Light scattering from normal and dysplastic cervical cells at different epithelial depths: finite-difference time-domain modeling with a perfectly matched layer boundary condition

The finite-difference time-domain (FDTD) method provides a flexible approach to studying the scattering that arises from arbitrarily inhomogeneous structures. We implemented a three-dimensional FDTD program code to model light scattering from biological cells. The perfectly matched layer (PML) boundary condition has been used to terminate the FDTD computational grid. We investigated differences in angle-dependent scattering properties of normal and dysplastic cervical cells. Specifically, the scattering patterns and phase functions have been computed for normal and dysplastic cervical cells at three different epithelial depths, namely, basal/parabasal, intermediate, and superficial. Construction of cervical cells within the FDTD computational grid is based on morphological and chromatin texture features obtained from quantitative histopathology. The results show that angle-dependent scattering characteristics are different not only for normal and dysplastic cells but also for cells at different epithelial depths. The calculated scattering cross-sections are significantly greater for dysplastic cells. The scattering cross-sections of cells at different depths indicate that scattering decreases in going from the superficial layer to the intermediate layer, but then increases in the basal/parabasal layer. This trend for epithelial cell scattering has also been observed in confocal images of ex vivo cervical tissue.     

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.     

Optical properties of the sclera

A relationship between the structure of the sclera and its light scattering properties is proposed. The relative optical density as a function of wavelength in the infrared was measured for three sclera and a good correlation with the predicted function was found. The angular distribution of the scattered light at 632.8 nm as well as the degree of polarisation of the scattered light at zero angle was measured for three sclera; from these results the total transmission of the sclera was determined.     

Koilocytotic lesions of the cervix: the interrelation of morphometric features, the presence of papillomavirus antigens, and the degree of koilocytosis

We studied the interrelation of morphometric features, the presence of human papilloma virus antigens (localized by the immunoperoxidase method), and the degree of koilocytosis in koilocytotic cervical intraepithelial neoplasia. We determined the morphometric features of the cells from the deep, the middle, and the superficial layers of the affected koilocytotic epithelium and in non-koilocytotic immature metaplasia and squamous cervical epithelium. This approach allows quantification of disturbances of maturation in cervical epithelium. Our quantitative findings support the contention of other authors that human papilloma virus infection is associated with a morphologically distinct lesion, which forms a morphological continuum with neoplasia. It can be argued that, in addition to the degree of koilocytosis, nuclear enlargement and excessive cellular enlargement in the middle layer of the affected epithelium are viral-related effects. With increasing immaturity of the cervical intraepithelial neoplasia all investigated viral-related changes are less prominent. These findings suggest that in neoplastic transformation the morphological and antigen expression of human papilloma virus infection is suppressed.     

Unified Mie and fractal scattering by cells and experimental study on application in optical characterization of cellular and subcellular structures

A unified Mie and fractal model for light scattering by biological cells is presented. This model is shown to provide an excellent global agreement with the angular dependent elastic light scattering spectroscopy of cells over the whole visible range (400 to 700 nm) and at all scattering angles (1.1 to 165 deg) investigated. Mie scattering from the bare cell and the nucleus is found to dominate light scattering in the forward directions, whereas the random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, is found to dominate light scattering at other angles. Angularly dependent elastic light scattering spectroscopy aided by the unified Mie and fractal model is demonstrated to be an effective noninvasive approach to characterize biological cells and their internal structures. The acetowhitening effect induced by applying acetic acid on epithelial cells is investigated as an example. The changes in morphology and refractive index of epithelial cells, nuclei, and subcellular structures after the application of acetic acid are successfully probed and quantified using the proposed approach. The unified Mie and fractal model may serve as the foundation for optical detection of precancerous and cancerous changes in biological cells and tissues based on light scattering techniques.     

Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.     

The tall cell variant of papillary carcinoma of the thyroid gland. Comparison with the common form of papillary carcinoma by DNA and morphometric analysis

The tall cell variant of papillary carcinoma of the thyroid manifests a more aggressive behavior than the usual form of papillary carcinoma of the thyroid. Morphometric analysis of nuclear features and DNA analysis may yield information predictive of aggressive behavior. Accordingly, the DNA content and morphometric features of the neoplastic cells of the tall cell variant were measured and compared with measurements obtained from neoplastic cells of the usual form of papillary carcinoma. Six of the 11 tall cell neoplasms were aneuploid, as were four of the eight usual papillary neoplasms. Although benign cells were separated from malignant cells in each case, differences between tall and usual papillary carcinoma cells were not observed regarding DNA content, chromatin texture, or nuclear size and shape. Differences in the clinical behavior of these neoplasms will likely need to be explained on the basis of other characteristics.     

Measurements of angular distributions of Rayleigh and Mie scattering events in biological models

Remission spectroscopy in living tissues can only be performed because scattering processes induce a pronounced amount of backscattered light. New types of scattering chambers were constructed in order to investigate the angular distribution of light intensities caused by scattering events typical for living tissues. Different solutions and suspensions containing microspheres, haemoglobin molecules, erythrocytes and liver homogenate were used in order to gain basic information applicable to remission spectrophotometry in tissue. Reflection spectroscopy in biological tissues is only possible because of the scattering properties of the material under observation. Light which penetrates the sample is remitted from the tissue. The remission is caused by the scattering of the light by different biological structures and particles. This scattered light gives information about the optical properties of the particles and structures in an integral form. A scattering chamber for small sample volumes has been developed for measurements of the optical properties of various particles in suspension. Measurements of scattering diagrams from microspheres with an average diameter of 7 microns, artificial particles, haemoglobin solutions, mixtures of microspheres and haemoglobin as well as erythrocytes and mitochondria have been performed. The different optical behaviour of the measured signals is qualitatively discussed.     

The relationship of quantitative nuclear morphology to molecular genetic alterations in the adenoma-carcinoma sequence of the large bowel.

The relationship of abnormal nuclear morphology to molecular genetic alterations that are important in colorectal tumorigenesis is unknown. Therefore, Feulgen-stained isolated nuclei from 22 adenomas and 42 carcinomas that had been analyzed for ras gene mutations and allelic deletions on chromosomes 5q, 18q, and 17p were characterized by computerized image analysis. Both nuclear area and the nuclear shape factor representing irregularity correlated with adenoma-carcinoma progression (r = 0.57 and r = 0.52, P < 0.0001), whereas standard nuclear texture, a parameter of chromatin homogeneity, was inversely correlated with progression (r = -0.80, P < 0.0001). The nuclear parameters were strongly interrelated (P < 0.0005). In multivariate analysis, the nuclear parameters were predominantly associated with adenoma-carcinoma progression (P < or = 0.0001) and were not influenced significantly by the individual molecular genetic alterations. Nuclear texture, however, was inversely correlated with fractional allelic loss, a global measure of genetic changes, in carcinomas (r = -0.39, P = 0.011). The findings indicate that nuclear morphology in colorectal neoplasms is strongly related to tumor progression. Nuclear morphology and biologic behavior appear to be influenced by accumulated alterations in cancer-associated genes.     

Detection and diagnosis of oral neoplasia with an optical coherence microscope

The use of high resolution, in vivo optical imaging may offer a clinically useful adjunct to standard histopathologic techniques. A pilot study was performed to investigate the diagnostic capabilities of optical coherence microscopy (OCM) to discriminate between normal and abnormal oral tissue. Our objective is to determine whether OCM, a technique combining the subcellular resolution of confocal microscopy with the coherence gating and heterodyne detection of optical coherence tomography, has the same ability as confocal microscopy to detect morphological changes present in precancers of the epithelium while providing superior penetration depths. We report our results using OCM to characterize the features of normal and neoplastic oral mucosa excised from 13 subjects. Specifically, we use optical coherence and confocal microscopic images obtained from human oral biopsy specimens at various depths from the mucosal surface to examine the optical properties that distinguish normal and neoplastic oral mucosa. An analysis of penetration depths achieved by the OCM and its associated confocal arm found that the OCM consistently imaged more deeply. Extraction of scattering coefficients from reflected nuclear intensity is successful in nonhyperkeratotic layers and shows differentiation between scattering properties of normal and dysplastic epithelium and invasive cancer.     

Flow cytometry of DNA content and other indicators of proliferative activity

Flow cytometric DNA content analysis, employing fluorescent stains that bind stoichiometrically to DNA, is widely used by researchers and clinicians to monitor cell growth kinetics and to detect abnormalities in nuclear DNA content in tumor cells. Measurement precision is critical because small differences in DNA content may be significant; plastic spheres and cell nuclei are used for instrument calibration and standardization. Preparative techniques must provide a uniformly stained sample representative of the cells or tissue under investigation; multiparameter gating may facilitate cell selection within samples. Flow cytometry of light scattering, surface or intracellular antigens, RNA, chromatin structure, and/or DNA synthesis may provide additional useful information about neoplastic cell behavior.     

Evolutionary somatic cell changes in cervical tumour progression quantitatively evaluated with morphological, histochemical and kinetic parameters.

The somatic cell changes which characterise malignancy evolution in human cervical preneoplastic and neoplastic lesions have been assessed on histological sections by means of a computerised image analyser. Many features have been simultaneously measured on each cell of the lesions studied, and the following results have been obtained: Some features, mainly kinetic, show continuously increasing values which express changes correlated to the increasing malignancy; other features, especially related to nuclear atypia, cellular heterogeneity and the degree of aneuploidy, have values dropping at the level of early stromal infiltration, which can be morphometrically characterised as composed of relatively homogeneous phenotypes; these features seem to express the degree of genetic instability and relate to the evolutionary somatic cell changes; tumour progression evolves through sequential discontinuous steps, each of them characterised by specific phenotypical features of the neoplastic cell population; the neoplastic cells in the foci of early stromal infiltration and vascular invasion, phenotypically more homogeneous than the parent cell populations of carcinoma in situ and infiltrating carcinoma, seem to possess a greater genetic stability.     

Cytomorphological criteria,subclassifications of endocervical glandular cell abnormalities,and histopathological outcome: A frequency study

The objective of this study was to evaluate the frequency and the significance of cytomorphological criteria defined in studies as being predictive of neoplasia in cervical smears of women with a cytological diagnosis of atypical glandular cells (AGC) or adenocarcinoma in situ (AIS). Women (n = 103) with cytological findings suggestive of AGC or AIS, whose diagnoses were later established by histopathology, were included in the study. The criteria analyzed and classified as present or absent in cervical smears previously classified as AGC‐NOS (not otherwise specified), AGC‐FN (favor neoplasia), or AIS were as follows: irregular nuclear membranes; scanty cytoplasm; dyskeratotic cells; increased nuclear/cytoplasmic ratio; nucleoli; overlapping; papillary clusters, feathering; loss of polarity; nuclear enlargement; coarsely granular chromatin; and pseudostratified strips. Histopathology resulted in neoplastic diagnoses in 55 cases (53.3%) and nonneoplastic diagnoses in 48 cases (46.6%). Coarsely granular chromatin was observed in 62.5% of cases with a diagnosis of neoplasia. Feathering was present in 80% of cases of histopathological AIS. Loss of polarity and coarsely granular chromatin were significantly associated with neoplastic diagnosis considering all subcategories of glandular abnormalities diagnosis. In AGC‐SOE subclassification, coarsely granular chromatin was significantly associated with neoplastic diagnosis. The presence of nucleoli was significantly associated with neoplastic diagnosis in cervical smears qualified as AGC‐FN and AIS. Nuclear enlargement, increased nuclear/cytoplasmic ratio, coarsely granular chromatin and overlapping cells were found in all the subclassifications of glandular cell abnormalities irrespective of the histopathological results. Chromatin aspects, polarity, and presence of nucleoli can predict neoplasia. Diagn. Cytopathol. 2010;38:806‐810. © 2010 Wiley‐Liss, Inc.     

Fractal dimension of chromatin texture of squamous intraepithelial lesions of cervix

The aim of this study was to explore the role of fractal dimension (FD) of chromatin texture in routinely stained Papanicolaou's smears and to distinguish the different grades of cervical intraepithelial lesions and normal cervical cells. We selected 14 each cases of normal, low grade cervical intra epithelial lesions (LSIL), and high-grade cervical intra epithelial lesions (HSIL) of Papanicolaou's stained cervical smears. Fractal dimension of the pseudo three-dimensional grey image of the nuclear chromatin was measured in 140 nuclei of each group. Mean FD of the normal cases, LSIL cases, and HSIL cases were 2.4225 ± 0.06410, 2.5159 ± 0.03291, and 2.5905 ± 0.06840, respectively. ANOVA test showed significant differences of mean FD in all these three groups (P < 0.000). Fractal dimension of the chromatin texture is easy to perform and can be done in routinely stained Papanicolaou's smear. It is reproducible and gives valuable information about the chromatin texture of the nucleus. In future, this promising variable can be incorporated along with other image morphometric features for accurate classification of dysplastic cells in cervical smear.     

Neoplastic non-papillary thyroid carcinoma lesions with a fine chromatin pattern

In papillary thyroid carcinoma (PTC) in cytological and surgical specimens, fine chromatin, nuclear grooves and nuclear pseudoinclusions are the hallmarks of diagnosis. We investigated the significance of these nuclear changes in neoplastic non-PTC lesions. Fine needle aspiration biopsies (FNAB) of thyroid lesions were reviewed with histologic correlation. Twenty-five low-grade PTC and 35 neoplastic non-PTC lesions with a fine chromatin pattern in cytology specimens were identified. These lesions were studied along with five multinodular goiters and five follicular adenomas with a coarse chromatin pattern. The neoplastic non-PTC lesions were selected from cases with a histopathologic diagnosis of follicular neoplasm (accompanied by cytopathologic examination) but lacking a coarse chromatin pattern. The nuclear changes were separated into three grades of nuclear atypia with a fine chromatin pattern, depending on the degree of nuclear enlargement and nuclear membrane thickening, or the presence of nuclear grooves or pseudoinclusions. Thyroid lesions with a higher grade of nuclear atypia with a fine chromatin pattern were associated with larger nuclei and more readily visible nucleoli. These lesions correlated histologically with PTC and follicular adenomas with a fine chromatin pattern. The latter could be divided into three grades: grade 1 lesions having a fine chromatin pattern similar to that of nuclei with open chromatin seen in areas of nodular goiter; grade 3 lesions having nuclear features closest to those of PTC; and grade 2 lesions showing intermediate changes. In conclusion, there is a spectrum of nuclear changes in neoplastic non-PTC lesions with a fine chromatin pattern. These lesions are often diagnosed as follicular adenomas in surgical pathology and pose cytopathologic diagnostic problems between nodular goiter, follicular adenoma and PTC. The significance of follicular adenomas with a fine chromatin pattern will be discussed.     

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.     

Follicular adenoma with papillary architecture: a lesion mimicking papillary thyroid carcinoma

AIMS: The purpose of this study was to investigate the significance of 'benign' encapsulated follicular thyroid nodules with papillary structures. METHODS AND RESULTS: Twenty-one cases of encapsulated neoplastic thyroid nodules with papillary structures and nuclear features not diagnostic of papillary thyroid carcinoma (PTC) were obtained. All cases were reviewed with particular attention to nuclear features (fine chromatin pattern, optical clearing, grooves and inclusions). Representative sections were submitted for measurement of the maximum diameter of 200 round or nearly round nuclei and for immunostaining for MIB1, CK19, HBME and Ret oncogene protein. Nine cases displayed scattered optically clear nuclei or nuclear grooves in less than 30% of total neoplastic cells. They were grouped in the category of thyroid nodules with limited nuclear features of papillary thyroid carcinoma (PTC), but not diagnostic of PTC. The other 12 cases had fine or coarse chromatin, but lacked other features of nuclei in PTC. The diameter of the nuclei ranged from 5.6 to 7.2 microm and were smaller than those of PTC (6.3-10.0 microm). Immunostaining revealed positive reactivity for MIB1 in the papillary structures. Immunostaining for CK19 and HBME varied from negative or focally weak to diffusely moderate reactivity. Ret oncogene protein immunostaining showed focal and weak reactivity in one case and was negative in other cases of the study. Clinical follow-up from 6 months to 15 years revealed no evidence of metastasis. CONCLUSIONS: The papillary structures in the study cases are unlikely to represent degenerative changes due to their proliferative activity. In view of (i) the encapsulation and the uniformity of the constituent cells, (ii) the varying degrees of immunoreactivity for CK19 and HBME and negative immunoreactivity for Ret oncogene protein, and (iii) the absence or insufficiency of nuclear criteria for the diagnosis of PTC and the absence of lymph node metastasis in all study cases, we believe that these lesions represent the papillary variant of follicular adenoma. Recognition of this pathological entity is important to avoid an over-diagnosis of PTC.     

Aspects of laser light scattering from skin tissue with application to laser Doppler blood flow measurement

Studies of both intensity and Doppler shifted linewidths of light scattered from skin tissue have been made using photon correlation spectroscopy and optical fibre techniques. Measurements as a function of the separation of input and detecting fibre positions show characteristic features. These features are interpreted in terms of the positions of the scattering red blood cells and the tissue structure. Evidence is given for an interpretation of the differences in the scattered light for scattering from superficial vessels including capillary loops and from deeper lying larger vessels and shunts. Measurements using various laser wavelengths are discussed. The results of this study are important in the development of laser light scattering instruments for the measurement of peripheral blood flow and microcirculation.