Tcs sp8 gated sted 3x microscope

Immunolabeled samples were imaged on either a custom built 2D FPALM setup as described previously (Huang et al., 2013 (link); Lin et al., 2015 (link)) or a custom built W-4PiSMSN microscope (Huang et al., 2016 ). For STED based imaging, we performed fixation and immunolabeling but used secondary antibodies conjugated to ATTO647N or ATTO594 dyes. These samples were imaged on a Leica TCS SP8 Gated STED 3x microscope.

HeLa cells were labeled with DiIC₁₆TCO or DiIC_16’TCO and SiR-Tz as described above. STED microscopy was performed on a Leica TCS SP8 Gated STED 3x microscope equipped with a tunable pulsed white light laser (460 – 660 nm) for excitation and two HyD detectors for tunable spectral detection. The microscope offers three STED depletion lasers (592 nm, 660 nm, and 775 nm). For all live-cell imaging, the microscope was equipped with a Tokai Hit stage top incubator (model: INUBG2A-GSI) with temperature and CO₂ control to maintain an environment of 37°C and 5% CO₂. In this work, SiR derivatives were excited at 633 nm (15% power: HeLa cell imaging, Figure 4a; 20% power: fibroblasts, Figure 4e, Figure 5a) and their emission was detected using a HyD detector from 650–737 nm. The 775 nm depletion laser was used for STED microscopy (20% power). Imaging was conducted with a 100x oil immersion objective (HC PL APO 100x/1.40 OIL) at 1000 Hz with 1-line accumulation and bi-directional x-scanning in a 19.38 μm² field of view (1024 × 1024 pixels at 18.94 nm/pixel). Raw microscopy data were Gaussian blurred (1.0 pixels) in ImageJ. The FWHM value were obtained by fitting line profiles to a Lorentz distribution using Origin 8.2 (www.originlab.com).