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19.1. NIR/IR multi-photon microscope setup. The beam of a mode-locked titanium-sapphire (TiSa) laser (80 MHz, 120 fsec) is divided using a beam splitter (BS). One part is used to pump an optical parametric oscillator (OPO; 80 MHz, 200 fsec), the other part is used for imaging the specimen directly. The OPO shifts the TiSa wavelength range from 680 to 1080 nm to a longer wavelength range of 1000 to 1600 nm. Both beams pass a beam-shaping device and are combined via a dichroic mirror (DM) before entering a scan head, which allows the simultaneous use of the TiSa and OPO beam. SHG and THG emission signals are collected in the forward direction by a condenser (C). Fluorescence light is collected in the backward direction through the objective lens. For spectral separation, dichroic mirrors and band pass filters (F) were used in front of the photomultiplier tube (PMT). LBF, laser-blocking filter; SL, scan lense; TL, tube lense.
Source publication
In this protocol, we combine two-photon excitation fluorescence to visualize in caenorhabditis elegans (C. elegans) dopaminergic (DAergic) neurons and their processes with non-linear optical measurements to reconstruct the three-dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid-body region. Femto seco...
Contexts in source publication
Context 1
... beam multi-photon microscope setup (Fig. 11.19.1 IR light source (e.g., Coherent Chameleon Compact OPO), capable of automated 1000 to 1600 nm wavelength extension, 200 fsec pulse width, 80 MHz repetition rate 20× IR objective lens (e.g., Olympus XLUMPlanFl 20×/1.0 W, working distance of 2.0 mm). Two x-y galvanometric mirrors, for scanning the sample at a rate of up to 1200 lines/s ...
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... Using the ImSpector software, assemble the MSR image files into one multi-color image stack, project the z-stack into a single image, and export the file as a highquality TIFF file (Fig. ...
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... files into a single multi-color image stack, and export the individual color channels as OME-TIFF files. 14. Import the files into image processing and analysis software (e.g., Imaris x64 7.7.2, Bitplane) for surface rendering according to the software manufacturer's recommended procedures, and export the surface-rendered plot as a TIFF file (Fig. ...
Citations
... Traditionally, light microscopy has been used in invertebrate neuroscience research to map the coarse architecture of the nervous system, with electron microscopy being employed for fine ultrastructural analysis (Schmidt-Rhaesa et al., 2016). More recently, confocal and multiphoton microscopy have facilitated the generation of highresolution 2D and 3D images of both thicker whole-mount and live specimens (Bixel et al., 2015). Laser microdissection tools provide an alternative to labor-intensive antibody-based experiments by enabling the post-capture profiling of neuropeptides (e.g. via RNAseq) in specific neurons or in tissues embedded in heterogeneous samples (Fricker, 2012). ...
Neuropeptides are one of the most diverse classes of signaling molecules and have attracted great interest over the years owing to their roles in regulation of a wide range of physiological processes. However, there are unique challenges associated with neuropeptide studies stemming from the highly variable molecular sizes of the peptides, low in vivo concentrations, high degree of structural diversity and large number of isoforms. As a result, much effort has been focused on developing new techniques for studying neuropeptides, as well as novel applications directed towards learning more about these endogenous peptides. The areas of importance for neuropeptide studies include structure, localization within tissues, interaction with their receptors, including ion channels, and physiological function. Here, we discuss these aspects and the associated techniques, focusing on technologies that have demonstrated potential in advancing the field in recent years. Most identification and structural information has been gained by mass spectrometry, either alone or with confirmations from other techniques, such as nuclear magnetic resonance spectroscopy and other spectroscopic tools. While mass spectrometry and bioinformatic tools have proven to be the most powerful for large-scale analyses, they still rely heavily on complementary methods for confirmation. Localization within tissues, for example, can be probed by mass spectrometry imaging, immunohistochemistry and radioimmunoassays. Functional information has been gained primarily from behavioral studies coupled with tissue-specific assays, electrophysiology, mass spectrometry and optogenetic tools. Concerning the receptors for neuropeptides, the discovery of ion channels that are directly gated by neuropeptides opens up the possibility of developing a new generation of tools for neuroscience, which could be used to monitor neuropeptide release or to specifically change the membrane potential of neurons. It is expected that future neuropeptide research will involve the integration of complementary bioanalytical technologies and functional assays.
... Neuropeptide localisation approaches are typically either antibody-mediated, or involve spatial mapping of gene transcripts/promoters, and are often coupled to high-resolution optical imaging platforms. For a detailed description microscopy have been used to create three-dimensional maps of C. elegans tissues, including pharynx, muscle, and neurons [35,36]. This type of advanced imaging tool could enable the construction of species-specific neuropeptide connectomes which, when overlaid with functional data, could significantly advance our understanding of nematode neuropeptide biology. ...
Expanding 'omics' datasets for parasitic nematodes have accelerated the identification of putative drug targets derived from the nematode nervous system. However, novel drug target validation is hampered by the absence of adequate localisation, functional characterisation, and receptor deorphanisation tools in key nematode pathogens. Reverse genetics techniques have advanced to encompass transgenic, targeted mutagenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9) approaches in Caenorhabditis elegans. Unfortunately the translation to nematode pathogens has been slow, such that parasite-focused toolbox development and optimisation is critical. Here we review the discovery, localisation, and functional characterisation toolkit available for parasitic nematode neuropeptide research, and assess the scope and limitations of the tools and techniques for novel nematicide discovery.
