Wc apochromat corr wd 0.28 m27 objective

Cells were plated prior to imaging on 10 μg/ml fibronectin-coated glass-bottomed dishes (MatTek Corporation). The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner. Appropriate filters, heated sample environment (+37°C), controlled CO₂, and 63×/1.2 WC-Apochromat Corr WD = 0.28 M27 objective (Zeiss) were used. The images were acquired via SlideBook 7.0 software (3I intelligent Imaging Innovations) and recorded via Neo sCMOS (Andor) camera. The recording was set as every 1 sec for 10 min and one focal plane was recorded for all live cell videos. For tracking and speed measurement of CAV-1 vesicles, the Imaris 9.2 (Bitplane) ‘Track’ module with globular-objects over time was used as in previous study (Jiu, 2018 (link)). Then, 2 μm estimated XY diameter, 5 μm max distance, and 3 max gap size were set for analyzing.

Cells were transfected with CAV-1-mEGFP and incubated for 24 h. Confocal images were acquired with a 3I Marianas imaging system (3I Intelligent Imaging Innovations, Denver, CO, United States), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss, Oberkochen, Germany), a Yokogawa CSU-X1 M1 confocal scanner and 63 × /1.2 WC-Apochromat Corr WD = 0.28 M27 objective (Zeiss). Heated sample environment (+37°C) and 5% CO₂ control were used. SlideBook 6.0 software (3I Intelligent Imaging Innovations) was used for the image acquirement. Five pre-bleach images were acquired followed by bleaching scans with 100% intensity laser lines over the region of interest. Recovery of fluorescence was monitored 50 times every 200 ms and 300 times every 1 s. The intensity of the bleached area was normalized to a neighboring non-bleached area. Mean scatter plots were calculated from different FRAP experiments and the means and standard deviations were calculated.