Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience.

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.

Genetically Encoded Self-Assembling Iron Oxide Nanoparticles as a Possible Platform for Cancer-Cell Tracking.

The study of growth and possible metastasis in animal models of tumors would benefit from reliable cell labels for noninvasive whole-organism imaging techniques such as magnetic resonance imaging. Genetically encoded cell-tracking reporters have the advantage that they are contrast-selective for viable cells with intact protein expression machinery. Besides, these reporters do not suffer from dilution during cell division. Encapsulins, which are bacterial protein nanocompartments, can serve as genetically controlled labels for multimodal detection of cells. Such nanocompartments can host various guest molecules inside their lumen. These include, for example, fluorescent proteins or enzymes with ferroxidase activity leading to biomineralization of iron oxide inside the encapsulin nanoshell. The aim of this work was to implement heterologous expression of encapsulin systems from Quasibacillus thermotolerans using the fluorescent reporter protein mScarlet-I and ferroxidase IMEF in the human hepatocellular carcinoma cell line HepG2. The successful expression of self-assembled encapsulin nanocompartments with functional cargo proteins was confirmed by fluorescence microscopy and transmission electron microscopy. Also, coexpression of encapsulin nanoshells, ferroxidase cargo, and iron transporter led to an increase in T2-weighted contrast in magnetic resonance imaging of HepG2 cells. The results demonstrate that the encapsulin cargo system from Q. thermotolerans may be suitable for multimodal imaging of cancer cells and could contribute to further in vitro and in vivo studies.