Rebekah A. Drezek

Light scattering from cells: finite-difference time-domain simulations and goniometric measurements

We have examined the light-scattering properties of inhomogeneous biological cells through a combination of theoretical simulations and goniometric measurements. A finite-difference time-domain (FDTD) technique was used to compute intensity as a function of scattering angle for cells containing multiple organelles and spatially varying index of refraction profiles. An automated goniometer was constructed to measure the scattering properties of dilute cell suspensions. Measurements compared favorably with FDTD predictions. FDTD and experimental results indicate that scattering properties are strongly influenced by cellular biochemical and morphological structure.

Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and implications.

OBJECTIVEnAt 380 nm excitation, cervical tissue fluorescence spectra demonstrate characteristic changes with both patient age and the presence of dysplasia. A Monte Carlo model was developed in order to quantitatively examine how intrinsic NADH and collagen fluorescence, in combination with tissue scattering and absorption properties, yield measured tissue spectra.nnnMETHODSnExcitation-emission matrices were measured for live cervical cells and collagen gel phantoms. Fluorescence microscopy of fresh tissue sections was performed to obtain the location and density of fluorophores as a function of patient age and the presence of dysplasia. A Monte Carlo model was developed which incorporated measurements of fluorophore line shapes and spatial distributions.nnnRESULTSnModeled spectra were consistent with clinical measurements and indicate that an increase in NADH fluorescence and decrease in collagen fluorescence create clinically observed differences between normal and dysplastic tissue spectra. Model predictions were most sensitive to patient age and epithelial thickness.nnnCONCLUSIONSnMonte Carlo techniques provide an important means to investigate the combined contributions of multiple fluorophores to measured emission spectra. The approach will prove increasingly valuable as a more sophisticated understanding of in vivo optical properties is developed.

Reflectance spectroscopy with polarized light: is it sensitive to cellular and nuclear morphology

We present a method for selective detection of size-dependent scattering characteristics of epithelial cells in vivo based on polarized illumination and polarization sensitive detection of scattered light. We illustrate the method using phantoms designed to simulate squamous epithelial tissue and progressing to epithelial tissue in vitro and in vivo. Elastic light scattering spectroscopy with polarized illumination/detection dramatically reduces background signals due to both diffuse stromal scattering and hemoglobin absorption. Resulting spectra can be described as a linear combination of forward and backscattering components determined from Mie theory. Nuclear sizes and refractive indices extracted by fitting experimental spectra to this model agree well with previous measurements. Reflectance spectroscopy with polarized light can provide quantitative morphological information which could potentially be used for non-invasive detection of neoplastic changes.

Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia ¶

Abstract Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 ± 39) relative to normal tissue (85 ± 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 ± 34 [dysplasia] vs 151 ± 44 [normal]) and 460 nm excitation (93 ± 35 [dysplasia] vs 137 ± 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17–40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Optical properties of gold-silica-gold multilayer nanoshells

The spectral and angular radiation properties of gold-silica-gold multilayer nanoshells are investigated using Mie theory for concentric multilayer spheres. The spectral tunability of multilayer nanoshells is explained and characterized by a plasmon hybridization model and a universal scaling principle. A thinner intermediate silica layer, scaled by particle size, red shifts the plasmon resonance. This shift is relatively insensitive to the overall particle size and follows the universal scaling principle with respect to the resonant wavelength of a conventional silica-gold core-shell nanoshell. The extra tunability provided by the inner core further shifts the extinction peak to longer wavelengths, which is difficult to achieve on conventional sub-100 nm nanoshells due to limitations in synthesizing ultrathin gold coatings. We found multilayer nanoshells to be more absorbing with a larger gold core, a thinner silica layer, and a thinner outer gold shell. Both scattering intensity and angular radiation pattern were found to differ from conventional nanoshells due to spectral modulation from the inner core. Multilayer nanoshells may provide more backscattering at wavelengths where silica-gold core-shell nanoshells predominantly forward scatter.

A Pulsed Finite-Difference Time-Domain (FDTD) Method for Calculating Light Scattering from Biological Cells Over Broad Wavelength Ranges

We combine the finite-difference time-domain method with pulse response techniques in order to calculate the light scattering properties of biological cells over a range of wavelengths simultaneously. The method we describe can be used to compute the scattering patterns of cells containing multiple heterogeneous organelles, providing greater geometric flexibility than Mie theory solutions. Using a desktop computer, we calculate the scattering patterns for common homogeneous models of biological cells and also for more complex representations of cellular morphology. We find that the geometry chosen significantly impacts scattering properties, emphasizing the need for careful consideration of appropriate theoretical models of cellular scattering and for accurate microscopic determination of optical properties.

Near real time confocal microscopy of cultured amelanotic cells: sources of signal, contrast agents and limits of contrast.

The use of high resolution, in vivo confocal imaging for noninvasive assessment of tissue pathology may offer a clinically important adjunct to standard histopathological techniques. To augment the present understanding of both the capabilities and limitations of in vivo confocal imaging, we investigated cellular sources of image contrast in amelanotic tissues, how contrast can be enhanced with external agents and how contrast is degraded by the scattering of overlying cells. A high-resolution reflected light confocal microscope was constructed and used to obtain images of various types of unstained amelanotic cells in suspension in real time before and after the addition of contrast agents. Reflectance images were compared to phase contrast images and electron micrographs to identify morphology visible with real time reflected light confocal microscopy. Mechanisms which decrease image contrast, including interference effects and scattering in overlying layers of cells, were considered. In amelanotic epithelial cells, fluctuations in the nuclear index of refraction provide signal which can be imaged even under several overlying cell layers. Acetic acid is an external contrast agent which can enhance this nuclear backscattering. Image contrast is degraded by the presence of multiple scattering in overlying cell layers. The degradation of image contrast by cell scattering depends on the scattering phase function; in vitro models which use polystyrene microspheres to approximate tissue underestimate the actual degradation caused by cell scattering. The loss in contrast can be explained using a finite difference time domain model of cellular scattering. We conclude that near real time reflected light confocal microscopy can be used to study cell morphology in vivo. Contrast degradation due to overlying tissue is a concern and cannot adequately be modeled using conventional tissue phantoms; however, acetic acid may be used to substantially increase intrinsic contrast, allowing imaging at significant depths despite distortion from overlying layers.

Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension

Normal and malignant human cervical cancer cells were imaged in vivo with confocal, phase contrast, and brightfield microscopies. Results were compared between cells in growth medium before and after addition of acetic acid, hypertonic saline solution, toluidine blue, and Lugols iodine. The exogenous agents changed the backscattering characteristics of the cells when measured with confocal microscopy at 808 nm. A tendency toward higher scattering was observed in treated cells. Acetic acid and toluidine blue increased the brightness of the nucleus with respect to the cytoplasm in normal and cancer cells. Hypertonic saline solution made the cytoplasm brighter than the nucleus in both types of cells. The results indicate that simple chemicals can be used to enhance confocal microscopys ability to differentiate intracellular components, such as nuclear size and shape. This can further confocal microscopys ability to assess disease in cells and tissues.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: Results from an animal model

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

ACTIVE FOURIER CONTOUR APPLIED TO REAL TIME 3D ULTRASOUND OF THE HEART

We describe an active contour based on the elliptical Fourier series, and its application to matrix-array ultrasound. Matrix-array, or Real Time 3D (RT3D), ultrasound is a relatively new medical imaging modality that scans a 3D-volume electronically without physically moving the transducer, allowing for real-time continuous 3D imaging of the heart. With the goal of automatically tracking the heart wall, an active contour has been developed using the elliptical Fourier series to find perpendicular lines intersecting an initial contour. The neighborhood defined by these perpendiculars is mapped into a rectangular space, called a swath, whose vertical axis represents the inside-vs-outside dimension of the contour (perpendicular to the contour), and whose horizontal axis represents parametric distance along the contour (tangent to the contour). A dynamic programming technique is then used to find the optimum error function traversing the rectangle horizontally, and this error function is mapped back into image space to yield a new contour. The method does not iterate, but rather simultaneously searches for the optimum contour within a limited domain. Results are presented applying the technique to RT3D ultrasound images of in vivo hearts.