Ixon3

Fluorescence images of gliomas in experiments with IR700, LysoTracker and a pH indicator were obtained using an inverted microscope (IX-70, Olympus) equipped with a phase-contrast objective (Olympus, PLAPON 60XOPH), a spinning disk confocal unit (CSU-10, Yokogawa), EMCCD cameras and an incubator (TOKAI HIT Co., Ltd.) to control the temperature at 37 °C and the CO₂ concentration at 5% in the glass-bottom dishes. For cell damage experiments, the cells were illuminated with a red laser (635 nm, 100 mW, EDMUND OPTICS) for 10 s and 30 s to photoactivate IR700. The fluorescence images of IR700 excited by the red laser were captured with an EMCDD camera (iXon 3, Andor Technology Ltd.) equipped with a 690–730 bandpass filter. After laser treatment, the cells were incubated with 5.0 nM EthD-1 and 1 μL Annexin-V-Cy5 to detect cell death. Epi-fluorescence images of EthD-1 and Annexin-V excited by the blue (20 mW, Showa Optronics) and green lasers (50 mW, Showa Optronics), respectively, were captured with EMCCD cameras.
Confocal images of LysoTracker and IR700 excited by the blue and red lasers were captured simultaneously by two EM-CCD cameras (iXon 3 and iXon+, Andor Technology Ltd.) at a rate of 10 frames/s. The confocal images of the pH indicator excited by the blue laser were observed with the EMCCD camera during irradiation of IR700 by the red laser.

In a typical reaction, 1 μL of 2.5 mM MgCl₂ and 10 mM EGTA were mixed with 5 μL of 9 μM 33% Oregon Green-labeled actin and incubated for 2 min. Four microliters of the actin solution were then added to 16 μL of a solution containing 1.25× TIRF buffer and any other proteins. Reactions were imaged on a Nikon TE2000 inverted microscope equipped with a 100 × 1.49 numerical aperture TIRF objective, 50 mW 488 nm and 561 nm Sapphire continuous wave solid state laser lines (Coherent), a dual band TIRF (zt488/561rpc) filter cube (Chroma C143315), and a 1× to 1.5× intermediate magnification module. Images were collected using an 512 × 512 pixel EM-CCD camera (iXon3, Andor). For two color reactions, typical imaging conditions were 50 ms exposures with the 488 nm laser (set to 5 mW) and 100 ms exposures with the 561 nm laser (set to 35 mW) for 1 s intervals. The camera EM gain was set to 200. The concentration of 568-Dip1 was kept in the low nanomolar range in all assays to prevent high backgrounds of nonspecifically adsorbed 568-Dip1 from obscuring Dip-Arp2/3 filament nucleation events.

Live-cell imaging of developing ovules was performed as previously described (Susaki et al., 2020 (link)). The ovules were dissected from stage 12 flowers of wild-type plants expressing GFP-GEX1 driven by the GEX1 promoter, and HISTONE H2B-tdTomato driven by the RPS5A promoter, and then mounted in a multi-well glass bottom dish containing 400 μL of ovule culture medium. Time-lapse confocal imaging was performed using an inverted microscope (IX-83; Olympus, Tokyo, Japan) equipped with a disk-scan confocal system (CSU-W1, Yokogawa Electric, Tokyo, Japan), 488 and 552 nm LD lasers (Sapphire; Coherent Japan, Tokyo, Japan), and an EM-CCD camera (iXon3, Andor Technology, Belfast, United Kingdom). Time-lapse images were acquired every 5 min with 21 z-planes at 1 μm intervals, using a silicone-immersion objective lens (UPLSAPO60XS2; Olympus, Tokyo, Japan). The images were analyzed using MetaMorph Version 7.8.10.0. (Molecular Devices Japan, Tokyo, Japan).