John W. Pyhtila

Determining nuclear morphology using an improved angle-resolved low coherence interferometry system

We outline the process for determining the morphology of subsurface epithelial cell nuclei using depth-resolved light scattering measurements. The measurements are accomplished using a second generation angle-resolved low coherence interferometry system. The new system greatly improves data acquisition and analysis times compared to the initial prototype system. The calibration of the new system is demonstrated in scattering studies to determine the size distribution of polystyrene microspheres in a turbid sample. The process for determining the size of cell nuclei is discussed by analyzing measurements of basal cells in a sub-surface layer of intact, unstained epithelial tissue.

Rapid, depth-resolved light scattering measurements using Fourier domain, angle-resolved low coherence interferometry

We present a novel angle-resolved low coherence interferometry scheme for rapid measurement of depth-resolved angular scattering distributions to enable determination of scatterer size via elastic scattering properties. Depth resolution is achieved using a superluminescent diode in a modified Mach-Zehnder interferometer with the mixed signal and reference fields dispersed by an imaging spectrograph. The spectrograph slit is located in a Fourier transform plane of the scattering sample, enabling angle-resolved measurements over a 0.21 radian range. The capabilities of the new technique are demonstrated by recording the distribution of light scattered by a sub-surface layer of polystyrene microspheres in 40 milliseconds. The data are used to determine the microsphere size with good accuracy. Future clinical application to measuring the size of cell nuclei in living epithelial tissues using backscattered light is discussed.

Prospective grading of neoplastic change in rat esophagus epithelium using angle-resolved low-coherence interferometry

Angle-resolved low-coherence interferometry (a/LCI) is used to obtain quantitative, depth-resolved nuclear morphology measurements. We compare the average diameter and texture of cell nuclei in rat esophagus epithelial tissue to grading criteria established in a previous a/LCI study to prospectively grade neoplastic progression. We exploit the depth resolution of a/LCI to exclusively examine the basal layer of the epithelium, approximately 50 to 100 microm beneath the tissue surface, without the need for exogenous contrast agents, tissue sectioning, or fixation. The results of two studies are presented that compare the performance of two a/LCI modalities. Overall, the combined studies show 91% sensitivity and 97% specificity for detecting dysplasia, using histopathology as the standard. In addition, the studies enable the effects of dietary chemopreventive agents, difluoromethylornithine (DFMO) and curcumin, to be assessed by observing modulation in the incidence of neoplastic change. We demonstrate that a/LCI is highly effective for monitoring neoplastic change and can be applied to assessing the efficacy of chemopreventive agents in the rat esophagus.

Fourier-domain angle-resolved low coherence interferometry through an endoscopic fiber bundle for light-scattering spectroscopy.

We present a novel endoscopic fiber bundle probe incorporated in a Fourier-domain angle-resolved low coherence interferometry system for the measurement of depth-resolved angular scattering distributions to permit the determination of scatterer size via elastic scattering properties. Depth resolution is achieved with a superluminescent diode via a Mach-Zehnder interferometer. The sample is illuminated with a collimated beam, and a Fourier plane image of the backscattered light is collected by a coherent fiber bundle. The angular scattering distribution relayed by the fiber bundle is mixed with the reference field and made to coincide with the input slit of an imaging spectrograph. The data collected are processed in real time, producing a depth-resolved angular scattering distribution in 0.37 s. The data are used to determine the sizes of polystyrene microspheres with subwavelength precision and accuracy.

Application of Mie theory to determine the structure of spheroidal scatterers in biological materials

We present here the results of a numerical study on light scattering from nonspherical particles with relevance to detecting precancerous states in epithelial tissues. In previous studies of epithelial cell nuclei, the experimental light scattering data have been analyzed by comparison with Mie theory. However, given the spheroidal shape of many cell nuclei, the validity of this assumption demands a thorough investigation. We investigate this assumption by using the T-matrix method to model light scattered from spheroids with parameters relevant to epithelial cell nuclei. In our previous studies, we have developed a data analysis procedure that extracts the oscillatory component of the angular-scattering distribution for an ensemble of epithelial cell nuclei for comparison with Mie theory. We demonstrate that application of our analysis procedure to the predictions of the T-matrix method for spheroids, oriented such that their axis of symmetry is aligned with the incident light propagation direction, generally yields the spheroid dimension that is transverse to the incident light propagation direction with subwavelength accuracy.

Review and Recent Development of Angle-Resolved Low-Coherence Interferometry for Detection of Precancerous Cells in Human Esophageal Epithelium

The combination of low-coherence interferometry with angle-resolved light scattering measurements has been shown to be a powerful method for determining the structure of cell nuclei within intact tissue samples. The nuclear morphology data have been used as a biomarker of neoplastic change in a wide range of settings. Here, we review the development of angle-resolved low-coherence interferometry (a/LCI) for assessing the health status of human esophageal epithelial tissues based on depth-resolved measurements of the morphology of cell nuclei. The design and implementation of clinical instrumentation are reviewed, and results from ex vivo human tissue measurements are presented to validate the capabilities of the technique. In addition to the review of earlier papers, new results are presented, which demonstrate the first application of a portable a/LCI system with a flexible endoscopic probe to assessing depth-resolved nuclear morphology in a clinical setting. High sensitivity for the detection of precancerous tissues is demonstrated.

In situ assessment of intraepithelial neoplasia in hamster trachea epithelium using angle-resolved low-coherence interferometry

Optical spectroscopy was used to evaluate the transformation of nuclear morphology associated with intraepithelial neoplasia in an animal model of carcinogenesis. In this pilot study, we have assessed the capability of angle-resolved low-coherence interferometry (a/LCI) to monitor in situ the neoplastic progression of hamster trachea epithelial tissue. By using the depth resolution made possible by coherence gating, the a/LCI system has been adapted to the unique geometry of the hamster trachea to allow us to extract useful nuclear morphometric information from cells in the epithelial layer without the need for exogenous staining or tissue fixation. Analysis of a/LCI nuclear morphology measurements has identified two important biomarkers of neoplastic transformation in hamster trachea epithelium, the size and the refractive index of epithelial cell nuclei. By comparing the a/LCI measurements of these two biomarkers to pathologic classification, we distinguished nuclear morphology changes for normal tissue, low-grade dysplasia, and high-grade dysplasia. Given its previous usefulness for tracking neoplastic change through nuclear morphometry measurements, the a/LCI technique may prove to be a useful tool in evaluating chemopreventive agents in future studies of hamster trachea epithelium. (Cancer Epidemiol Biomarkers Prev 2007;16(2):223–7)

Polarization effects on scatterer sizing accuracy analyzed with frequency-domain angle-resolved low-coherence interferometry

Angle-resolved low-coherence interferometry (a/LCI) enables us to make depth-resolved measurements of scattered light that can be used to recover subsurface structural information such as the size of cell nuclei. Endoscopic frequency-domain a/LCI (fa/LCI) acquires data by using a novel fiber probe in a fraction of a second, making it a clinically practical system. However, birefringent effects in fiber-based systems can alter the polarization state of the incident light and potentially skew the collected data. We analyze the effect the polarization state of the incident light has on scattering data collected from polystyrene microsphere tissue phantoms and in vitro cell samples and examine the subsequent accuracy of the determined sizes. It is shown that the endoscopic fa/LCI system accurately determines the size of polystyrene microspheres without the need to control the polarization of the incident beam, but that epithelial cell nuclear sizes are accurately determined only when the polarization state of the incident light is well characterized.

Analysis of long range correlations due to coherent light scattering from in-vitro cell arrays using angle-resolved low coherence interferometry

Angle-resolved low coherence interferometry (a/LCI) enables depth-resolved measurements of scattered light that can be used to recover subsurface structural information, such as the size of cell nuclei. Measurements of nuclear morphology, however, can be complicated by coherent scattering between adjacent cell nuclei. Previous studies have eliminated this component by applying a window filter to Fourier transformed angular data, based on the justification that the coherent scattering must necessarily occur over length scales greater than the cell size. To fully study this effect, results of experiments designed to test the validity of this approach are now presented. The a/LCI technique is used to examine light scattered by regular cell arrays, created using stamped adhesive micropatterned substrates. By varying the array spacing, it is demonstrated that cell-to-cell correlations have a predictable effect on light scattering distributions. These results are compared to image analysis of fluorescence micrographs of the cell array samples. The a/LCI results show that the impact of coherent scattering on nuclear morphology measurements can be eliminated through data filtering.

In situ nuclear morphology measurements using light scattering as biomarkers of neoplastic change in animal models of carcinogenesis.

Light scattering spectroscopy measurements can be used to determine the structure of tissue samples. Through refined data acquisition and signal processing techniques, quantitative nuclear morphology measurements may be obtained from light scattering data. These data have been used primarily as a biomarker of neoplastic change in a wide range of settings. Here, we review the application of light scattering to assessing the health status of tissues drawn from animal models of carcinogenesis, in particular, the rat esophagus and the golden Syrian hamster trachea carcinogenesis models. In addition, we present results from ex vivo human tissues to demonstrate the relevance of the use of animal models which are excellent surrogates for several human cancers. These models provide the opportunity to develop biomarkers and test chemopreventive and therapy strategies before application in humans.