Maryana Escalante

Membrane invagination in Rhodobacter sphaeroides is initiated at curved regions of the cytoplasmic membrane, then forms both budded and fully detached spherical vesicles.

The purple phototrophic bacteria synthesize an extensive system of intracytoplasmic membranes (ICM) in order to increase the surface area for absorbing and utilizing solar energy. Rhodobacter sphaeroides cells contain curved membrane invaginations. In order to study the biogenesis of ICM in this bacterium mature (ICM) and precursor (upper pigmented band – UPB) membranes were purified and compared at the single membrane level using electron, atomic force and fluorescence microscopy, revealing fundamental differences in their morphology, protein organization and function. Cryo‐electron tomography demonstrates the complexity of the ICM of Rba. sphaeroides. Some ICM vesicles have no connection with other structures, others are found nearer to the cytoplasmic membrane (CM), often forming interconnected structures that retain a connection to the CM, and possibly having access to the periplasmic space. Near‐spherical single invaginations are also observed, still attached to the CM by a ‘neck’. Small indents of the CM are also seen, which are proposed to give rise to the UPB precursor membranes upon cell disruption. ‘Free‐living’ ICM vesicles, which possess all the machinery for converting light energy into ATP, can be regarded as bacterial membrane organelles.

Assembly of Bionanostructures onto β-Cyclodextrin Molecular Printboards for Antibody Recognition and Lymphocyte Cell Counting

The assembly of complex bionanostructures onto beta-cyclodextrin (betaCD) monolayers has been investigated with the aims of antibody recognition and cell adhesion. The formation of these assemblies relies on host-guest, protein-ligand, and protein-protein interactions. The buildup of a structure consisting of a divalent bis(adamantyl)-biotin linker, streptavidin (SAv), biotinylated protein A (bt-PA), and an Fc fragment of a human immunoglobin G (IgG-Fc) was studied with surface plasmon resonance (SPR) spectroscopy. Patterns of this bionanostructure were obtained via microcontact printing of the divalent linker at the molecular printboard, followed by the subsequent attachment of the proteins. Fluorescence microscopy showed that the buildup of these bionanostructures on the betaCD monolayers is highly specific. On the basis of these results, bionanostructures were made in which whole antibodies (ABs) were used instead of the IgG-Fc. These ABs were bound to the SAv layer via biotinylated protein G (bt-PG) or via a biotinylated AB. These constructions yielded specifically bound ABs with a less than maximal density, as shown by SPR spectroscopy and atomic force microscopy (AFM). Finally, the immobilization of ABs to the molecular printboard was used to create platforms for lymphocyte cell count purposes. Monoclonal ABs (MABs) were attached to the SAv layer using bt-PG, an engineered biotin functionality, or through nonspecific adsorption. The binding specificity of the immobilized cells was the highest on the buildup made from bt-PG, which is attributed to an optimized orientation of the antibodies. An approximately linear relationship between the numbers of seeded cells and counted cells was demonstrated, rendering the platform potentially suitable for lymphocyte cell counting.

Nanometer arrays of functional light harvesting antenna complexes by nanoimprint lithography and host--guest interactions

We show an approach based on a combination of site-directed mutagenesis, NIL and multivalent host-guest interactions for the realization of engineered ordered functional arrays of purified components of the photosynthetic system, the membrane-bound LH2 complex. In addition to micrometer-scale patterned structures, we demonstrated the use of nanometer-scale hard NIL stamps to generate functional protein arrays approaching molecular dimensions.

Long-Range Energy Propagation in Nanometer Arrays of Light Harvesting Antenna Complexes

Here we report the first observation of long-range transport of excitation energy within a biomimetic molecular nanoarray constructed from LH2 antenna complexes from Rhodobacter sphaeroides. Fluorescence microscopy of the emission of light after local excitation with a diffraction-limited light beam reveals long-range transport of excitation energy over micrometer distances, which is much larger than required in the parent bacterial system. The transport was established from the influence of active energy-guiding layers on the observed fluorescence emission. We speculate that such an extent of energy migration occurs as a result of efficient coupling between many hundreds of LH2 molecules. These results demonstrate the potential for long-range energy propagation in hybrid systems composed of natural light harvesting antenna molecules from photosynthetic organisms.

Elastocapillary filling of deformable nanochannels

The capillary filling speed of wetting liquids of varying viscosity and surface tension in hydrophilic nanochannels with an elastic capping layer has been analyzed. The channels, with a height just below 80 nm, are suspended by a thin flexible membrane that easily deforms due to the negative pressure which develops behind the moving meniscus. In the elastocapillary filling of the channels, two opposite effects compete: the decreased cross channel sections increase the flow resistance, while the Laplace pressure that acts as the driving force becomes more negative due to the increased meniscus curvature. Although the meniscus position shows a square root of time behavior as described by the Washburn relation, the net result of the induced bending of the membranes is a definite increase of the filling speed. We propose a relatively straightforward model for this elastocapillary process and present experimental results of the filling speed of ethanol, water, cyclohexane and acetone that are found to be in good agreement with the presented model, for membrane deflections of up to 80 percent of the original channel height.

Multimode microscopy: spectral and lifetime imaging

Multimode microscopy exploits the measurement of multiple spectroscopic parameters to yield a wealth of spatially resolved spectroscopic detail about the sample under study. Here, we describe the realization of a multimode microscope capable of wide-field transmission, reflectivity and emission imaging. The instrument also incorporates confocal spectral and lifetime imaging enabling convenient high-content imaging of complex samples, allowing the direct correlation of the data obtained from the different modes. We demonstrate the versatility of this imaging platform by reviewing applications to the modulation of fluorescent protein emission by inverse opal photonic crystals, to the detection and visualization of J-aggregate coupling of small molecule dyes intercalated into nanochannels in zeolites and to the visualization of fluorescent proteins micropatterned onto surfaces. In all cases, the combination of different microspectroscopic modes is essential for the resolution of specific photophysical details of the complex systems in question.

Capillary Negative Pressure Measured by Nanochannel Collapse

A new method is presented to measure capillarity-induced negative pressure. Negative pressures of several bars have been measured for five different liquids (ethanol, acetone, cyclohexane, aniline, and water) over a range of surface tension. Capillary negative pressure was measured in 79 +/- 3 nm silica nanochannels on the basis of the determination of the critical channel width for elastocapillary collapse of the flexible plate covering the channels. The results are consistent with the Young-Laplace equation.

FRET Pair Printing of Fluorescent Proteins

We report for the first time the directed assembly and characterization of FRET pairs on micrometer patterned surfaces. We used visible fluorescent proteins expressing a hexahistidine affinity tag as component molecules for the construction of the FRET constructs, where His(6)-EGFP served as donor fluorophore and His(6-)DsRed-FT as the acceptor. We created 2D and 3D structures that exhibit fluorescence resonance energy transfer at the interfaces between the donor and acceptor patterns in the lateral or axial directions, respectively. We quantitatively visualized the energy transfer by multiparameter optical microscopy.

Supporting data of spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo

This data article contains seven figures and two tables supporting the research article entitled: spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo[1]. The data explain the culture of stromal cells in vitro in three culture systems: discs, scaffolds and scaffolds in a perfusion bioreactor system. Also, quantification of extracellular matrix components (ECM) in vitro and staining of ECM components in vivo can be found here. Finally the quantification of blood vessels dimensions from CD31 signals and representative histograms of stanniocalcin-1 fluorescent signals in negative controls and experimental conditions in vivo are presented.

Erratum: “Elastocapillary filling of deformable nanochannels” [J. Appl. Phys. 101, 094310 (2007)]

In the Experimental section, a mistake has been made in the mentioned value of the advancing contact angle for water ring capillary filling. The contact angle mentioned is the contact angle that was measured before the 1100 °C anneal of the