Jeffery S. Reynolds

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.

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.

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.

Three-dimensional Bayesian optical diffusion tomography with experimental data

Reconstructions of a three-dimensional absorber embedded in a scattering medium by use of frequency domain measurements of the transmitted light in a single source-detector plane are presented. The reconstruction algorithm uses Bayesian regularization and iterative coordinate descent optimization, and it incorporates estimation of the detector noise level, the source-detector coupling coefficient, and the background diffusion coefficient in addition to the absorption image. The use of multiple modulation frequencies is also investigated. The results demonstrate the utility of this algorithm, the importance of a three-dimensional model, and that out-of-plane scattering permits recovery of three-dimensional features from measurements in a single plane.

Investigation of static structure factor in dense suspensions by use of multiply scattered light.

Near-infrared, frequency-domain photon migration measurements of phase shift are used to derive accurate values of isotropic scattering coefficients in concentrated, interacting suspensions of aqueous polystyrene microspheres with volume concentrations ranging from 1% to 45% by solids and mean diameters ranging from 135 to 500 nm. Under conditions of high ionic strength, the isotropic scattering coefficient can be quantitatively predicted by the Percus-Yevick model for hard-sphere interactions and Mie theory. In addition, the attractive interactions between scatterers arising from the addition of soluble poly(ethylene glycol) with molecular weights of 100 and 600 K cause hindered scattering. The increases in static structure and decreases in isotropic scattering coefficient agree with that predicted by Mie theory and the depletion interaction model developed by Asakura and Oosawa [J. Chem. Phys. 22, 1255 (1954)]. These results demonstrate the success of monitoring interaction between particles by use of multiple-scattered light and the necessity of incorporating models for these interactions when predicting scattering of dense, concentrated suspensions.

Optical diffusion imaging: a comparative numerical and experimental study

We present a low-cost photodiode-LED apparatus for making broadband frequency domain photon migration measurements. We compare measured data to finite-difference frequency domain solutions of the diffusion approximation of the Boltzmann transport equation. Specific comparisons include the influence of boundary conditions on simulations and the effect of finite source size on resolution.

Comparison of sensitivity for single-source and dual-interfering-source configurations in optical diffusion imaging

A numerical study of single-source and dual-interfering-source modalities for optical imaging through biological media with a view to practical implications is given. Our comparisons are based on multigrid finite-difference frequency-domain solutions of the diffusion equation, for example, inhomogeneous imaging problems. Detector noise levels, the influence of boundaries, and ease of measurement are considered. This investigation indicates that a dual source, or dual-source data synthesized from single-source measurements, offers significant imaging opportunities when compared with single-source data.

Frequency domain modeling of reradiation in highly scattering media

We present a straightforward procedure for frequency domain modeling of reradiation in a highly scattering medium with an arbitrary, finite three-dimensional geometry. We use a finite difference numerical solver to determine the fluence distribution at the excitation wavelength, which is then coupled to the emission wavelength with an array of equivalent reradiating sources. We then calculate the fluence distribution at the emission wavelength with a second, independent numerical simulation with new optical parameters appropriate to the emission wavelength, using the distributed reradiating sources as the excitation. We compare three-dimensional simulations of a fluorophore distributed in a scattering medium with experimental data. We also compare simulations of the Raman reradiation of small diamonds in a scattering medium with experiment.

Raman spectroscopic studies of diamond in Intralipid

Raman spectroscopic measurements were performed to determine the presence of a diamond located within an Intralipid scattering medium. The Raman signature of the diamond was recorded for varying thicknesses and concentrations of the Intralipid by use of a 514-nm argon-ion laser and a cw 785-nm Ti:sapphire laser. Results show how Raman spectroscopy can be used as a diagnostic tool for identifying objects embedded within scattering media. In particular, future applications could include tissue diagnostics performed in vivo.