Simulation-based optimisation of thermodynamic conditions in the ESEM for dynamical in-situ study of spherical polyelectrolyte complex particles in their native state.

We present a complex analysis and optimisation of dynamic conditions in the environmental scanning electron microscope (ESEM) to allow in-situ observation of extremely delicate wet bio-polymeric spherical particles in their native state. According to the results of gas flow and heat transfer simulations, we were able to develop an improved procedure leading to thermodynamic equilibrium between the sample and chamber environment. To quantify and hence minimise the extent of any sample deformation during specimen chamber pumping, a strength-stress analysis is used. Monte Carlo simulations of beam-gas, -water, and -sample interactions describe beam scattering, absorbed energy, interaction volume and the emission of signal electrons in the ESEM. Finally, we discuss sample damage as a result of drying and the production of beam-induced free radicals. Based on all experimental and simulation results we introduce a Delicate Sample Observation Strategy for the ESEM. We show how this strategy can be applied to the characterization of polyelectrolyte complex spherical particles containing immobilized recombinant cells E. coli overexpressing cyclohexanone monooxygenase, used as a model biocatalyst. We present the first native-state electron microscopy images of the viscous core of a halved polyelectrolyte complex capsule containing living cells.

Vitrification and increase of basicity in between ice Ih crystals in rapidly frozen dilute NaCl aqueous solutions.

The freezing of ionic aqueous solutions is common in both nature and human-conducted cryopreservation. The cooling rate and the dimensions constraining the solution are known to fundamentally influence the physicochemical characteristics of the sample, including the extent of vitrification, morphology, and distribution of ions. The presence of some salts in an aqueous solution often suppresses the ice crystallization, allowing bulk vitrification during relatively slow cooling. Such a process, however, does not occur in NaCl solutions, previously observed to vitrify only under hyperquenching and/or in sub-micrometric confinements. This work demonstrates that, at freezing rates of ≥100 K min-1, crystallized ice Ih expels the freeze-concentrated solution onto the surfaces of the crystals, forming lamellae and veins to produce glass, besides eutectic crystallization. The vitrification covers (6.8% ± 0.6%) and (17.9% ± 1.5%) of the total eutectic content in 0.06M and 3.4 mM solutions, respectively. The vitrified solution shows a glass-to-liquid transition succeeded by cold crystallization of NaCl · 2H2O during heating via differential scanning calorimetry. We establish that ice crystallization is accompanied by increased basicity in freeze-concentrated solutions, reflecting preferential incorporation of chloride anions over sodium cations into the ice. After the sample is heated above the glass transition temperature, the acidity gradually returns towards the original value. The morphology of the samples is visualized with an environmental scanning electron microscope. Generally, the method of vitrifying the freeze-concentrated solution in between the ice Ih crystals via fast cooling can be considered a facile route towards information on vitrified solutions.