Our Research

MEASUREMENTS OF CELL MEMBRANE ORGANIZATION AND DYNAMICS

Fluorescence Analysis Techniques used: Fluorescence Resonance Energy Transfer (FRET); Fluorescence Recovery After Photobleaching (FRAP); Single Particle Tracking (SPT); Sub-diffraction Stimulated Emission Depletion (STED) Imaging; Stochastic Optical Reconstruction Microscopy (STORM)

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Overview: 

The cell membrane is a complex organization of lipids, proteins, carbohydrates and small molecules that has a dynamic interface with its environment. We aim to measure the small-scale organization of the cell membrane in live cells, as well as the extracellular and intracellular cues that cause a rearrangement of this organization. We seek to develop the relationship between cell membrane organization and cell signaling across the membrane.

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Some Recent Findings:

We previously developed a noninvasive FRET assay for measuring the clustering of wild-type and mutant cell membrane receptors, termed integrins. The assay uses donor and acceptor FRET reporter peptides that cluster with integrins. Energy transfer from donor to acceptor FRET reporter peptides, when the two are in close proximity, is used to measure integrin clustering within the cell membrane of cultured cells.

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We have also developed a method to measure the role of cytoplasmic or membrane proteins in altering the clustering and diffusion of integrin cell membrane receptors. The method involves the combination of FRET or FRAP and methods to selectively reduce the expression of a target protein. We have identified a number of cytoplasmic and membrane proteins that alter integrin diffusion and clustering.

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We also developed SPT methods to measure the role of ligands on RAGE diffusion. We used quantum dots specifically attached to hemagglutinin-tagged RAGE on the cell membrane. We have shown methylglyoxal modified-bovine serum albumin ligand (MGO-BSA) containing advanced glycation endproducts modifications changes the RAGE diffusion. We also identified that the mechanism of MGO-BSA ligand-induced RAGE diffusion alteration is cholesterol dependant.

We have also studied the effect of the cytoplasmic protein diaphanous-1 (Diaph1) on the nanoscale clustering and diffusion of RAGE. SPT used to measure the diffusion and STORM was used to measure the clustering. Reduced Diaph1 expression on the cell membrane increased the RAGE diffusion but the Diaph1 with altered binding sites resulted in converse effect. We also identified the decreased number and size of RAGE clusters resulted from reduced Diaph1 expression or altered binding sites on Diaph1 protein.

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In collaboration with Professor Arthur Winter, Department of Chemistry at Iowa State University, we are measuring the photoactivation of BODIPY-based photocages in the cellular environment and developing these photocages for imaging of cellular structures that are smaller in size than the diffraction limit of light using localization-based super-resolution microscopy.

TOTAL INTERNAL REFLECTION RAMAN MICROSCOPY

Techniques Used:

Total Internal Reflection (TIR); Scanning Angle (SA) Raman Microscopy; Directional Raman Spectroscopy (DRS); Surface-plasmon-polariton (SPP) cone; Surface plasmon resonance (SPR); Plasmon waveguide resonance (PWR)

Overview:

Total internal reflection spectroscopies are surface sensitive and nondestructive techniques that provide information about the chemical content at an interface and can determine adsorbate thickness or the dielectric constant parameters. Under conditions of total internal reflection, an evanescent wave is generated in a dielectric layer when a beam enters a high refractive index prism at or above the critical angle for internal reflection to occur.

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The excitation of surface plasmons under total internal reflection on the gold surface generates a surface-plasmon-polariton cone due to two momentum conserving processes called optical and roughness coupling. The directional emission of the cone at the sharply defined angle on the prism side of the Kretschmann configuration allows one to two orders of magnitude enhancement under total internal reflection conditions, and the signals are very reproducible and can be well modelled with simple calculations.   

We have developed several instruments that can provide chemical specific depth-profiling data with sub-diffraction spatial resolution and are applying the instruments to study heterogeneous catalytic systems, polymer films, and to monitor biomolecule interactions for biosensing applications.

Recent Findings:

The SA-TIR Raman spectroscopy method is used to measure the chemical composition and extract the location of buried interfaces in bilayer and trilayer thin waveguide films consisting of polystyrene-block-poly(methyl methacrylate) and homopolymer poly(methyl methacrylate) (PS-b-PMMA:PMMA), and poly(2-vinylnapthalene)-block-poly(methyl methacrylate) (P2VN-b-PMMA). We show that the SA-TIR Raman method can be modeled with a recursive/iterative FDTD script of the electric field intensity within the polymer layers to extract the total thickness and interface locations of the multilayer system. We demonstrated that SA-TIR Raman method can be used to determine individual layers in a multilayer system with a total thickness ranging from 300 to 1000 nm, and with 7–80-nm axial spatial resolution.

We have measured the directional Raman scattering signal from an interface containing a thin gold film to obtain surface plasmon resonance and enhanced Raman scattering measurements simultaneously. We developed the DRS method and instrumentation that enables the simultaneous collection of three angle-dependent parameters: (1) the SPP cone intensity, (2) cone diameter and (3) Raman scattering as a function of incident angle. The data demonstrate a method for collecting SPR data using images of the SPP cones from thin polymer films, and self-assembled monolayers on a noble metal surface. We demonstrate that the SPP cone encompasses the thickness, refractive index and chemical composition of the adsorbed molecules. In addition, the directional Raman spectrometer utilizes two TIR configurations on a single instrument set up. The Kretschmann and reverse-Kretschmann configurations are used to study polymer waveguide films. Specifically, the data demonstrates a method for collecting PWR data using images of the SPP cones in both configurations.

ANALYTICAL METHODS FOR BIOFUELS RESEARCH

Techniques Used:

Raman Spectroscopy and Imaging, Coherent Anti-Stokes Raman Spectroscopy (CARS)

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Goals & Importance:

The goals and importance of our work is to reveal an in situ cellular level understanding of various plant and biomass materials in order to determine better lignocellulosic and other plant biomass fuels.

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Recent Findings and Current Research:

The plant cell wall is highly heterogenous, complex and highly dynamic structure within plants and consists of sugars (cellulose and hemicellulose) and lignin and other components. The cell wall provides structure, strength, and a defensive barrier against predation, fungi and bacterial infection. Biofuel production is extremely complex and improvements for higher conversion are always needed. We aim to determine a deeper understanding of plants and biomass materials by utilizing analytical techniques including Raman and fluorescence imaging.

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We demonstrated spontaneous Raman imaging would useful to measure resulting biochemical changes from silencing plant genes that alter the synthesis of lignin, cellulose, carotenoids and other biochemicals in multiple plant tissues at the cellular level. Raman imaging is advantageous with capabilities to do in situ studies with little or no sample preparation and reveal the spatial distribution, relative content, and localized accumulation.

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In a collaboration across Iowa State University campus that includes imaging scientists, plant biochemists, microbiologists, and computation and theory scientists that is currently focused on overcoming sub-diffraction imaging problems while revealing further information about the plant cell wall that can assist in biofuel production. The Smith group goal was to build a coherent anti-stokes Raman spectroscopy (CARS) imaging set-up for super resolution imaging of plant samples. This instrumentation is extremely complex and requires a vast array of components. Currently we are optimizing the CARS instrumentation to measure plant samples with high reproducibility to reveal a deeper understanding of the plant cell wall.