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Biomedical subjects

Ramachandra R Dasari

Publications and source records attributed to Ramachandra R Dasari.

At least 19 recordsLinked to original sources
PubMed2006

Optical measurement of cell membrane tension.

Using a novel noncontact technique based on optical interferometry, we quantify the nanoscale thermal fluctuations of red blood cells (RBCs) and giant unilamellar vesicles (GUVs). The measurements reveal a nonvanishing tension coefficient for RBCs, which increases as cells transition from a discocytic shape to a spherical shape. The tension coefficient measured for GUVs is, however, a factor of 4-24 smaller. By contrast, the bending moduli for cells and vesicles have similar values. This is consistent with the cytoskeleton confinement model, in which the cytoskeleton inhibits membrane fluctuations [Gov et al., Phys. Rev. Lett. 90, 228101, (2003).

Biophysical Phenomena↗
PubMed2006

Assessing epithelial cell nuclear morphology by using azimuthal light scattering spectroscopy.

We describe azimuthal light scattering spectroscopy (phi/LSS), a novel technique for assessing epithelial-cell nuclear morphology. The difference between the spectra measured at azimuthal angles phi = 0 degrees and phi = 90 degrees preferentially isolates the single backscattering contribution due to large (approximately 10 microm) structures such as epithelial cell nuclei by discriminating against scattering from smaller organelles and diffusive background. We demonstrate the feasibility of using phi/LSS for cancer detection by showing that spectra from cancerous colon tissue exhibit significantly greater azimuthal asymmetry than spectra from normal colonic tissues.

Cell Nucleus↗
PubMed2006

Live cell refractometry using microfluidic devices.

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

Cell Shape↗
PubMed2006

In vivo margin assessment during partial mastectomy breast surgery using raman spectroscopy.

We present the first demonstration of in vivo collection of Raman spectra of breast tissue. Raman spectroscopy, which analyzes molecular vibrations, is a promising new technique for the diagnosis of breast cancer. We have collected 31 Raman spectra from nine patients undergoing partial mastectomy procedures to show the feasibility of in vivo Raman spectroscopy for intraoperative margin assessment. The data was fit with an established model, resulting in spectral-based tissue characterization in only 1 second. Application of our previously developed diagnostic algorithm resulted in perfect sensitivity and specificity for distinguishing cancerous from normal and benign tissues in our small data set. Significantly, we have detected a grossly invisible cancer that, upon pathologic review, required the patient to undergo a second surgical procedure. Had Raman spectroscopy been used in a real-time fashion to guide tissue excision during the procedure, the additional reexcision surgery might have been avoided. These preliminary findings suggest that Raman spectroscopy has the potential to lessen the need for reexcision surgeries resulting from positive margins and thereby reduce the recurrence rate of breast cancer following partial mastectomy surgeries.

Breast Neoplasms↗
PubMed2006

Diffraction phase microscopy for quantifying cell structure and dynamics.

We have developed diffraction phase microscopy as a new technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.

Cell Movement↗
PubMed2005

Diagnosing breast cancer by using Raman spectroscopy.

We employ Raman spectroscopy to diagnose benign and malignant lesions in human breast tissue based on chemical composition. In this study, 130 Raman spectra are acquired from ex vivo samples of human breast tissue (normal, fibrocystic change, fibroadenoma, and infiltrating carcinoma) from 58 patients. Data are fit by using a linear combination model in which nine basis spectra represent the morphologic and chemical features of breast tissue. The resulting fit coefficients provide insight into the chemical/morphological makeup of the tissue and are used to develop diagnostic algorithms. The fit coefficients for fat and collagen are the key parameters in the resulting diagnostic algorithm, which classifies samples according to their specific pathological diagnoses, attaining 94% sensitivity and 96% specificity for distinguishing cancerous tissues from normal and benign tissues. The excellent results demonstrate that Raman spectroscopy has the potential to be applied in vivo to accurately classify breast lesions, thereby reducing the number of excisional breast biopsies that are performed.

Adipose Tissue↗
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↗