Magnetic particle imaging (Mpi) is a non-invasive, non-ionizing imaging technique for the visualization and quantification of magnetic nanoparticles (MNPs). The technique is especially suitable for cell imaging as it offers zero background contribution from the surrounding tissue, high sensitivity, and good spatial and temporal resolutions. Previous studies have demonstrated that the dynamic magnetic behaviour of MNPs changes during cellular binding and internalization. In this study, we demonstrate how this information is encoded in the Mpi imaging signal. through Mpi imaging we are able to discriminate between free and cell-bound MNPs in reconstructed images. This technique was used to image and quantify the changes that occur in-vitro when free Mnps come into contact with cells and undergo cellular-uptake over time. The quantitative MPI results were verified by colorimetric measurements of the iron content. The results showed a mean relative difference between the MPI results and the reference method of 23.8% for the quantification of cell-bound MNPs. With this technique, the uptake of MNPs in cells can be imaged and quantified directly from the first MNP cell contact, providing information on the dynamics of cellular uptake. Magnetic particle imaging (MPI) is a non-invasive technique capable of determining the spatial distribution of magnetic nanoparticles (MNPs) both in-vivo and in-vitro 1. MPI achieves imaging and quantification detecting the non-linear dynamic magnetic response of MNPs exposed to multiple superimposed static and dynamic magnetic fields with a submillimetre spatial resolution. No background signals are generated by bone or tissue. The technique uses non-ionizing radiation and non-toxic nanoparticles to prevent tissue damage. MPI shows great potential for different biomedical applications, such as angiography, stem cell tracking, diagnosis of inflammatory diseases and cancer 2-11. In inflammation-associated diseases, including cancer, MNPs accumulate preferentially in diseased tissue as a result of leaky vasculature thereby enabling imaging with MRI and MPI 12-16. In diseased tissue, MNPs accumulate mainly in macrophages 17,18. These phagocytic cells are a hallmark of tissue inflammation and their quantity is considered a marker of the severity of the disease 19-21. The magnetic response generated by MNPs is severely influenced by their local environment. In this respect, the changing magnetic properties of MNPs induced by their interaction with cells are a relevant factor for MPI. Previous studies using magnetic particle spectroscopy (MPS), have described and quantified the changes that occur in the dynamic magnetization of certain MNP systems upon interaction with living cells 22-26. These effects may be caused by a variety of factors including the aggregation of particles in the surrounding solution, within the extra-cellular matrix or within various intracellular compartments. "Size-filtering" during cellular uptake and an increase in dipole-dipole interactions caused by a lower separati...

The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.