Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-µm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

Molecular dynamic-secondary ion mass spectrometry (D-SIMS) ionized by co-sputtering with C60+ and Ar+.

Dynamic secondary ion mass spectrometry (D-SIMS) analysis of poly(ethylene terephthalate) (PET) and poly(methyl methacrylate) (PMMA) was conducted using a quadrupole mass analyzer with various combinations of continuous C(60)(+) and Ar(+) ion sputtering. Individually, the Ar(+) beam failed to generate fragments above m/z 200, and the C(60)(+) beam generated molecular fragments of m/z ~1000. By combining the two beams, the auxiliary Ar(+) beam, which is proposed to suppress carbon deposition due to C(60)(+) bombardment and/or remove graphitized polymer, the sputtering range of the C(60)(+) beam is extended. Another advantage of this technique is that the high sputtering rate and associated high molecular ion intensity of the C(60)(+) beam generate adequate high-mass fragments that mask the damage from the Ar(+) beam. As a result, fragments at m/z ~900 can be clearly observed. As a depth-profiling tool, the single C(60)(+) beam cannot reach a steady state for either PET or PMMA at high ion fluence, and the intensity of the molecular fragments produced by the beam decreases with increasing C(60)(+) fluence. As a result, the single C(60)(+) beam is suitable for profiling surface layers with limited thickness. With C(60)(+)-Ar(+) co-sputtering, although the initial drop in intensity is more significant than with single C(60)(+) ionization because of the damage introduced by the auxiliary Ar(+), the intensity levels indicate that a more steady-state process can be achieved. In addition, the secondary ion intensity at high fluence is higher with co-sputtering. As a result, the sputtered depth is enhanced with co-sputtering and the technique is suitable for profiling thick layers. Furthermore, co-sputtering yields a smoother surface than single C(60)(+) sputtering.

The role of the auxiliary atomic ion beam in C60(+)-Ar+ co-sputtering.

Cluster ion sputtering has been proven to be an effective technique for depth profiling of organic materials. In particular, C(60)(+) ion beams are widely used to profile soft matter. The limitation of carbon deposition associated with C(60)(+) sputtering can be alleviated by concurrently using a low-energy Ar(+) beam. In this work, the role of this auxiliary atomic ion beam was examined by using an apparatus that could analyze the sputtered materials and the remaining target simultaneously using secondary ion mass spectrometry (SIMS) and X-ray photoelectron spectrometry (XPS), respectively. It was found that the auxiliary 0.2 kV Ar(+) stream was capable of slowly removing the carbon deposition and suppresses the carbon from implantation. As a result, a more steady sputtering condition was achieved more quickly with co-sputtering than by using C(60)(+) alone. Additionally, the Ar(+) beam was found to interfere with the C(60)(+) beam and may lower the overall sputtering rate and secondary ion intensity in some cases. Therefore, the current of this auxiliary ion beam needs to be carefully optimized for successful depth profiling.

ToF-SIMS imaging of the nanoscale phase separation in polymeric light emitting diodes: effect of nanostructure on device efficiency.

The nanostructure of the light emissive layer (EL) of polymer light emitting diodes (PLEDs) was investigated using force modulation microscopy (FMM) and scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) excited with focused Bi(3)(2+) primary beam. Three-dimensional nanostructures were reconstructed from high resolution ToF-SIMS images acquired with different C(60)(+) sputtering times. The observed nanostructure is related to the efficiency of the PLED. In poly(9-vinyl-carbazole) (PVK) based EL, a high processing temperature (60 °C) yielded less nanoscale phase separation than a low processing temperature (30 °C). This nanostructure can be further suppressed by replacing the host polymer with poly[oxy(3-(9H-9-carbazol-9-ilmethyl-2-methyltrimethylene)] (SL74) and poly[3-(carbazol-9-ylmethyl)-3-methyloxetane] (RS12), which have similar chemical structures and energy levels as PVK. The device efficiency increases when the phase separation inside the EL is suppressed. While the spontaneous formation of a bicontinuous nanostructure inside the active layer is known to provide a path for charge carrier transportation and to be the key to highly efficient polymeric solar cells, these nanostructures are less efficient for trapping the carrier inside the EL and thus lower the power conversion efficiency of the PLED devices.

Effect of the chemical composition on the work function of gold substrates modified by binary self-assembled monolayers.

This study demonstrated that the work function (Φ) of Au substrates can be fine-tuned by using series ratios of binary self-assembled monolayers (SAMs). By using pure amine- and carboxylic acid-bearing alkanethiol SAM on gold substrates, Φ of Au changed from 5.10 to 5.16 and 5.83, respectively, as determined by ultra-violet photoelectron spectrometry (UPS). The shift in Φ due to the use of different functional groups was rationalized by considering the dipole moments of the molecules anchored on the Au surface. A series of binary SAMs were fabricated by mixing carboxylic acid- and amine-terminated alkanethiols in the deposition solution. By mixing these functional groups in SAMs, a linear correlation between Φ with respect to chemical composition (hence the effective dipole moment on the Au surface) was observed. It was found that arbitrary Φ between extremes (5.16 and 5.83) controlled by respective functional groups can be obtained by changing the chemical composition of SAMs. The Scanning Kelvin Probe (SKP) was also used to measure the contact potential difference (CPD) between SAMs and referencing Au on a patterned substrate prepared by photo-lithography. It was found that the CPD of SAMs with different chemical compositions correlates to their Φ. However, the magnitude of the CPD was smaller than the difference in Φ measured by UPS that was possibly due to the adsorption of contaminants in air.

Adsorption behavior of plasmid DNA on binary self-assembled monolayers modified gold substrates.

Gold is known to have good biocompatibility because of its inert activity and the surface property can be easily tailored with self-assembled monolayers (SAMs). In previous works, gold surfaces were tailored with homogeneously mixed amine and carboxylic acid functional groups to generate surfaces with a series of isoelectronic points (IEPs). In other words, by tailoring the chemical composition in binary SAMs, different surface potentials can be obtained under controlled pH environments. To understand how the surface potentials affect the interaction at the interface, a binary-SAMs-modified Au electrode on a quartz crystal microbalance with dissipation detection (QCM-D) was used owing to the high weight sensitivity of QCM-D. In QCM-D, the frequency shift and the energy dissipation are monitored simultaneously to determine the adsorption behaviors of the plasmid DNA to surfaces of various potentials in Tris-buffered NaCl solutions of different pH. The results revealed that the plasmid DNA can be adsorbed on the SAM-modified surfaces electrostatically; thus, in general, the amount of adsorbed plasmid DNA decreased with increasing environmental pH and the decreasing ratio of the amine functional groups on the surfaces owing to weaker positive potentials on the surface. For the high amine-containing surfaces, due to the strong electrostatic attraction, denser films were observed, and thus, the apparent thickness decreased slightly. The negatively charged carboxylic acid surfaces can still adsorb the negatively charged plasmid DNA at some conditions. In other words, the electrostatic model cannot explain the adsorption behavior completely, and the induced dipole (Debye) interaction between the charged and polarizable molecules needs to be considered as well.

Parallel detection, quantification, and depth profiling of peptides with dynamic-secondary ion mass spectrometry (D-SIMS) ionized by C60(+)-Ar(+) co-sputtering.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed C(60)(+) primary ions is a promising technique for analyzing biological specimens with high surface sensitivities. With molecular secondary ions of high masses, multiple molecules can be identified simultaneously without prior separation or isotope labeling. Previous reports using the C(60)(+) primary ion have been based on static-SIMS, which makes depth profiling complicated. Therefore, a dynamic-SIMS technique is reported here. Mixed peptides in the cryoprotectant trehalose were used as a model for evaluating the parameters that lead to the parallel detection and quantification of biomaterials. Trehalose was mixed separately with different concentrations of peptides. The peptide secondary ion intensities (normalized with respect to those of trehalose) were directly proportional to their concentration in the matrix (0.01-2.5 mol%). Quantification curves for each peptide were generated by plotting the percentage of peptides in trehalose versus the normalized SIMS intensities. Using these curves, the parallel detection, identification, and quantification of multiple peptides was achieved. Low energy Ar(+) was used to co-sputter and ionize the peptide-doped trehalose sample to suppress the carbon deposition associated with C(60)(+) bombardment, which suppressed the ion intensities during the depth profiling. This co-sputtering technique yielded steadier molecular ion intensities than when using a single C(60)(+) beam. In other words, co-sputtering is suitable for the depth profiling of thick specimens. In addition, the smoother surface generated by co-sputtering yielded greater depth resolution than C(60)(+) sputtering. Furthermore, because C(60)(+) is responsible for generating the molecular ions, the dosage of the auxiliary Ar(+) does not significantly affect the quantification curves.

Effect of fabrication parameters on three-dimensional nanostructures of bulk heterojunctions imaged by high-resolution scanning ToF-SIMS.

Solution processable fullerene and copolymer bulk heterojunctions are widely used as the active layers of solar cells. In this work, scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) is used to examine the distribution of [6,6]phenyl-C61-butyric acid methyl ester (PCBM) and regio-regular poly(3-hexylthiophene) (rrP3HT) that forms the bulk heterojunction. The planar phase separation of P3HT:PCBM is observed by ToF-SIMS imaging. The depth profile of the fragment distribution that reflects the molecular distribution is achieved by low energy Cs(+) ion sputtering. The depth profile clearly shows a vertical phase separation of P3HT:PCBM before annealing, and hence, the inverted device architecture is beneficial. After annealing, the phase segregation is suppressed, and the device efficiency is dramatically enhanced with a normal device structure. The 3D image is obtained by stacking the 2D ToF-SIMS images acquired at different sputtering times, and 50 nm features are clearly differentiated. The whole imaging process requires less than 2 h, making it both rapid and versatile.