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Biomedical subjects

Ramachandra R Dasari

Publications and source records attributed to Ramachandra R Dasari.

16 recordsLinked to original sources
PubMed2005

Hilbert phase microscopy for investigating fast dynamics in transparent systems.

We introduce Hilbert phase microscopy (HPM) as a novel optical technique for measuring high transverse resolution quantitative phase images associated with optically transparent objects. Because of its single-shot nature, HPM is suitable for investigating rapid phenomena that take place in transparent structures such as biological cells. The potential of this technique for studying biological systems is demonstrated with measurements of red blood cells, and its ability to quantify dynamic processes on a millisecond scale is exemplified with measurements of evaporating micrometer-sized water droplets.

Computer Systems↗
PubMed2005

Harmonic phase-dispersion microscope with a Mach-Zehnder interferometer.

Harmonic phase-dispersion microscopy (PDM) is a new imaging technique in which contrast is provided by differences in refractive index at two harmonically related wavelengths. We report a new configuration of the harmonic phase-dispersion microscope in a Mach-Zehnder geometry as an instrument for imaging biological samples. Several improvements on the earlier design are demonstrated, including a single-pass configuration and acousto-optic modulators for generating the heterodyne signals without mechanical arm scanning. We demonstrate quantitative phase-dispersion images of test structures and biological samples.

Equipment Design↗
PubMed2004

Fourier phase microscopy for investigation of biological structures and dynamics.

By use of the Fourier decomposition of a low-coherence optical image field into two spatial components that can be controllably shifted in phase with respect to each other, a new high-transverse-resolution quantitative-phase microscope has been developed. The technique transforms a typical optical microscope into a quantitative-phase microscope, with high accuracy and a path-length sensitivity of lambda/5500, which is stable over several hours. The results obtained on epithelial and red blood cells demonstrate the potential of this instrument for quantitative investigation of the structure and dynamics associated with biological systems without sample preparation.

Cells, Cultured↗
PubMed2004

Noncontact measurement of nerve displacement during action potential with a dual-beam low-coherence interferometer.

We have used a novel phase-referenced heterodyne dual-beam low-coherence interferometer to perform what we believe are the first noncontact measurements of surface motion in a nerve bundle during the action potential. Nerve displacements of approximately 5-nm amplitude and approximately 10-ms duration are measured without signal averaging. This interferometer may find general application in measurement of small motion in cells and other weakly scattering samples.

Action Potentials↗
PubMed2004

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.

Aorta↗
PubMed2004

Spectroscopic diagnosis and imaging of invisible pre-cancer.

The theme of this paper is the use of optical spectroscopy to diagnose invisible pre-cancer in patients undergoing endoscopy and similar medical procedures. We describe three techniques that provide diagnostic information and two instruments to implement them, the FastEEM for studying small regions of tissue and the LSS (light scattering spectroscopy) imaging system for wide-area surveillance. The FastEEM is an optical fiber clinical device that collects spectra of reflected light and fluorescence at multiple excitation wavelengths from the tissue, all in a fraction of a second. Quantitative information is obtained in real time, without removing the tissue and without the need for staining and fixation. Three types of spectral information are extracted intrinsic fluorescence, diffuse reflectance and elastic light scattering. Each of the three analyses is based on a biophysical model, and each provides complementary quantitative physical and chemical information about cellular/tissue structures. This information is used to make a combined spectral diagnosis, a method we call tri-modal spectroscopy (TMS). Promising clinical studies are being carried out on patients undergoing routine pre-cancer surveillance in the oral cavity, the uterine cervix and the gastrointestinal tract. The LSS imaging system provides wide-area spectroscopic images of the epithelium, typically 2 cm in each dimension, depicting the size distribution and chromatin content of the cell nuclei, which are key parameters in diagnosing pre-cancer. This instrument is in preclinical stages of development, although a laboratory prototype has been used to create diagnostic images in resected colon polyp samples. The combination of the TMS/FastEEM and LSS imaging instrument will constitute a powerful new diagnostic tool, with LSS imaging to provide wide area surveillance and the TMS probe to provide detailed information on suspect tissue sites.

Cervix Uteri↗
PubMed2003

In situ detection of neoplastic transformation and chemopreventive effects in rat esophagus epithelium using angle-resolved low-coherence interferometry.

We present a quantitative study of the nuclear morphometry of epithelial cells in an animal model of esophageal carcinogenesis. Changes in the size and texture of cell nuclei as a result of neoplastic transformation and chemopreventive action are observed in situ using a new optical technique, angle-resolved low-coherence interferometry (a/LCI). The capabilities of a/LCI are demonstrated via quantitative in situ measurements of the nuclear morphometry of basal epithelial cells, approximately 50-100 microm beneath the tissue surface without the need for exogenous contrast agents or tissue fixation. The measurements quantify changes in nuclear size, characterized by average diameter, and nuclear texture, characterized by fractal dimension of the subcellular structures. Using this technique, we observed changes in the morphometry of rat esophageal epithelial cells in response to treatment with the carcinogen N-nitrosomethylbenzylamine. In addition, morphometric changes were observed in the esophagi of rats treated with N-nitrosomethylbenzylamine and two chemopreventive agents, difluoromethylornithine and perillyl alcohol. These agents induced either apoptosis in the basal epithelium (difluoromethylornithine) or both apoptosis and vacuolation of basal epithelial cells (perillyl alcohol). Vacuolation was associated with cellular toxicity. The light-scattering measurements were compared with histological images of the same tissues. The potential of a/LCI as a noninvasive means to investigate the development of epithelial neoplasia and for tracking the efficacy of chemopreventive agents appears high. This technique also may provide a new screening tool for intraepithelial neoplasia.

Animals↗
PubMed2003

Spectroscopic detection and evaluation of morphologic and biochemical changes in early human oral carcinoma.

BACKGROUND: Understanding the development and progression of head and neck squamous cell carcinoma is key in the quest for the early diagnosis and prevention of this type of malignancy. The current study correlated early biochemical and histologic changes in oral tissue with spectral features in fluorescence, reflectance, and light scattering spectra acquired in vivo to diagnose early stages of oral malignancies. METHODS: A total of 91 tissue sites from 15 patients with varying degrees of malignancy (normal, dysplastic, and cancerous sites) and 8 healthy volunteers were analyzed with 3 spectroscopic techniques. Direct biochemical information regarding oral tissue native fluorophores was obtained with intrinsic fluorescence spectroscopy by fitting a linear combination of collagen and the reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence spectra to the intrinsic tissue fluorescence spectra excited with 337 nanometer (nm) and 358-nm laser light. Diffuse reflectance spectroscopy was used to provide information regarding tissue absorption and structure, such as hemoglobin concentration and stroma density, by measuring the wavelength-dependent absorption and scattering coefficients. By subtracting the diffusely reflected component from the measured reflectance, light scattering spectroscopy (LSS) information resulting from single backscattering from epithelial cell nuclei was obtained. LSS provides information concerning the size distribution of cell nuclei. RESULTS: These optically extracted tissue parameters provide biochemical or structural information in vivo without the need for tissue excision, and can be used to diagnose tissue abnormalities. By combining the information provided by the three techniques, a method known as trimodal spectroscopy, a sensitivity and specificity of 96% and 96%, respectively, in distinguishing cancerous/dysplastic (mild, moderate, and severe) from normal tissue was achieved. In addition, the authors were able to distinguish dysplastic from cancerous tissue with a sensitivity of 64% and a specificity of 90%. CONCLUSIONS: The results of the current study demonstrated that Trimodal spectroscopy is a highly sensitive and specific technique with which to diagnose tissue abnormalities.

Carcinoma↗
PubMed2003

Quantitative detection of benzo[alpha]pyrene diolepoxide-DNA adducts by cryogenic laser induced fluorescence.

In the present report, we describe a fluorescence-based method capable of measuring benzo[alpha]pyrene diolepoxide (BPDE) adducts in intact genomic DNA, with a sensitivity of a few hundreds copies per cell. The assay is based on cryogenic laser-induced fluorescence technology at liquid nitrogen temperatures, coupled with an intensified charge-coupled device camera, and incorporates several enhancements to existing methodologies. One important modification was the incorporation of terbium(III)nitrate pentahydrate, Tb(NO3)3, as an internal fluorescence standard to correct for differences in light scattering and fluctuations in instrument parameters. Since the fluorescence spectrum of Tb(NO3)3 does not overlap with those of BPDE-DNA adducts, use of this lanthanide salt markedly improved the sensitivity of cryogenic laser-induced fluorescence. The limit of quantification of the assay is 6.4 BPDE-DNA adducts/10(8) nucleotides, or 776 adducts/cell, using 22.5 micrograms of genomic DNA. This assay is rapid, highly sensitive, and economical and has been applied to monitor DNA adduct levels as a function of time after exposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.

Benzo(a)pyrene↗
PubMed2002

Identifying microcalcifications in benign and malignant breast lesions by probing differences in their chemical composition using Raman spectroscopy.

We have applied Raman spectroscopy to analyze the chemical composition of microcalcifications occurring in benign and malignant lesions in the human breast. Microcalcifications were initially separated into two categories based on their Raman spectrum: type I, calcium oxalate dihydrate, and type II, calcium hydroxyapatite. Type I microcalcifications were diagnosed as benign, whereas type II were subdivided into benign and malignant categories using principal component analysis, a statistical technique. Although type II microcalcifications are primarily composed of calcium hydroxyapatite, they also contain trace amounts of several biological impurities. Using principal component analysis, we were able to highlight subtle chemical differences in type II microcalcifications that correlate with breast disease. On the basis of these results, we believe that type II microcalcifications formed in benign ducts typically contain a larger amount of calcium carbonate and a smaller amount of protein than those formed in malignant ducts. Using this diagnostic strategy, we were able to distinguish microcalcifications occurring in benign and malignant ducts with a sensitivity of 88% and a specificity of 93%. This is a significant improvement over current X-ray mammography techniques, which are unable to reliably differentiate microcalcifications in benign and malignant breast lesions.

Adult↗
PubMed2002

NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes.

During the development of neoplasia, epithelial tissues undergo biochemical and structural changes that can manifest in tissue fluorescence. There have been several reports on different in vivo fluorescence characteristics between normal and precancerous (dysplastic) tissues. However, it has been difficult to identify and quantify the origins of these changes, mainly because of distortions introduced in measured tissue fluorescence spectra by tissue scattering and absorption. Such distortions can be removed by combining information in simultaneously measured fluorescence and reflectance spectra. Thus, we can recover the intrinsic (undistorted) tissue fluorescence. In this report, we show that extraction of the intrinsic fluorescence allows us: (a) to determine the fluorescence spectra of NAD(P)H and collagen in an in vivo environment, and (b) to use these NAD(P)H and collagen spectra to describe, quantitatively, diagnostically significant biochemical changes between normal and dysplastic tissues. Specifically, by analyzing intrinsic fluorescence of human epithelial tissue as it becomes deoxygenated in vivo, we can resolve the fluorescence spectra of NAD(P)H and collagen, two of the major tissue fluorophores. This is important because fluorescence depends on the local environment of the chromophore. Then, we extract the intrinsic fluorescence spectra of sites from 35 patients with suspected cervical lesions and 7 patients with Barrett's esophagus and describe them accurately as a linear combination of NAD(P)H and collagen contributions. In both tissue cases, we find that low collagen and high NAD(P)H fluorescence characterizes the high-grade dysplastic lesions when compared with nondysplastic tissues. These data present evidence for the presence of detectable levels of NAD(P)H fluorescence in human epithelial tissues in an in vivo setting and demonstrate that NAD(P)H and collagen may be used as quantitative fluorescence biomarkers for in vivo detection of dysplasia in the cervix and the esophagus.

Barrett Esophagus↗
PubMed2002

Model-based biological Raman spectral imaging.

Raman spectral imaging is a powerful tool for determining chemical information in a biological specimen. The challenge is to condense the large amount of spectral information into an easily visualized form with high information content. Researchers have applied a range of techniques, from peak-height ratios to sophisticated models, to produce interpretable Raman images. The purpose of this article is to review some of the more common imaging approaches, in particular principal components analysis, multivariate curve resolution, and Euclidean distance, as well as to present a new technique, morphological modeling. How to best extract meaningful chemical information using each imaging approach will be discussed and examples of images produced with each will be shown.

Algorithms↗
PubMed2002

Cellular organization and substructure measured using angle-resolved low-coherence interferometry.

We measure the organization and substructure of HT29 epithelial cells in a monolayer using angle-resolved low-coherence interferometry. This new technique probes cellular structure by measuring scattered light, as in flow cytometry, but offers an advantage in that the structure can be examined in situ, avoiding the need to disrupt the cell monolayer. We determine the size distribution of the cell nuclei by fitting measured light-scattering spectra to the predictions of Mie theory. In addition, we obtain information about the cellular organization and substructure by examining the spatial correlations within the monolayer. A remarkable finding is that the spatial correlations over small length scales take the form of an inverse power law, indicating the fractal nature of the packing of the subcellular structures. We also identify spatial correlations on a scale large compared with the size of a cell, indicating an overlying order within the monolayer.

Biophysics↗
PubMed2002

Trimodal spectroscopy for the detection and characterization of cervical precancers in vivo.

OBJECTIVE: The objective of this study was to assess the potential of 3 spectroscopic techniques (intrinsic fluorescence, diffuse reflectance, and light scattering) individually and in combination (trimodal spectroscopy) for the detection of cervical squamous intraepithelial lesions. STUDY DESIGN: The study was conducted with 44 patients who underwent colposcopy for the evaluation of an abnormal Papanicolaou smear. Fluorescence and reflectance spectra were collected from colposcopically normal and abnormal sites and analyzed to extract quantitative information about tissue biochemistry and morphologic condition. This information was compared with histopathologic classification, and diagnostic algorithms were developed and validated with the use of logistic regression and cross-validation. RESULTS: Diagnostically significant differences exist in the composition of fluorescing biochemicals, the scattering properties, and the epithelial cell nuclear morphology of cervical squamous intraepithelial lesions and non-squamous intraepithelial lesions. Trimodal spectroscopy is a superior tool for the detection of cervical squamous intraepithelial lesions than any 1 of the techniques alone. CONCLUSION: Trimodal spectroscopy has the potential to improve the in vivo detection of precancerous cervical changes.

Diagnosis, Differential↗
PubMed2002

Determination of particle size by using the angular distribution of backscattered light as measured with low-coherence interferometry.

We employ a novel interferometer to measure the angular distribution of light backscattered by a turbid medium. Through comparison of the measured data with the predictions of Mie theory, we are able to determine the size of the scatterers comprising the medium with subwavelength precision. As the technique is based on low-coherence interferometry, we are able to examine the evolution of the angular distribution of scattered light as it propagates into the medium. The effects of multiple scattering as a function of penetration depth in the medium are analyzed. We also present various considerations for extending this technique to determining structural information in biological tissues, such as the effects of a distribution of particle sizes and the need to average out speckle contributions.

Interferometry↗