Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom STED microscope

摘要

We performed super-resolution imaging of isolated olfactory sensory neurons (OSNs) using a custom-built Stimulated Emission Depletion (STED) microscope. The design for the STED microscope is based on the system developed in the laboratory of Dr. Stefan Hell. Our system is capable of imaging with sub-diffraction limited resolution simultaneously in two color channels (at Atto 590/Atto 647N wavelengths). A single, pulsed laser source (ALP; Fianium, Inc.) generates all four laser beams, two excitation and two STED. The two STED beams are coupled into one polarization maintaining (PM) fiber and the two excitation beams into another. They are then collimated and both STED beams pass through a vortex phase plate (RPC Photonics) to allow shaping into a donut at the focus of the objective lens. The beams are then combined and sent into an inverted research microscope (IX-71; Olympus Inc.) allowing widefield epifluorescence, brightfield and DIC imaging on the same field of view as STED imaging. A fast piezo stage scans the sample during STED and confocal imaging. The fluorescent signals from the two color channels are detected with two avalanche photodiodes (APD) after appropriate spectral filtering. The resolution of the system was characterized by imaging 40 nm fluorescent beads as ～60 nm (Atto 590) and ～50 nm (Atto 647N). We performed STED imaging on immunolabeled isolated OSNs tagged at the CNGA2 and ANO2 proteins. The STED microscope allows us to resolve ciliary CNGA2 microdomains of ～54 nm that were blurred in confocal.