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A fast, flexible algorithm for calculating correlations in Fluorescence Correlation Spectroscopy

Description: A new algorithm is introduced for computing correlations of photon arrival time data acquired in single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). The correlation is first rewritten as a counting operation on photon pairs. For each photon, the contribution to the correlation function for each subsequent photon is calculated for arbitrary bin spacings of the correlation time lag. By retaining the bin positions in the photon sequence after each photon, the correlation can be performed efficiently. Example correlations for simulations of FCS experiments are shown, with comparable execution speed to the commonly used multiple-tau correlation technique. Also, wide bin spacings are possible that allow for real-time software calculation of correlations even for high count rates ({approx}350 kHz). The flexibility and broad applicability of the algorithm is demonstrated using results from single molecule photon antibunching experiments.
Date: October 13, 2005
Creator: Laurence, T; Fore, S & Huser, T
Partner: UNT Libraries Government Documents Department

Using Femtosecond Laser Subcellular Surgery as a Tool to Study Cell Biology

Description: Research on cellular function and regulation would be greatly advanced by new instrumentation using methods to alter cellular processes with spatial discrimination on the nanometer-scale. We present a novel technique for targeting submicrometer sized organelles or other biologically important regions in living cells using femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we can vaporize cellular material inside the cell through nonlinear optical processes. This technique enables non-invasive manipulation of the physical structure of a cell with sub-micrometer resolution. We propose to study the role mitochondria play in cell proliferation and apoptosis. Our technique provides a unique tool for the study of cell biology.
Date: February 27, 2007
Creator: Shen, N; Colvin, M E & Huser, T
Partner: UNT Libraries Government Documents Department

Counting constituents in molecular complexes by fluorescence photon antibunching

Description: Modern single molecule fluorescence microscopy offers new, highly quantitative ways of studying the systems biology of cells while keeping the cells healthy and alive in their natural environment. In this context, a quantum optical technique, photon antibunching, has found a small niche in the continuously growing applications of single molecule techniques to small molecular complexes. Here, we review some of the most recent applications of photon antibunching in biophotonics, and we provide a guide for how to conduct photon antibunching experiments at the single molecule level by applying techniques borrowed from time-correlated single photon counting. We provide a number of new examples for applications of photon antibunching to the study of multichromophoric molecules and small molecular complexes.
Date: April 17, 2007
Creator: Fore, S; Laurence, T; Hollars, C & Huser, T
Partner: UNT Libraries Government Documents Department

Non-destructive Identification of Individual Leukemia Cells by Optical Trapping Raman Spectroscopy

Description: Currently, a combination of technologies is typically required to assess the malignancy of cancer cells. These methods often lack the specificity and sensitivity necessary for early, accurate diagnosis. Here we demonstrate using clinical samples the application of laser trapping Raman spectroscopy as a novel approach that provides intrinsic biochemical markers for the noninvasive detection of individual cancer cells. The Raman spectra of live, hematopoietic cells provide reliable molecular fingerprints that reflect their biochemical composition and biology. Populations of normal T and B lymphocytes from four healthy individuals, and cells from three leukemia patients were analyzed, and multiple intrinsic Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for cancer cell identification. A combination of two multivariate statistical methods, principal component analysis (PCA) and linear discriminant analysis (LDA), was used to confirm the significance of these markers for identifying cancer cells and classifying the data. The results indicate that, on average, 95% of the normal cells and 90% of the patient cells were accurately classified into their respective cell types. We also provide evidence that these markers are unique to cancer cells and not purely a function of differences in their cellular activation.
Date: March 5, 2007
Creator: Chan, J W; Taylor, D S; Lane, S; Zwerdling, T; Tuscano, J & Huser, T
Partner: UNT Libraries Government Documents Department

Micro-Raman spectroscopy Detects Individual Neoplastic and Normal Hematopoietic Cells

Description: Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often non-specific, slow, biologically perturbing, or a combination, thereof. Here, we show that single-cell micro-Raman spectroscopy averts these shortcomings and can be used to discriminate between unfixed normal human lymphocytes and transformed Jurkat and Raji lymphocyte cell lines based on their biomolecular Raman signatures. We demonstrate that single-cell Raman spectra provide a highly reproducible biomolecular fingerprint of each cell type. Characteristic peaks, mostly due to different DNA and protein concentrations, allow for discerning normal lymphocytes from transformed lymphocytes with high confidence (p << 0.05). Spectra are also compared and analyzed by principal component analysis (PCA) to demonstrate that normal and transformed cells form distinct clusters that can be defined using just two principal components. The method is shown to have a sensitivity of 98.3% for cancer detection, with 97.2% of the cells being correctly classified as belonging to the normal or transformed type. These results demonstrate the potential application of confocal micro-Raman spectroscopy as a clinical tool for single cell cancer detection based on intrinsic biomolecular signatures, therefore eliminating the need for exogenous fluorescent labeling.
Date: January 18, 2005
Creator: Chan, J W; Taylor, D; Zwerdling, T; Lane, S M; Ihara, K & Huser, T
Partner: UNT Libraries Government Documents Department

Raman Spectroscopic Analysis of Biochemical Changes in Individual Triglyceride-Rich Lipoproteins in the Pre- and Postprandial State

Description: Individual triglyceride-rich lipoprotein (TGRL) particles derived from human volunteers are non-destructively analyzed by laser tweezers Raman microspectroscopy and information on their composition and distribution is obtained. The Raman signature of single optically trapped very low-density lipoproteins (VLDL), a subclass of TGRL, which play an important role in cardiovascular disease, exhibits distinct peaks associated with molecular vibrations of fatty acids, proteins, lipids, and structural rearrangements of lipids. Our analysis of pre- and postprandial VLDL exhibits the signature of biochemical changes in individual lipoprotein particles following the consumption of meals. Interaction of VLDL with endothelium leads to the breakdown of complex triacylglycerols and the formation of a highly ordered core of free saturated fatty acids in the particle. A particle distribution analysis reveals trends in the degree to which this process has occurred in particles at different times during the postprandial period. Differences in particle distributions based on the different ratios of polyunsaturated to saturated fats in the consumed meals are also easily discerned. Individual lipoprotein particles hydrolyzed in-vitro through addition of lipoprotein lipase (LpL) exhibit strikingly similar changes in their Raman spectra. These results demonstrate the feasibility of monitoring the dynamics of lipid metabolism of individual TGRL particles as they interact with LpL in the endothelial cell wall using Raman spectroscopy.
Date: September 13, 2004
Creator: Chan, J; Motton, D; Rutledge, J; Keim, N & Huser, T
Partner: UNT Libraries Government Documents Department

Nanoparticle Based Surface-Enhanced Raman Spectroscopy

Description: Surface-enhanced Raman scattering is a powerful tool for the investigation of biological samples. Following a brief introduction to Raman and surface-enhanced Raman scattering, several examples of biophotonic applications of SERS are discussed. The concept of nanoparticle based sensors using SERS is introduced and the development of these sensors is discussed.
Date: January 3, 2005
Creator: Talley, C E; Huser, T R; Hollars, C W; Jusinski, L; Laurence, T & Lane, S M
Partner: UNT Libraries Government Documents Department

Spectroscopy of Single Free Standing Quantum Wells

Description: We investigated the interaction of quantum confined exciton states GaAs quantum wells with native surface states. Single molecule photoluminescence (PL) spectroscopy, developed by T. Huser at LLNL was used to probe the unique bare quantum wells in the free standing quantum well structure. The latter was developed by the M. D. Williams at Clark Atlanta University. The goals of the project during this budget cycle were to procure samples containing GaAs free standing QWs, identify suitable regions for PL analysis at Lawrence Livermore, analyze the structures at room temperature and at liquid nitrogen temperatures. The specific regions of interest on the sample structures were identified by scanning electron microscopy at Clark Atlanta prior to transport to LLNL. Previous attempts at other facilities using NSOM, cathodoluminescence, and conventional PL showed little luminescence activity at room temperature from the 200 {angstrom} thick wells. This suggested either excess recombination due to surface states in the quantum well region or insufficient absorption length for photoluminescence. The literature suggested that the effect of the defects could be eliminated by reducing the sample temperature below their associated activation energies. In our previous subcontract work with LLNL, a significant amount of effort was expended to modify the apparatus to allow low temperature measurements. The modifications were not successful and we concluded that in order to do the measurements at low temperature we would need to purchase a commercial optical cryostat to get reliable results. Ms. Rochelle Bryant worked during the summer as an intern at LLNL on the project under the supervision of C. Hollars and in collaboration with T. Huser and found that PL emission could be obtained at room temperature. This was a surprising result as the literature and our experience shows that there is no PL emission from GaAs at room temperature. We speculate ...
Date: March 14, 2006
Creator: Williams, M D; Hollars, C W; Huser, T; Jallow, N; Cochran, A & Bryant, R
Partner: UNT Libraries Government Documents Department

Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

Description: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available ...
Date: January 9, 2007
Creator: Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R et al.
Partner: UNT Libraries Government Documents Department

Single Fluorescent Molecule Confocal Microscopy: A New Tool for Molecular Biology Research and Biosensor Development

Description: Our original proposal was presented to the LDRD committee on February 18, 1999. The revised proposal that followed incorporated changes that addressed the issues, concerns, and suggestions put forth by the committee members both during the presentation and in subsequent discussions we've had with individual committee members. The goal of the proposal was to establish an SMD confocal microscopy capability and technology base at LLNL. Here we report on our progress during the 6-month period for which funding was available.
Date: March 9, 2000
Creator: Darrow, C.; Huser, T.; Campos, C.; Yan, M.; Lane, S. & Balhorn, R.
Partner: UNT Libraries Government Documents Department

Using Vulcan to Recreate Planetary Cores

Description: An accurate equation of state (EOS) for planetary constituents at extreme conditions is the key to any credible model of planets or low mass stars. However, experimental validation has been carried out on at high pressure (>few Mbar), and then only on the principal Hugoniot. For planetary and stellar interiors, compression occurs from gravitational force so that material states follow a line of isentropic compression (ignoring phase separation) to ultra-high densities. An example of the predicted states for water along the isentrope for Neptune is shown in a figure. The cutaway figure on the left is from Hubbard, and the phase diagram on the right is from Cavazzoni et al. Clearly these states lie at quite a bit lower temperature and higher density than single shock Hugoniot states but they are at higher temperature than can be achieved with accurate diamond anvil experiments. At extreme densities, material states are predicted to have quite unearthly properties such as high temperature superconductivity and low temperature fusion. High density experiments on Earth are achieved with either static compression techniques (i.e.diamond anvil cells) or dynamic compression techniques using large laser facilities, gas guns, or explosives. A major thrust of this work is to develop techniques to create and characterize material states that exists primarily at the core of giant planets and brown dwarf stars. Typically, models used to construct planetary isentropes are constrained by only the planet radius, outer atmospheric spectroscopy, and space probe gravitational moment and magnetic field data. Thus any data, which provide rigid constraints for these models will have a significant impact on a broad community of planetary and condensed matter scientists. Recent laser shock wave experiments have made great strides in recreating material states that exist in the outer 25% (in radius) of the Jovian planets and at the exterior ...
Date: August 15, 2001
Creator: Collins, G.W.; Celliers, P.M.; Hicks, D.G.; Mackinnon, A.J.; Moon, S.J.; Cauble, R. et al.
Partner: UNT Libraries Government Documents Department