Language：Others  

Abstract

Resolution enhancement in structured illumination microscopy is hindered when volumetric samples are observed owing to background signals from out-of-focus planes. In this study, we utilized selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. We demonstrated high-resolution fluorescence imaging of volumetric samples, including dense fluorescent objects in a cell spheroid.

DOI： 10.1101/2023.06.19.545558

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jpcb.2c07291

Language：English   Publishing type：Research paper (scientific journal)  

We demonstrate the use of Bessel beams for side illumination slit-scanning Raman imaging for label-free and hyperspectral analysis of cell spheroids. The background elimination by the side illumination and the aberration-resistant Bessel beam drastically improves the image contrast in Raman observation, allowing label-free investigation of intracellular molecules in thick biological samples. Live cell spheroids were observed to confirm the improvement in image contrast and background reduction with Bessel illumination compared to conventional epi-line illumination.

DOI： 10.1364/BOE.456138

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1364/oe.455621

Language：English   Publishing type：Research paper (scientific journal)  

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm²), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.

DOI： 10.1364/boe.446273

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/jmi.13033

Language：English   Publishing type：Research paper (scientific journal)  

© 2020 American Chemical Society. Light-matter interactions in the deep-ultraviolet (DUV) wavelength region exhibit a variety of optical effects such as luminescence, photoisomerization, and polymerization in many materials. Despite the rich photochemistry and high spatial resolution due to the short wavelength, the notorious lack of DUV-compatible optical components and devices precludes the use of DUV light in microscopy and lithography as a routine laboratory tool. Here, we present the use of two-photon excitation with visible laser light to realize photopolymerization of molecules with an excitation energy equivalent to DUV light. Using standard optics for visible light, we polymerized methacrylate oligomers with 400 nm femtosecond pulses without any addition of photoinitiators and sensitizers. By scanning the laser focus in 3D, we created a series of fine 3D structures with the smallest resolved line-space features of 80 nm. We found that photopolymerizations induced by two-photon absorption at DUV is surprisingly efficient and requires laser intensity only on the order of 100 kW/cm2. The use of DUV absorption sites natively existing in monomers simplifies polymerization reactions without sacrificing material purities by adding initiators and facilitates applications to diverse materials. We have applied two-photon DUV polymerizations to metal-oxo clusters and the amino acid cysteine. With the variety of successful demonstrations including organic- and inorganic-material-made-structures presented, multiphoton DUV polymerization offers a distinct tool for fabrications of nanodevices in 3D.

DOI： 10.1021/acsanm.0c02519

Language：English   Publishing type：Research paper (scientific journal)  

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

DOI： 10.1117/1.jbo.25.1.014502

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/1.5039567

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1364/boe.9.000202

Language：Others   Publishing type：Research paper (scientific journal)  

We demonstrated resolution improvement in two-photon excitation microscopy by combining saturated excitation (SAX) of fluorescence and pupil manipulation. We theoretically estimated the resolution improvement and the sidelobe effect in the point spread function with various pupil designs and found that the combination of SAX and core-ring illumination can effectively enhance the spatial resolution in 3D and suppress sidelobe artifacts. The experimental demonstration shows that the proposed technique is effective for observation with a depth of 100 μm in a tissue phantom and can be applied to 3D observations of tissue samples with higher spatial resolution than conventional two-photon excitation microscopy.

DOI： 10.1364/ol.42.000571

Language：English   Publishing type：Research paper (scientific journal)  

Nonlinear optical effects play key roles to communication, sensing, imaging, and so on. Recently, nonlinear scattering (saturation and reverse saturation) was discovered in gold nanospheres, providing a novel approach to nonbleaching super-resolution microscopy. However, the nonlinearity was previously limited to green-orange plasmonic band. It is highly desirable to extend the applicable wavelength range. In this work, we demonstrated nonlinear scattering in near-infrared with gold nanorods and in blue-violet with silver nanospheres. Besides, the nonlinear mechanism is clarified via different material/geometry. By spectrally decoupling the contributions of plasmonic absorption/scattering and interband/intraband transitions, we have verified plasmonic absorption, and the subsequent thermal effects to be the dominating source of nonlinearity. Our work not only provides the physical mechanism of the nonlinear scattering, but also paves the way toward multicolor super-resolution imaging based on plasmonic scattering.

DOI： 10.1021/acsphotonics.6b00025

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep24293

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3791/53338

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1364/oe.22.026016

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/ph4000218

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1103/physrevlett.112.017402

Language：English  

Country：Other  

Language：Japanese  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：English  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：English  

Country：Other  

Language：Japanese  

Country：Other  

Language：Japanese  

Country：Other  

Language：English   Presentation type：Oral presentation (invited, special)  

Country：United States  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan