For analysis of the simulated images, all simulations were performed ten times in impartial runs, and images were scaled to mean image intensity. Statistical analysis Statistical analyses were performed with BAIAP2 GraphPad Prism version 4.0 for windows (GraphPad Software, San Diego, USA). in the field of cell therapies1,2 and the increasing understanding of the complex interplay between different cell populations3C5 have created a demand for novel methods to longitudinally study the fate of specific cell populations or even individual cells. Optical techniques Thalidomide-O-amido-PEG2-C2-NH2 (TFA) such as confocal or two-photon microscopy are well established for cell tracking, but require invasive procedures such as installation of cranial windows or skin-fold chambers6,7. This approach is usually therefore not suitable for all animal models, and has limited potential for clinical translation. Non-invasive cell tracking is possible by a number of different methods such as fluorescence or radionuclide imaging8,9 and different Magnetic Resonance Imaging (MRI) approaches using T2*w MRI of iron nanoparticle (ION)-labelled cells, 19F-MRI, or highly shifted proton MRI10C12. All of these methods have unique advantages which, however, are accompanied by drawbacks such as limited tissue penetration, instability of the marker, Thalidomide-O-amido-PEG2-C2-NH2 (TFA) low spatial resolution, high background signal or limited sensitivity. With regards to potential clinical translation, T2*w MRI using ION-labelled cells offers the advantages of unlimited tissue penetration, stability of the marker material, high spatial resolution, and additional morphological information13C20. However, due to the long image acquisition times, MRI and other noninvasive imaging methods could only acquire a static snap shot of labelled cells until recently. Although migration of cells has been detected by identifying cells at different locations at different time points, the actual movement remained concealed17,21. However, the direct observation of individual moving cells by MRI still seemed challenging until the concept of MRI time-lapse imaging was successfully implemented18. In this method, the established fluorescence microscopy time-lapse concept6,7, which collates sequentially acquired individual images into a movie that tracks migrating cells, was applied to MRI through repetitive acquisition of a series of static T2*w images. The time-lapse concept has recently been extended by performing real-time MRI acquisitions to visualize and assess the inflow and distribution of labelled cells in brain and spine in different animal models22. However, this approach did not aim at resolving single Thalidomide-O-amido-PEG2-C2-NH2 (TFA) cells, but detected bulk signal of grafted cells from the vasculature directly after injection with a temporal resolution of two seconds. The detection of single monocytes was previously shown to be feasible with time frames of 20 minutes18. Multi-slice time-lapse acquisitions with whole-brain coverage provided movies tracking individual labelled monocytes in the vasculature of rat brain non-invasively. Yet, the strengths of such dynamic cell tracking has not been exploited in a clinical disease model18, and the temporal range of single cell motion that could be potentially resolved Thalidomide-O-amido-PEG2-C2-NH2 (TFA) by time-lapse MRI was not addressed previously. The range of cellular velocities is usually of particular interest. Without any inflammatory stimulus, monocytes have been shown to patrol the endothelium at a velocity of approximately 0.2?m/s, before being eventually dragged away in the blood stream with Thalidomide-O-amido-PEG2-C2-NH2 (TFA) much higher velocity6,23. Upon inflammatory stimuli, monocytes start rolling around the endothelium at approximately 40? m/s and potentially extravasate into the surrounding tissue6. Here, we aim to determine the velocity range that can be resolved with time-lapse MRI and to assess whether altered motion patterns of labelled leukocytes upon an immune response can be detected with this methodology. We use a murine model of experimental autoimmune encephalomyelitis (EAE)24,25 and compare it to healthy mice to assess whether time-lapse MRI is able to resolve different leukocyte motion patterns in the na?ve and inflammatory state. Results Development of time-lapse MRI protocol A time-lapse MRI protocol with frame rate of 8?min 12?s was implemented to cover the whole mouse brain with a spatial resolution of 61?m by 55?m in 0.3?mm contiguous slices. To verify that this protocol was able to detect single labelled cells, measurements in agar gel phantoms with and without embedded ION-labelled monocytes were performed. The protocol provided images with a mean signal-noise ratio (SNR) of 35??5. Inspecting the individual signal voids showed that signal was decreased in one central voxel by ~70%, slowly recovering over the two to three neighbouring voxels in all four directions (Fig.?1a,b). Quantitative analysis showed a significantly increased number of signal voids, depending on the number of ION-labelled cells embedded in the gel (Fig.?1c): an average of.