Quantifying molecular specificity of alphavbeta3 integrin-targeted optical contrast agents with dynamic optical imaging.

Dynamic fluorescence images were obtained from a subcutaneous human Kaposi's sarcoma tumor (KS1767) model immediately following the intravenous injection of an integrin-targeting cyanine dye conjugate, Cy5.5-c(KRGDf). The fluorescence images, acquired via an intensified charge-coupled device detection system, were used in conjunction with a pharmacokinetic (PK) model to determine kinetic properties of target binding in the presence and absence of a competitive ligand, free c(KRGDf). The results indicate that the conjugate dye behaves similarly in normal tissue to the free Cy5.5 dye while it possesses increased uptake in tumor tissue. The change in pharmacokinetic parameters obtained from dynamic imaging of Cy5.5-c(KRGDf) after administration of c(KRGDf) as a competitive ligand to the integrin receptor suggests that (i) the increased uptake of Cy5.5-c(KRGDf) is molecularly specific and that (ii) receptor turnover occurs within 24 h. In addition, PK analysis enables quantification of an in vivo c(KRGDf) binding constant attributable to integrin binding. In vivo pharmacokinetic analysis based on rapid and dynamic optical imaging may be potentially useful for evaluating the presence and turnover rate of disease markers that are potential targets of molecular medicine.

Determination of optical properties in semi-infinite turbid media using imaging measurements of frequency-domain photon migration obtained with an intensified charge-coupled device.

Frequency-domain photon migration measurements across the surface of a tissue-mimicking, semi-infinite phantom are acquired via an intensified charge-coupled device (ICCD) detection system and used in conjunction with the diffusion approximation to determine the optical properties. The absorption and reduced scattering coefficients are determined least accurately when relative measurements of average light intensity I(rel)dc are employed either alone or in a combination with relative modulation amplitude data I(rel)ac and/or relative phase shift data theta(rel). The absorption and reduced scattering coefficients are found accurate to within 15 and 11%, respectively, of the values obtained from standard single-pixel measurements when theta(rel) measurements are employed alone or in combination with I(rel)ac data.

Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts.

The specificity of a novel epidermal growth factor (EGF)-Cy5.5 fluorescent optical probe in the detection of EGF receptor (EGFr) was assessed using continuous-wave fluorescence imaging accomplished via an intensified charge-coupled device (CCD) camera. Human mammary MDA-MB-468 (EGFr+) and MDA-MB-435 (EGFr-) cancer cells were incubated with Cy5.5, EGF-Cy5.5, or the anti-EGFr monoclonal antibody C225 or EGF followed by EGF-Cy5.5 and examined under a fluorescence microscope. In vivo imaging was performed on mice with s.c. MDA-MB-468 and MDA-MB-435 tumors. Images were obtained every 6 s for 20 min after i.v. injection of each agent and every 24 h after injection for up to 192 h. Additionally, mice with MDA-MB-468 tumors were injected i.v. with C225 24 h before injection of EGF-Cy5.5. EGF-Cy5.5, but not Cy5.5 or indocyanine green dye (ICG), bound to MDA-MB-468 cells. Binding of EGF-Cy5.5 was blocked by C225 and by EGF. In contrast, binding of EGF-Cy5.5 to MDA-MB-435 cells was not observed. Monitoring of the time-fluorescence intensity in mice confirmed that ICG and Cy5.5 had no favorable binding to tumor regardless of EGFr expression level. In contrast, EGF-Cy5.5 accumulated only in MDA-MB-468 tumors. Moreover, tumor uptake of EGF-Cy5.5 was blocked by C225. ICG and Cy5.5 fluorescence was completely absent from the tumor site, regardless of EGFr expression level, 24 h after injection. Little EGF-Cy5.5 fluorescence was detected in MDA-MB-435 tumors 24 h after injection. In MDA-MB-468 tumors, our data suggest that EGF-Cy5.5 may be used as a specific NIR contrast agent for noninvasive imaging of EGFr expression and monitoring of responses to molecularly targeted therapy.

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.

Sensitivity and depth penetration of continuous wave versus frequency-domain photon migration near-infrared fluorescence contrast-enhanced imaging.

The development of near-infrared fluorescent contrast agents and imaging techniques depends on the deep penetration of excitation light through several centimeters of tissue and the sensitive collection of the re-emitted fluorescence. In this contribution, the sensitivity and depth penetration of various fluorescence-enhanced imaging studies is surveyed and compared with current studies using continuous wave (CW) and frequency-domain photon migration (FDPM) measurements with planar wave illumination of modulated excitation light at 100 MHz and area collection of reemitted fluorescent light using a previously developed modulated intensified charge-coupled device camera system. Fluorescence was generated from nanomolar to micromolar solutions of indocyanine green (ICG) in a 100 microL volume submerged at 1-4 cm depths in a 1% Liposyn solution to mimic tissue scattering properties. Enhanced depth penetration and sensitivity are achieved with optimal filter rejection of excitation light, and FDPM rejection of background light is not achieved using CW methods. We show the ability to detect as few as 100 fmol of ICG from area illumination of 785 nm light (5.5 mW/cm2) and FDPM area collection of 830 nm fluorescent light generated from 3 cm below the phantom surface. The lowered noise floor of FDPM measurements enables greater sensitivity and penetration depth than comparable CW measurements.

Near-infrared fluorescence optical imaging and tomography.

The advent of recent advances in near-infrared laser diodes and fast electro-optic detection has spawned a new research field of diagnostic spectroscopy and imaging based on targeting and reporting exogenous fluorescent agents. This review seeks to concisely address the physics, instrumentation, advancements in tomography, and near-infrared fluorescent contrast agent development that promises selective and specific molecular targeting of diseased tissues. As an example of one area of the field, recent work focusing on pharmacokinetic analysis of fluorophores targeting the epidermal growth factor receptor (EGFR) is presented in a human breast cancer xenograft mouse model to demonstrate specificity of molecularly targeted contrast agents. Finally, a critical evaluation of the limitations and the opportunities for future translation of fluorescence-enhanced optical imaging of deep tissues is presented.

Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents.

The deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers new opportunities for diagnostic imaging when employing sensitive detection techniques and NIR excitable fluorescent agents that target and report disease and metabolism. Herein, we highlight approaches for illuminating tissues and monitoring the re-emitted fluorescence for tomographic reconstruction, strategies for developing fluorescent dye constructs, and clinical opportunities for fluorescence-enhanced NIR optical imaging.