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We demonstrate that morphological features pertinent to a tissues pathology may be ascertained from localized measures of broadband reflectance, with a mesoscopic resolution (100-m lateral spot size) that permits scanning of an entire margin for residual disease. The technical aspects and optimization of a k-nearest neighbor classifier for automated diagnosis of pathologies are presented, and its efficacy is validated in 29 breast tissue specimens. When discriminating between benign and malignant pathologies, a sensitivity and specificity of 91 and 77 was achieved. Furthermore, detailed subtissue-type analysis was performed to consider how diverse pathologies influence scattering response and overall classification efficacy. The increased sensitivity of this technique may render it useful to guide the surgeon or pathologist where to sample pathology for microscopic assessment. [ABSTRACT FROM AUTHOR]

We demonstrate quantitative functional imaging using image-guided near-infrared spectroscopy (IG-NIRS) implemented with the boundary element method (BEM) for reconstructing 3-D optical property estimates in breast tissue in vivo. A multimodality MRI-NIR system was used to collect measurements of light reflectance from breast tissue. The BEM was used to model light propagation in 3-D based only on surface discretization in order to reconstruct quantitative values of total hemoglobin (HbT), oxygen saturation, water, and scatter. The technique was validated in experimental measurements from heterogeneous breast-shaped phantoms with known values and applied to a total of seven subjects comprising six healthy individuals and one participant with cancer imaged at two time points during neoadjuvant chemotherapy. Using experimental measurements from a heterogeneous breast phantom, BEM for IG-NIRS produced accurate values for HbT in the inclusion with a <3 error. Healthy breast tissues showed higher HbT and water in fibroglandular tissue than in adipose tissue. In a subject with cancer, the tumor showed higher HbT compared to the background. HbT in the tumor was reduced by 9 M during treatment. We conclude that 3-D MRI-NIRS with BEM provides quantitative and functional characterization of breast tissue in vivothrough measurement of hemoglobin content. The method provides potentially complementary information to DCE-MRI for tumor characterization. [ABSTRACT FROM AUTHOR]

Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy (PDT) of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated. Our objective is to study the kinetics of protoporphyrin IX (PpIX) accumulation in AK after short contact ALA and relate this to erythemal responses. Using a noninvasive dosimeter, PpIX fluorescence measurements (5 replicates) were taken at 20-minintervals for 2 hfollowing ALA application, in 63 AK in 20 patients. Data were analyzed for maximal fluorescent signal obtained, kinetic slope, and changes in erythema. Our results show that PpIX accumulation was linear over time, becoming statistically higher than background in 48 of all lesions by 20 min, 92 of lesions by 1 h, and 100 of lesions by 2 h. PpIX accumulation was roughly correlated with changes in lesional erythema post-PDT. We conclude that significant amounts of PpIX are produced in all AK lesions by 2 h. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Noninvasive fluorescence monitoring of PpIX may be useful to delineate areas of high PpIX accumulation within precancerous areas of the skin. [ABSTRACT FROM AUTHOR]

Fluorescence molecular tomography (FMT) systems coupled to conventional imaging modalities such as magnetic resonance imaging (MRI) and computed tomography provide unique opportunities to combine data sets and improve image quality and content. Yet, the ideal approach to combine these complementary data is still not obvious. This preclinical study compares several methods for incorporating MRI spatial prior information into FMT imaging algorithms in the context of in vivotissue diagnosis. Populations of mice inoculated with brain tumors that expressed either high or low levels of epidermal growth factor receptor (EGFR) were imaged using an EGF-bound near-infrared dye and a spectrometer-based MRI-FMT scanner. All data were spectrally unmixed to extract the dye fluorescence from the tissue autofluorescence. Methods to combine the two data sets were compared using student’s t-tests and receiver operating characteristic analysis. Bulk fluorescence measurements that made up the optical imaging data set were also considered in the comparison. While most techniques were able to distinguish EGFR(+) tumors from EGFR(-) tumors and control animals, with area-under-the-curve values=1, only a handful were able to distinguish EGFR(-) tumors from controls. Bulk fluorescence spectroscopy techniques performed as well as most imaging techniques, suggesting that complex imaging algorithms may be unnecessary to diagnose EGFR status in these tissue volumes. [ABSTRACT FROM AUTHOR]

An approach to quantitatively image targeted-agent binding rate in vivois demonstrated with dual-probe injection of both targeted and nontargeted fluorescent dyes. Images of a binding rate constant are created that reveal lower than expected uptake of epidermal growth factor in an orthotopic xenograft pancreas tumor (2.3×10−5 s−1), as compared to the normal pancreas (3.4×10−5 s−1). This approach allows noninvasive assessment of tumor receptor targeting in vivoto determine the expected contrast, spatial localization, and efficacy in therapeutic agent delivery. [ABSTRACT FROM AUTHOR]

This study investigates differences in the response of breast tumor tissue versus healthy fibroglandular tissue to inspired gases. Cycles of carbogen and oxygen gas are administered while measuring the changes with magnetic-resonance-guided near-infrared imaging in a pilot study of breast cancers. For two patients, analyses are performed with cross-correlation techniques, which measure the strength of hemodynamic modulation. The results show that the overall vasoresponse, indicated by total hemoglobin, of healthy tissue has approximately a 72 and 41 greater correlation to the gas stimulus than the tumor region, in two patients respectively, when background physiological changes are controlled. These data support the hypothesis that tumor vasculature has a poorly functioning vasodilatory mechanism, most likely caused by dysfunctional smooth muscle cells lining the vasculature. This study presents a methodology to quantitatively analyze inspired gas changes in human breast tumors, and demonstrates this technique in a pilot patient population. [ABSTRACT FROM AUTHOR]

A high frequency ultrasound-coupled fluorescence tomography system, primarily designed for imaging of protoporphyrin IX production in skin tumors in vivo, is demonstrated for the first time. The design couples fiber-based spectral sampling of the protoporphyrin IX fluorescence emission with high frequency ultrasound imaging, allowing thin-layer fluorescence intensities to be quantified. The system measurements are obtained by serial illumination of four linear source locations, with parallel detection at each of five interspersed detection locations, providing 20 overlapping measures of subsurface fluorescence from both superficial and deep locations in the ultrasound field. Tissue layers are defined from the segmented ultrasound images and diffusion theory used to estimate the fluorescence in these layers. The system calibration is presented with simulation and phantom validation of the system in multilayer regions. Pilot in-vivodata are also presented, showing recovery of subcutaneous tumor tissue values of protoporphyrin IX in a subcutaneous U251 tumor, which has less fluorescence than the skin. [ABSTRACT FROM AUTHOR]

An automated algorithm and methodology is presented to identify tumor-tissue morphologies based on broadband scatter data measured by raster scan imaging of the samples. A quasi-confocal reflectance imaging system was used to directly measure the tissue scatter reflectance in situ, and the spectrum was used to identify the scattering power, amplitude, and total wavelength-integrated intensity. Pancreatic tumor and normal samples were characterized using the instrument, and subtle changes in the scatter signal were encountered within regions of each sample. Discrimination between normal versus tumor tissue was readily performed using a K-nearest neighbor classifier algorithm. A similar approach worked for regions of tumor morphology when statistical preprocessing of the scattering parameters was included to create additional data features. This type of automated interpretation methodology can provide a tool for guiding surgical resection in areas where microscopy imaging cannot be realized efficiently by the surgeon. In addition, the results indicate important design changes for future systems. [ABSTRACT FROM AUTHOR]

Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivodemonstration of deep tissue tomographic imaging with protoporphyrin IX. [ABSTRACT FROM AUTHOR]

Highly localized reflectance measurements can be used to directly quantify scatter changes in tissues. We present a microsampling approach that is used to raster scan tumors to extract parameters believed to be related to the tissue ultrastructure. A confocal reflectance imager was developed to examine scatter changes across pathologically distinct regions within tumor tissues. Tissue sections from two murine tumors, AsPC-1 pancreas tumor and the Mat-LyLu Dunning prostate tumor, were imaged. After imaging, histopathology-guided region-of-interest studies of the images allowed analysis of the variations in scattering resulting from differences in tissue ultra-structure. On average, the median scatter power of tumor cells with high proliferation index (HPI) was about 26 less compared to tumor cells with low proliferation index (LPI). Necrosis exhibited the lowest scatter power signature across all the tissue types considered, with about 55 lower median scatter power than LPI tumor cells. Additionally, the level and maturity of the tumor’s fibroplastic response was found to influence the scatter signal. This approach to scatter visualization of tissue ultrastructure in situcould provide a unique tool for guiding surgical resection, but this kind of interpretation into what the signal means relative to the pathology is required before proceeding to clinical studies. [ABSTRACT FROM AUTHOR]

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 hafter ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situat ex vivoanalysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm)is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups. [ABSTRACT FROM AUTHOR]

Multispectral near-infrared (NIR) tomographic imaging has the potential to provide information about molecules absorbing light in tissue, as well as subcellular structures scattering light, based on transmission measurements. However, the choice of possible wavelengths used is crucial for the accurate separation of these parameters, as well as for diminishing crosstalk between the contributing chromophores. While multispectral systems are often restricted by the wavelengths of laser diodes available, continuous-wave broadband systems exist that have the advantage of providing broadband NIR spectroscopy data, albeit without the benefit of the temporal data. In this work, the use of large spectral NIR datasets is analyzed, and an objective function to find optimal spectral ranges (windows) is examined. The optimally identified wavelength bands derived from this method are tested using both simulations and experimental data. It is found that the proposed method achieves images as qualitatively accurate as using the full spectrum, but improves crosstalk between parameters. Additionally, the judicious use of these spectral windows reduces the amount of data needed for full spectral tomographic imaging by 50%, therefore increasing computation time dramatically. [ABSTRACT FROM AUTHOR]

A tutorial introduction to diffuse light transport is presented. The basic analytic equations of time-resolved, steady-state and modulated light transport are introduced. The perturbation method for handling slight heterogeneities in optical properties is outlined. The treatment of boundary conditions such as an air/tissue surface is described. Finite mesh-based numerical methods are introduced to calculate the diffuse light field in complex tissues with arbitrary boundaries. Applications in tissue spectroscopy and imaging illustrate these theoretical and computational tools. [ABSTRACT FROM AUTHOR]

Near-infrared (NIR) region-based spectroscopy is examined for accuracy with spectral recovery using frequency domain data at a discrete number of wavelengths, as compared to that with broadband continuous wave data. Data with more wavelengths in the frequency domain always produce superior quantitative spectroscopy results with reduced noise and error in the chromophore concentrations. Performance of the algorithm in the situation of doing region-guided spectroscopy within the MRI is also considered, and the issue of false positive prior regions being identified is examined to see the effect of added wavelengths. The results indicate that broadband frequency domain data are required for maximal accuracy. A broadband frequency domain experimental system was used to validate the predictions, using a mode-locked Ti:sapphire laser for the source between 690- and 850-nm wavelengths. The 80-MHz pulsed signal is heterodyned with photomultiplier tube detection, to lower frequency for data acquisition. Tissue-phantom experiments with known hemoglobin absorption and tissue-like scatter values are used to validate the system, using measurements every 10 nm. More wavelengths clearly provide superior quantification of total hemoglobin values. The system and algorithms developed here should provide an optimal way to quantify regions with the goal of image-guided breast tissue spectroscopy within the MRI. [ABSTRACT FROM AUTHOR]

Fluorescence measurements have been used to track the dosimetry of photodynamic therapy (PDT) for many years, and this approach can be especially important for treatments with aminolevulinic-acid-induced protoporphyrin IX (ALA-PpIX). PpIX photobleaches rapidly, and the bleaching is known to be oxygen dependent, and at the same time, fractionation or reduced irradiance treatments have been shown to significantly increase efficacy. Thus, in vivo measurement of either the bleaching rate and/or the total bleaching yield could be used to track the deposited dose in tissue and determine the optimal treatment plans. Fluorescence in rat esophagus and human Barrett’s esophagus are measured during PDT in both continuous and fractionated light delivery treatment, and the bleaching is quantified. Reducing the optical irradiance from 50 to 25 mW/cm did not significantly alter photobleaching in rat esophagus, but fractionation of the light at 1-min on and off intervals did increase photobleaching up to 10% more (p value=0.02) and up to 25% more in the human Barrett’s tissue (p value<0.001). While two different tissues and two different dosimetry systems are used, the data support the overall hypothesis that light fractionation in ALA-PpIX PDT esophageal treatments should have a beneficial effect on the total treatment effect. [ABSTRACT FROM AUTHOR]

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images. [ABSTRACT FROM AUTHOR]

Receiver operating characteristic (ROC) analysis was performed on simulated near-infrared tomography images, using both human observer and contrast-to-noise ratio (CNR) computational assessment, for application in breast cancer imaging. In the analysis, a nonparametric approach was applied for estimating the ROC curves. Human observer detection of objects had superior capability to localize the presence of heterogeneities when the objects were small with high contrast, with a minimum detectable threshold of CNR near 3.0 to 3.3 in the images. Human observers were able to detect heterogeneities in the images below a size limit of 4 mm, yet could not accurately find the location of these objects when they were below 10 mm diameter. For large objects, the lower limit of a detectable contrast limit was near 10% increase relative to the background. The results also indicate that iterations of the nonlinear reconstruction algorithm beyond 4 did not significantly improve the human detection ability, and degraded the overall localization ability for the objects in the image, predominantly by increasing the noise in the background. Interobserver variance performance in detecting objects in these images was low, suggesting that because of the low spatial resolution, detection tasks with NIR tomography is likely consistent between human observers. [ABSTRACT FROM AUTHOR]

Rapid near-infrared (NIR) diffuse optical tomography is implemented using a low-coherence source. The spectral bandwidth of the low-coherence source is dispersed and coupled to linearly bundled fibers, such that “spread”-spectral encoding among the bundled fibers is formed, and could be used for parallel source illumination onto tissue. In comparison with a previous spectral-encoding technique that employed multiple laser diodes, the use of a low-coherence source for spread-spectral encoding presents a few unique characteristics: (1) it provides shift-free spectral encoding; (2) it reduces the reconstruction uncertainty significantly owing to the minimization of spontaneous channel-to-channel intensity fluctuation; and (3) it enables the implementation of NIR tomography into an endoscopic imaging mode. A 20-mW superluminescent diode centered at 840 nm with a 40-nm bandwidth is used as the source, and a sampling speed of 5 Hz is obtained in a 27-mm imaging array consisting of eight sources and eight detection channels. The principles of using a low-coherence source for spread-spectral encoding are elaborated, the characteristic performances are demonstrated, and the preliminary results of imaging hemoglobin absorption variations during 10 s of voluntary breath-holding are presented. [ABSTRACT FROM AUTHOR]