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Landes Research Group
Rice University
6100 Main Street MS-60
Houston, TX 77005
Christy's Office: Dell Butcher 352
Student Office: Dell Butcher 133
Lab: Dell Butcher 130
Lab Phone: 713-348-4437

Phone: 713-348-4232
Fax: 713-348-5155

Research Topics


Hardware and Computational Methods for Data Collection and Processing in Single Molecule Spectroscopy

Measuring the fluorescence from a single molecule allows us to study protein conformation dynamics, single molecule diffusion and adsorption on surfaces, and many other processes that bulk studies fail to capture. However, lower signals, photobleaching, the diffraction limit of light, and many other factors make single molecule fluorescence hard to measure experimentally. Large sample size and heterogeneity also make data analysis very challenging. The Landes lab develops methods to improve experimental and data processing techniques for SMS with the ultimate goals of improving space and time resolution, reducing errors, and extending measurement time.

Extending Measurement Time

Traditional laser excitation constantly excites a dye molecule, but a detector is not able to collect all of the light emitted because it takes time to store information. To maximize the amount of collection time before a dye molecule was photobleached, the laser excitation was synchronized with collection time. We extended the single molecule fluorophore lifetime by up to 1500% and super- localization by 47% by modulating laser excitation using an acousto-optic modulator (AOM) synchronized to the data collection and inherent data storage time of the detector.

extending measurement time

We showed by simulations and by experiments that fluorescence fluctuations from diffusing probes within porous spaces can be analyzed to yield information about pore sizes and diffusion coefficients. Our method does not require extensive development of experimental protocol to directly label the material compared to other super-resolution techniques; radiant probes only must be able explore the porous space. 

Improving Space Resolution with fcsSOFI

We developed a super-resolution analytical method that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as “fluorescence correlation spectroscopy super-resolution optical fluctuation imaging” or “fcsSOFI”.

langmuirads

We showed by simulations and by experiments that fluorescence fluctuations from diffusing probes within porous spaces can be analyzed to yield information about pore sizes and diffusion coefficients. Our method does not require extensive development of experimental protocol to directly label the material compared to other super-resolution techniques; radiant probes only must be able explore the porous space.

3D Super-Resolution Microscopy

Traditional super-resolution methods are only able to detect molecules in two dimenstions, but molecular diffusion occurs in three dimensions. We pushed the limit of detection dimension from 2D to 3D using double helix point spread function (PSF) microscopy. A phase mask in the Fourier plane converts the point spread function of the instrument from a gaussian to a double helix. In this technique one emitter corresponds to two lobes and the two lobes rotate when the emitters are located at different z-position. The orientation of two lobes then encodes the depth of the emitters, offering depth detection of several micrometers. The figure below shows both the instruments and raw images at different depths.

langmuirads

Due to the complexity and flexibility of the 3D point spread functions, well-accepted recovery algorithms independent of point spread functions are still missing. We introduced a general 3D super-resolution recovery algorithm that works for arbitrary phase masks generating 3D point spread functions.

langmuirads

We have demonstrated in simulation that our advanced algorithm can recover 3D super-resolution images measured by a 3D microscope using phase masks. In development of our algorithm, we have made use of the state-of-art techniques in signal processing and optimization, as well as advanced computation resources to achieve better algorithm performance and faster computation speed.

State Identification from Noisy Data

Förster Resonance Energy Transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. However, the information embedded in efficiency traces can be easily missing as a result of low signal to noise (SNR) level as well as events of photoblinking. We applied a wavelet denoising method and Bayesian inference to boost the SNR and identify fluorophore photoblinking in the time trajectories. We also proposed an efficient state detection method Fast Step Transition and State Identification (STaSI) based on minimum description length (MDL).

langmuirads

The denoising method is applied to simulated and observed smFRET data, and it is found that the denoised data retain their underlying dynamic properties, but with increased resolution.

langmuirads

The STaSI method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis.

Kisley, L.; Brunetti, R.; Tauzin, L. J.; Shuang, B.; Yi, X.; Kirkeminde, A. W.; Higgins, D. A.; Weiss, S; Landes, C.F. Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging (fcsSOFI). ACS Nano, 2015, 9,9158–9166.

Taylor, J. N.; Li, C. B.; Cooper, D. R.; Landes, C. F.; Komatsuzaki, T. Error-based Extraction of Effective Free Energy Landscapes from Experimental Single-Molecule Time-Series. Sci. Rep. 2015, 5, 9174

Chen, J; Poddar, N. K.; Tauzin, L. J.; Cooper, D.; Kolomeisky, A. B.; Landes, C. F. Single-Molecule FRET Studies of HIV TAR- DNA Hairpin Unfolding Dynamics.J. Phys. Chem. B, 2014, 118(42),12130–12139

Shuang, B.; Cooper, D.; Taylor, J. N.; Kisley, L.; Chen, J.; Wang, W.; Li, C. B.; Komatsuzaki, T.; Landes, C. F. Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis J. Phys Chem Lett., 2014, 5 (18),3157-3161

Shuang, B.; Chen, J.; Kisley, L.; Landes, C. F. Troika of Single Particle Tracking Programing: SNR Enhancement, Particle Identification, and Mapping Phys. Chem. Chem. Phys., 2014, 16, 624-634

Hu, Z.; Adachi, T.; Haws, R.T.; Shuang, B.; Ono, R.J.; Bielawski, C.W.; Landes, C.F.; Rossky, P.J.; Vanden Bout, D.A. Excitonic Energy Migration in Conjugated Polymers: The Critical Role of Interchain Morphology JACS, 2014, 136 (45),16023–16031

Shuang, B.; Byers, C.P.; Kisley, L.; Wang, L. Y.; Zhao, J.; Morimura, H.; Link, S.; Landes, C. F. Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking. Langmuir, 2013,29(1), 228-234.

Kisley, L.; Chang, W. -S.; Cooper, D.; Mansur, A. P.; Landes, C. F. Extending single molecule fluorescence observation time by amplitude modulated excitation. Methods Appl. Fluoresc., 2013, 1, 037001.

Landes, C.F.; Rambhadran, A; Taylor, J. N.; Salatan, F.; Jayaraman, V. Structural landscape of isolated agonist-binding domains form single AMPA receptors. Nature Chemical Biology, 2011,7, 168-173

Taylor, J. N.; Landes, C.F. Improved Resolution of Complex Single-Molecule FRET Systems via Wavelet Shrinkage. J. Phys. Chem. B, 2011, 115, 1105-1114.

Taylor, J. N.; Makarov, D.E.; Landes, C.F. Denoising Single-Molecule FRET Trajectories with Wavelets and Bayesian Inference. Biophys. J., 2010, 98, 164-173.

Interfacial Chemistry and Dynamics

Catalysis, pharmaceutical separations, water purification, and enzyme activity all rely on interfacial physical and chemical processes such as adsorption, redox, and diffusion. Local structural features across a sample interface create differences in reactivity and adsorption between specific sites that can only be uncovered using single molecule spectroscopy. In the Landes lab, we study chemistry at a wide variety of interfaces, from adsorption of protein on porous films to sensing reactions on metal nanoparticle surfaces.

Interfacial Adsorption of Biomolecules

We have investigated the tunable adsorption dynamics of small molecules in weak polyelectrolyte films and multilayers. Currently we are applying our experience with single-molecule fluorescence microscopy to study the adsorption of different proteins (such as α-lactalbumin (α-LA)) labeled with dye molecules (such as Alexa 555) on chemically identical but morphologically heterogeneous polymer surfaces. The adsorption kinetics of spatially-resolved single molecules to polymer films can be quantified by kinetic adsorption models. Such single-molecule based kinetic studies may be extended to various protein polymer interactions.

   proteinads

The adsorption of single molecules on a polymer film is described by the Langmuir adsorption model.

langmuirads

With this adsorption model we did experiments on two polymer films with different surface profiles. Single protein molecule adsorption based on pore size and protein concentration are shown below.

   adsdata

Protein Separation By Charged Particles

Traditional chromatography uses single charged peptides to separate proteins, but we showed that protein adsorption occurs primarily at sites with clustered peptides using super-resolution microscopy. In the images below, two stationary phases for protein separations were compared – one with isolated single-charged peptides and one with engineered clustered-charges. Using super-resolution wide field imaging, we showed only recurrent, long-lived localized protein association/dissociation occurred at intentionally clustered-charge adsorbents that only happen accidentally when using traditional single-charges. In addition, by fluorescence correlation spectroscopy we have quantified heterogeneous diffusion dynamics at two separate locations at a clustered-charge peptide interface due to variation in peptide distribution.

superresads

Electrochemistry at Nanoparticle Interfaces

Active control of the optical and electronic properties of nanoparticles is critical for many future technological applications (give examples), yet remains largely elusive. We focus on the study of charge transfer between nanoparticles and work on the active control over the nanoparticle optical properties.

We are applying electrochemical methods to tune both the plasmon response and activate specific chemical reactions in single nanoparticles. Electrochemical methods afford unrivaled control of surface chemistry at metal electrodes but are classically built upon bulk electrochemical current, potential, and charge relationships. Changes in surface charge density of gold nanoparticles can also be detected through changes in the surface plasmon resonance energy. The use of these spectral characteristics to infer electrochemical processes is the subject of nanoparticle plasmon spectroelectrochemistry.

echemsetup

The optical properties of metallic nanoparticles, particularly when in close proximity to each other, are highly sensitive to interparticle distance, frequently giving rise to dramatic, irreversible color changes. By electrochemical modification of individual nanoparticles and nanoparticle pairs, we induced equally dramatic, yet reversible, changes in their optical properties. We achieved reversible plasmon tuning by oxidation - reduction chemistry of Ag- AgCl shells on the surfaces of both individual and strongly coupled Au nanoparticle pairs, resulting in extreme but reversible changes in scattering line shape.

echemdimer

Byers, C. P.; Zhang, H.; Swearer, D. F.; Yorulmaz, M.; Hoener, B. S.; Huang, D.; Hoggard, A.; Chang, W.-S.; Mulvaney, P.; Ringe, E.; Halas, N. J.; Nordlander, P.; Link, S.; Landes, C.F. From tunable core-shell nanoparticles to plasmonic drawbridges: Active control of nanoparticle optical properties Science Advances, 2015, 111

Landes, C.F. Single-molecule tracking and super-resolution imaging shed light on cholera toxin transcription activation. Molecular Microbiology. 2015, 96, 1-3

Kisley, L.; Landes, C. F.; Molecular Approaches to Chromatography Using Single Molecule Spectroscopy. Anal. Chem. 2015, 87,83-98.

Kisley, L.; Poongavanam, M. -V.; Kourentzi, K.; Willson, R.C.; Landes, C. F. pH-dependence of single-protein adsorption and diffusion at a liquid chromatographic interface. J. Sep. Sci. Accepted.

Poongavanam, M. -V.; Kisley, L.; Kourentzi, K.; Willson, R.C.; Landes, C. F. Ensemble and Single-Molecule Biophysical Characterization of D17.4 DNA Aptamer-IgE Interactions. BBA Proteins Proteom. ASAP.

Landes, C.F.; Link. S.; Wine, P.H.; Zhang, Z.J. 2013 Southeastern Regional ACS Meeting. Nanochemistry and Spectroscopy: Symposium Honoring Mostafa El-Sayed. J. Phys. Chem. C. 2014. 14009-14009.

Kisley, L.; Chen, J.; Mansur, A. P.; Dominguez-Medina, S.; Kulla, E.; Kang, M.; Shuang, B.; Kourentzi, K.; Poongavanam, M. -V.; Dhamane, S.; Willson, R.C.; Landes, C. F. High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study. J. Chromatogr. A., 2014, 1343, 135-142.

Kisley, L.; Chen, J.; Mansur, A. P.; Shuang, B.; Kourentzi, K.; Poongavanam, M. -V.; Chen, W. -H.; Dhamane, S.; Willson, R.C.; Landes, C. F. Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations. Proc. Natl. Acad. Sci., 2014, 111, 2075-2080.

Tauzin, L. J.; Shuang, B.; Kisley, L. M.; Mansur, A. P.; Chen, J.; Leon, A.; Advincula, R. C.; Landes, C. F. Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer. Langmuir, 2014, 30, 8391-8399

Byers, C. P.; Hoener, B. S.; Chang, W.S.; Yorulmaz, M.; Link, S.; Landes, C. F. Single-Particle Spectroscopy Reveals Heterogeneity in Electrochemical Tuning of the Localized Surface Plasmon. J. Phys. Chem. B, 2014, 118 (49), pp 14047–14055

Daniels C. R.; Tauzin L. J.; Foster E.; Advincula R. C.; Landes, C. F. On the pH-Responsive, Charge-Selective, Polymer-Brush-Mediated Transport Probed by Traditional and Scanning Fluorescence Correlation Spectroscopy. J. Phys. Chem. B,2013, 117(16), 4284–4290

Chen, J.; Bremauntz, A.; Kisley, L.; Shuang, B.; Landes, C. F. Super-Resolution mbPAINT for Optical Localization of Single- Stranded DNA. ACS Appl. Mater. Interfaces, 2013, 5, 9338-9343.

Biophysics for Conformational Dynamics of Biomolecules

In the Landes lab we use single molecule spectroscopy to study the structure and dynamics of biomolecules. Understanding the structure and conformation dynamics of protein, DNA, and other common biomolecules found in the human body as they interact with natural and man-made targets is important to for understanding complex diseases such as Alzheimer’s and HIV. To observe transitions between structural states of biomolecules in situ, single molecule measurements are required. Therefore, we use Förster resonance energy transfer (FRET) to determine structures of protein molecules and dynamics of DNA molecules.

Glutamate Receptors

Glutamate receptors perform a critical role in the nervous system, mediating synaptic transmission through cellular membranes. These proteins have developed a quick response to agonist presence and, when activated, undergo a conformational shift that allows calcium ion transport across the membrane.

In collaboration with Prof. Vasanthi Jayaraman and her group at UTHSC, we are studying structural dynamics of single soluble domain glutamate receptor proteins, to search for heterogeneity in structure/transport mechanisms, using single molecule fluorescence resonance energy transfer to observe the bound form of the glutamate receptors and subsequent desensitization process. For a complete understanding of the desensitization mechanism we would like to determine if the desensitization pathway is conserved between the different types of glutamate receptors. Mutations in the binding domain could change the dynamics of the glutamate receptors and should be investigated. Full and partial agonist response will also help to provide a complete picture of the possible heterogeneous mechanisms for activation and to determine the correlation between binding affinity and the desensitization process.

glutamate

DNA Sequencing Using Super-Resolution Techniques

While the Human Genome Project to determine the full human DNA sequence was successfully completed in 2003, there is still much work required to fully understand how mutations in DNA sequences contribute to large scale functional problems and diseases. Genome optical mapping has previously been used as a tool for genome sequencing, but until recently the resolution of genome optical mapping was limited by the diffraction limit of light. While a postdoc in the Landes lab, Dr. Jixin Chen, now an Assistant Professor at Ohio University,developed a super-resolution method to map single strand DNA called motion blur point accumulation for imaging in nanoscale topography (mbPAINT).

dnasuperres

Single strand target DNA (tDNA) was immobilized on a glass substrate and probe DNA (pDNA), having a complementary sequence to the tDNA and labeled with fluorescent dye (Alexa 532), was flowed over the substrate. A homebuilt microscope using total internal reflection fluorescence (TIRF) excitation geometry was used to excite single Alexa 532 molecules and measure single pDNA adsorption events. The fluorescence intensity from each excitation even was fit to a 2D-Gaussian and the peak was marked in the x and y plane to build a super resolution image of the single strand tDNA location with the complementary sequence to the pDNA. This technique to improve the spatial resolution of genome optical mapping is currently being developed by Dr. Chen at Ohio University, and we are excited to see what he discovers!

TAR-DNA

The melting and annealing of DNA hairpins are essential in many biological processes such as replication, transcription, recombination, gene expression, and DNA transposition for both prokaryotic and eukaryotic systems. Furthermore, hairpins with multiple loops are known to play specific roles in viral replication.

We directly measured the dynamics of the HIV trans-activation response (TAR)–DNA hairpin with multiple loops using single-molecule Förster resonance energy transfer (smFRET) methods. Multiple FRET states were identified that correspond to intermediate melting states of the hairpin. In our research in DNA molecules we figured out that hairpin unfolding obeys a “fraying and peeling” mechanism, and evidence for the collapse of the ends of the hairpin during folding is observed.

TARDNA

Cooper, D.; Dolino, D.; Jaurich, H.; Shuang, B.; Ramaswamy, S.; Nurik, C.E.; Chen, J.; Jayaraman, V.; Landes, C. F. Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET Biophys.J, 2015, 109, 66 - 75

Dolino, D.; Cooper, D.; Ramaswamy, S.; Henriette Jaurich, H.; Landes, C. F.; Jayaraman, V. Structural Dynamics of the Glycine-Binding Domain of the N-Methyl-D-Aspartate Receptor. J. Biol. Chem., 2015, 290, 797-804.

Shuang, B.; Cooper, D.; Taylor, J. N.; Kisley, L.; Chen, J.; Wang, W.; Li, C. B.; Komatsuzaki, T.; Landes, C. F. Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis J. Phys Chem Lett., 2014, 5 (18), 3157-3161

Chen, J; Poddar, N. K.; Tauzin, L. J.; Cooper, D.; Kolomeisky, A. B.; Landes, C. F. Single-Molecule FRET Studies of HIV TAR- DNA Hairpin Unfolding Dynamics.J. Phys. Chem. B, 2014, 118(42),12130–12139

Chen, J.; Bremauntz, A.; Kisley, L.; Shuang, B.; Landes, C. F. Super-Resolution mbPAINT for Optical Localization of Single- Stranded DNA. ACS Appl. Mater. Interfaces, 2013, 5, 9338-9343.

Ramaswamy, S.; Cooper, D; Poddar, N.; Rambhadran, A.; Taylor, J. N.; Uhm, H.; Landes, C.F.; Jayaraman, V. Partial agonism of AMPA receptors studied by single molecule FRET investigations. J. Biol. Chem., 2012, 287, 43557-43564.

Landes, C.F.; Rambhadran, A; Taylor, J. N.; Salatan, F.; Jayaraman, V. Structural landscape of isolated agonist-binding domains form single AMPA receptors. Nature Chemical Biology, 2011 , 7, 168-173