Fig. 1. Schematic diagram of the experimental setup. FI, Faraday isolator; P, SF10 prism; M mirror; L, lens; ND, neutral density filter; DM, dichroic mirror; OB, objective lens; GM, pair of galvanometer mirrors; PMT, photomultiplier tube

Fig. 2. Stacked three-dimensional confocal images (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). Stacked images were obtained by translating the objective lens by 1 μm in the depth direction in steps of 250 nm. Yellow fluorescence shows mitochondria of HeLa cells visualized by EYFP. A target mitochondrion is indicated by a red arrow. Scale bar: 10 μm. Confocal cross-sectional images at different depths (c) before and (d) after irradiation of the femtosecond laser pulses. The femtosecond laser pulses were focused at a depth of z = 0. Scale bar: 3 μm.

Fig. 3. Mitochondrial fragmentation in HeLa cells induced by femtosecond laser pulses. Stacked three-dimensional confocal images obtained (a) before and (b) after irradiation with 0.53 nJ/pulse (exposure time: 32 ms). The red arrow indicates the irradiation point. Scale bar: 10 μm. (c) Time-lapse confocal images after irradiation. Scale bar: 5 μm.

Fig. 4. Dependence of disruption of mitochondria with EYFP and MitoTracker Red on femtosecond laser pulse energy and irradiation time. (a–c) The rate of disruption and fragmentation of the EYFP-mitochondria at irradiation times of (a) 32 ms, (b) 16 ms, and (c) 8 ms for various laser energies. (d) The rate of disruption and fragmentation of the mitochondria stained with MitoTracker Red at an irradiation time of 32 ms for various laser energies. The number of measurements was 10 for each pulse energy and irradiation time.

Fig. 5. Cell division after femtosecond laser disruption of a mitochondrion labeled with EYFP. Confocal images (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). The red arrow indicates the irradiation point. (c–f) Time-lapse confocal images. The process of cell division finished successfully 12 h after laser irradiation (c–d). The migration of daughter cells was observed from 12 to 16.5 h after laser irradiation (f). Scale bar: 10 μm.

Fig. 6. Mitotic events of cell division after disruption of mitochondria in the histone EGFP-H1 expressed cell. The disruption of a single mitochondrion by femtosecond laser irradiation had no influence on cell division or cell activity. The cell nuclei and mitotic chromosomes in HeLa cells were visualized using histone EGFP-H1. Mitochondria were stained with MitoTracker Red. Confocal fluorescence image and transmission image (a) before and (b) after femtosecond laser irradiation with 0.39 nJ/pulse (exposure time: 32 ms). The yellow arrow indicates the irradiation point. (c)–(f) Time-lapse confocal images and transmission images. The mitotic events of cell division in the irradiated cells proceeded normally. Scale bar: 20 μm.