title: Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model.
abstract: High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Rohola
authors.initials: R
authors.lastname: Hosseini
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Gerda Em
authors.initials: GE
authors.lastname: Lamers
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Zlatan
authors.initials: Z
authors.lastname: Hodzic
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Annemarie H
authors.initials: AH
authors.lastname: Meijer
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Marcel Jm
authors.initials: MJ
authors.lastname: Schaaf
authors.affiliation: Institute of Biology; Leiden University; Leiden, The Netherlands.
authors.forename: Herman P
authors.initials: HP
authors.lastname: Spaink
mesh_terms: D058500:Animal Fins / Q000382:microbiology / Q000473:pathology / Q000648:ultrastructure; D000818:Animals; D001343:Autophagy; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007814:Larva / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008854:Microscopy, Electron / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009165:Mycobacterium Infections, Nontuberculous / Q000175:diagnosis* / Q000473:pathology*; D019910:Mycobacterium marinum / Q000502:physiology* / Q000648:ultrastructure; D015027:Zebrafish / Q000382:microbiology*; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D008869:Microtubule-Associated Proteins; D029961:Zebrafish Proteins; C542260:map1lc3b protein, zebrafish; D049452:Green Fluorescent Proteins
keywords: TEM; autophagic vacuole; confocal laser scanning microscopy; correlative; imaging; infection; mycobacterium; tail fin; zebrafish