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.
Highlights
For over a century of optical microscopy, the diffraction of light limited the study of cellular structures to a resolution of ∼200 nm
We demonstrate use of the two-color stimulated emission depletion (STED) microscope by imaging transduction proteins on the cilia of olfactory sensory neurons (OSNs) to reveal sub-diffraction-limited structures involved in olfactory transduction
We describe construction of a custom-built two-color STED microscope and demonstrate capabilities for imaging protein– protein localization in biological samples
Summary
For over a century of optical microscopy, the diffraction of light limited the study of cellular structures to a resolution of ∼200 nm. There are several widely used SR techniques including stimulated emission depletion (STED),[2,3] photo-activated localization microscopy (PALM),[4] stochastic optical reconstruction microscopy (STORM),[5] and structured illumination microscopy (SIM).[6] Other SR techniques have been developed including microlenses[7] for white light imaging or contact microspheres for fluorescence imaging of biological samples.[8]. One of the limitations of STED microscopy compared with other SR techniques is that it requires higher laser powers than standard fluorescence imaging to saturate the stimulated emission transition. High STED laser powers can potentially lead to sample heating, photobleaching, and photodamage that can pose a problem when imaging
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