All images were acquired at room temperature (23–25°C) with a VT-Hawk two-dimensional array laser scanning confocal microscopy system (Visitech International, Sunderland, UK) with an Olympus IX-83 inverted microscope with a 100×/numerical aperture 1.49 UAPO lens (Olympus, Tokyo, Japan) and electron-multiplying charge-coupled device digital camera (Hamamatsu, Hamamatsu City, Japan).
For still, z-series, and time-lapse images (<5 min), the cells were mounted directly on glass slides with a #1.5 coverslip (Fisher Scientific, Waltham, MA) and imaged immediately. For z-series, images were acquired with a depth interval of 0.4 μm. For time-lapse images >5 min, the cells were placed in 3.5-mm glass-bottom culture dishes (MatTek, Ashland, MA) and overlaid with YE medium plus 1% agar. Ascorbic acid (100 μM) as an antioxidant was added to the culture to minimize fluorescence toxicity to the cell, as reported previously (Frigault et al., 2009 (link)). Images were acquired by MetaMorph (Molecular Devices, Sunnyvale, CA) and analyzed by ImageJ (National Institutes of Health, Bethesda, MD). Statistically significant difference between two groups of cells was determined by p value from Student’s t test.
For still, z-series, and time-lapse images (<5 min), the cells were mounted directly on glass slides with a #1.5 coverslip (Fisher Scientific, Waltham, MA) and imaged immediately. For z-series, images were acquired with a depth interval of 0.4 μm. For time-lapse images >5 min, the cells were placed in 3.5-mm glass-bottom culture dishes (MatTek, Ashland, MA) and overlaid with YE medium plus 1% agar. Ascorbic acid (100 μM) as an antioxidant was added to the culture to minimize fluorescence toxicity to the cell, as reported previously (Frigault et al., 2009 (link)). Images were acquired by MetaMorph (Molecular Devices, Sunnyvale, CA) and analyzed by ImageJ (National Institutes of Health, Bethesda, MD). Statistically significant difference between two groups of cells was determined by p value from Student’s t test.