Hc pl apo cs2 100 1.40 oil objective

STED images with a 660 nm STED laser were acquired on a Leica TCS SP8 STED 3X scanning microscope (Leica Microsystems) equipped with an HC PL APO CS2 100×/1.40 oil objective and the incubator set to 37 °C and 5% CO₂. The 4-TMR-LTX probe was excited with the 561 nm, 80 MHz pulsed line of a white light laser, de-excited with a continuous wave 660 nm STED laser and detected in the 600/50 nm window. In this setup, the pixel size was set to 23 nm in the xy plane. The STED effect was estimated as described above.

We used a gated STED-3X-WLL SP8 microscope (Leica Microsystems, Wetzlar, Germany) and a HC Pl Apo CS2 100×/1.40 oil objective for all experiments. The microscope was equipped with 592-nm and 660-nm depletion lasers, and the excitation was provided by a pulsed white laser. The acquisition software was LAS X 3.5.6.21594 (Leica Microsystems, Wetzlar, Germany). Cells were stained with anti-myosin-Alexa-532 Ab and/or anti-actin-Alexa-568 Ab. STED images were acquired under X,Y depletion at 660 nm, deconvolved (Huygens software, Scientific Volume Imaging BV, Hilversum, The Netherlands) and analyzed by ImageJ/Fiji software (Image.net).^{32 (link)} For details see Supplemental methods section and previous protocol optimization.²⁴