Plan apochromat 40 1.3 na

FRAP experiments were performed using an inverted ZEISS 980 confocal microscope (Carl ZEISS) with a Plan-Apochromat ×40/1.3 NA oil-immersion objective. Cells were kept at 37 °C and allowed equilibrate for 30 min before data acquisition. Fifteen image scans were conducted, followed by a bleach pulse of 5 ms on a spot of one pixel within a reference area. Bleaching was performed using a 10-mW 488 nm laser operating at 80% laser power. A series of 385 single-focal-plane images was then collected at low laser intensity (0.02% of the 10 mW laser), with 270-ms intervals for the M120I mutant and 860-ms intervals for other groups. Image size was 926 × 263 pixels and the pixel width was 92 nm. To quantify the dynamic fluorescence signals, a region outside of the cell labeled as “background” and a region with fluorescent signal but not affected by photobleaching labeled as “reference” were first identified to correct the data at each time-point for any bleaching artifacts that occurred during the imaging process. Mean fluorescence intensities within the regions were used. Mobile and immobile fractions within selected bleached regions were then quantified. All images were processed in a blinded manner using the FRAP function in ZEN (black edition) with a mono-exponential model for data-fitting.