Decimating Spatial Frequency Components in Periodically Modulated Nanoscale Surface Structures for Sensing of Ambient Refractive Index Changes

In our previous study, we developed an array of unique porous structures (an array of barnacle-like porous structures) to apply to biosensing chips. The porous structure was formed by an internal swelling phenomenon of a polystyrene colloidal particle monolayer, which was surrounded by a poly­(vinyl alcohol) layer, for the duration of the monolayer’s immersion in a toluene bath. Barnacle-like porous structures were formed when polystyrene particles that had rapidly swelled broke the outer layer around the top of the particles. However, after the surface was coated with a thin Ag layer, the porous structure showed a relatively broad extinction spectrum that was undesirable for sensing chips based on both surface plasmon extinction and grating coupling. In this paper, we propose an approach to obtain relatively sharp extinction spectra based on the decimation of the spatial frequencies of the porous structures. This study also investigates formation properties in more detail to control the structural features of the resultant porous structures. A relatively sharp peak in the extinction spectrum was ultimately obtained

Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton

Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton

Multicolor High-Speed Tracking of Single Biomolecules with Silver, Gold, and Silver–Gold Alloy Nanoparticles

Gold nanoparticles have been used as imaging probes to track the motions of single biomolecules. To investigate behaviors of various biomolecules simultaneously, increase of the color palette is necessary. Here we developed a multicolor high-speed single-particle tracking system using silver, gold, and silver–gold alloy (5:5 composition ratio) nanoparticles. The peak wavelengths of the plasmon resonances for 30 nm silver, 30 nm silver–gold alloy, and 40 nm gold nanoparticles were around 410, 460, and 530 nm, respectively, and we constructed multicolor total internal reflection dark-field microscope with multiple lasers at 404 nm for silver, 473 nm for silver–gold alloy, and 561 nm for gold nanoparticles. By the use of a spectrophotometer in the detection optics, scattering images at each wavelength were projected onto different portions of a single two-dimensional detector. High-contrast images of silver, silver–gold alloy, and gold nanoparticles were simultaneously obtained in different color channels. After correction of positional shifts among different color channels by affine transformation, a maximum shift less than 17 nm was achieved. Furthermore, an additional 649 nm laser enabled the detection of plasmon coupling by transient dimer formation of two nanoparticles. With this system, diffusional motions of phospholipids in a supported membrane and stepping motions of kinesins along microtubules were successfully observed with a localization precision of 2 nm and a time resolution of 100 μs at each channel. Our method will pave the way to investigate the operation mechanisms of complex biomolecular systems

Multicolor High-Speed Tracking of Single Biomolecules with Silver, Gold, and Silver–Gold Alloy Nanoparticles

Gold nanoparticles have been used as imaging probes to track the motions of single biomolecules. To investigate behaviors of various biomolecules simultaneously, increase of the color palette is necessary. Here we developed a multicolor high-speed single-particle tracking system using silver, gold, and silver–gold alloy (5:5 composition ratio) nanoparticles. The peak wavelengths of the plasmon resonances for 30 nm silver, 30 nm silver–gold alloy, and 40 nm gold nanoparticles were around 410, 460, and 530 nm, respectively, and we constructed multicolor total internal reflection dark-field microscope with multiple lasers at 404 nm for silver, 473 nm for silver–gold alloy, and 561 nm for gold nanoparticles. By the use of a spectrophotometer in the detection optics, scattering images at each wavelength were projected onto different portions of a single two-dimensional detector. High-contrast images of silver, silver–gold alloy, and gold nanoparticles were simultaneously obtained in different color channels. After correction of positional shifts among different color channels by affine transformation, a maximum shift less than 17 nm was achieved. Furthermore, an additional 649 nm laser enabled the detection of plasmon coupling by transient dimer formation of two nanoparticles. With this system, diffusional motions of phospholipids in a supported membrane and stepping motions of kinesins along microtubules were successfully observed with a localization precision of 2 nm and a time resolution of 100 μs at each channel. Our method will pave the way to investigate the operation mechanisms of complex biomolecular systems

Multicolor High-Speed Tracking of Single Biomolecules with Silver, Gold, and Silver–Gold Alloy Nanoparticles

Gold nanoparticles have been used as imaging probes to track the motions of single biomolecules. To investigate behaviors of various biomolecules simultaneously, increase of the color palette is necessary. Here we developed a multicolor high-speed single-particle tracking system using silver, gold, and silver–gold alloy (5:5 composition ratio) nanoparticles. The peak wavelengths of the plasmon resonances for 30 nm silver, 30 nm silver–gold alloy, and 40 nm gold nanoparticles were around 410, 460, and 530 nm, respectively, and we constructed multicolor total internal reflection dark-field microscope with multiple lasers at 404 nm for silver, 473 nm for silver–gold alloy, and 561 nm for gold nanoparticles. By the use of a spectrophotometer in the detection optics, scattering images at each wavelength were projected onto different portions of a single two-dimensional detector. High-contrast images of silver, silver–gold alloy, and gold nanoparticles were simultaneously obtained in different color channels. After correction of positional shifts among different color channels by affine transformation, a maximum shift less than 17 nm was achieved. Furthermore, an additional 649 nm laser enabled the detection of plasmon coupling by transient dimer formation of two nanoparticles. With this system, diffusional motions of phospholipids in a supported membrane and stepping motions of kinesins along microtubules were successfully observed with a localization precision of 2 nm and a time resolution of 100 μs at each channel. Our method will pave the way to investigate the operation mechanisms of complex biomolecular systems

Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells

Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure–Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2′-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated

Imaging of EdU, an Alkyne-Tagged Cell Proliferation Probe, by Raman Microscopy

Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by means of Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. This approach may eliminate the need for click reactions in the detection of alkyne-labeled molecules

Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein

Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography–mass spectrometry (LC–MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site. The use of SERS realizes high sensitivity (detection limit: ∼100 femtomole) and reproducibility in the peptide screening. By using an automated ATRaS system, we successfully identified the inhibitor-binding site in cysteine protease cathepsin B, a potential drug target and prognostic marker for tumor metastasis. We further showed that the ATRaS system works for complex mixtures of trypsin-digested cell lysate. The ATRaS technology, which provides high molecular selectivity to LC–MS analysis, has potential to contribute in various research fields, such as drug discovery, proteomics, metabolomics and chemical biology

Validation and reliability of current guidelines for the treatment of essential thrombocythemia under real-world clinical settings in Japan

Current guidelines for essential thrombocythemia (ET) patients recommend different treatment approaches based on thrombosis risk stratification models. However, these recommendations may not be applicable to some patients under real clinical settings. Therefore, we carried out a retrospective real-world validation study. Thrombosis-free survival (TFS) was compared between treatment naïve ET patients receiving different treatment approaches. ET patients were stratified by three representative risk models, the conventional, the International Prognostic Score for thrombosis in ET (IPSET-thrombosis), and revised IPSET-thrombosis. Treatment decisions were largely made by individual physicians, taking into account patient preferences and backgrounds. A total of 179 ET patients were included, and thrombotic events were observed in 26 patients. TFS was significantly longer in high-risk patients of all risk models receiving a combination of cytoreductive therapy (CRT) and antiplatelet therapy (APT) compared to CRT alone. Similar results were seen in intermediate-risk patients stratified by IPSET-thrombosis. In contrast, in very low- and low-risk patients of all risk models, TFS was not affected by addition of CRT, indicating that observation or APT alone is an appropriate treatment approach for these patients. We demonstrate that current guidelines provide optimal treatment approaches for Japanese ET patients under real-world clinical settings.</p