Tsc sp8 microscope

HeLa cells were cultivated on 35-mm dishes with a glass-bottom (µ-Dish 35 mm, high, Ibidi) and transfected with EGFP-tagged SRSF2 [43 (link)]. For FRAP analysis, cells on cultivation dishes were placed into a cultivation hood (EMBL, Heidelberg, Germany), maintained at 37 °C and 5% CO₂. Time-lapse imaging of live cells was carried out in a confocal Leica TSC SP8 microscope, equipped with an argon laser and objective with 64× magnification, and the numerical aperture NA = 1.4. The time of pre-bleaching was 2.61 s, during which fluorescence intensity (FI) was measured. Bleaching of defined regions (2 μm²) was performed using an argon laser at 100% laser power (laser line 488 was used). Bleaching was performed for 0.783 s, and post-bleaching observation for 20.88 s, with an interval of 0.261 s. During the whole procedure, the fluorescence intensity was measured. Scanning was done at 0.5% power of the 488-nm argon laser and with a frame resolution of 512 × 512 pixels. The scanning rate was 1000 Hz, and an 8x zoom was used. We analyzed data using LEICA LAS AF lite 4 software, and line graphs were created by Excel software.

Confocal images in Figures 2,3,4,5 were captured with a Leica TSC SP8 microscope using LAS X software (Leica, Wetzlar, Germany). Confocal images in Figures 1,6 and 7 and Supplementary Figures 1 and 2 were taken with a Nikon eclipse TE 2000-U microscope using the EZ-C1 3.30 software (Nikon, Tokyo, Japan). Confocal images stacks were exported in original format and processed using Fiji, an image processing package - a "batteries-included" distribution of ImageJ, bundling a lot of plugins which facilitate scientific image analysis. All images were then processed further using Adobe Photoshop CS5 software (Adobe Systems Canada, Ottawa, ON, Canada).