title: Scanning transmission electron microscopy through-focal tilt-series on biological specimens.
abstract: Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.
authors.affiliation: Institut Curie, Centre de Recherche, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; INSERM U1196, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; CNRS UMR9187, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France. Electronic address: sylvain.trepout@curie.fr.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Trepout
authors.affiliation: Institut Curie, Centre de Recherche, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; INSERM U1196, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; CNRS UMR9187, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Messaoudi
authors.affiliation: Institut Pasteur, Trypanosome Cell Biology Unit, 25 rue du Docteur Roux, 75015 Paris, France; INSERM U1201, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Perrot
authors.affiliation: Institut Pasteur, Trypanosome Cell Biology Unit, 25 rue du Docteur Roux, 75015 Paris, France; INSERM U1201, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Bastin
authors.affiliation: Institut Curie, Centre de Recherche, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; INSERM U1196, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France; CNRS UMR9187, Bât. 112, Centre Universitaire, 91405 Orsay Cedex, France.
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
mesh_terms: D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods; D005407:Flagella / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017348:Microscopy, Electron, Scanning Transmission / Q000295:instrumentation* / Q000379:methods*; D059629:Signal-To-Noise Ratio; D014057:Tomography, X-Ray Computed; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000648:ultrastructure
keywords: Depth of field; Flagella; Scanning transmission electron microscopy; Through-focal tilt-series; Tomography; Transmission electron microscopy; Trypanosome