Anikitos Garofalakis

Noncontact optical imaging in mice with full angular coverage and automatic surface extraction

During the past decade, optical imaging combined with tomographic approaches has proved its potential in offering quantitative three-dimensional spatial maps of chromophore or fluorophore concentration in vivo. Due to its direct application in biology and biomedicine, diffuse optical tomography (DOT) and its fluorescence counterpart, fluorescence molecular tomography (FMT), have benefited from an increase in devoted research and new experimental and theoretical developments, giving rise to a new imaging modality. The most recent advances in FMT and DOT are based on the capability of collecting large data sets by using CCDs as detectors, and on the ability to include multiple projections through recently developed noncontact approaches. For these to be implemented, we have developed an imaging setup that enables three-dimensional imaging of arbitrary shapes in fluorescence or absorption mode that is appropriate for small animal imaging. This is achieved by implementing a noncontact approach both for sources and detectors and coregistering surface geometry measurements using the same CCD camera. A thresholded shadowgrammetry approach is applied to the geometry measurements to retrieve the surface mesh. We present the evaluation of the system and method in recovering three-dimensional surfaces from phantom data and live mice. The approach is used to map the measured in vivo fluorescence data onto the tissue surface by making use of the free-space propagation equations, as well as to reconstruct fluorescence concentrations inside highly scattering tissuelike phantom samples. Finally, the potential use of this setup for in vivo small animal imaging and its impact on biomedical research is discussed.

Three-Dimensional in Vivo Imaging of Green Fluorescent Protein-Expressing T Cells in Mice with Noncontact Fluorescence Molecular Tomography:

Given that optical tomography is capable of quantitatively imaging the distribution of several important chromophores and fluorophores in vivo, there has been a great deal of interest in developing optical imaging systems with increased numbers of measurements under optimal experimental conditions. In this article, we present a novel system that enables three-dimensional imaging of fluorescent probes in whole animals using a noncontact setup, in parallel with a three-dimensional surface reconstruction algorithm. This approach is directed toward the in vivo imaging of fluorophore or fluorescent protein concentration in small animals. The system consists of a rotating sample holder and a lens-coupled charge-coupled device camera in combination with a fiber-coupled laser scanning device. By measuring multiple projections, large data sets can be obtained, thus improving the accuracy of the inversion models used for quantitative three-dimensional reconstruction of fluorochrome distribution, as well as facilitating a higher spatial resolution. In this study, the system was applied to determining the distribution of green fluorescent protein (GFP)-expressing T lymphocytes in a transgenic mouse model, thus demonstrating the potential of the system for studying immune system function. The technique was used to image and reconstruct fluorescence originating from 32 × 106 T cells in the thymus and 3 × 105 T cells in the spleen.

Imaging changes in lymphoid organs in vivo after brain ischemia with three-dimensional fluorescence molecular tomography in transgenic mice expressing green fluorescent protein in T lymphocytes

Stroke induces a strong inflammatory reaction in the brain and depresses the immune system. We sought to assess longitudinal changes in T-cell numbers in the lymphoid organs of living mice after brain ischemia. Middle cerebral artery occlusion was carried out in transgenic mice expressing green fluorescent protein (GFP+) in the T-cell population under the control of the hCD2 locus control region. Imaging was performed by three-dimensional fluorescence molecular tomography (FMT) before and at several time points after ischemia or sham operation and in controls. At day 7, GFP+ cell content in lymphoid organs was measured postmortem by flow cytometry. GFP+ cell numbers and in vivo FMT signal intensity were reduced at day 7 after ischemia and, to a lesser extent, after sham operation. Linear regression analysis demonstrated that postmortem GFP+ cell numbers and corresponding in vivo FMT data were significantly correlated in the thymus (r2 = .65, p < .0001) and lymph nodes (r2 = .67, p < .0001). These relationships allowed inferring the number of GFP+ T cells from in vivo FMT data. The results show the time course reduction of T-cell content in the lymphoid organs of living mice, providing in vivo evidence of lymphoid organ atrophy after stroke and, to a lesser extent, after head surgery with craniectomy and dura mater opening in sham-operated mice.

Optical characterization of thin female breast biopsies based on the reduced scattering coefficient.

One of the main goals in optical characterization of biopsies is to discern between tissue types. Usually, the theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, biopsies are usually small in size compared to the transport mean free path and thus cannot be treated with standard diffusion theory. To account for this, an improved theory was developed, by the authors, that can correctly describe light propagation in small geometries (Garofalakis et al 2004 J. Opt. A: Pure Appl. Opt. 6 725-35). The theorys limit was validated by both Monte Carlo simulations and experiments performed on tissue-like phantoms, and was found to be two transport mean free paths. With the aid of this theory, we have characterized 59 samples of breast tissue including cancerous samples by retrieving their reduced scattering coefficients from time-resolved transmission data. The mean values for the reduced scattering coefficients of the normal and the tumour tissue were measured to be 9.7 +/- 2.2 cm(-1) and 10.8 +/- 1.8 cm(-1), respectively. The correlation with age was also investigated.

A study of photon propagation in free-space based on hybrid radiosity-radiance theorem

Noncontact optical imaging has attracted increasing attention in recent years due to its significant advantages on detection sensitivity, spatial resolution, image quality and system simplicity compared with contact measurement. However, photon transport simulation in free-space is still an extremely challenging topic for the complexity of the optical system. For this purpose, this paper proposes an analytical model for photon propagation in free-space based on hybrid radiosity-radiance theorem (HRRT). It combines Lamberts cosine law and the radiance theorem to handle the influence of the complicated lens and to simplify the photon transport process in the optical system. The performance of the proposed model is evaluated and validated with numerical simulations and physical experiments. Qualitative comparison results of flux distribution at the detector are presented. In particular, error analysis demonstrates the feasibility and potential of the proposed model for simulating photon propagation in free-space.

Characterization of the reduced scattering coefficient for optically thin samples: theory and experiments

Diffusion theory is currently used to characterize highly scattering media from time and/or spatially resolved measurements. However, many of the limits of validity of the diffusion approximation in its standard form have been set through assumptions taken during its derivation, and it is generally believed that the diffusion equation holds only for length scales much larger than the transport mean free path (ltr). We here present several modifications to the standard diffusion approximation to provide a simple expression for characterizing optically thin scattering slabs. The improvements concern mainly the correct modelling of the temporal and spatial distribution of the source term and the use of a frequency dependent diffusion coefficient and boundary conditions. This novel expression is put to the test against Monte Carlo simulations and experimental data taken from measurements with phantoms of known optical properties. The reduced scattering coefficient can be retrieved accurately for slab widths of the order of the mean free path.

Autofluorescence removal from fluorescence tomography data using multispectral imaging

Autofluorescence has been a significant disadvantage when dealing with tomographic imaging of biological samples or tissue phantoms. Consequently, the accurate removal of autofluorescence signal has been a major concern in fluorescence tomography. Here we present a study on three-dimensional mapping and removal of autofluorescence from fluorescence molecular tomography (FMT) data, both for phantoms and small animal in vivo. The technique is based on the recording of tomographic data in multiple spectral regions with different excitation light and on the application of a linear unmixing algorithm for targeting multiple fluorescent probes. Two types of measurements are taken, one with the excitation being in the region of the maximum absorption of the targeted fluorophore and one in a region away from the maximum. The relative strengths of the different spectra are employed to calculate the signal to be removed from the tomographic reconstruction. Autofluorescence spectra are recorded using identical reflection geometry as during the FMT acquisitions allowing for the correct mapping of the autofluorescence signal. Results from phantoms exhibiting different background autofluorescence strengths are presented and discussed. In this work we have also studied in vivo fluorescent activity in mice, involving both subcutaneously implanted fluorescent phantoms and b10 transgenic mice.

Accounting for Point Source Propagation Properties in 3D Fluorescence OPT

� Abstract- Reconstructing images from projections in fluorescence Optical Projection Tomography (OPT) is a relatively new problem requiring attention from researchers in this emerging field. There are several aspects of fluorescence OPT which require different treatment compared with non-fluorescence tomography. One significant problem, and the subject of this paper, is that fluorophores emit light isotropically and so the intensity of light captured at a microscope lens is inversely proportional to the square of the distance of the fluorophore from the focal point of the lens. Standard back-projection techniques do not account for this phenomenon and we show numerically that net effect is that fluorophores close to the centre of a body are assigned relatively low values in standard reconstructions. This is an inherent limitation of the standard back-projection method for quantitative fluorescence applications (e.g. in molecular imaging). In this paper we present a working, though computationally intensive, method to account for this, and discuss how more sophisticated (and less computationally intense) methods could be derived in the near future.

3D in vivo imaging of GFP-expressing T-cells in mice with non-contact fluorescence molecular tomography

Optical tomography has been proposed as a promising technique for probing deep in tissue with many medical applications. Recently, the adaptation of fluorescent probes by the radiologists, gave rise to a new imaging tool in the area of molecular imaging. Optical tomography can, provide three-dimensional images of fluorescent concentrations inside living systems of sizes in the order of many cm. Our optical tomographer was based on a technique which is called Fluorescence Molecular Tomography (FMT) and can quantify fluorescent signals in mice. The imaging procedure is performed in a non-contact geometry so that living subjects of arbitrary shapes can be imaged with no fibers attached to them. We have developed a way to reconstruct the 3D surface of the subject and we use theoretical models to account for the propagation of the emerging signal in the free space. The system consists of a rotating sample holder and a CCD camera in combination with a laser-scanning device. An Argon-ion laser is used as the source and different filters are used for the detection of various fluorophores or fluorescing proteins. So far, we have observed of the distribution of GFP expressing T-lymphocytes in-vivo for the study of the function of the immune system in a murine model. Then we investigated the performance of the FMT setup to quantify the different amounts of migrated cells in the different organs by comparing our results with the FACS measurements. Further experiments included the measurement of the variations of the T cells concentration in-vivo, over time.

A multi-projection non-contact tomography setup for imaging arbitrary geometries

Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores in-vivo. Due to this fact, there has been great interest in developing imaging systems offering quantitative information on the location and concentration of chromophores and fluorescent probes. However, most imaging systems currently used in reasearch make use of fiber technology for delivery and detection, which restricts the size ofthe photon collecting arrays leading to insufficient spatial sampling and field of view. To enable large data sets and full 3600angular measurements, we developed a novel imaging system that enables 3D imaging of fluorescent signals in bodies of arbitrary shapes in a non-contact geometry in combination with a 3D surface reconstruction algorithm. The system is appropriate for in-vivo small animal imaging of fluorescent probes. The system consists of a rotating sample holder and a lens coupled CCD camera in combination with a fiber coupled scanning device. The accuracy of the system in obtaining the surface reconstruction was measured to be in the order of 1μm.