High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators.

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10-15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.

Internalization and Viability Studies of Suspended Nanowire Silicon Chips in HeLa Cells.

Micrometer-sized silicon chips have been demonstrated to be cell-internalizable, offering the possibility of introducing in cells even smaller nanoelements for intracellular applications. On the other hand, silicon nanowires on extracellular devices have been widely studied as biosensors or drug delivery systems. Here, we propose the integration of silicon nanowires on cell-internalizable chips in order to combine the functional features of both approaches for advanced intracellular applications. As an initial fundamental study, the cellular uptake in HeLa cells of silicon 3 µm × 3 µm nanowire-based chips with two different morphologies was investigated, and the results were compared with those of non-nanostructured silicon chips. Chip internalization without affecting cell viability was achieved in all cases; however, important cell behavior differences were observed. In particular, the first stage of cell internalization was favored by silicon nanowire interfaces with respect to bulk silicon. In addition, chips were found inside membrane vesicles, and some nanowires seemed to penetrate the cytosol, which opens the door to the development of silicon nanowire chips as future intracellular sensors and drug delivery systems.

Imaging low-dimensional nanostructures by very low voltage scanning electron microscopy: ultra-shallow topography and depth-tunable material contrast.

We demonstrate the implications of very low voltage operation (<1 kV) of a scanning electron microscope for imaging low-dimensional nanostructures where standard voltages (2-5 kV) involve a beam penetration depth comparable to the cross-section of the nanostructures. In this common situation, image sharpness, contrast quality and resolution are severely limited by emission of secondary electrons far from the primary beam incidence point. Oppositely, very low voltage operation allows reducing the beam-specimen interaction to an extremely narrow and shallow region around the incidence point, enabling high-resolution and ultra-shallow topographic contrast imaging by high-angle backscattered electrons detection on the one hand, and depth-tunable material contrast imaging by low-angle backscattered electrons detection on the other. We describe the performance of these imaging approaches on silicon nanowires obtained by the vapor-liquid-solid mechanism. Our experimental results, supported by Monte Carlo simulations of backscattered electrons emission from the nanowires, reveal the self-assembly of gold-silica core-shell nanostructures at the nanowire tips without any ad-hoc thermal oxidation step. This result demonstrates the capacity of very low voltage operation to provide optimum sharpness, contrast and resolution in low-dimensional nanostructures and to gather information about nanoscaled core-shell conformations otherwise impossible to obtain by standard scanning electron microscopy alone.

Suspended mechanical structures based on elastic silicon nanowire arrays.

We demonstrate a bottom-up/top-down combined method for the fabrication of horizontally suspended, well-oriented and size-controlled Si nanowire arrays. Mechanical beamlike structures composed of multiple ordered arrays consecutively linked by transversal microspacers are obtained by this method. Such structures are used to investigate the mechanical elasticity of the nanowire arrays by atomic force microscopy. Our results point out important differences in the morphology and mechanical behavior of the fabricated nanowire-based structures with respect to equivalent bulk material structures.