Tamara L. Troy

IMAGING OF SPONTANEOUS CANINE MAMMARY TUMORS USING FLUORESCENT CONTRAST AGENTS

Abstract— We present near‐infrared frequency‐domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue‐simulating phantoms and actual tissues. We employ intensity‐modulated excitation light that is expanded and delivered to the surface of a tissue or tissue‐simulating phantom. The intensity‐modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain‐modulated image‐intensified charge‐coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of dieth‐ylthiatricarbocyanine iodide (π= 1.17 ns) and indocyanine green (ICG) (π= 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency‐domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.

Fluorescence and Absorption Contrast Mechanisms for Biomedical Optical Imaging Using Frequency‐Domain Techniques

Abstract— The ability to optically image or detect diseased tissue volumes located deep within tissues depends upon the degree of contrast provided by differences in local optical properties. In this report, we show that the exogenous contrast offered by fluorescent compounds is superior to that provided by nonfluorescing, light‐absorbing compounds when time‐dependent measurements are employed. In addition, we show that the induced contrast is not only moderated by the preferential uptake of fluorescent agents into diseased tissue volumes of interest but also by the fluorescent optical properties and the fluorescence dynamics in the specific tissue volume. Using tissue phantom studies, we demonstrated experimentally that near‐infrared‐absorbing and fluorescent dyes such as in‐docyanine green can provide detection of diseased tissue volumes from fluorescence measurements made at the periphery of tissue when there is perfect, 100‐fold and 10‐fold partitioning in diseased tissues over that in surrounding normal tissues. Experimental results of common laser dyes show the contrast is also mediated by the quantum yield and lifetime parameters that may be dependent upon the local tissue environment.

Optical properties of normal and diseased breast tissues: prognosis for optical mammography

The use of near-infrared (NIR) measurements of photon migration has been recently demonstrated for the detection of breast cancer in Europe. Yet the clinical success of this potential screening tool depends upon consistent detection of the disease at earlier stages than is currently possible with conventional x-ray mammography. In this paper, we present the optical property measurements of 115 histologically classified breast tissue specimens in order to determine whether consistent and significant optical contrast exists for detection of the disease. Our in vitro optical properties measured with a double integrating sphere technique show consistent changes (yet statistically insignificant) in effective scattering coefficients, ms8, with tissue classification of infiltrating carcinoma (n=48), ductal carcinoma in situ (n=5), mucinous carcinoma (n=3), normal fatty (n=23), and normal fibrous tissues (n=35). However, there is little change in the in vitro tissue absorption coefficient, ma , measured at 749, 789, and 836 nm. For normal and diseased tissue specimens extracted from the same patient, we found differences in optical properties, indicating optical contrast. Using a finite-element prediction of light propagation, we evaluated this optical contrast for photon migration detection of ductal carcinoma in situ tissues using these optical properties measured in vitro.

Pharmacokinetics of ICG and HPPH-car for the Detection of Normal and Tumor Tissue Using Fluorescence, Near-infrared Reflectance Imaging: A Case Study¶

Abstract We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg−1 ICG or 0.3 mg kg−1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5–1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.

BIOMEDICAL OPTICAL TOMOGRAPHY USING DYNAMIC PARAMETERIZATION AND BAYESIAN CONDITIONING ON PHOTON MIGRATION MEASUREMENTS

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.

Multipixel techniques for frequency-domain photon migration imaging.

The ability to map interior optical properties of a highly scattering medium from exterior measurements of light propagation is afforded by optical tomography. In this communication, we describe the problem of optical tomography, the techniques of photon migration measurements necessary to accomplish it, and the development of multipixel measurements for rapid collection of optical signals. These multipixel measurements are shown to provide detection of contrast owing to the optical properties of absorption and fluorescence associated with dye‐laden heterogeneities embedded in a tissue‐like scattering medium. From these rapid measurements, successful reconstruction of an interior optical property map may now be possible with clinically realistic data acquisition times. Applications for the technology arise for biomedical optical imaging for the in vivo detection of disease and the diagnosis of tissue (bio‐) chemistry.

Role of higher-order scattering in solutions to the forward and inverse optical-imaging problems in random media

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.

Photon migration imaging using multipixel measurements

In order to reconstruct images of heterogeneities embedded deep within tissues, multiple-pixel measurements of frequency domain photon migration are necessary. Typically, these measurements are performed at multiple locations on the periphery of the sample using individual fiber optics. However, measurements using multiple fiber optics are both tedious and time consuming, especially as one increases spatial measurement resolution. In our research, we acquire multi-pixel measurements by employing a gain-modulated image intensified CCD camera and near infrared milliwatt laser diodes to monitor the propagation characteristics of sinusoidally modulated light as it passes through tissue-like phantoms containing 0.5% intralipid solutions. Our results show that images of millimeter sized absorbing objects located greater than 1 cm inside an ideal tissue phantom an be successfully obtained. In addition, we have been able to generate fluorescent images locating portions of micromolar concentrations of indocyanine green embedded within an intralipid solution contained within a glass cylindrical vessel (10 mm by 4 mm diameter).

Fluorescence lifetime spectroscopy and imaging in random media

Tissue fluorescence, whether from endogenous or exogenous probes, provides an opportunity for interrogation on the basis of structure and function. However to date, there has been little understanding of the localization of signal origin of fluorescence signals re-emitted from tissues. Previously, we have shown using finite element computations that the origin or re- emitted fluorescence signal depends upon the lifetime of the optical probe. The signal arising from long-lived phosphorescent probes may predominately come from the tissue-air interface rather than from deep within the tissue. Therefore, the use of short-lived optical probes may be desirable for fluorescence spectroscopy and as contrast agents for biomedical optical lived probes embedded within scattering media. Our results show that upon proper referencing of frequency-domain measurements, lifetime measurements can be made in the presence of uniform and nonuniform distribution of optical probes. The implications for biomedical optical imaging on the basis of probe lifetime are discussed.

Multipixel imaging of interfering photon density waves

A gain-modulated image intensifier with a CCD array was developed and used to simultaneously acquire multi-pixel modulation amplitude and phase images of the interference pattern within 0.5% intralipid solution and resulting from two 5 mW near infrared sources modulated 180 degrees out-of-phase at frequencies between 10 and 120 MHz. Gain modulation was achieved by modulating the voltage between the image intensifiers photocathode and multichannel plate input. Homodyning the gain modulation and source signals enabled images of the interference pattern to be obtained with total exposure times on the order of 3 seconds or less. A strongly absorbing, embedded heterogeneity could be localized in two dimensions by its deflection of the interference plane between two out-of-phase sources.