Iron oxide nanoparticles have attracted a great deal of research interest and have been widely used in bioscience and clinical research including as contrast agents for magnetic resonance imaging, hyperthermia and magnetic field assisted radionuclide therapy. It is therefore important to develop methods, which can provide high-throughput screening of biological responses that can predict toxicity. The use of nanoelectrodes for single cell analysis can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. We have developed a new method for in vitro study of the toxicity of magnetic nanoparticles (NP) based on the measurement of intracellular reactive oxygen species (ROS) by a novel nanoelectrode. Previous studies have suggested that ROS generation is frequently observed with NP toxicity. We have developed a stable probe for measuring intracellular ROS using platinized carbon nanoelectrodes with a cavity on the tip integrated into a micromanipulator on an upright microscope. Our results show a significant difference for intracellular levels of ROS measured in HEK293 and LNCaP cancer cells before and after exposure to 10 nm size iron oxide NP. These results are markedly different from ROS measured after cell incubation with the same concentration of NP using standard methods where no differences have been detected. In summary we have developed a label-free method for assessing nanoparticle toxicity using the rapid (less than 30 minutes) measurement of ROS with a novel nanoelectrode.

High-quality, 25 nm octahedral-shaped Fe3O4 magnetite nanocrystals are epitaxially grown on 9 nm Au seed nanoparticles using a modified wet-chemical synthesis. These Fe3O4-Au Janus nanoparticles exhibit bulk-like magnetic properties. Due to their high magnetization and octahedral shape, the hybrids show superior in vitro and in vivo T2 relaxivity for magnetic resonance imaging as compared to other types of Fe3O4-Au hybrids and commercial contrast agents. The nanoparticles provide two functional surfaces for theranostic applications. For the first time, Fe3O4-Au hybrids are conjugated with two fluorescent dyes or the combination of drug and dye allowing the simultaneous tracking of the nanoparticle vehicle and the drug cargo in vitro and in vivo. The delivery to tumors and payload release are demonstrated in real time by intravital microscopy. Replacing the dyes by cell-specific molecules and drugs makes the Fe3O4-Au hybrids a unique all-in-one platform for theranostics.

Multi-colored gene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes 1,2 and uncovering the network architecture of cell-circuits 3,4 , are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals 5-9 which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization. The latter technical constraints also affect EM immunolabeling techniques 10-13 which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core 14-16 , but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system of Quasibacillus thermotolerans (Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtIMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin of Myxococcus xanthus 17 (Mx,~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells. Encapsulins are a class of proteinaceous spherical nanocompartments naturally occurring in bacteria and archaea, so far described as icosahedral structures with either T=1 (60 subunits,~18 nm diameter) or T=3 (180 subunits,~30 nm) symmetry, which can encapsulate cargo proteins with a wide range of functions 18-21. It has also been shown that foreign cargos such as fluorescent proteins or