N storm super resolution microscope system

Fluorescent liposomes were imaged in both 2D and 3D experiments at an exposure time of 100 ms. 2D intersection experiments were performed using a customized Nikon Eclipse Ti U microscope and labelled actin and liposomes were excited using a Prior Lumen 200 epi-fluorescent lamp. The microscope was equipped with a Nikon 100 × /1.49 N.A. Plan Apo objective lens and a CCD camera (model Turbo 620 G; Stanford photonics) and image intensifier (model VS4-1845; Video Scope International). Experiments investigating the 3D helical trajectory of motor–cargo complexes moving on suspended actin filaments were performed on a modified Nikon TE-2000 inverted microscope equipped with a Nikon 100 × /1.49 N.A. Plan Apo objective lens and a Stanford Photonics XR/Turbo G intensified CCD camera, using Piper software for image acquisition. A removable 1 m focal length cylindrical lens was installed between the camera relay lens and the camera for 3D imaging, and a Custom Z-axis focus lock controller using optical feedback piezo stage control was used for all 3D helical trajectory experiments.
For 3D intersection assay, 3D STORM images were acquired using a Nikon N-STORM super-resolution microscope system using a 647 and 405 nm laser for excitation of Alexa 647-phalloidin-labelled actin and 3D cylindrical lens added to the light path.

High-resolution fluorescence microscopy by dSTORM imaging was performed on a TIRF-based Nikon N-STORM Super-Resolution microscope system according to published protocols^{20 (link),23 (link)}. All imaging was performed using the SR Apochromat TIRF 100 × 1.49 N.A. objective lens. We used the MEA-based imaging buffer as previously described, consisting of 10 mM 2-aminoethanethiol (MEA; Fluka) in 50 mM Tris, pH 8.0, 10 mM NaCl, 10% (w/v) glucose, and 0.5 mg/ml glucose oxidase (Sigma-Aldrich) and 40 μg/ml catalase (Sigma-Aldrich) as the oxygen scavenger system^{20 (link)}. PLIN1 in human primary adipocytes was detected by immunofluorescence staining using a primary PLIN1-specific primary antibody and the Alexa Fluor® 647 conjugated secondary antibody. This approach can visualize cellular structures with a resolution of approximately 20 nm, which is well below the Abbe diffraction limit^{22 (link)}. Another advantage with this approach is that the adjustable TIRF angle only penetrates to a depth of only approximately 100 nm into the sample medium, which enables selective visualization of surface regions (reviewed in ref.^{20 (link)}). The Nikon NIS-Elements software was used for data acquisition, image processing and analysis.