Giannis Zacharakis

Fluorescent protein tomography scanner for small animal imaging

Microscopy of fluorescent proteins has enabled unprecedented insights into visualizing gene expression in living systems. Imaging deeper into animals, however, has been limited due to the lack of accurate imaging methods for the visible. We present a novel system designed to perform tomographic imaging of fluorescent proteins through whole animals. The tomographic method employed a multiangle, multiprojection illumination scheme, while detection was achieved using a highly sensitive charge-coupled device camera with appropriate filters. Light propagation was modeled using a modified solution to the diffusion equation to account for the high absorption and high scattering of tissue at the visible wavelengths. We show that the technique can quantitatively detect fluorescence with sub millimeter spatial resolution both in phantoms and in tissues. We conclude that the method could be applied in tomographic imaging of fluorescent proteins for in vivo targeting of different diseases and abnormalities.

Random laser action in organic–inorganic nanocomposites

Random laser action is demonstrated in organic–inorganic, disordered hybrid materials consisting of ZnO semiconductor nanoparticles dispersed in an optically inert polymer matrix. The ZnO particles provide both the gain and the strong scattering power that leads to light trapping due to multiple elastic scattering, whereas the polymer matrix offers ease of material fabrication and processability in view of potential applications. Excitation of the nanohybrids by a laser pulse with duration shorter than the ZnO photoluminescence lifetime leads to a dramatic increase in the emitted light intensity accompanied by a significant spectral and temporal narrowing above a certain threshold of the excitation energy density. Critical laser and material parameters that influence the observed laser-like emission behavior are investigated in a series of nanocomposites.

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.

Experimental determination of photon propagation in highly absorbing and scattering media

Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores. Several theoretical models have been validated for diffuse photon propagation in highly scattering and low-absorbing media that describe the optical appearance of tissues in the near-infrared (NIR) region. However, these models are not generally applicable to quantitative optical investigations in the visible because of the significantly higher tissue absorption in this spectral region compared with that in the NIR. We performed photon measurements through highly scattering and absorbing media for ratios of the absorption coefficient to the reduced scattering coefficient ranging approximately from zero to one. We examined experimentally the performance of the absorption-dependent diffusion coefficient defined by Aronson and Corngold [J. Opt. Soc. Am. A 16, 1066 (1999)] for quantitative estimations of photon propagation in the low- and high-absorption regimes. Through steady-state measurements we verified that the transmitted intensity is well described by the diffusion equation by considering a modified diffusion coefficient with a nonlinear dependence on the absorption. This study confirms that simple analytical solutions based on the diffusion approximation are suitable even for high-absorption regimes and shows that diffusion-approximation-based models are valid for quantitative measurements and tomographic imaging of tissues in the visible.

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.

Photon statistics of laserlike emission from polymeric scattering gain media.

The coherent properties of the temporally and spectrally narrowed emission of laser-induced fluorescence of organic dyes hosted inside artificial scattering matrices (random lasers) were investigated. The excitation source was a frequency-doubled 200-fs pulsed laser emitting at 400 nm. Spectral and temporal features were simultaneously recorded with a spectrograph and a streak camera operating in photon-counting mode. Photon-number distributions were thus created. The temporal coherence of the laserlike emission above and below the excitation energy threshold was investigated from the photon-number distribution that was obtained.

Random lasing following two-photon excitation of highly scattering gain media

We present experimental evidence of laser emission following two-photon excitation of dye-infiltrated random gain media with optical properties similar to biological tissue. The excitation was performed with femtosecond laser pulses at 800 nm and the emission (at 480 nm) was recorded with a spectrograph streak camera system. The coherent properties of the random lasing emission were also investigated by performing single photon counting. The applications of coherent laser light that can be emitted deep inside a random medium are far reaching, particularly for imaging and therapeutic purposes.

Artificial neural networks for discriminating pathologic from normal peripheral vascular tissue

The identification of the state of human peripheral vascular tissue by using artificial neural networks is discussed in this paper. Two different laser emission lines (He-Cd, Ar+) are used to excite the chromophores of tissue samples. The fluorescence spectrum obtained, is passed through a nonlinear filter based on a high-order (HO) neural network neural network (NN) [HONN] whose weights are updated by stable learning laws, to perform feature extraction. The values of the feature vector reveal information regarding the tissue state. Then a classical multilayer perceptron is employed to serve as a classifier of the feature vector, giving 100% successful results for the specific data set considered. Our method achieves not only the discrimination between normal and pathologic human tissue, but also the successful discrimination between the different types of pathologic tissue (fibrous, calcified). Furthermore, the small time needed to acquire and analyze the fluorescence spectra together with the high rates of success, proves our method very attractive for real-time applications.

A New Optical-CT Apparatus for 3-D Radiotherapy Dosimetry: Is Free Space Scanning Feasible?

In this paper, we present a new optical computed tomography (Optical-CT) scanner for the verification of the radiation dose schemes delivered in modern radiotherapy applications. The optical-CT scanner is capable of providing rapid relative 3-D dosimetry with high spatial resolution with the use of normoxic N-Vinylpyrrolidone based polymer gel dosimeter. The scanner employs a diffuse uncollimated light illumination beam, a computer controlled motorized rotation stage and a charge-coupled device (CCD) camera. Various test experiments were performed to determine the performance characteristics of the optical-CT apparatus. Attenuation coefficient (¿ ) versus dose calibration data were generated from two calibration experiments using gel containers of two different diameters. All irradiations were performed using a 6 MV linear accelerator. A comparison of the reconstructed images between optical-CT scans using refractive index (RI) matching fluid and corresponding scans performed in free space was demonstrated. The dose readout of a test irradiation model was found to be in good agreement with independent readout performed by MR imaging. The findings presented in this study suggest that polymer dosimeters combined with the new optical-CT scanner constitute a potentially feasible method capable of measuring complex 3-D dose distributions with high resolution and in a wide dose range.

Investigation of the laserlike behavior of polymeric scattering gain media under subpicosecond laser excitation

The narrowing effects of scatterers on the lifetime and the spectral width of the laser-induced fluorescence of organic dyes hosted in poly(methyl methacrylate) polymer sheets were studied. The excitation source was a distributed-feedback dye laser emitting 0.5-ps pulses at 496 nm. Spectral and temporal features were recorded simultaneously on a spectrograph-streak-camera detection system. The results were then compared with those obtained from dye solutions in methanol that were recorded in previous studies. The effects of the different host environments on the fluorescence characteristics of the dye were thus investigated. These effects are currently studied when the dye is inserted into human tissue in an attempt to boost tumor detection and photodynamic-therapy efficiency. Some initial results are presented.