Imaging cell-type-specific dynamics of mRNAs in living mouse brain.

We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose ß-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.

Label-free imaging of Schwann cell myelination by third harmonic generation microscopy.

Understanding the dynamic axon-glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt-Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis.

Shaping field for 3D laser scanning microscopy.

Imaging deep tissue can be extremely inefficient when the region of interest is nonplanar and buried in a thick sample, yielding a severely limited effective field-of-view (FOV). Here we describe a novel technique, namely adaptive field microscopy, which improves the efficiency of 3D imaging by controlling the image plane. The plane of scanning laser focus is continuously reshaped in situ to match the conformation of the sample. The practicality is demonstrated for ophthalmic imaging, where a large area of the corneal epithelium of intact mouse eye is captured in a single frame with subcellular resolution.

Protective effects of nicotinamide in a mouse model of glaucoma DBA/2 studied by second-harmonic generation microscopy.

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The morphology and integrity of microtubules of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2, which received a dietary supplement of nicotinamide to reduce metabolic stress. It was compared with control DBA/2, which did not receive nicotinamide, and non-glaucomatous DBA/2-Gpnmb+. We found that morphology but not microtubules are significantly protected by nicotinamide. Furthermore, from co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, indicating that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopy.

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The integrity of axonal microtubules and the morphology of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2J, which received a dietary supplement of nicotinamide (NAM) for reducing metabolic stress. It was compared with control DBA/2J, which did not receive NAM, and non-glaucomatous DBA/2J-Gpnmb+. We found that the morphology of the retinal nerve fibers, but not axonal microtubules, are significantly protected by NAM. The decoupling is analogous to microtubule deficit, a glaucoma pathology in which axonal microtubules exhibit rapid degradation compared to the morphology of the retinal nerve fibers. Understanding microtubule deficit could provide insights into the divergent responses to NAM. From co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Furthermore, microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, suggesting that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

Label-free morphometry of retinal nerve fiber bundles by second-harmonic-generation microscopy.

We present an application of second-harmonic generation (SHG) microscopy for label-free visualization and quantification of the morphology of nerve fibers in the retina. We show that SHG arises from the retinal nerve fiber layer and that it is specifically associated with uniformly oriented microtubules in the axons. The utility of axonal SHG is demonstrated for imaging the neuroanatomy of fresh ex vivo retina, and the three-dimensional structure of the axons of retinal ganglion cells is quantitatively analyzed.

Axonal architecture of the mouse inner retina revealed by second harmonic generation.

We describe a novel method for visualizing the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e., photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG+ axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG+ axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied to testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb+ as the nonglaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the presynaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retinas in 3D.

High-pulse-energy multiphoton imaging of neurons and oligodendrocytes in deep murine brain with a fiber laser.

Here we demonstrate high-pulse-energy multiphoton microscopy (MPM) for intravital imaging of neurons and oligodendrocytes in the murine brain. Pulses with an order of magnitude higher energy (~ 10 nJ) were employed from a ytterbium doped fiber laser source at a 1-MHz repetition rate, as compared to the standard 80-MHz Ti:Sapphire laser. Intravital imaging was performed on mice expressing common fluorescent proteins, including green (GFP) and yellow fluorescent proteins (YFP), and TagRFPt. One fifth of the average power could be used for superior depths of MPM imaging, as compared to the Ti:Sapphire laser: A depth of ~ 860 µm was obtained by imaging the Thy1-YFP brain in vivo with 6.5 mW, and cortical myelin as deep as 400 µm ex vivo by intrinsic third-harmonic generation using 50 mW. The substantially higher pulse energy enables novel regimes of photophysics to be exploited for microscopic imaging. The limitation from higher order phototoxicity is also discussed.

Fabrication and Electrochemical Characterization of Sulfurized-Polyacrylonitrile Nanofiber Electrodes for Na/S Batteries Using Various Polyacrylonitrile Solutions.

A polyacrylonitrile (PAN) nanofiber web was prepared by electrospinning PAN solutions with dimethyl sulfoxide (DMSO) or Dimethylformamide (DMF) as solvent. The PAN web was heated with sulfur to synthesize a sulfurized polyacrylonitrile (SPAN) nanofiber web. The shape of the SPAN web was found to depend on concentration of the PAN solution and properties of the solvent. The SPAN web synthesized using 8 wt% PAN solution in DMSO showed the highest capacity of 1053.3 mAh g-1sulfur under a current density of 526 mA g-1sulfur. Thus, we determined that DMSO could be a potential solvent for preparation of SPAN web electrodes.

Harmonic Generation Microscopy 2.0: New Tricks Empowering Intravital Imaging for Neuroscience.

Optical harmonic generation, e.g., second- (SHG) and third-harmonic generation (THG), provides intrinsic contrasts for three-dimensional intravital microscopy. Contrary to two-photon excited fluorescence (TPEF), however, they have found relatively specialized applications, such as imaging collagenous and non-specific tissues, respectively. Here we review recent advances that broaden the capacity of SHG and THG for imaging the central nervous system in particular. The fundamental contrast mechanisms are reviewed as they encode novel information including molecular origin, spectroscopy, functional probes, and image analysis, which lay foundations for promising future applications in neuroscience.

A Method to Measure Myeloarchitecture of the Murine Cerebral Cortex in vivo and ex vivo by Intrinsic Third-Harmonic Generation.

A new label-free method is presented for measuring myeloarchitecture of the murine cerebral cortex in vivo and ex vivo. Growing evidence suggests that cortical myelination plays significant roles in neuronal plasticity and pathologies, such as multiple sclerosis (MS), but illuminating the mechanism requires longitudinal imaging of the same brains. Here we demonstrate imaging unlabeled myelinated fibers in a live mouse brain by third-harmonic generation (THG). Contrary to other label-free microscopies based on reflectance, fibers of all orientations could be visualized, i.e., radial and tangential to the pia, which is suitable for revealing the three-dimensional connectivity. The depth of THG imaging in an intact brain was approximately 200 µm, so the network of myelinated fibers could be captured into layers 2/3 in vivo. THG provides a novel base for reconstruction of morphology. Semi-automatic tracing of THG-positive axons unraveled the depth-dependent distribution of the myelin lattice. Finally, a unique light property of THG was exploited for the estimation of the g-ratio. The demonstrated THG morphometry of the length density, orientation, and sheath thickness of cortical myelin could be useful for elucidating its function and how it is modulated during learning and disease.

Microtubule Imaging Reveals Cytoskeletal Deficit Predisposing the Retinal Ganglion Cell Axons to Atrophy in DBA/2J.

Purpose: Glaucoma is characterized by progressive loss of the retinal ganglion cells (RGCs) and their axons. Here we test an outstanding notion that microtubules (MTs) within RGC axons degrade before the loss of morphology ("MT hypothesis"). Methods: The integrity of axonal MTs was interrogated by intrinsic second-harmonic generation (SHG) microscopy. Using DBA/2J mice as a model of glaucoma and DBA/2J-Gpnmb+ as a nonglaucomatous control, the relationship between MT disruption and morphology was quantitatively examined as a function of age and sex in the fresh retinal wholemounts. Results: The mean SHG density (i.e., the mean SHG intensity per thickness) was significantly lower in DBA/2J than in DBA/2J-Gpnmb+ and also depended on sex and age. The loss of SHG density, indicating MT disruption within intact RGC axons, occurred in a sectorial manner near the loss of the retinal nerve fiber bundles. The decay rate of SHG density was approximately 97% higher than that of thickness. Conclusions: Collectively, the results indicate that the breakdown of MTs is pathology of glaucoma and likely a precursor of morphological atrophy. Based on a new finding that SHG density is highly variable and spatially discrete, a new model of RGC degeneration is proposed. This study validates SHG retinal imaging for elucidating the role and mechanism of MT deficiency in the course of glaucoma pathogenesis.

Montelukast inhibits RANKL­induced osteoclast formation and bone loss via CysLTR1 and P2Y12.

Osteoclasts (OCs) are resorptive cells responsible for bone erosion in diseases, including osteoporosis, periodontitis and rheumatoid arthritis. Montelukast is a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist clinically used for the treatment of asthma. In the present study, the role of CysLTR1 on OC formation and bone loss was investigated using montelukast. Montelukast inhibited receptor activator of nuclear factor­κB ligand (RANKL)­induced OC formation in cultures of mouse bone marrow macrophages. Additionally, montelukast suppressed actin ring formation and bone resorption activity of differentiated OCs. The inhibitory effect of montelukast was associated with impaired activation of extracellular signal­regulated kinase, AKT serine/threonine kinase, and/or phospholipase Cγ2 signaling pathways downstream of RANK, followed by decreased expression of nuclear factor of activated T cells c1. Notably, OC formation was efficiently restored by addition of adenosine diphosphate, a P2Y12 agonist, as well as by addition of CysLT. Furthermore, similar to montelukast, P2Y12 blockade by a pharmacological inhibitor or siRNAs suppressed OC differentiation. These data indicate the involvement of the P2Y12 receptor in the inhibitory effect of montelukast on osteoclastogenesis. In vivo, montelukast significantly inhibited inflammation­induced osteoclastogenesis in the calvarial model. Montelukast also served a protective role in a murine ovariectomy (OVX)­ and unloading­induced bone loss model. Altogether, these results confirmed that the CysLTR1 antagonist exerted an inhibitory effect on OC formation in vitro and in vivo. It may be useful for the treatment of bone diseases associated with excessive bone resorption.

The farnesoid X receptor negatively regulates osteoclastogenesis in bone remodeling and pathological bone loss.

Farnesoid X receptor (FXR, NR1H4) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Since the role of FXR in osteoclast differentiation remains ill-defined, we investigated the biological function of FXR on osteoclastogenesis, using FXR-deficient mice. We demonstrated that FXR deficiency increases osteoclast formation in vitro and in vivo. First, FXR deficiency was found to accelerate osteoclast formation via down-regulation of c-Jun N-terminal kinase (JNK) 1/2 expression. Increased expression of peroxisome proliferator-activated receptor (PPAR)γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1)ß seems to mediate the pro-osteoclastogenic effect of FXR deficiency via the JNK pathway. In addition, we found that FXR deficiency downregulated the expression of interferon-ß (IFN-ß), a strong inhibitor of osteoclastogenesis, via receptor activator of nuclear factor-kappaB ligand (RANKL). We further suggested that interference of IFN-ß expression by FXR deficiency impaired the downstream JAK3-STAT1 signaling pathways, which in turn increased osteoclast formation. Finally, FXR deficiency accelerated unloading- or ovariectomy-induced bone loss in vivo. Thus, our findings demonstrate that FXR is a negative modulator in osteoclast differentiation and identify FXR as a potential therapeutic target for postmenopausal osteoporosis and unloading-induced bone loss.

In vivo optical frequency domain imaging of human retina and choroid.

Optical frequency domain imaging (OFDI) using swept laser sources is an emerging second-generation method for optical coherence tomography (OCT). Despite the widespread use of conventional OCT for retinal disease diagnostics, until now imaging the posterior eye segment with OFDI has not been possible. Here we report the development of a highperformance swept laser at 1050 nm and an ophthalmic OFDI system that offers an A-line rate of 18.8 kHz, sensitivity of >92 dB over a depth range of 2.4 mm with an optical exposure level of 550 muW, and deep penetration into the choroid. Using these new technologies, we demonstrate comprehensive human retina, optic disc, and choroid imaging in vivo. This advance enables us to view choroidal vasculature in vivo without intravenous injection of fluorescent dyes and may provide a useful tool for evaluating choroidal as well as retinal diseases.

Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina.

To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α - CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

Environmentally-stable femtosecond ytterbium fiber laser with birefringent photonic bandgap fiber.

We demonstrate an environmentally-stable mode-locked ytterbium fiber laser. The large birefringence of hollow-core photonic bandgap fiber allows it to control polarization in the laser while it provides the anomalous dispersion necessary for stretched-pulse operation. The laser generates 1-nJ pulses, which are dechirped to 70 fs.

Visualization of dynamics of single endogenous mRNA labeled in live mouse.

The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all ß-actin mRNA is fluorescently labeled. We found that ß-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple ß-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of ß-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

Effect of axonal micro-tubules on the morphology of retinal nerve fibers studied by second-harmonic generation.

Many studies suggest that the degradation of microtubules in the retinal ganglion cells may be an early event in the progression of glaucoma. Because reflectance and birefringence of the retinal nerve fibers arise primarily from microtubules, the optical properties have been intensively studied for early detection of the disease. We previously reported a novel nonlinear optical signal from axonal microtubules for visualizing the retinal nerve fibers, namely second-harmonic generation (SHG). We demonstrate the use of axonal SHG to investigate the effect of microtubules on the morphology of the retinal nerve fiber bundles. Time-lapse SHG imaging of ex vivo rat retinal flat mounts was performed during pharmacological treatment of nocodazole, and the intensity of axonal SHG and the changes in nerve fiber bundle morphology were monitored. We found that the microtubule disruption does not lead to immediate modification in the morphology of the nerve fibers. Our results indicate that microtubular SHG may provide a useful means for sensitive detection of axonal injuries. Since the intrinsic radiation depends on the regular architecture of the cytoskeleton element as maintained by active cellular regulations, the intensity of signal reflects the health of the retinal ganglion cell axons.