Irene Georgakoudi

Imaging human epithelial properties with polarized light-scattering spectroscopy

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.

Bioactive Silk Protein Biomaterial Systems for Optical Devices

Silk-based biomaterial systems have been previously explored for a variety of medical and nonmedical materials needs. The unique biophysical features of silks provide options to generate highly tailored structures and morphologies with this unique family of fibrous proteins. To exploit these features, we have optimized the all aqueous processing of silk fibroin into novel surface nanopatterned protein materials. We have exploited control of this nanomorphology to optimize the optical features of these silk protein systems. We demonstrate control of surface morphology down to 125 nm, with fidelity over large length scales. This surface nanopatterning allows the silk protein to be formed into diffractive optics such as diffraction gratings, pattern generators, and lenses due to novel aqueous processing into optically clear materials via control of beta sheet crystallinity. Further, we incorporate biological components, such as hemoglobin and the enzyme peroxidase, during the process of forming the silk diffraction gratings. The ambient processing of the silk protein in water, in combination with these bioactive components, allows these entrained molecules to retain activity and provide added functions and selectivity to the optically active silk films. Thus, combinations of biochemical and optical readout is feasible and provides in a single, disposable/all degradable element with both spectral discrimination and biological function. These new surface nanopatterned, bioactive silk protein-based material systems offer a unique combination of features potentially useful for a range of biosensor needs, particularly when considered in concert with the remarkable mechanical properties of these proteins, their biocompatibility, and controllable biodegradation.

Spectroscopic detection and evaluation of morphologic and biochemical changes in early human oral carcinoma

Understanding the development and progression of head and neck squamous cell carcinoma is key in the quest for the early diagnosis and prevention of this type of malignancy. The current study correlated early biochemical and histologic changes in oral tissue with spectral features in fluorescence, reflectance, and light scattering spectra acquired in vivo to diagnose early stages of oral malignancies.

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption

The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.

The Mechanism of Photofrin Photobleaching and Its Consequences for Photodynamic Dosimetry

Abstract— We report experimental results that support a theory of self‐sensitized singlet oxygen‐mediated bleaching of the porphyrin photosensitizer Photofrin. Microelectrode measurements of photodynamic oxygen consumption were made near the surface of individual, Photofrin‐sensitized EMT6 spheroids during laser irradiation. The progressive decrease in photochemical oxygen consumption with sustained irradiation is consistent with a theory in which bleaching occurs via self‐sensitized singlet oxygen reaction with the photosensitizer ground state. A bleaching model based solely on absorbed optical energy density is inconsistent with the data. Photobleaching has a significant effect on calculated photodynamic dose distributions in 500 pin diameter spheriods. Dose distributions corrected for the effects of bleaching produce a new estimate (12.1 ± 1.2 mM) for the threshold dose of reacting singlet oxygen in this system.

In vivo flow cytometer for real-time detection and quantification of circulating cells

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.

Singlet Oxygen‐Versus Nonsinglet Oxygen‐Mediated Mechanisms of Sensitizer Photobleaching and Their Effects on Photodynamic Dosimetry

We report the effects of singlet oxygen (1O2) and non‐1O2‐mediated sensitizer photobleaching on oxygen consumption and dosimetry during photodynamic therapy (PDT) of sensitized multicell tumor spheroids. We develop a theoretical model for the description of non‐1O2‐mediated photobleaching resulting from irreversible reactions of the excited singlet or triplet sensitizer populations with cell substrate. We show that the fluence‐dependent simple exponential decay expression of sensitizer degradation is not consistent with these mechanisms and, therefore, with any reasonable mechanism that we consider, because we have shown previously that 1O2‐mediated photobleaching cannot be described by a simple exponential with a constant photobleaching coefficient (I. Georgakoudi et al., Photochem. Photobiol. 65, 135–144, 1997). Analysis of oxygen microelectrode measurements performed at the edge of Nile blue selenium (EtNBSe)‐and protoporphyrin IX (PpIX)‐sensitized spheroids during PDT demonstrates that the former drug photobleaches via a non‐1O2‐mediated mechanism, while the latter is degraded via a 1O2‐mediated mechanism. Comparisons of the cytotoxic effects of EtNBSe with those of Photofrin® (a drug that is degraded via a 1O2‐mediated mechanism) indicate that the lower threshold 1O2 dose and the higher extinction coefficient and 1O2 yield for EtNBSe do not necessarily result in improved photodynamic effects, thus emphasizing the importance of the sensitizer photo‐bleaching mechanism for dosimetry.

Interferometric phase-dispersion microscopy

We describe a new scanning microscopy technique, phase-dispersion microscopy (PDM). The technique is based on measuring the phase difference between the fundamental and the second-harmonic light in a novel interferometer. PDM is highly sensitive to subtle refractive-index differences that are due to dispersion (differential optical path sensitivity, 5 nm). We apply PDM to measure minute amounts of DNA in solution and to study biological tissue sections. We demonstrate that PDM performs better than conventional phase-contrast microscopy in imaging dispersive and weakly scattering samples.

Effect of processing on silk-based biomaterials: Reproducibility and biocompatibility†

Silk fibroin has been successfully used as a biomaterial for tissue regeneration. To prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or β-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0 and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.

In vivo flow cytometry: a new method for enumerating circulating cancer cells.

The fate of circulating tumor cells is an important determinant of their ability to form distant metastasis. Here, we demonstrate the use of in vivo flow cytometry as a powerful new method for detecting quantitatively circulating cancer cells. We specifically examine the circulation kinetics of two prostate cancer cell lines with different metastatic potential in mice and rats. We find that the cell line and the host environment affect the circulation kinetics of prostate cancer cells, with the intrinsic cell line properties determining the initial rate of cell depletion from the circulation and the host affecting cell circulation at later time points.