Martin Hunter

Optical fiber probe for biomedical Raman spectroscopy

In vitro experiments have demonstrated the ability of Raman spectroscopy to diagnose a wide variety of diseases. Recent in vivo investigations performed with optical fiber probes were promising but generally limited to easily accessible organs, often requiring relatively long collection times. We have implemented an optical design strategy to utilize system throughput fully by characterizing the Raman distribution from tissue. This scheme optimizes collection efficiency, minimizes noise, and has resulted in small-diameter, highly efficient Raman probes that are capable of collecting high-quality data in 1 s. Performance has been tested through simulations and experiments with tissue models and several in vitro tissue types, demonstrating that this new design can advance Raman spectroscopy as a clinically practical technique.

Atmospheric fate of several alkyl nitrates Part2UV absorption cross-sectionsand photodissociation quantum yields

The UV absorption cross-sections of methyl, ethyl and isopropyl nitrate between 233 and 340 nm have been measured using a diode array spectrometer in the temperature range 240–360 K. The absorption cross-sections of these alkyl nitrates decrease with increasing wavelength and decrease with decreasing temperature for λ>280 nm. The photodissociation quantum yield for CH 3 ONO 2 to produce NO 2 and CH 3 O was found to be essentially unity at 248 nm using transient UV absorption methods. Production of O and H atoms in the photodissociation of methyl nitrate at 248 and 308 nm were found to be negligible using resonance fluorescence detection of the atoms. High quantum yields for O atoms were measured following 193 nm photolysis of methyl nitrate. The OH radical was measured to be a photoproduct with a very small quantum yield. Using the OH rate coefficients reported in the accompanying paper and the UV absorption cross-sections and the photodissociation quantum yields measured here, the first-order rate constants for atmospheric loss of methyl, ethyl and isopropyl nitrate were calculated. Photolysis was found to be the dominant atmospheric loss process for the three alkyl nitrates.

Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy.

Designing biomaterial scaffolds remains a major challenge in tissue engineering. Key to this challenge is improved understanding of the relationships between the scaffold properties and its degradation kinetics, as well as the cell interactions and the promotion of new matrix deposition. Here we present the use of non-linear spectroscopic imaging as a non-invasive method to characterize not only morphological, but also structural aspects of silkworm silk fibroin-based biomaterials, relying entirely on endogenous optical contrast. We demonstrate that two photon excited fluorescence and second harmonic generation are sensitive to the hydration, overall beta sheet content and molecular orientation of the sample. Thus, the functional content and high resolution afforded by these non-invasive approaches offer promise for identifying important connections between biomaterial design and functional engineered tissue development. The strategies described also have broader implications for understanding and tracking the remodeling of degradable biomaterials under dynamic conditions both in vitro and in vivo.

Endogenous optical biomarkers of normal and human papillomavirus immortalized epithelial cells

Cellular transformation is associated with a number of phenotypic, cell biological, biochemical and metabolic alterations. The detection and classification of morphological cellular abnormalities represents the foundation of classical histopathology and remains an important mainstay in the clinic. More recently, significant effort is being expended towards the development of noninvasive modalities for the detection of cancer at an early stage, when therapeutic interventions are highly successful. Methods that rely on the detection of optical signatures represent one class of such approaches that have yielded promising results. In our study, we have applied two spectroscopic imaging approaches to systematically identify in a quantitative manner the fluorescence and light scattering signatures of subcellular abnormalities that are associated with cellular transformation. Notably, we find that tryptophan images reveal not only intensity but also localization differences between normal and human papillomavirus immortalized cells, possibly originating from changes in the expression, 3D packing and organization of proteins and protein‐rich subcellular organelles. Additionally, we detect alterations in cellular metabolism through quantitative evaluation of the NADH, FAD fluorescence and the corresponding redox ratio. Finally, we use light scattering spectroscopy to identify differences in nuclear morphology and subcellular organization that occur from the nanometer to the micrometer scale. Thus, these optical approaches provide complementary biomarkers based on endogenous fluorescence and scattering cellular changes that occur at the molecular, biochemical and morphological level. Since they obviate the need for staining and tissue removal and can be easily combined, they provide desirable options for further clinical development and assessment.

Diagnostic cellular organization features extracted from autofluorescence images

Depth-resolved NADH autofluorescence images are shown to differentiate between normal and precancerous engineered tissues. An inverse power law behavior of the power spectral density (PSD) of these images is observed, indicating a self-affine organization of mitochondrial NADH at length scales 1-10 microm. Power exponents of the PSD functions vary significantly with tissue depth and precancerous state, giving insight into the morphological changes associated with precancerous lesions and providing substantial potential for noninvasive clinical diagnosis of squamous epithelial lesions and tumors.

Assessing epithelial cell nuclear morphology by using azimuthal light scattering spectroscopy

We describe azimuthal light scattering spectroscopy (phi/LSS), a novel technique for assessing epithelial-cell nuclear morphology. The difference between the spectra measured at azimuthal angles phi = 0 degrees and phi = 90 degrees preferentially isolates the single backscattering contribution due to large (approximately 10 microm) structures such as epithelial cell nuclei by discriminating against scattering from smaller organelles and diffusive background. We demonstrate the feasibility of using phi/LSS for cancer detection by showing that spectra from cancerous colon tissue exhibit significantly greater azimuthal asymmetry than spectra from normal colonic tissues.

NON-INVASIVE OPTICAL CHARACTERIZATION OF BIOMATERIAL MINERALIZATION

Current approaches to study biomaterial mineralization are invasive and prevent dynamic characterization of this process within the same sample. Polarized light scattering spectroscopy (LSS) may offer a non-invasive alternative for assessing the levels of mineralization as well as some aspects of the organization of the mineral deposits. Specifically, we used LSS to characterize the formation of hydroxyapatite deposits on three types of silk films (water-annealed, methanol-treated and polyaspartic acid (PAA)-mixed) following 1, 3, 5 and 7 cycles of mineralization. We found that the total light scattering intensity provided a quantitative measure of the degree of mineralization as confirmed by thermal gravimetric analysis (TGA). The PAA-mixed silk films yielded the highest level of mineral deposition and the water-annealed ones the least, consistent with the beta sheet content of the films prior to the onset of mineralization. The wavelength dependence of the singly backscattered light was consistent with a self-affine fractal morphology of the deposited films within scales in the range of 150-300nm; this was confirmed by Fourier analysis of scanning electron microscopy (SEM) images of the corresponding films. The deposits of minerals in the water-annealed films were predominantly flake-like, with positively correlated density fluctuations (Hurst parameter, H>0.5), whereas methanol-treated and PAA-mixed silk films resulted in densely-packed, bulk mineral deposits with negatively correlated density fluctuations (H<0.5). Therefore, LSS could serve as a valuable tool for understanding the role of biomaterial properties in mineral formation, and, ultimately, for optimizing biomaterial designs that yield mineral deposits with the desired organization.

Improved Fourier-based characterization of intracellular fractal features

A novel Fourier-based image analysis method for measuring fractal features is presented which can significantly reduce artifacts due to non-fractal edge effects. The technique is broadly applicable to the quantitative characterization of internal morphology (texture) of image features with well-defined borders. In this study, we explore the capacity of this method for quantitative assessment of intracellular fractal morphology of mitochondrial networks in images of normal and diseased (precancerous) epithelial tissues. Using a combination of simulated fractal images and endogenous two-photon excited fluorescence (TPEF) microscopy, our method is shown to more accurately characterize the exponent of the high-frequency power spectral density (PSD) of these images in the presence of artifacts that arise due to cellular and nuclear borders.

Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three‐dimensional (3D) tissues using exclusively endogenous two‐photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre‐cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

Confocal light scattering spectroscopic imaging system for in situ tissue characterization

We report on the design and construction of a confocal light scattering spectroscopic imaging system aimed ultimately to conduct depth-resolved characterization of biological tissues. The confocal sectioning ability of the system is demonstrated using a two-layer sample consisting of a 200 microm thick cancer cell layer on top of a scattering layer doped with a green absorber. The measurement results demonstrate that distinct light scattering signals can be isolated from each layer with an axial and a lateral resolution of 30 and 27 microm, respectively. Such a system is expected to have significant applications in the areas of tissue engineering and disease diagnostics and monitoring.