Sp8 sted 3 microscope

Specimen slides were prepared as before. Following overnight incubation with primary antibodies, samples were incubated with two secondary antibodies (goat anti-chicken IgY Alexa Flour Plus 647 [Thermo Fischer Scientific, Waltham, MA] and goat anti-rabbit IgG Alexa Flour 488 [Abcam, Cambridge MA]), rinsed, and then stained with DAPI using the TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA) as outlined by the manufacturer. Images were captured using a Leica SP8 STED 3 × microscope (Leica Microsystems Inc., Buffalo Grove, IL). Acquired Z-stacks were further processed to generate figure images using Fiji ImageJ software (version 1.0, https://imagej.net/Fiji)^{57 (link)}.

Live-cell STED microscopy was conducted on a Leica SP8 STED 3× microscope (Leica Microsystems, Wetzlar, Germany) equipped with a SuperK EXTREME EXW-12 pulsed white light laser (NKT Photonics Inc., Portland, OR) as an excitation source, a Katana-08HP pulsed depletion laser as a depletion light source (775 nm; NKT Photonics), and a Leica high-contrast plan apochromat 93× 1.30 numerical aperture (NA) glycerol CS2 objective with motorized correction collar (Leica Microsystems); imaging was controlled by Leica Application Suite X software (LAS X, Leica Microsystems). Cells were imaged in a temperature-controlled Tokai Hit microscope stage top incubator (Amuza Inc., San Diego, CA) set to 37°C with 5% CO₂. mNeon-KDEL was imaged with 505-nm excitation and 775-nm depletion wavelengths. The detection gate was set at 0.3 to 6 ns with a hybrid detector. Forty-nanometer pixel size was chosen on the basis of the Nyquist sampling criterion. All live-cell image stacks were acquired with 2× line averaging and 0.1-μm z-steps with 30 ms per frame. Image deconvolution was performed to remove noise via the classic maximum likelihood estimation deconvolution algorithm using Huygens Professional software (version 16.10, Scientific Volume Imaging, Hilversum, the Netherlands) as described previously (78 (link)). Deconvolution of the original raw image data was limited to 20 iterations of processing.

CTL were seeded on poly-l-lysin-coated high-performance coverslips and fixed in 3% PFA (10 min, 37°C). Permeabilization and staining were performed in PBS 3% BSA, 0.1% saponin (Sigma) for 5 min and 60 min, respectively. Cells were stained with an anti-human Granzyme B antibody (10 µg/ml, clone GB11, Thermo Scientific) followed by a goat anti-mouse IgG Abberior Star 580 (Abberior Instruments). Coverslips (high-performance D = 0.17 mm ± 0.005, ZEISS, Germany) were mounted on microscopy slides using Mowiol-DABCO.
STED images were acquired with a Leica SP8 STED 3× microscope (Leica Microsystems, Germany) using a HC PL APO CS2 100×/1.4 oil immersion objective. To optimize resolution without bleaching in 3D, the 775 nm STED laser line was applied at the lowest power that can provide sufficient improvement in resolution compared to confocal. Z-stack series were acquired sequentially with the pulsed 532 nm laser. For image acquisition, we used the following parameters: three time average/line, 400 Hz scan speed. STED images were subsequently deconvoluted with Huygens Professional (SVI, USA) using the CMLE algorithm, with a signal to noise ratio (SNR) of 7. 3D image visualization was performed using the Fiji software.