Anabela Da Silva

Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging

Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields Phi (respectively, 0.38 and 0.25) and longer fluorescence lifetimes tau (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (Phi=0.28, tau=1.0 ns) and Cy7 (Phi=0.13, tau=0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.

In vivo mice lung tumor follow-up with fluorescence diffuse optical tomography

We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.

Optical calibration protocol for an x-ray and optical multimodality tomography system dedicated to small-animal examination

A small-animal multimodality tomography system dedicated to the coregistration of fluorescence optical signal and x-ray measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility of getting in vivo anatomical and functional information simultaneously. Moreover, anatomical measurements can be used as a regularization factor to achieve more accurate reconstructions of the biodistribution of fluorochromes and to speed up treatment. A dedicated acquisition protocol has been established, and the methodology of the reconstruction of the three-dimensional distribution of the biomarkers under cylindrical geometry consistent with classic computed tomography has been implemented. A phantom study was conducted to evaluate and to fix the parameters for the coregistration. These test experiments were reproduced by considering anesthetized mice that had thin glass tubes containing fluorochromes inserted into their esophagus. The instrument is also used for an in vivo biological study conducted on mice with lung tumors, tagged with near-infrared optical probes (targeting probes such as Transferin-AlexaFluor750).

Effects of the surface boundary on the determination of the optical properties of a turbid medium with time-resolved reflectance.

Solutions of the time-dependent diffusion equation were developed to take into account the depth of the source and the detector inside a semi-infinite medium. These solutions permitted an evaluation of optical properties at different depths below the surface by fitting time-resolved data. Measurements were performed on liquid optical phantoms with optical fibers for delivering and collecting light. A time-correlated single-photon-counting chain was used for electronic detection. The determination of optical properties underlines the continuity between the surface model and the infinite model and shows the depth at which the derived solutions can be applied.

Considering sources and detectors distributions for quantitative photoacoustic tomography

Photoacoustic tomography (PAT) is a hybrid imaging modality that takes advantage of high optical contrast brought by optical imaging and high spatial resolution brought by ultrasound imaging. However, the quantification in photoacoustic imaging is challenging. Multiple optical illumination approach has proven to achieve uncoupling of diffusion and absorption effects. In this paper, this protocol is adopted and synthetic photoacoustic data, blurred with some noise, were generated. The influence of the distribution of optical sources and transducers on the reconstruction of the absorption and diffusion coefficients maps is studied. Specific situations with limited view angles were examined. The results show multiple illuminations with a wide field improve the reconstructions.

A comprehensive study of the use of temporal moments in time-resolved diffuse optical tomography: part I. Theoretical material

The problem of fluorescence diffuse optical tomography consists in localizing fluorescent markers from near-infrared light measurements. Among the different available acquisition modalities, the time-resolved modality is expected to provide measurements of richer information content. To extract this information, the moments of the time-resolved measurements are often considered. In this paper, a theoretical analysis of the moments of the forward problem in fluorescence diffuse optical tomography is proposed for the infinite medium geometry. The moments are expressed as a function of the source, detector and markers positions as well as the optical properties of the medium and markers. Here, for the first time, an analytical expression holding for any moments order is mathematically derived. In addition, analytical expressions of the mean, variance and covariance of the moments in the presence of noise are given. These expressions are used to demonstrate the increasing sensitivity of moments to noise. Finally, the newly derived expressions are illustrated by means of sensitivity maps. The physical interpretation of the analytical formulae in conjunction with their map representations could provide new insights into the analysis of the information content provided by moments.

Elliptically polarized light for depth resolved optical imaging

It is shown that using elliptically polarized light permits selecting well-defined subsurface volumes in a turbid medium. This suggests the possibility of probing biological tissues at specific depths. First, we present the method and preliminary results obtained on an Intralipid phantom. We next report on the method’s performance on a biological phantom (chicken breast) and, finally, on the exposed cortex of an anesthetized rat.

A comprehensive study of the use of temporal moments in time-resolved diffuse optical tomography: part II. Three-dimensional reconstructions

This paper addresses the inverse problem of time-resolved (fluorescence) diffuse optical tomography from temporal moments of the measurements. A methodology that enables one to provide fairly comparable reconstructions is presented. The proposed reconstruction methodology is applied to infinite medium synthetic phantoms in the transmission geometry. Reconstructions are performed for moment orders increasing from 0 to 3. The reconstruction quality is shown to be increasing when higher moment orders are added. However, the value of the highest useful moments order strongly depends on the number of photons that can be acquired. In particular, it can be considered that the benefit of using higher order moments vanishes when fewer than 10(8) photons are detected. The evolution of the reconstruction quality with respect to the optical properties of the medium and fluorescence lifetime is also shown.

Approximations of the measurable quantity in diffuse optical problems: theoretical analysis of model deviations

Photon density and photon flux are widely used to model the measurable quantity in diffuse optical tomography problems. However, it is not these two quantities that are actually measured, but rather the radiance accepted by the detection system. We provide a theoretical analysis of the model deviations related to the choice of the measurable quantity-either photon density or flux. By using the diffusion approximation to the radiative transfer equation and its solution with extrapolated boundary conditions, an exact analytical expression of the measurable quantity has been obtained. This expression has been employed as a reference to assess model deviation when considering the photon density or the photon flux as the measurable quantity. For the case of semi-infinite geometry and for both continuous wave and time domains, we show that the photon density approximates the measurable quantity better than the photon flux. We also demonstrate that the validity of this approximation strongly depends on the optical parameters.

Depth probing of diffuse tissues controlled with elliptically polarized light

Abstract. Polarization gating is a popular technique in biomedical optics. It is widely used to inspect the surface of the tissues (under colinear or cocircular detection) or instead to probe the volume (cross-linear detection), without information on the probed depth. Elliptical polarization is introduced to explore the possibility of probing diffuse tissues at selective depths. A thorough Monte Carlo simulation study shows complete correlation between the probed depths and the ellipticity of the polarized light, for a medium with known optical properties. Within a wide range of optical parameters, a linear relation between the backscattered intensity and the depth extension of the probed volume was found whatever the polarization used, but with a controlled extension depending on the ellipticity.