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Femtosecond laser disruption of subcellular organelles in a living cell

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Abstract

Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.

©2004 Optical Society of America

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Figures (5)

Fig. 1.
Fig. 1. Schematic of the experimental setup. OB, DM, BP, and PMT denote objective lens, dichroic mirror, bandpass filter, and photomulutiplier tube, respectively.
Fig. 2.
Fig. 2. Confocal images of the cells (a) before and (b) after laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. The white circles and arrows indicate individual HeLa cells and target mitochondria, respectively. Additional red fluorescence in Fig. 2b is derived from propidium iodide (PI). The laser pulses were focused inside cells α and β at energies of 7 nJ/pulse and 3 nJ /pulse, respectively.
Fig. 3
Fig. 3 Confocal images of HeLa cell before and after fs-laser irradiation. (a) and (b) confocal images of the HeLa cell before and after femtosecond laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. A target mitochondrion is indicated by a white arrow. (c) and (d) magnified views of square areas indicated in (a) and (b), respectively. The center of the dotted circles show target mitochondria.
Fig. 4.
Fig. 4. (a) and (b) stacked three-dimensional confocal images at different depths before and after femtosecond laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. A target mitochondrion is indicated by a white arrow. Twenty-one images were obtained by translation of the objective lens by 6 μm in steps of 300 nm. (c) and (d) magnified views of square areas indicated in (a) and (b), respectively. The center of the dotted circles show target mitochondria.
Fig. 5.
Fig. 5. (a) Confocal images of laser-irradiated cells before and after restaining. Yellow fluorescence shows mitochondria visualized by EYFP. Target mitochondrion is indicated by a white arrow. (c) magnified view of square area indicated in (a) before femtosecond laser irradiation. (b) confocal image obtained after femtosecond laser irradiation. (d) magnified view of square area in (b). (e) and (f) confocal images obtained after restaining by MitoTracker Red. (e) confocal image obtained by excitation with the Ar+ laser. (f) confocal image obtained by excitation with the He-Ne laser. Dotted circles show target mitochondria.
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