Selective detection of protein crystals by second harmonic microscopy.

The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.

An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles.

Controlled assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. Here we present a novel electrochemical alignment technique to control the assembly of type-I collagen molecules into highly oriented and densely packed elongated bundles at the macroscale. The process involves application of electric currents to collagen solutions which in turn generate a pH gradient. Through an isoelectric focusing process, the molecules migrate and congregate within a plane. It was possible to fabricate collagen bundles with 50-400 microm diameter and several inches length via this process. The current study assessed the orientational order, and the presence of fibrillar assembly in such electrochemically oriented constructs by polarized optical microscopy, small angle X-ray scattering, second harmonic generation, and electron microscopy. The mechanical strength of the aligned crosslinked collagen bundles was 30-fold greater than its randomly oriented-crosslinked counterpart. Aligned crosslinked collagen bundles had about half the strength of the native tendon. Tendon-derived fibroblast cells were able to migrate and populate multiple macroscopic bundles at a rate of 0.5mm/day. The anisotropic order within biocompatible collagenous constructs was conferred upon the nuclear morphology of cells as well. These results indicate that the electrochemically oriented collagen scaffolds carry baseline characteristics to be considered for tendon/ligament repair.

Discrete retardance second harmonic generation ellipsometry.

A new instrument was constructed to perform discrete retardance nonlinear optical ellipsometry (DR-NOE). The focus of the design was to perform second harmonic generation NOE while maximizing sample and application flexibility and minimizing data acquisition time. The discrete retardance configuration results in relatively simple computational algorithms for performing nonlinear optical ellipsometric analysis. NOE analysis of a disperse red 19 monolayer yielded results that were consistent with previously reported values for the same surface system, but with significantly reduced acquisition times.

Assembly of dithiocarbamate-anchored monolayers on gold surfaces in aqueous solutions.

Dithiocarbamates (DTCs) can be formed by the in situ condensation of polar alkylamines with CS 2, and assembled into dithiocarbamate-anchored monolayers (DAMs) on Au substrates in aqueous solutions. Primary and secondary amines can both be used to prepare DTCs, but have significant differences in their reactivities and product stabilities. Ultraviolet absorption spectroscopy provides a convenient method for monitoring in situ DTC formation as well as the formation of potential byproducts. The kinetics of DAM assembly on Au substrates as measured by second harmonic generation (SHG) indicated first-order rate processes and saturation coverages similar to those of alkanethiols on Au. However, the rate of adsorption did not change with DTC concentration in a manner expected of Langmuir kinetics, and is attributed to the competitive adsorption of alkylammonium counterions to the freshly oxidized Au substrate. These analyses establish a practical range of conditions for preparing DAMs from polar amines using in situ DTC formation.