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Nirmala Ramanujam

Publications and source records attributed to Nirmala Ramanujam.

At least 19 recordsLinked to original sources
PubMed2006

Sequential estimation of optical properties of a two-layered epithelial tissue model from depth-resolved ultraviolet-visible diffuse reflectance spectra.

A method for estimating the optical properties of two-layered media (such as squamous epithelial tissue) over a range of wavelengths in the ultraviolet-visible spectrum is proposed and tested with Monte Carlo modeling. The method first used a fiber-optic probe with angled illumination and the collection fibers placed at a small separation ( or=1000 microm) was used to detect diffuse reflectance preferentially from the bottom layer. A second Monte Carlo-based inverse model for a two-layered medium was applied to estimate the bottom layer optical properties, as well as the top layer thickness, given that the top layer optical properties have been estimated. The results of Monte Carlo validation show that this method works well for an epithelial tissue model with a top layer thickness ranging from 200 to 500 microm. For most thicknesses within this range, the absorption coefficients were estimated to within 15% of the true values, the reduced scattering coefficients were estimated to within 20% and the top layer thicknesses were estimated to within 20%. The application of a variance reduction technique to the Monte Carlo modeling proved to be effective in improving the accuracy with which the optical properties are estimated.

Algorithms↗
PubMed2006

Monte Carlo-based inverse model for calculating tissue optical properties. Part I: Theory and validation on synthetic phantoms.

A flexible and fast Monte Carlo-based model of diffuse reflectance has been developed for the extraction of the absorption and scattering properties of turbid media, such as human tissues. This method is valid for a wide range of optical properties and is easily adaptable to existing probe geometries, provided a single phantom calibration measurement is made. A condensed Monte Carlo method was used to speed up the forward simulations. This model was validated by use of two sets of liquid-tissue phantoms containing Nigrosin or hemoglobin as absorbers and polystyrene spheres as scatterers. The phantoms had a wide range of absorption (0-20 cm(-1)) and reduced scattering coefficients (7-33 cm(-1)). Mie theory and a spectrophotometer were used to determine the absorption and reduced scattering coefficients of the phantoms. The diffuse reflectance spectra of the phantoms were measured over a wavelength range of 350-850 nm. It was found that optical properties could be extracted from the experimentally measured diffuse reflectance spectra with an average error of 3% or less for phantoms containing hemoglobin and 12% or less for phantoms containing Nigrosin.

Algorithms↗
PubMed2006

Monte Carlo-based inverse model for calculating tissue optical properties. Part II: Application to breast cancer diagnosis.

The Monte Carlo-based inverse model of diffuse reflectance described in part I of this pair of companion papers was applied to the diffuse reflectance spectra of a set of 17 malignant and 24 normal-benign ex vivo human breast tissue samples. This model allows extraction of physically meaningful tissue parameters, which include the concentration of absorbers and the size and density of scatterers present in tissue. It was assumed that intrinsic absorption could be attributed to oxygenated and deoxygenated hemoglobin and beta-carotene, that scattering could be modeled by spheres of a uniform size distribution, and that the refractive indices of the spheres and the surrounding medium are known. The tissue diffuse reflectance spectra were evaluated over a wavelength range of 400-600 nm. The extracted parameters that showed the statistically most significant differences between malignant and nonmalignant breast tissues were hemoglobin saturation and the mean reduced scattering coefficient. Malignant tissues showed decreased hemoglobin saturation and an increased mean reduced scattering coefficient compared with nonmalignant tissues. A support vector machine classification algorithm was then used to classify a sample as malignant or nonmalignant based on these two extracted parameters and produced a cross-validated sensitivity and specificity of 82% and 92%, respectively.

Algorithms↗
PubMed2006

Diagnosis of breast cancer using diffuse reflectance spectroscopy: Comparison of a Monte Carlo versus partial least squares analysis based feature extraction technique.

BACKGROUND AND OBJECTIVE: We explored the use of diffuse reflectance spectroscopy in the ultraviolet-visible (UV-VIS) spectrum for the diagnosis of breast cancer. A physical model (Monte Carlo inverse model) and an empirical model (partial least squares analysis) based approach, were compared for extracting diagnostic features from the diffuse reflectance spectra. STUDY DESIGN/METHODS: The physical model and the empirical model were employed to extract features from diffuse reflectance spectra measured from freshly excised breast tissues. A subset of extracted features obtained using each method showed statistically significant differences between malignant and non-malignant breast tissues. These features were separately input to a support vector machine (SVM) algorithm to classify each tissue sample as malignant or non-malignant. RESULTS AND CONCLUSIONS: The features extracted from the Monte Carlo based analysis were hemoglobin saturation, total hemoglobin concentration, beta-carotene concentration and the mean (wavelength averaged) reduced scattering coefficient. Beta-carotene concentration was positively correlated and the mean reduced scattering coefficient was negatively correlated with percent adipose tissue content in normal breast tissues. In addition, there was a statistically significant decrease in the beta-carotene concentration and hemoglobin saturation, and a statistically significant increase in the mean reduced scattering coefficient in malignant tissues compared to non-malignant tissues. The features extracted from the partial least squares analysis were a set of principal components. A subset of principal components showed that the diffuse reflectance spectra of malignant breast tissues displayed an increased intensity over wavelength range of 440-510 nm and a decreased intensity over wavelength range of 510-600 nm, relative to that of non-malignant breast tissues. The diagnostic performance of the classification algorithms based on both feature extraction techniques yielded similar sensitivities and specificities of approximately 80% for discriminating between malignant and non-malignant breast tissues. While both methods yielded similar classification accuracies, the model based approach provided insight into the physiological and structural features that discriminate between malignant and non-malignant breast tissues.

Adipose Tissue↗
PubMed2006

Effect of optical clearing agents on the in vivo optical properties of squamous epithelial tissue.

BACKGROUND AND OBJECTIVES: Optical clearing agents (OCAs) have previously been shown to increase depth penetration within turbid tissue ex vivo. This paper quantifies tissue optical properties of the hamster cheek pouch model in order to provide a means to assess the effect of OCAs quantitatively in vivo. STUDY DESIGN/MATERIALS AND METHODS: Diffuse reflectance spectra were obtained from both cheeks of 12 hamsters before and after immersion in dimethyl sulfoxide (DMSO), glycerol or a phosphate buffer saline (PBS) control for 20 minutes. A Monte Carlo model was then utilized to derive the wavelength dependent reduced scattering and absorption coefficients. RESULTS: DMSO caused a statistically significant decrease in the absorption and reduced scattering coefficients derived by the model. Glycerol caused a statistically significant increase in the wavelength dependent absorption coefficient, but no statistically significant changes in the reduced scattering coefficient. CONCLUSIONS: DMSO and glycerol act upon tissues differently as reflected by the tissue optical properties, implying that not all OCAs are equally effective in optically clearing tissues.

Absorption↗
PubMed2005

Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH.

Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD+ reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of protein-bound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD+. Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD+ ratio.

Cell Line↗
PubMed2005

Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues.

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Endoscopically compatible near-infrared photon migration probe.

We have developed a 2.3-mm-diameter fiber-optic probe for near-infrared photon migration spectroscopy that can be inserted into the body through an endoscope or biopsy needle. This probe is specifically designed to be inserted into a core biopsy needle to facilitate optical sampling of lesions during breast needle biopsy. This probe was tested on tissue phantoms containing heterogeneities (to stimulate breast lesions) of various sizes and optical properties. Under the conditions tested, the probe can measure the absorption coefficient to within 30% for heterogeneities with radii as small as 10 mm.

Biomarkers, Tumor↗
PubMed2004

Experimental proof of the feasibility of using an angled fiber-optic probe for depth-sensitive fluorescence spectroscopy of turbid media.

An angled fiber-optic probe that facilitates depth-sensitive fluorescence measurements was developed for enhancing detection of epithelial precancers. The probe was tested on solid, two-layered phantoms and proved to be effective in selectively detecting fluorescence from different layers. Specifically, a larger illumination angle provides greater sensitivity to fluorescence from the top layer as well as yielding an overall higher fluorescence signal. Monte Carlo simulations of a theoretical model of the phantoms demonstrate that increasing the illumination angle results in an increased excitation photon density and, thus, in increased fluorescence generated in the top layer.

Biomarkers, Tumor↗
PubMed2004

Investigation of fiber-optic probe designs for optical spectroscopic diagnosis of epithelial pre-cancers.

BACKGROUND AND OBJECTIVES: The first objective of this study was to evaluate the performance of fluorescence spectroscopy for diagnosing pre-cancers in stratified squamous epithelial tissues in vivo using two different probe geometries with (1) overlapping versus (2) non-overlapping illumination and collection areas on the tissue surface. Probe (1) and probe (2) are preferentially sensitive to the fluorescence originating from the tissue surface and sub-surface tissue depths, respectively. The second objective was to design a novel, angled illumination fiber-optic probe to maximally exploit the depth-dependent fluorescence properties of epithelial tissues. STUDY DESIGN/MATERIALS AND METHODS: In the first study, spectra were measured from epithelial pre-cancers and normal tissues in the hamster cheek pouch and analyzed with a non-parametric classification algorithm. In the second study, Monte Carlo modeling was used to simulate fluorescence measurements from an epithelial tissue model with the angled illumination probe. RESULTS: An unbiased classification algorithm based on spectra measured with probes (1) and (2), classified pre-cancerous and normal tissues with 78 and 94% accuracy, respectively. The angled illumination probe design provides the capability to detect fluorescence from a wide range of tissue depths in an epithelial tissue model. CONCLUSIONS: The first study demonstrates that fluorescence originating from sub-surface tissue depths (probe (2)) is more diagnostic than fluorescence originating from the tissue surface (probe (1)) in the hamster cheek pouch model. However in general, it is difficult to know a priori the optimal probe geometry for pre-cancer detection in a particular epithelial tissue model. The angled illumination probe provides the capability to measure tissue fluorescence selectively from different depths within epithelial tissues, thus obviating the need to select a single optimal probe design for the fluorescence-based diagnosis of epithelial pre-cancers.

Algorithms↗
PubMed2004

Autofluorescence and diffuse reflectance properties of malignant and benign breast tissues.

BACKGROUND: Fluorescence spectroscopy is an evolving technology that can rapidly differentiate between benign and malignant tissues. These differences are thought to be due to endogenous fluorophores, including nicotinamide adenine dinucleotide, flavin adenine dinucleotide, and tryptophan, and absorbers such as beta-carotene and hemoglobin. We hypothesized that a statistically significant difference would be demonstrated between benign and malignant breast tissues on the basis of their unique fluorescence and reflectance properties. METHODS: Optical measurements were performed on 56 samples of tumor or benign breast tissue. Autofluorescence spectra were measured at excitation wavelengths ranging from 300 to 460 nm, and diffuse reflectance was measured between 300 and 600 nm. Principal component analysis to dimensionally reduce the spectral data and a Wilcoxon ranked sum test were used to determine which wavelengths showed statistically significant differences. A support vector machine algorithm compared classification results with the histological diagnosis (gold standard). RESULTS: Several excitation wavelengths and diffuse reflectance spectra showed significant differences between tumor and benign tissues. By using the support vector machine algorithm to incorporate relevant spectral differences, a sensitivity of 70.0% and specificity of 91.7% were achieved. CONCLUSIONS: A statistically significant difference was demonstrated in the diffuse reflectance and fluorescence emission spectra of benign and malignant breast tissue. These differences could be exploited in the development of adjuncts to diagnostic and surgical procedures.

Algorithms↗
PubMed2003

Transabdominal near infrared oximetry of hypoxic stress in fetal sheep brain in utero.

The feasibility of transabdominal near-infrared (NIR) spectroscopy for detecting and quantifying fetal hypoxia in utero is demonstrated in a pregnant ewe model. A frequency domain NIR spectroscopy probe, consisting of two detectors and six sources operating at three wavelengths (675, 786, and 830 nm), was placed on the maternal abdomen directly above the fetal head. Fetal hypoxia was indirectly induced through occlusion of uterine blood flow for approximately 3 min. NIR photon diffusion measurements were made during a baseline period, during hypoxia of the fetus, and during recovery. Fetal blood samples were drawn from the fetal brachial artery and jugular veins at several time points during the cycle. Seven hypoxic cycles were induced in a total of five pregnant ewes. The NIR measurements were analyzed by using a two-layer diffusion model to deconvolve the fetal blood saturation from that of the pregnant ewe. Fetal hypoxia was detected. Good agreement was found between fetal blood saturation determined by the transabdominal NIR method and arterial and venous fetal blood saturation quantified from fetal blood samples by using a hemoximeter.

Abdomen↗
PubMed2003

Comparison of multiexcitation fluorescence and diffuse reflectance spectroscopy for the diagnosis of breast cancer (March 2003).

Nonmalignant (n = 36) and malignant (n = 20) tissue samples were obtained from breast cancer and breast reduction surgeries. These tissues were characterized using multiple excitation wavelength fluorescence spectroscopy and diffuse reflectance spectroscopy in the ultraviolet-visible wavelength range, immediately after excision. Spectra were then analyzed using principal component analysis (PCA) as a data reduction technique. PCA was performed on each fluorescence spectrum, as well as on the diffuse reflectance spectrum individually, to establish a set of principal components for each spectrum. A Wilcoxon rank-sum test was used to determine which principal components show statistically significant differences between malignant and nonmalignant tissues. Finally, a support vector machine (SVM) algorithm was utilized to classify the samples based on the diagnostically useful principal components. Cross-validation of this nonparametric algorithm was carried out to determine its classification accuracy in an unbiased manner. Multiexcitation fluorescence spectroscopy was successful in discriminating malignant and nonmalignant tissues, with a sensitivity and specificity of 70% and 92%, respectively. The sensitivity (30%) and specificity (78%) of diffuse reflectance spectroscopy alone was significantly lower. Combining fluorescence and diffuse reflectance spectra did not improve the classification accuracy of an algorithm based on fluorescence spectra alone. The fluorescence excitation-emission wavelengths identified as being diagnostic from the PCA-SVM algorithm suggest that the important fluorophores for breast cancer diagnosis are most likely tryptophan, NAD(P)H and flavoproteins.

Algorithms↗
PubMed2003

Experimental validation of Monte Carlo modeling of fluorescence in tissues in the UV-visible spectrum.

The goal of the work is to experimentally verify Monte Carlo modeling of fluorescence and diffuse reflectance measurements in turbid, tissue phantom models. In particular, two series of simulations and experiments, in which one optical parameter (absorption or scattering coefficient) is varied while the other is fixed, are carried out to assess the effect of the absorption coefficient (mu(a)) and scattering coefficient (mu(s)) on the fluorescence and diffuse reflectance measured from a turbid medium. Moreover, simulations and experiments are carried out for several fiber optic probe geometries that are designed to sample small tissue volumes. Additionally, a group of conversion expressions are derived to convert the optical properties and fluorescence quantum yield measured from tissue phantoms for use in Monte Carlo simulations. The conversions account for the differences between the definitions of the absorption coefficient and fluorescence quantum yield of fluorophores in a tissue phantom model and those in a Monte Carlo simulation. The results indicate that there is good agreement between the simulated and experimentally measured results in most cases. This dataset can serve as a systematic validation of Monte Carlo modeling of fluorescent light propagation in tissues. The simulations are carried out for a wide range of absorption and scattering coefficients as well as ratios of scattering coefficient to absorption coefficient, and thus would be applicable to tissue optical properties over a wide wavelength range (UV-visible/near infrared). The fiber optic probe geometries that are modeled in this study include those commonly used for measuring fluorescence from tissues in practice.

Absorption↗
PubMed2003

Effect of fiber optic probe geometry on depth-resolved fluorescence measurements from epithelial tissues: a Monte Carlo simulation.

Developing fiber optic probe geometries to selectively measure fluorescence spectra from different sublayers within human epithelial tissues will potentially improve the endogenous fluorescence contrast between neoplastic and nonneoplastic tissues. In this study, two basic fiber optic probe geometries, which are called the variable aperture (VA) and multidistance (MD) approaches, are compared for depth-resolved fluorescence measurements from human cervical epithelial tissues. The VA probe has completely overlapping illumination and collection areas with variable diameters, while the MD probe employs separate illumination and collection fibers with a fixed separation between them. Monte Carlo simulation results show that the total fluorescence detected is significantly higher for the VA probe geometry, while the probing depth is significantly greater for the MD probe geometry. An important observation is that the VA probe is more sensitive to the epithelial layer, while the MD probe is more sensitive to the stromal layer. The effect of other factors, including numerical aperture (NA) and tissue optical properties on the fluorescence measurements with VA and MD probe geometries, are also evaluated. The total fluorescence detected with both probe geometries significantly increases when the fiber NA is changed from 0.22 to 0.37. The sensitivity to different sublayers is found to be strongly dependent on the tissue optical properties. The simulation results are used to design a simple fiber optic probe that combines both the VA and MD geometries to enable fluorescence measurements from the different sublayers within human epithelial tissues.

Absorption↗
PubMed2003

Relationship between collagen autofluorescence of the human cervix and menopausal status.

The goal of this study was to evaluate the effect of different menopausal states (pre- and post-) on the endogenous fluorescence of normal cervical tissues. In particular, the average fluorescence as well as the interpatient and intrasample variability in the average fluorescence of the epithelium and stroma were evaluated as a function of pre- and postmenopausal states. High-resolution fluorescence images at excitation-emission wavelengths of 440, 520 nm and 365, 465 nm were obtained from epithelia and stroma of freeze-trapped cervical tissue blocks maintained at -196 degrees C. The fluorescence images were recorded using a low temperature optical scanner. Fluorescence images from a normal sample population (n = 27) were quantitatively analyzed, and the average epithelial and stromal fluorescence intensities were obtained. Data grouped according to menopausal status (pre- vs post-) showed statistically significant differences (P < 0.002) in stromal fluorescence. In particular, the cervical stroma of postmenopausal women showed (1) significantly greater average fluorescence and (2) greater interpatient and intrasample variability in the fluorescence, relative to that of premenopausal women. These results provide evidence for changes in collagen cross-linking with menopause.

Adult↗
PubMed2003

Autofluorescence spectroscopy of normal and malignant human breast cell lines.

The fluorescence of tryptophan, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized in normal human breast cells as well as in malignant human breast cells of similar and dissimilar genetic origins. Fluorescence measurements of each cell line were made over a wide range of cell concentrations, and the fluorescence per cell was determined from the slope in the linear range of the fluorescence intensity vs cell concentration plot. All of the malignant cells showed a statistically significant decrease in the tryptophan fluorescence per cell relative to that of the normal cells. No statistically significant differences were observed in the NAD(P)H or FAD fluorescence per cell between the normal and any of the malignant cell types. NAD(P)H fluorescence was also imaged from monolayers of the normal and malignant cells (of similar genetic origin) using two-photon fluorescence microscopy. A statistically significant decrease in the NAD(P)H fluorescence with malignancy was observed, suggesting that fluorescence imaging of single cells or the cell monolayer preparation may provide more contrast than volume-averaged fluorescence measurements of cells in suspension. In conclusion, the differences in normal and malignant human breast tissue fluorescence spectra may be attributed in part to differences in the intrinsic cellular fluorescence of normal and malignant breast epithelial cells.

Breast↗