Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

Stephanie A. Meyer; Baris N. Ozbay; Mariana Potcoava; Ernesto Salcedo; Diego Restrepo; Emily A. Gibson

doi:10.1117/1.JBO.21.6.066017

28 June 2016 Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

Stephanie A. Meyer, Baris N. Ozbay, Mariana Potcoava, Ernesto Salcedo, Diego Restrepo, Emily A. Gibson

Author Affiliations +

Journal of Biomedical Optics, Vol. 21, Issue 6, 066017 (June 2016). https://doi.org/10.1117/1.JBO.21.6.066017

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.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Citation Download Citation

Stephanie A. Meyer, Baris N. Ozbay, Mariana Potcoava, Ernesto Salcedo, Diego Restrepo, and Emily A. Gibson "Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope," Journal of Biomedical Optics 21(6), 066017 (28 June 2016). https://doi.org/10.1117/1.JBO.21.6.066017

Published: 28 June 2016

ACCESS THE FULL ARTICLE

JOURNAL ARTICLE
8 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY