Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Objectives: This study was carried out to reveal functions and mechanisms of MEK ⁄ ERK and p38 pathways in chondrogenesis of rat bone marrow mesenchymal stem cells (BMSCs), and to investigate further any interactions between the mitogen-activated protein kinase (MAPK) and transforming growth factor-b1 (TGF-b1) ⁄ Smads pathway in the process. Materials and methods: Chondrogenic differentiation of rat BMSCs was initiated in micromass culture, in the presence of TGF-b1, for 2 weeks. ERK1 ⁄ 2 and p38 kinase activities were investigated by Western Blot analysis. Specific MAPK inhibitors PD98059 and SB20350 were employed to investigate regulatory effects of MEK ⁄ ERK and p38 signals on gene expression of chondrocyte-specific markers, and TGF-b1 downstream pathways of Smad2 ⁄ 3. Results: ERK1 ⁄ 2 was phosphorylated in a rapid but transient manner, whereas p38 was activated in a slow and sustained way. The two MAPK subtypes played opposing roles in mediating transcription of cartilage-specific genes for Col2a and aggrecan. TGF-b1-stimulated gene expression of chondrogenic regulators, Sox9, Runx2 and Ihh, was also affected by activity of PD98059 and SB203580, to different degrees. However, influences of MAPK inhibitors on gene expression were relatively minor when not treated with TGF-b1. In addition, gene transcription of Smad2 ⁄ 3 was significantly upregulated by TGF-b1, but was regulated more subtly by treatment with MAPK inhibitors. Conclusions: MAPK subtypes seemed to regulate chondrogenesis with a delicate balance, interacting with the TGF-b1 ⁄ Smads signalling pathway.

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