Fibronectin coated glass bottomed dishes

U2OS cells were transiently transfected with siRNA and Mito–DsRed, a plasmid encoding red fluorescent protein fused to a mitochondrial targeting peptide from subunit VIII of human cytochrome c oxidase (MSVLTPLLLRGLTGSARRLPVPRAKIHSLPP), prior to 72 h and 48 h, respectively. Cells were then replated on 10 μg/ml fibronectin-coated glass-bottomed dishes (MatTek). The dish was placed in a heated sample chamber with 5% CO₂. The time-lapse images were acquired with a Marianas imaging system (3I) equipped with an inverted spinning-disk confocal microscope (Axio-Observer Z1; Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner. A 63$ 1.2 W C-Apochromat Corr working distance=0.28 M27 objective was used, and all the images were acquired by an sCMOS (Andor) Neo camera and Slidebook 5.0 software (3I) or a laser scanning confocal microscope (Leica SP8 TCS) with built-in LAS X software (Leica Biosystem). Quantification of fission and fusion events was manually performed in the ImageJ.

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, incubated for 24 hr, and re-plated prior to imaging on 10 μg/ml fibronectin–coated glass-bottomed dishes (MatTek Corporation). The time-lapse images were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), with an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner, or with an inverted microscope (IX-71; Olympus) equipped with a Polychrome IV monochromator (TILL Photonics). Both systems have appropriate filters, heated sample chamber (+37°C), and controlled CO2. With 3I Marianas, a 63x/1.2 W C-Apochromat Corr WD = 0.28 M27 objective was used. SlideBook 5.0 software (3I intelligent Imaging Innovations) and sCMOS (Andor) Neo camera were used for the image acquirement and recording. With Olympus, a 60x water objective with 1.6× magnification was used. TILL Vision 4 software (TILL Photonics) and Imago QE (TILL Photonics) and Andor iXon (Andor) cameras were used for the image acquirement and recording. Deconvolution of the time-lapse videos was performed with AutoQuant AutoDeblur 2D non-blind Deconvolution (AutoQuant Imaging, Inc.). Further analyses of the video frames were performed with Image Pro Plus 6.0.