In this paper we present the absorption coefficient mu(a) and the isotropic scattering coefficient mu(s)(') for 22 human skin samples measured using a double integrating sphere apparatus in the wavelength range of 1000-2200 nm. These in vitro results show that values for mua) follow 70% of the absorption coefficient of water and values for mu(s)(') range from 3 to 16 cm(-1). From the measured optical properties, it was found that a 2% Intralipid solution provides a suitable skin tissue phantom.
Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging.
A new method is described for obtaining a 3-D reconstruction of a bioluminescent light source distribution inside a living animal subject, from multispectral images of the surface light emission acquired on charge-coupled device (CCD) camera. The method uses the 3-D surface topography of the animal, which is obtained from a structured light illumination technique. The forward model of photon transport is based on the diffusion approximation in homogeneous tissue with a local planar boundary approximation for each mesh element, allowing rapid calculation of the forward Green's function kernel. Absorption and scattering properties of tissue are measured a priori as input to the algorithm. By using multispectral images, 3-D reconstructions of luminescent sources can be derived from images acquired from only a single view. As a demonstration, the reconstruction technique is applied to determine the location and brightness of a source embedded in a homogeneous phantom subject in the shape of a mouse. The technique is then evaluated with real mouse models in which calibrated sources are implanted at known locations within living tissue. Finally, reconstructions are demonstrated in a PC3M-luc (prostate tumor line) metastatic tumor model in nude mice.
Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A highsensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLaluc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging. Mol Imaging (2004) 3, 9-23.
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.
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 indocyanine 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.
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 diethylthiatricarbocyanine iodide (tau = 1.17 ns) and indocyanine green (ICG) (tau = 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.
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, s Ј, 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, a , 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.
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