Amir H. Gandjbakhche

Use of polarized light to discriminate short-path photons in a multiply scattering medium.

We describe a method for discriminating short- and long-path photons transmitted through a multiply scattering medium that is based on the relationship between the polarization states of the incident and forward-scattered light. Results of Monte Carlo simulations and experiments show that if the scattering anisotropy of the scatterers is sufficiently small, absorbing barriers embedded in optically dense suspensions of polystyrene spheres can be resolved with good contrast by selectively detecting a component of the scattered-light intensity that has preserved its incident circular polarization state.The principles of operation of a polarization-modulation system capable of measuring small polarization fractions are explained. Using this system we were able to measure polarized light in a depolarized background over 1000 times as large.

Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging

Purpose: HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal. Experimental Design: We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors. Results: The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Conclusion: Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.

Whole-body fluorescence lifetime imaging of a tumor- targeted near-infrared molecular probe in mice

Fluorescence lifetime imaging can provide valuable diagnostic information relating to the functional status of diseases. In this study, a near-infrared (NIR) dye-labeled hexapeptide (abbreviated Cyp-GRD) was synthesized. In vitro, Cyp-GRD internalized in nonsmall cell lung cancer cells (A549) without observable cytotoxic or proliferative effects to the cells at a concentration up to 1x10(-4) M. Time-domain fluorescence intensity and lifetime imaging of Cyp-GRD injected into A549 tumor-bearing mice revealed that the probe preferentially accumulated in the tumor and the major excretion organs. The fluorescence lifetime of the conjugate at the tumor site was mapped, showing the spatial distribution of the lifetime related to its environment. Additionally, fluorescence intensity image reconstruction obtained by integrating the time-resolved intensities enabled the contrast ratios of tumor-to-kidney or liver in slices at different depths to be displayed. The mean lifetime was 1.03 ns for the tumor and 0.80 ns for the liver when averaging those pixels exhibiting adequate signal-to-noise ratio, showing the tumor had a higher lifetime average and reflecting the altered physiopathology of the tumor. This study clearly demonstrated the feasibility of whole-body NIR fluorescence lifetime imaging for tumor localization and its spatial functional status in living small animals.

Light-scattering technique for the study of orientation and deformation of red blood cells in a concentrated suspension

The backscattered and transmitted diagrams of He-Ne laser light illuminating a concentrated suspension of red blood cells (RBCs) are investigated. The shapes of these diagrams are closely related to the state of the suspension (at rest or submitted to a simple shear flow) and to the parameters that govern the non-Newtonian behavior of the blood suspension (such as the viscosity of the suspending medium and the volume concentration of the cells). An asymmetry in the backscattering diagram, which is absent on transmitted diagrams, is observed when the suspension is in a simple shear flow. This asymmetry is related to the deformation and orientation of the RBCs. The propagation of light through the suspension is modeled and a set of Monte Carlo simulations is performed to substantiate the inference that the relative variation of the backscattered flux is proportional to the gradients of deformation of the RBCs, and that such gradients must be known in order to apply a rheological model describing the non-Newtonian behavior of RBC membranes.

Resolution limits for optical transillumination of abnormalities deeply embedded in tissues

Random walk theory is used to calculate the line spread function (LSF) of photons as they cross the midplane of a slab of finite thickness. The relationship between the LSF and the photon transit time in transillumination time-resolved experiments is investigated. It is found that the LSF is approximately Gaussian distributed, with a standard deviation, sigma, which can be used as a criterion of the spatial resolution of the imaging system. Results are substantiated by comparison with actual data in the literature. Any given resolution can be improved by reducing the excess transit time delta t, but heterogeneity of the scattering medium and low levels of detected light enormously complicate the achievement of subcentimeter spatial resolution. The latter point is discussed by using optical parameters of breast tissues for visible and near-infrared radiation (NIR) light.

Fluorescence Lifetime Imaging System for In Vivo Studies

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.

The use of the Henyey–Greenstein phase function in Monte Carlo simulations in biomedical optics

Monte Carlo (MC) simulations are often at the heart of the testing procedure in biomedical optics. One of the critical points in MC simulations is to define the new photon direction after each scattering event. One of the most popular solutions is to use the Henyey-Greenstein phase function or some linear combinations of it. In this note, we demonstrate that randomly generating the angle defining the new direction of a photon after a collision, by means of the Henyey-Greenstein phase function, is not equivalent to generating the cosine of this angle, as is classically done. In practice, it is demonstrated that for a nearly isotropic medium (asymmetry parameter g approximately 0) this discrepancy is not large, however for an anisotropic medium as is typically found in vivo (e.g. g = 0.98) the two methods give completely different results.

V: Random Walk and Diffusion-Like Models of Photon Migration in Turbid Media

Publisher Summary The chapter focuses on the properties of multiply-scattering or turbid media, and emphasizes results obtained using random walk theory of photon migration in a turbid medium. Methodology based on the theory of random walks is a ubiquitous tool in the analysis of polymer configurations and is central to many of the techniques used to transform data taken from X-ray scattering experiments into useful structural parameters. The types of random walk models that are distinguished by the space in which the walk occurs include the continuum random walk and the lattice random walk. The chapter derives the joint probabilities required for translating optical data into physical parameters for a slab whose properties are homogeneous on a macroscopic scale. There are several approaches for modelling the use of optical techniques by random walk and/or diffusion theory to determine internal structure and inclusions in a multiply-scattering medium. Three categories of applications of the theory will be described (i) laser Doppler flowmetry, (ii) random walk results applied to spectroscopic methods, and (iii) the characterization of the performance of an optical imaging system.

Scaling relationships for theories of anisotropic random walks applied to tissue optics.

Monte Carlo simulations are used to discern scaling relationships for photon migration occurring within homogeneous, anisotropic scattering media of semi-infinite extent. Special attention is given to events associated with short path lengths. Empirical scaling relationships for path lengths and surface intensities are shown to agree with a consistency equation derived in an earlier study of anisotropic random walks. They are augmented here by a procedure that accounts for concomitant scaling of optical absorption coefficients. Results then are used to transform expressions that were obtained previously by analytical random-walk theory developed for an isotropic scattering model of photon migration. Quantities that are studied include the diffuse surface reflectance, the depth distribution of the fluence, and the time-resolved intensity of backreflected photons.

Using noninvasive multispectral imaging to quantitatively assess tissue vasculature

This research describes a noninvasive, noncontact method used to quantitatively analyze the functional characteristics of tissue. Multispectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposis sarcoma lesions and the surrounding tissue before and during experimental therapies. The multispectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.