Ultra 897 emccd camera

Cells were grown in 96-well glass bottom dishes (Matriplate, Brooks). Images were acquired using a Yokogawa CSU-X1 spinning disk confocal attached to an inverted Nikon TI microscope with Nikon Perfect Focus system, 100× NA 1.49 objective, an Andor iXon Ultra 897 EM-CCD camera, and Micro-Manager software (Edelstein et al., 2010 ). Single z-plane images were acquired every 30 s unless noted otherwise. During image acquisition, cells were maintained at a constant temperature of 36°C–37°C. Camera exposure times were generally set to 500 ms, unless noted otherwise. We note that stable expression of PP7-mCherry, either with or without the CAAX domain, also resulted in an accumulation of mCherry signal in lysosomes, but lysosomes could be readily distinguished from mRNA foci based on signal intensity and mobility.

Microscopy was performed on spinning disk confocal microscope (Yokogawa CSU-X1) set up on a Nikon Eclipse Ti inverted microscope with a 100× ApoTIRF 1.4 NA objective (Nikon) in line with 2× amplification. BODIPY 493/503 fluorophore was exited on 561 nm laser line. Fluorescence was detected by an iXon Ultra 897 EMCCD camera (Andor). Acquired images were processed using FIJI software (http://fiji.sc/Fiji).

All microscopy experiments were carried out with a custom-made superresolution setup based on an inverted Zeiss Axiovert 200 body equipped with a Zeiss Apochromat 100×/1.45 NA oil-immersion objective (Zeiss, Oberkochen, Germany). We used a 637 nm Coherent OBIS laser for excitation and an iXon Ultra 897 EM-CCD camera (Andor, Belfast, UK). Fluorescence microscopy measurements were carried out with a freshly prepared dSTORM buffer according to van de Linde et al. [21 (link)]: 500 µL 20% glucose, 200 µL 10× PBS, 230 µL distilled water, 50 µL 1 M cysteamine, 10 µL 50 mg/mL glucose oxidase, 10 µL 12.6 mg/mL catalase. The buffer was added to each chamber immediately before imaging. For each SMLM image, we recorded 40,000 frames in epi-configuration, whereby the first 10,000 were not included in the analysis. The illumination time was 1 ms and the delay between consecutive frames was 7 ms. The raw data was pre-processed with temporal filtering to separate the slowly bleaching background from the rapidly flashing signals [10 (link)], and single-molecule signals were localized using Daostorm [22 (link)]. Finally, images were displayed utilizing a Gaussian kernel density estimator.