Dataset Information

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.

ABSTRACT: Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

SUBMITTER: Gogoi P

PROVIDER: S-EPMC9374259 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.

Gogoi Prerana P Shiozaki Momoko M Gouaux Eric E

PloS one 20220812 8

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory s ...[more]

PMID: 35960737

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Project description:PurposeTo determine the diagnostic accuracy of in vivo confocal microscopy (IVCM) for moderate to severe microbial keratitis (MK).DesignDouble-masked prospective cohort study.ParticipantsConsecutive patients presenting to Aravind Eye Hospital, Madurai, India, between February 2012 and February 2013 with MK (diameter ≥3 mm, excluding descemetocele, perforation, or herpetic keratitis).MethodsFollowing examination, the corneal ulcer was scanned by IVCM (HRT3/RCM, Heidelberg Engineering, Heidelberg, Germany). Images were graded for the presence or absence of fungal hyphae or Acanthamoeba cysts by the confocal microscopist who performed the scan (masked to microbial diagnosis) and 4 other experienced confocal graders (masked to clinical features and microbiology). The regrading of the shuffled image set was performed by 3 graders, 3 weeks later. Corneal-scrape samples were collected for microscopy and culture.Main outcome measuresThe main outcome measures were sensitivity, specificity, and positive and negative predictive values of IVCM compared with those of a reference standard of positive culture or light microscopy. Sensitivities and specificities for multiple graders were pooled and 95% confidence intervals calculated using a bivariate random-effects regression model.ResultsThe study enrolled 239 patients with MK. Fungal infection was detected in 176 (74%) and Acanthamoeba in 17 (7%) by microbiological methods. IVCM had an overall pooled (5 graders) sensitivity of 85.7% (95% confidence interval [CI]: 82.2%-88.6%) and pooled specificity of 81.4% (95% CI: 76.0%-85.9%) for fungal filament detection. For Acanthamoeba, the pooled sensitivity was 88.2% (95% CI: 76.2%-94.6%) and pooled specificity was 98.2% (95% CI: 94.9%-99.3%). Intergrader agreement was good: κ was 0.88 for definite fungus; κ was 0.72 for definite Acanthamoeba. Intragrader repeatability was high for both definite fungus (κ: 0.88-0.95) and definite Acanthamoeba classification (κ: 0.63-0.90). IVCM images from 11 patients were considered by all 5 graders to have a specific organism present (10 fungus, 1 Acanthamoeba) but had negative results via culture and light microscopy.ConclusionsLaser scanning IVCM performed with experienced confocal graders has high sensitivity, specificity, and test reproducibility for detecting fungal filaments and Acanthamoeba cysts in moderate to large corneal ulcers in India. This imaging modality was particularly useful for detecting organisms in deep ulcers in which culture and light microscopy results were negative.

Dataset Information

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.

Publications

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.

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