Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy.

Chemical characterisation of sodium starch glycolate particles.

The internal and surface chemical compositions of three sodium starch glycolate (SSG) products, Explotab, Primojel and Vivastar P were studied using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), time of flight secondary ion mass spectrometry (TOF-SIMS) and 23Na nuclear magnetic resonance spectroscopy (NMR). The surfaces of Explotab and Primojel contained very distinct features containing Na and Cl, however, Primojel also contained features which contained Na which may reflect the presence of Na glycolate and/or Na citrates. Vivastar P contained relatively few surface Cl containing features. Analysis of cross-sections of the particles showed that Na appeared to be uniformly distributed throughout the particles of all the products. Additionally, there was a significant concentration of Cl in the periphery of Explotab and Primojel. In the case of Vivastar P, significant levels of Na and Cl were detected in the internal regions of the particles which, together with 23Na NMR, suggests that NaCl is uniformly distributed within Vivastar P. 23Na NMR also suggested that the ratio of organic Na to NaCl was considerably lower in Vivastar P than Primojel and Explotab. Overall, even though all these three products satisfy the pharmacopeial descriptions of SSG, these studies suggest that Primojel and Explotab exhibit different chemical compositions to Vivastar P. Since the three products studied are reported to be prepared from potato starch, the apparent differences in chemical composition probably reflect the different manufacturing processes used, however, batch to batch variations may account for some of the subtle differences.

Enhanced TOF-SIMS imaging of a micropatterned protein by stable isotope protein labeling.

Patterning of biomolecules on surfaces is an increasingly important technological goal. Because the fabrication of biomolecule arrays often involves stepwise, spatially resolved derivatization of surfaces, spectroscopic imaging of these arrays is important in their fabrication and optimization. Although imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a powerful method for spatially resolved surface analysis, TOF-SIMS images of micropatterned proteins on organic substrates can be difficult to acquire, because of the lack of high intensity, protein-specific molecular ions that are essential for imaging under static conditions. In contrast, low-mass ions are of suitable intensity for imaging, but can originate from different chemical species on the surface. A potential solution to this problem is to utilize stable isotope labeled proteins, an approach that has heretofore not been explored in TOF-SIMS imaging of micropatterned proteins and peptides. To investigate the feasibility of stable isotope enhanced TOF-SIMS imaging of proteins, we synthesized 15N-labeled streptavidin by labeling of the protein during expression from a recombinant gene. The spatial distribution of streptavidin bound to biotin micropatterns, fabricated on a polymer and on a self-assembled monolayer on gold, was imaged by TOF-SIMS. Imaging of high-intensity, low-m/z secondary ions (e.g., C15N-) unique to streptavidin enabled unambiguous spatial mapping of the micropatterned protein with a lateral resolution of a few micrometers. TOF-SIMS imaging of micropatterned 15N-labeled streptavidin also illustrated the exquisite sensitivity of TOF-SIMS to low fractional coverage of protein (5 A effective thickness) in the background regions of the protein micropattern.

TOF-SIMS characterization and imaging of controlled-release drug delivery systems.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used for the analysis of multilayer drug beads that serve as controlled-release drug delivery systems. TOF-SIMS analysis of a cross section of each bead system allowed molecular chemical information to be gained from all of the layers simultaneously, in situ. The integrity of each of the layers was evaluated through imaging of specific ion species for the drug, excipient, and coating materials. The three beads in this study each showed a unique distribution of ingredients. Images of the parent molecular ion for each drug (theophylline, paracetamol, prednisolone) showed their distribution ranged from micrometer-sized particles in one bead cross section to almost homogeneous in another bead cross section. The chemical composition of each of the layers in the beads was evaluated through mass spectrometry; the ingredients did not always match the manufacturer's specification. In addition, many common drug bead ingredients were analyzed as pure substances, providing TOF-SIMS reference spectra of these materials for the first time.

Characterization of combinatorially designed polyarylates by time-of-flight secondary ion mass spectrometry.

A series of 16 polyarylates, with well-controlled and systematically varying chemistry, has been characterized by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The polymers are structurally identical except for the incremental additions of C2H4 units to the backbone and sidechain. From the spectra, peaks characteristic of all polyarylates are identified. Furthermore, evaluation of the spectra and identification of unique signals allow classification of the polyarylates according to sidechain and backbone chemistry.

A new method for straightening DNA molecules for optical restriction mapping.

We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.

Evaluation of matrix-assisted laser desorption ionization mass spectrometry for polymer characterization.

A protocol for the preparation of polymeric samples for time-of-flight matrix-assisted laser desorption ionization mass spectrometry (TOF-MALDI-MS) analysis was developed. Dithranol was identified as a good matrix for polystyrene (PS), and the addition of silver for cationization of molecules was determined to be necessary. Based on this preparative method, low molecular weight samples of other polymers [polyisoprene, polybutadiene, poly(ethylene oxide), poly(methyl methacrylate), and polydimethylsiloxane] were analyzed with molecular weights up to 49 ku. The effects of laser intensity were determined to influence the molecular weight distribution of intact oligomers, most significantly for low molecular weight polymers. Linear and reflectron modes of analysis were evaluated; better signal intensity and resolution were obtained in the reflectron mode. The TOF-MALDI-MS measurements are compared with time-of-flight secondary ion mass spectrometry (TOF-SIMS) and gel permeation chromatography (GPC) for the same polymers. The M n values calculated by TOF-MALDI-MS consistently are higher than values calculated by TOF-SIMS for all classes of polymers with molecular weights up to 8 ku. The molecular weights of the PS calculated from TOF-MALDI-MS are in good agreement with GPC (±10%). The composition of the terminal group on a polymer chain may affect the ion yields. The ion yields of intact oligomers were evaluated as a function of end group composition for both TOF-MALDI-MS and TOF-SIMS. The slight disparity of results between TOF-SIMS and TOF-MALDI-MS for the perfluoroalkyl-terminated PS suggests that the oligomers are desorbed preferentially from the surface in the TOF-SIMS analysis, rather than having an increased ionization probability.