Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

T antigen (Galß1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 ß1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAß1-3Galß1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila ß1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.

Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila.

T antigen (Galß1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 ß1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O-glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis.

Label-free visualization of acetaminophen-induced liver injury by high-speed stimulated Raman scattering spectral microscopy and multivariate image analysis.

We recently established a high-speed, label-free, spectral imaging method based on stimulated Raman scattering (SRS). This method enables examination of cellular features within relatively short periods, thus enabling new imaging applications in pathology. Previously, we reported on label-free visualization of mouse tissue using SRS spectral microscopy combined with multivariate image analysis, but the feasibility of applying this approach to diseased tissues with diverse morphology and irregular chemical species has not been examined. We, therefore, assessed acetaminophen-induced liver injury to evaluate the potential use of Raman spectral microscopy for visualizing histopathologic specimens. Acetaminophen-overdosed mouse liver was prepared and the pathologic changes including centrilobular necrosis were confirmed. Multi-colored images were reconstructed through principal component analysis (PCA) of a multi-band SRS dataset, which provided rich information compared with a monochrome single-band SRS dataset. A wide view of the multi-colored principal component (PC) images showed the distribution of cellular constituents, which was similar to that observed by fat staining. In addition, different types of cells in liver parenchyma were also demonstrated. In conclusion, the combination of SRS spectral microscopy and PCA has the potential to reveal both the morphological and chemical features of specimens and therefore has potential utility in diagnostic pathology.

Sensitivity enhancement of fiber-laser-based stimulated Raman scattering microscopy by collinear balanced detection technique.

We propose the collinear balanced detection (CBD) technique for noise suppression in fiber laser (FL)-based stimulated Raman scattering (SRS) microscopy. This technique reduces the effect of laser intensity noise at a specific frequency by means of pulse splitting and recombination with a time delay difference. We experimentally confirm that CBD can suppress the intensity noise of second harmonic (SH) of Er-FL pulses by 13 dB.The measured noise level including the thermal noise is higher by only ~1.4 dB than the shot noise limit. To demonstrate SRS imaging, we use 4-ps SH pulses and 3-ps Yb-FL pulses, which are synchronized subharmonically with a jitter of 227 fs. The effectiveness of the CBD technique is confirmed through SRS imaging of a cultured HeLa cell.

Stimulated Raman hyperspectral imaging based on spectral filtering of broadband fiber laser pulses.

We demonstrate a technique of hyperspectral imaging in stimulated Raman scattering (SRS) microscopy using a tunable optical filter, whose transmission wavelength can be varied quickly by a galvanometer mirror. Experimentally, broadband Yb fiber laser pulses are synchronized with picosecond Ti:sapphire pulses, and then spectrally filtered out by the filter. After amplification by fiber amplifiers, we obtain narrowband pulses with a spectral width of <3.3 cm(-1) and a wavelength tunability of >225 cm(-1). By using these pulses, we accomplish SRS imaging of polymer beads with spectral information.

Five-bit parallel operation of optical quantization and coding for photonic analog-to-digital conversion.

We report the attempt of optical quantization and coding in 5-bit parallel format for photonic A/D conversion. The proposed system is designed to realize generation of 32 different optical codes in proportion to the corresponding signal levels when fed a certain range of amplitude-varied input pulses to the setup. Optical coding in a bit-parallel format made it possible, that provides 5 bit optical codes from 32 optical quantized pulses. The 5-bit parallel operation of an optical quantization and coding module with 5 multi-ports was tested in our experimental setup.

Mucin-Type O-Glycosylation in the Drosophila Nervous System.

Mucin-type O-glycosylation, a predominant type of O-glycosylation, is an evolutionarily conserved posttranslational modification in animals. Mucin-type O-glycans are often found on mucins in the mucous membranes of the digestive tract. These glycan structures are also expressed in other cell types, such as blood cells and nephrocytes, and have crucial physiological functions. Altered expression of mucin-type O-glycans is known to be associated with several human disorders, including Tn syndrome and cancer; however, the physiological roles of mucin-type O-glycans in the mammalian brain remains largely unknown. The functions of mucin-type O-glycans have been studied in the fruit fly, Drosophila melanogaster. The basic structures of mucin-type O-glycans, including Tn antigen (GalNAcα1-Ser/Thr) and T antigen (Galß1-3GalNAcα1-Ser/Thr), as well as the glycosyltransferases that synthesize them, are conserved between Drosophila and mammals. These mucin-type O-glycans are expressed in the Drosophila nervous system, including the central nervous system (CNS) and neuromuscular junctions (NMJs). In primary cultured neurons of Drosophila, mucin-type O-glycans show a characteristic localization pattern in axons. Phenotypic analyses using mutants of glycosyltransferase genes have revealed that mucin-type O-glycans are required for CNS development, NMJ morphogenesis, and synaptic functions of NMJs in Drosophila. In this review, we describe the roles of mucin-type O-glycans in the Drosophila nervous system. These findings will provide insight into the functions of mucin-type O-glycans in the mammalian brain.

Ultrashort pulse generation from continuous wave by pulse trapping in birefringent fibers.

We investigated the phenomenon of orthogonally polarized pulse trapping between a continuous-wave beam and an ultrashort soliton pulse in birefringent fibers both experimentally and numerically. Using pulse trapping and amplification, we demonstrated ultrashort pulse generation from a continuous-wave beam. The generated pulse had a nearly transform-limited sech(2)-shape and a temporal width of 350 fs. The obtained maximum pulse energy was 300 pJ using a 400 m-long low-birefringence fiber, and the corresponding gain was as large as 41 dB.

Stimulated Raman scattering microscope with shot noise limited sensitivity using subharmonically synchronized laser pulses.

We propose and demonstrate the use of subharmonically synchronized laser pulses for low-noise lock-in detection in stimulated Raman scattering (SRS) microscopy. In the experiment, Yb-fiber laser pulses at a repetition rate of 38 MHz are successfully synchronized to Ti:sapphire laser pulses at a repetition rate of 76 MHz with a jitter of <8 fs by a two-photon detector and an intra-cavity electro-optic modulator. By using these pulses, high-frequency lock-in detection of SRS signal is accomplished without high-speed optical modulation. The noise level of the lock-in signal is found to be higher than the shot noise limit only by 1.6 dB. We also demonstrate high-contrast, 3D imaging of unlabeled living cells.

Quasi-supercontinuum generation using 1.06 µm ultrashort-pulse laser system for ultrahigh-resolution optical-coherence tomography.

We demonstrate quasi-supercontinuum (quasi-SC) generation around the 1.3 µm wavelength region using ultrahigh-speed, wavelength-tunable, femtosecond soliton pulses based on an ultrashort-pulse laser system operating at a wavelength of 1.0 µm. The wavelength tuning range was from 1.0 to 1.9 µm, and the scanning speed was up to 1.3 MHz. A Gaussian-like quasi-SC with a bandwidth of 220 nm was generated at 1220 nm. The generated quasi-SC was used in an optical-coherence tomography system. High axial resolutions of 5.1 µm in air and 3.7 µm in tissue were obtained. A maximum sensitivity of 100 dB was achieved, and ultrahigh-resolution images of a hamster cheek pouch were observed.

Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy.

We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.

Broadband group delay dispersion compensation for a microscope objective lens with a specially-designed mechanical deformable mirror.

We propose that the bending profile of a mechanical deformable mirror can be designed by shaping its form, realizing a simple, compact, and broadband group delay dispersion compensator in a 4-f pulse shaper arrangement. By using the proposed compensator, spectral phase distortion of a microscope objective lens is successfully pre-compensated for to generate a sub-8 fs pulse at the focus of the lens.

In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation.

Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein.

Optical coding scheme using optical interconnection for high sampling rate and high resolution photonic analog-to-digital conversion.

We propose and demonstrate an optical coding scheme using optical interconnection for a photonic analog-to-digital conversion. It allows us to convert a multi-power level signal into a multiple-bit binary code so as to detect it in a bit-parallel format by binary photodiode array. The proposed optical coding is executed after optical quantization using self-frequency shift. Optical interconnection based on a binary conversion table generates a multiple-bit binary code by appropriate allocation of a level identification signal which is provided as a result of optical quantization. Experimental results show that 8-levels analog pulses are converted into 3-bit parallel binary codes.

All-optical M-ary ASK signal demultiplexer based on a photonic analog-to-digital conversion.

An all-optical M-ary amplitude shift keying (ASK) signal demultiplexer is proposed and demonstrated. It allowed us to seamlessly demultiplex a high bit-rate optical M-ary ASK signal into on-off keying (OOK) signals without O/E conversion. It is composed of multilevel thresholding using self-frequency shift and OOK signal generation using optical interconnection. A level identification signal is provided as a result of multilevel thresholding and it is fed to an optical interconnection circuit which can generate corresponding OOK signals. We demonstrate the quadrature ASK signal demultiplexing at 100 Gsymbol/s and its error free operation at 10 Gb/s.

Single-organelle tracking by two-photon conversion.

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

Laser micro-welding of transparent materials by a localized heat accumulation effect using a femtosecond fiber laser at 1558 nm.

We report on laser micro-welding of materials based on a localized heat accumulation effect using an amplified femtosecond Er-fiber laser with a wavelength of 1558 nm and a repetition rate of 500 kHz. We demonstrated the welding of non-alkali alumino silicate glass substrates, resulting in a joint strength of 9.87 MPa. We also welded a non-alkali glass substrate and a silicon substrate using the 1558-nm laser pulses, resulting in a joint strength of 3.74 MPa. Our laser micro-welding technique can be extended to welding of semiconductor materials and has potential for various applications, such as three-dimensional stacks and assembly of electronic devices and microelectromechanical system devices.

Symmetric waveguides in poly(methyl methacrylate) fabricated by femtosecond laser pulses.

We report on the fabrication of symmetric waveguides in bulk poly(methyl methacrylate) (PMMA) by femtosecond laser pulses. A waveguide with a circular transverse profile can be obtained by using a slit beam shaping method. The refractive index in the core increases by up to 4.6 x 10(-4) and the waveguide works as single-mode waveguide at a wavelength of 632.8 nm. This writing technique is applied to the fabrication of a directional coupler to split a coupled beam with a 1:1 splitting ratio at 632.8 nm.

Structural modification in fused silica by a femtosecond fiber laser at 1558 nm.

We report on structural modification in fused silica by a novel commercial femtosecond fiber laser with a fundamental wavelength of 1558 nm. The refractive-index change was induced by laser pulses at a repetition rate of 173 kHz and pulse duration of 870 fs. The refractive index change with a magnitude of 1.2 x 10(-3) was estimated from the diffraction efficiencies of an internal grating.

Stimulated parametric emission microscopy.

We propose a novel microscopy technique based on the four-wave mixing (FWM) process that is enhanced by two-photon electronic resonance induced by a pump pulse along with stimulated emission induced by a dump pulse. A Ti:sapphire laser and an optical parametric oscillator are used as light sources for the pump and dump pulses, respectively. We demonstrate that our proposed FWM technique can be used to obtain a one-dimensional image of ethanol-thinned Coumarin 120 solution sandwiched between a hole-slide glass and a cover slip, and a two-dimensional image of a leaf of Camellia sinensis.