Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.

No abstract

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.

Single particle tracking (SPT) of transmembrane receptors in the plasma membrane often reveals heterogeneous diffusion. A thorough interpretation of the displacements requires an extensive analysis suited for discrimination of different motion types present in the data. Here the diffusion pattern of the homomeric alpha3-containing glycine receptor (GlyR) is analyzed in the membrane of HEK 293 cells. More specifically, the influence of the alpha3 RNA splice variants alpha3K and alpha3L on lateral membrane diffusion of the receptor is revealed in detail. Using a combination of ensemble and local SPT analysis, free and anomalous diffusion parameters are determined. The GlyR alpha3 free diffusion coefficient is found to be 0.13 +/- 0.01 microm2/s and both receptor variants display confined motion. The confinement probability level and residence time are significantly elevated for the alpha3L variant compared to the alpha3K variant. Furthermore, for the alpha3L GlyR, the presence of directed motion was also established, with a velocity matching that of saltatory vesicular transport. These findings reveal that alpha3 GlyRs are prone to different types of anomalous diffusion and reinforce the role of RNA splicing in determining lateral membrane trafficking.

Image correlation and cross-correlation spectroscopy are most appropriate to unravel the motion of the PLLA nanocarrier and to demonstrate that the rate of the directed motion of organelles is influenced by their interaction with the nanocarriers. Not all PLLA-hypericin NPs are associated with organelles.

Abstract. Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M ¼ 0.12 μm for single-band intensity profiles when fixing the A-band length (A) to A ¼ 1.6 μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn ¼ n 2ω − n ω ) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn ¼ 0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A-and M-band sizes yielded A ¼ 1.40 AE 0.04 μm and M ¼ 0.07 AE 0.05 μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle.