Laser-induced Wound Imaging in Cells

Cells expressing GFP-tagged proteins were observed under a total internal reflection fluorescence (TIRF) microscope (based on the IX71 microscope, Olympus, Tokyo, Japan), as previously described [51 (link)]. The cells were wounded with a nanosecond-pulsed laser (FDSS532-Q, CryLas, Berlin, Germany), and the wound diameter was set as 0.5 µm, as previously described [13 (link)]. Time-lapse fluorescence images were acquired at 40–100 ms exposure and 130–500 ms intervals using a cooled CCD camera (Orca ER, Hamamatsu Photonics, Shizuoka, Japan). The time courses of the fluorescence intensities within the circle (3 µm in diameter), including the wound site, were examined using ImageJ software (http://rsbweb.nih.gov/ij, accessed on 1 September 2022). The fluorescence intensities were normalized by setting the value before wounding to 1 after subtracting the background.