POLYMERS Vol.64 No.9
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Super-Resolution Fluorescence Microscopy and Polymers
COVER STORY: Highlight Reviews
Photoswitchable Fluorescent Proteins for PALM: Molecular Basis and Tips for Labeling Hideaki MIZUNO
<Abstract> Fluorescent proteins have become indispensable tools for non-invasive fluorescence labeling in living cells. A series of fluorescent proteins that change spectral properties upon light illumination is called photoswitchable fluorescent proteins (PSFP). By using this series of fluorescent proteins, a super-resolution microscopic modality, photoactivated localization microscopy (PALM) has been developed. PSFPs are categorized into three groups based on the mode of switching: photoactivatable (PAFP), photoconvertible (PCFP), and reversibly photoswitchable (RSFP). PAFP is originally non-fluorescent and becomes fluorescent upon the illumination with the activation light. The photoactivation is caused by decarboxylation of the glutamate residue nearby the chromophore. PCFP changes the fluorescence color upon light illumination; photo-induced formal β-elimination expands the π-conjugation at the histidine residue next to the chromophore, and as a result, the absorption and emission spectra shift to the red. RSFP shows photo-induced reversible switching between fluorescent and non-fluorescent states due to photochromic configuration change. Here I introduce properties of PSFP and tips on selection for PALM.
Keywords: Photoswitchable Fluroescent Protein / Super-Resolution Microscopy / PALM
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Super-Resolution Localization Microscopy Reveals Nanoscale Dynamics of Polymers and Soft Matter Shuzo HIRATA, Syoji ITO, Martin VACHA, Hiroshi MIYASAKA
<Abstract> Super-resolved astigmatic microscopy allows 3D spatial localization of individual molecules of fluorescent dyes with nanometer accuracy. Apart from imaging, such localization has found applications in the study of nanometer-scale physical properties of polymers and complex soft matter. Here, we introduce the principle of the astigmatic imaging and present examples of its use in the study of polymers and liquid crystals. Study of relaxation dynamics across thin polymer films shows that the effect of substrate is dominant over the surface effect in determining the thin film properties below the glass transition temperature. Monitoring of diffusion in nematic liquid crystals reveals position-dependent changes in tilt angle, confined diffusion close to the liquid crystal cell wall and reorientation of the liquid crystal director close to a nanoparticle. Study of diffusion in polymer films above the glass transition shows that the film substrate causes considerable slow-down in the diffusion and that this effect extends up to 200 nm into the bulk of the film.
Keywords: Super-Resolution Microscopy / Single Molecule / Polymer Film / Relaxation Dymanics / Diffusion / Liquid Crystal
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Conformational Analysis of Polymer Chain by Single Molecule Localization Microscopy Hiroyuki AOKI
<Abstract> The direct observation of individual polymer chains would provide valuable information to understand the fundamental properties of polymer materials. Fluorescence imaging is the most effective method to detect a single molecule embedded in a bulk medium; however, the imaging of the conformation of a single chain has been impossible because the spatial resolution is more than 200 nm by the diffraction limit of light. Single-molecule localization microscopy has been developed as a fluorescence imaging technique with nanometric resolution. The localization microscopy sequentially observes the position of the dye molecules on a sample with the nanometric accuracy, and a high-resolution fluorescence image is reconstructed from the coordinate data. The single polymer chain in a bulk medium can be visualized with this methodology, and the conformational of individual chains is analyzed in three dimensions. The single molecule localization microscopy would be a powerful tool to discuss the fundamental process at the single chain level in various phenomena of polymeric materials.
Keywords: Single Molecule Localization Microscopy / Super-Resolution / Single Polymer Chain / Conformation / Deformation / Fluorescence
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COVER STORY: Topics and Products
Principle and Application of STED Microscopy Jun-ichi HOTTA
<Abstract> Stimulated emission depletion (STED) microscopy is one of the super-resolution fluorescence microscopy techniques, which realize better resolution than the diffraction limit of light. In order to squeeze a fluorescent spot, STED microscopy utilize fluorescence depletion by stimulated emission process for switching off the fluorescence molecules except for the center of the donut STED beam. In this paper, principle and application of STED microscopy are discussed. The important role of patterned polymer thin films as spatical phase modulators is explained. Generation of two- and three-dimensional donut beams using polymer thin films are described. As examples of STED microscopy in the field of polymers, single molecule imaging of dendrimers, visualization of nanoscale block copolymer morphology, and STED lithography are discussed.
Keywords: STED Microscopy / Super-Resolution / Dendrimer / Block Copolymer / STED Lithography
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Super-Resolution Fluorescence Imaging Based on Molecular Adsorption Satoshi HABUCHI
<Abstract> While super-resolution fluorescence imaging has been revolutionizing the way we study nanoscale structures, applications of the imaging technique is still limited, partly due to limited fluorophores and labeling methods available for the imaging. Here, I introduce a localization microscopy technique (PALM/STORM) based on an adsorption of fluorophores to a target structure. This method does not require a covalent labeling of a target structure with a special fluorophore whose fluorescent state can be switched, which is not a trivial issue. In this approach, the fluorophores adsorb selectively to the target structure. Fluorescent states of the fluorophore are then spatiotemporally controlled by adsorption/desorption kinetics of the fluorophore. Based on the adsorption/desorption-induced fluorescence switching, a super-resolution image is reconstructed, similar to PALM/STORM imaging. I show several examples of the super-resolution fluorescence imaging based on this approach, including micro phase separation of phospholipids in a lipid bilayer, DNA nanostructures, and polymer nanostructures.
Keywords: Super-Resolution / Fluorescence Imaging / STORM / Single-Molecule / Adsorption / Nano-Structure / Thermal Nanoimprinting
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The Principle and the Applications of Structured Illumination Super Resolution Microscopy Yoshiro OIKAWA
<Abstract> Optical microscopy has the theoretical resolution limit of 200 nm which is defined as d=λ/2NA. Recently some ideas of breaking this limit are employed in the commercially available product, known as “super resolution microscopy”. “Structured Illumination Microscopy (SIM)” is one of those technologies that provides approx. 100 nm resolution. In the SIM method, a stripe illumination pattern is applied to capture the diffraction light. Several images with such illumination pattern are captured and they are converted into Fourier space. Then high frequency components are separated and relocated. The image frequency range has become twice bigger. Then they are converted back into the real space, and a super resolution image with some 100 nm resolution is generated. The Electron microscope is useful to observe a very small sample, but not applicable to a living sample. SIM is used in the fields of neuro-science, immunology research or regenerative medicine research. Super resolution microscopy is expanding its application field not only for bioscience, but material science and other fields.
Keywords: Microscope / Super Resolution / SIM / Structured Illumination
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IR Super-resolution Microscopy Makoto SAKAI, Masaaki FUJII
<Abstract> For many years, spatial resolution was the most critical problem in IR microscopy. This is because the spatial resolution of a conventional IR microscope is restricted by the diffraction limit, which is almost the same as the wavelength of IR light, ranging from 2.5 to 25 μm. In the recent years, we have developed two novel types of far-field IR super-resolution microscopes using 2-color laser spectroscopies, transient fluorescence detected IR spectroscopy and vibrational sum-frequency generation (VSFG) spectroscopy. Applying these methods, in which both transient fluorescence and VSFG signal have a wavelength in the visible region, the image is observed at the resolution of visible light, which is about 10 times smaller than that of IR light, that is, IR super-resolution. By using these techniques, we can map the specific IR absorption with a sub-micrometer spatial resolution, visualization of the molecular structure includung molecular orientations in a non-uniform environment becomes a possibility.
Keywords: IR Super-Resolution / Microscope / 2-Color Laser Spectroscopy / IR Imaging / IR Spectroscopy / Molecular Structure / Molecular Orientation
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Polymer Science and I: A Personal Account
Passive but Positive Takuya NAKASHIMA
<Abstract> I had great opportunities to be involved in the startup of new laboratories by chance. These experiences made me open to new research fields and a witty player. I’m still struggling with science to achieve a great discovery.
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Front-Line Polymer Science
Synthesis of Inorganic Hollow Nanoparticles Templated by Polymeric Micelles Kenichi NAKASHIMA
<Abstract> Inorganic hollow nanoparticles have many applications from use as heat insulation and lightweight materials to drug carriers for medicinal purposes. Two synthesis techniques have been used so far; one relies on a template and the other is template-free. Template-free processes have a drawback that it is difficult to control particle size. The templates are further classified into two; one is hard and the other is soft. Hard template techniques also suffer from low product yield and shell weakness. Thus, researchers have turned to soft templates, especially polymeric micelles. In this review, we introduce recent progress in the synthesis of inorganic hollow nanoparticles focusing on a strategy of polymeric micelle templates. We demonstrate advantages of ABC triblock copolymer micelles with core-shell-corona architecture. By tailoring experimental parameters, we can readily obtain a variety of hollow nanoparticles including silica, metal-oxides, metal-phosphates, etc. Finally, we highlight the potential use of these hollow particles in drug delivery and bio-imaging.
Keywords: Inorganic Hollow Nanopartilce / Template Syntehsis / Polymeric Micelle
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