title: Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope.
abstract: We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44  nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48  nm and 124±43  nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Bioengineering, MS 8607, 12700 East 19th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Meyer
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Bioengineering, MS 8607, 12700 East 19th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Baris N
authors.initials: BN
authors.lastname: Ozbay
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Bioengineering, MS 8607, 12700 East 19th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Mariana
authors.initials: M
authors.lastname: Potcoava
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Cell and Developmental Biology, MS 8108, 12801 East 17th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Salcedo
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Cell and Developmental Biology, MS 8108, 12801 East 17th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Diego
authors.initials: D
authors.lastname: Restrepo
authors.affiliation: University of Colorado Denver Anschutz Medical Campus, Department of Bioengineering, MS 8607, 12700 East 19th Avenue, Aurora, Colorado 80045-2560, United States.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Gibson
mesh_terms: D000818:Animals; D002923:Cilia / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D007834:Lasers; D051379:Mice; D008856:Microscopy, Fluorescence; D018034:Olfactory Receptor Neurons / Q000648:ultrastructure*; D061848:Optical Imaging / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes