Abstract
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.
Highlights
Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of biocomplexes
Discussion powerful for resolving the dipole orientation, conventional fluorescence polarization microscopy (FPM) techniques were previously hindered by their poor spatial resolution due to the diffraction limit
We have demonstrated that polarized structured illumination microscopy (pSIM) is compatible with a variety of Structured illumination microscopy (SIM) modalities, such as 2D-SIM, total internal reflection fluorescence-SIM (TIRFSIM), and 3D-SIM
Summary
Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of biocomplexes. 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. Super-resolution FPM is essential for imaging sub-diffraction dipole assemblies with hidden geometries, preferably at high speeds, to capture the complex dynamics of live cells. With minutes to hours required for a typical image acquisition procedure, it is difficult to measure the structural dynamics of living cells Another category of superresolution FPM, termed polarization demodulation, achieves super-resolution with sparse deconvolution, which results in a fast imaging speed (~5 fps) and applies to regular labeling strategies[2,3,4,5]. The pSIM technique successfully images the dipole orientation of cytoskeletal filaments with superresolution in fixed cells, tissue sections, and live cells
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