In Vivo Imaging of Myelin in the Vertebrate Central Nervous System Using Third Harmonic Generation Microscopy

Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.
Biophysical Journal (Impact Factor: 3.97). 03/2011; 100(5):1362-71. DOI: 10.1016/j.bpj.2011.01.031
Source: PubMed


Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy-a coherent, nonlinear, dye-free imaging modality-provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery.

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Available from: Chris Schaffer, Oct 05, 2015
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    • "The tunica vaginalis forms a compact myelin structure at 7 dpf (Brösamle and Halpern, 2002). Most myelin-associated mammalian genes have homologies with zebrafish, including dm20, mbp, sox10, etc. (Emery, 2010; Farrar et al., 2011; Schweitzer et al., 2006). Chemical methods have often been used to induce demyelination in mouse models (Merrill, 2009). "
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    ABSTRACT: Demyelinating diseases consist of a variety of autoimmune conditions in which the myelin sheath is damaged due to genetic and/or environmental factors. During clinical treatment, some patients undergo partial remyelination, especially during the early disease stages. However, the mechanisms that regulate demyelination remain unclear. The myelin structure, myelin formation and myelin-related gene expression are highly conserved between mammals and zebrafish. Therefore, the zebrafish is an ideal model organism to study myelination. In this study, we generated a transgenic zebrafish Tg(mbp:nfsB-egfp) expressing a fusion protein composed of enhanced green fluorescent protein (EGFP) and NTR from the myelin basic protein (mbp) promoter. Tg(mbp:nfsB-egfp) expressed NTR-EGFP reproducibly and hereditarily in oligodendrocytes along the spinal cord. Treatment of zebrafish larvae Tg(mbp:nfsB-egfp) with metronidazole (Mtz) resulted in the selective ablation of oligodendrocytes and led to demyelination, accompanied by behavioral changes, including decreased total movement distance, velocity, total movement time and fast movement time. After withdrawal of Mtz for a seven day recovery period, the expression of EGFP and MBP protein was observed again which indicates remyelination. Additionally, locomotor capacity was restored. Collectively, Tg(mbp:nfsB-egfp), a heritable and stable transgenic line, provides a novel, powerful tool to study the mechanisms of demyelination and remyelination. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 11/2014; 4(1). DOI:10.1242/bio.201410736 · 2.42 Impact Factor
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    • "Fluorophores are essential for 2PM, either by injectable tracers, like intravenous bolus injection of fluorescein-conjugated dextran to target blood vessels and blood flow (Shih et al., 2012), or FPs expressed by specific cell types (Belluscio, 2005). Label-free 2PM of the living mouse brain has been reported by Witte et al. (2011) by using intrinsic non-linear light interactions, referred to second- and third-harmonic generation, which can be applied to visualize, e.g., myelin (Farrar et al., 2011). Zhang and Murphy (2007) and Sigler and Murphy (2010) could identify with 2PM to which extent reduced blood flow in stroke leads to changes in synaptic circuitry. "
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    ABSTRACT: Transplanted stem cells can induce and enhance functional recovery in experimental stroke. Invasive analysis has been extensively used to provide detailed cellular and molecular characterization of the stroke pathology and engrafted stem cells. But post mortem analysis is not appropriate to reveal the time scale of the dynamic interplay between the cell graft, the ischemic lesion and the endogenous repair mechanisms. This review describes non-invasive imaging techniques which have been developed to provide complementary in vivo information. Recent advances were made in analyzing simultaneously different aspects of the cell graft (e.g., number of cells, viability state, and cell fate), the ischemic lesion (e.g., blood-brain-barrier consistency, hypoxic, and necrotic areas) and the neuronal and vascular network. We focus on optical methods, which permit simple animal preparation, repetitive experimental conditions, relatively medium-cost instrumentation and are performed under mild anesthesia, thus nearly under physiological conditions. A selection of recent examples of optical intrinsic imaging, fluorescence imaging and bioluminescence imaging to characterize the stroke pathology and engrafted stem cells are discussed. Special attention is paid to novel optimal reporter genes/probes for genetic labeling and tracking of stem cells and appropriate transgenic animal models. Requirements, advantages and limitations of these imaging platforms are critically discussed and placed into the context of other non-invasive techniques, e.g., magnetic resonance imaging and positron emission tomography, which can be joined with optical imaging in multimodal approaches.
    Frontiers in Cellular Neuroscience 08/2014; 8:226. DOI:10.3389/fncel.2014.00226 · 4.29 Impact Factor
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    • "Therefore, a pulsed laser source with a wavelength of 1200 nm or higher is required to generate THG within the visible range (400–800 nm). THG signals with shorter wavelengths have been successfully recorded [23], [24] but since most optical glasses absorb UV light, such signals are attenuated if not all glass elements are UV transmissible. "
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    ABSTRACT: Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG) microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200-1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label.
    PLoS ONE 06/2014; 9(6):e99615. DOI:10.1371/journal.pone.0099615 · 3.23 Impact Factor
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