Alan B. Thompson

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.

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.

Fluorescence-enhanced optical imaging in large tissue volumes using a gain-modulated ICCD camera

A novel image-intensified charge-coupled device (ICCD) imaging system has been developed to perform 3D fluorescence tomographic imaging in the frequency-domain using near-infrared contrast agents. The imager is unique since it (i) employs a large tissue-mimicking phantom, which is shaped and sized to resemble a female breast and part of the extended chest-wall region, and (ii) enables rapid data acquisition in the frequency-domain by using a gain-modulated ICCD camera. Diffusion model predictions are compared to experimental measurements using two different referencing schemes under two different experimental conditions of perfect and imperfect uptake of fluorescent agent into a target. From these experimental measurements, three-dimensional images of fluorescent absorption were reconstructed using a computationally efficient variant of the approximate extended Kalman filter algorithm. The current work represents the first time that 3D fluorescence-enhanced optical tomographic reconstructions have been achieved from experimental measurements of the time-dependent light propagation on a clinically relevant breast-shaped tissue phantom using a gain-modulated ICCD camera.

Diagnostic imaging of breast cancer using fluorescence-enhanced optical tomography: phantom studies

Molecular targeting with exogenous near-infrared excitable fluorescent agents using time-dependent imaging techniques may enable diagnostic imaging of breast cancer and prognostic imaging of sentinel lymph nodes within the breast. However, prior to the administration of unproven contrast agents, phantom studies on clinically relevant volumes are essential to assess the benefits of fluorescence-enhanced optical imaging in humans. Diagnostic 3-D fluorescence-enhanced optical tomography is demonstrated using 0.5 to 1 cm(3) single and multiple targets differentiated from their surroundings by indocyanine green (micromolar) in a breast-shaped phantom (10-cm diameter). Fluorescence measurements of referenced ac intensity and phase shift were acquired in response to point illumination measurement geometry using a homodyned intensified charge-coupled device system modulated at 100 MHz. Bayesian reconstructions show artifact-free 3-D images (3857 unknowns) from 3-D boundary surface measurements (126 to 439). In a reflectance geometry appropriate for prognostic imaging of lymph node involvement, fluorescence measurements were likewise acquired from the surface of a semi-infinite phantom (8x8x8 cm(3)) in response to area illumination (12 cm(2)) by excitation light. Tomographic 3-D reconstructions (24,123 unknowns) were recovered from 2-D boundary surface measurements (3194) using the modified truncated Newtons method. These studies represent the first 3-D tomographic images from physiologically relevant geometries for breast imaging.

Near-infrared fluorescence contrast-enhanced imaging with intensified charge-coupled device homodyne detection: measurement precision and accuracy

Fluorescence frequency-domain photon migration (FDPM) through tissue refers to the propagation of intensity-modulated fluorescent light that originates from tissue-laden fluorophores following illumination with an intensity-modulated excitation light source. FDPM measurements of modulation amplitude and phase are ultimately employed in an inversion algorithm for tomographic reconstruction of interior optical and fluorescent property maps that delineate disease enhanced with fluorescent contrast agent. Because the inverse problem is underdetermined, measurement precision and accuracy crucially impact its solution. Reported here are the precision and accuracy of FDPM measurements acquired using an intensified CCD homodyne detection system. By introducing 32 phase delays between the oscillators used to modulate the intensifier gain and light source intensity at 100 MHz, mean precision is maximized at +/-0.46% and +/-0.26 deg for measurements of modulation amplitude and phase, respectively. Measurement precision improves when the number of phase delays increases. Measurements of fluorescence modulation amplitude and phase, acquired from the surface of a tissue phantom at distances ranging between 0.71 and 3.6 cm from an incident excitation point source, exhibit a mean accuracy of 17% and 1.9 deg, respectively. Measurement accuracy deteriorates with increasing distance from the point source, but for distances up to 1.0 cm from the point source, measurements of fluorescence modulation amplitude and phase exhibit a mean accuracy of 5.4% and 0.30 deg, respectively.

Adaptive finite element methods for fluorescence enhanced frequency domain optical tomography: forward imaging problem

In this contribution we introduce adaptive finite element methods for forward modeling in fluorescence optical tomography. Adaptive local mesh refinement increases the accuracy of the solutions of coupled photon diffusion equations in a computationally optimal manner and when implemented in the inverse problem, can impact the resolution of fluorescence enhanced tomography. An adaptive Galerkin finite element scheme is implemented and the simulation results are compared with experimental data obtained from a tissue phantom by an area illumination and area detection scheme.

Penalty/modified barrier function method for diagnostic imaging using area and point illumination geometries in fluorescence-enhanced optical tomography

A novel imaging technique is developed for (i) area illumination and area collection and (ii) point illumination and point collection geometries in 3D fluorescence-enhanced optical tomography. The target reconstruction problem was formulated as a solution to a nonlinear least-squares-type simple bounds constrained optimization problem. The numerical technique for target reconstruction is based on the penalty/modified barrier function method and the parameter estimates were minimized by the gradient based constrained truncated Newton with trust region method. Three dimensional targets were reconstructed from experimental data under two different experimental conditions of (i) perfect uptake (1:0, target to background ratio) and (ii) imperfect uptake (100:1, target to background ratio).

Image reconstruction for diagnosis and prognosis of breast cancer using fluorescence measurements: phantom studies

Fluorescence-enhance optical tomography is performed using (i) point illumination and point collection and (ii) area illumination and area collection geometrics in 3D. In both measurement techniques, an image-intensified charge-coupled (ICCD) imaging system is used in the frequency-domain to image near-infrared contrast agents. The experimental measurements are compared to diffusion model predictions in least squares form in the inverse problem. For image recovery for both area and point illumination geometries, an efficient gradient-based optimization technique based on the Penalty/modified barrier function (PMBF) method and the constrained truncated Newton with trust region (CONTN) method is developed. Targets in 3D were reconstructed from experimental data under two conditions of (i) perfect uptake (1:0, target to background ratio) and (ii) imperfect uptake (100:1, target to background ratio). Parameters of absorption cross section due to fluorophore and lifetimes are reconstructed. The present work demonstrates that 3D fluorescence enhanced optical tomography reconstructions can be successfully performed from both point/area illumination and collection experimental measurements of the time-dependent light propagation on clinically relevant tissue phantoms using a gain-modulated ICCD camera.

Adaptive finite element methods for forward modeling in fluorescence enhanced frequency domain optical tomography

An adaptive finite element scheme for forward modeling in fluorescence optical tomography is implemented and the simulation results are compared with experimental data obtained from a tissue phantom by an area-illumination and area-detection scheme.

Multipixel assessment of fluorescence uptake and lifetime in the detection of heterogeneous tissue volumes

The objective of this work is to identify an imaging modality which can best locate heterogeneous tissue volumes when designer contrast agents are used. We used a multi- pixel, homodyne, frequency-domain photon migration detection system to acquire images of 830 nm fluorescent heterogeneities immersed within a tissue-simulating phantom that contained 0.5 percent Intralipid solution. An expanded beam of 25 mW, 778 nm light modulated at 100 MHz illuminated the phantom surface. Specifically, we monitor fluorescence average intensity, modulation amplitude, phase, and modulation ratio resulting from micromolar concentrations of indocyanine green and DTTCI embedded within tissue- mimicking, highly scattering media. The results indicate that under conditions of perfect uptake, only phase and modulation distinguish dye solutions that possess equivalent fluorescence yield but unequal lifetime when both heterogeneities are located 0.5 cm from the illumination surface. Enhanced phase contrast was observed for fluorescent solutions with short lifetimes located within a surrounding of longer lived fluorophore and visa versa. These results have important implications for the development of contrast agents whose lifetimes depend on the local biochemical environment.