Jerilyn A. Timlin

3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

Localizing and Quantifying Carotenoids in Intact Cells and Tissues

Raman spectroscopy provides detailed information about the molecular structure of carot‐ enoids. Advances in detector sensitivity and acquisition speed have driven the expansion of Raman spectroscopy from a bulk analytical tool to a powerful method for mapping carotenoid abundance in cells and tissues. In many applications, the technique is compat‐ ible with living organisms, providing highly specific molecular structure information in intact cells and tissues with subcellular spatial resolution. This leads to spatial‐temporal‐ chemical resolution critical to understanding the complex processes in the life cycle of carotenoids and other biomolecules.

Advanced Optical Imaging of Endocytosis

Endocytosis is the highly controlled and complex process by which a portion of the plasma membrane, including its lipids, proteins, and local extracellular fluid becomes internalized in a cell. Endocytosis serves to mediate a multitude of interactions between a cell and its environment, including nutrient uptake, mitosis, motility, as well as adaptive and innate immune response, among many others. There are multiple routes of endocytotic uptake into cells, with the most studied being clathrin mediated endocytosis (CME). Although CME differs significantly on a molecular level from the clathrin-independent endocytosis mechanisms (e.g. macropinocytosis, phagocytosis), all of the endocytic mechanisms involve a sequence of changes in morphology, molecular composition, and protein interactions at the plasma membrane, as well as throughout the bulk of the cell. Further, each of these changes is tightly regulated in space and time. To fully characterize endocytic pathways and their intertwined relationship to other signalling pathways, there is a need to visualize the dynamics of multiple species at the plasma membrane and within the cell with high threedimensional spatial resolution.

Biomolecular Interactions and Responses of Human Epithelial and Macrophage Cells to Engineered Nanomaterials

Engineered nanomaterials (ENMs) are increasingly being used in commercial products, particularly in the biomedical, cosmetic, and clothing industries. For example, pants and shirts are routinely manufactured with silver nanoparticles to render them wrinkle-free. Despite the growing applications, the associated environmental health and safety (EHS) impacts are completely unknown. The significance of this problem became pervasive within the general public when Prince Charles authored an article in 2004 warning of the potential social, ethical, health, and environmental issues connected to nanotechnology. The EHS concerns, however, continued to receive relatively little consideration from federal agencies as compared with large investments in basic nanoscience RD Kwon et al., 2008, J. Occup. Health 50, 1) has spurred a recent realization within the NNI and other federal agencies that the EHS impacts related to nanotechnology must be addressed now. In our study we proposed to address critical aspects of this problem by developing primary correlations between nanoparticle properties and their effects on cell health and toxicity. A critical challenge embodied within this problem arises from the ability to synthesize nanoparticles with a wide array of physical properties (e.g., size, shape, composition, surface chemistry, etc.), which in turn creates an immense, multidimensional problem in assessing toxicological effects. In this work we first investigated varying sizes of quantum dots (Qdots) and their ability to cross cell membranes based on their aspect ratio utilizing hyperspectral confocal fluorescence microscopy. We then studied toxicity of epithelial cell lines that were exposed to different sized gold and silver nanoparticles using advanced imaging techniques, biochemical analyses, and optical and mass spectrometry methods. Finally we evaluated a new assay to measure transglutaminase (TG) activity; a potential marker for cell toxicity.

Fluorescence measurements for evaluating the application of multivariate analysis techniques to optically thick environments.

Laser-induced fluorescence measurements of cuvette-contained laser dye mixtures are made for evaluation of multivariate analysis techniques to optically thick environments. Nine mixtures of Coumarin 500 and Rhodamine 610 are analyzed, as well as the pure dyes. For each sample, the cuvette is positioned on a two-axis translation stage to allow the interrogation at different spatial locations, allowing the examination of both primary (absorption of the laser light) and secondary (absorption of the fluorescence) inner filter effects. In addition to these expected inner filter effects, we find evidence that a portion of the absorbed fluorescence is re-emitted. A total of 688 spectra are acquired for the evaluation of multivariate analysis approaches to account for nonlinear effects.

Resolving dynamics of cell signaling via real-time imaging of the immunological synapse.

This highly interdisciplinary team has developed dual-color, total internal reflection microscopy (TIRF-M) methods that enable us to optically detect and track in real time protein migration and clustering at membrane interfaces. By coupling TIRF-M with advanced analysis techniques (image correlation spectroscopy, single particle tracking) we have captured subtle changes in membrane organization that characterize immune responses. We have used this approach to elucidate the initial stages of cell activation in the IgE signaling network of mast cells and the Toll-like receptor (TLR-4) response in macrophages stimulated by bacteria. To help interpret these measurements, we have undertaken a computational modeling effort to connect the protein motion and lipid interactions. This work provides a deeper understanding of the initial stages of cellular response to external agents, including dynamics of interaction of key components in the signaling network at the immunological synapse, the contact region of the cell and its adversary.

Development and Integration of Raman Imaging Capabilities to Sandia National Laboratories Hyperspectral Fluorescence Imaging Instrument

Raman spectroscopic imaging is a powerful technique for visualizing chemical differences within a variety of samples based on the interaction of a substances molecular vibrations with laser light. While Raman imaging can provide a unique view of samples such as residual stress within silicon devices, chemical degradation, material aging, and sample heterogeneity, the Raman scattering process is often weak and thus requires very sensitive collection optics and detectors. Many commercial instruments (including ones owned here at Sandia National Laboratories) generate Raman images by raster scanning a point focused laser beam across a sample--a process which can expose a sample to extreme levels of laser light and requires lengthy acquisition times. Our previous research efforts have led to the development of a state-of-the-art two-dimensional hyperspectral imager for fluorescence imaging applications such as microarray scanning. This report details the design, integration, and characterization of a line-scan Raman imaging module added to this efficient hyperspectral fluorescence microscope. The original hyperspectral fluorescence instrument serves as the framework for excitation and sample manipulation for the Raman imaging system, while a more appropriate axial transmissive Raman imaging spectrometer and detector are utilized for collection of the Raman scatter. The result is a unique and flexible dual-modalitymorexa0» fluorescence and Raman imaging system capable of high-speed imaging at high spatial and spectral resolutions. Care was taken throughout the design and integration process not to hinder any of the fluorescence imaging capabilities. For example, an operator can switch between the fluorescence and Raman modalities without need for extensive optical realignment. The instrument performance has been characterized and sample data is presented.«xa0less