Abstract

Mitochondria play a key role in all cellular physiology, processes, and behaviors. It is very difficult to precisely stimulate single mitochondria noninvasively in traditional biomedical research. In this study, we report that femtosecond laser can stimulate fragmentation or swelling of single mitochondria in human mesenchymal stem cells rather than physical disruption or ablation. In experiments, fragmented mitochondria can recover normal very soon but swelling ones cannot. At the same time, laser-induced generation of mitochondrial reactive oxygen species and opening of mitochondria permeability transition pores are involved in mitochondrial responses to photostimulation. Furthermore, the localized translocation of proapoptotic molecules are found in those stimulated mitochondria. Those results suggest femtosecond-laser photostimulation as a noninvasive and precise method for mitochondrial manipulation and related research.

© 2015 Optical Society of America

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References

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  1. E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics. 7(2), 93–101 (2013).
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    [Crossref] [PubMed]
  4. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13(10), 3690–3696 (2005).
    [Crossref] [PubMed]
  5. W. Watanabe, N. Arakawa, S. Matsunaga, T. Higashi, K. Fukui, K. Isobe, and K. Itoh, “Femtosecond laser disruption of subcellular organelles in a living cell,” Opt. Express 12(18), 4203–4213 (2004).
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  6. T. Shimada, W. Watanabe, S. Matsunaga, T. Higashi, H. Ishii, K. Fukui, K. Isobe, and K. Itoh, “Intracellular disruption of mitochondria in a living HeLa cell with a 76-MHz femtosecond laser oscillator,” Opt. Express 13(24), 9869–9880 (2005).
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
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  19. W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
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2015 (1)

J. Yoon, S. W. Ryu, S. Lee, and C. Choi, “Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-Like Cell Death via Intrinsic Reactive Oxygen Cascades,” Sci. Rep. 5, 8231 (2015).
[Crossref] [PubMed]

2014 (1)

Y. Sano, W. Watanabe, and S. Matsunaga, “Chromophore-assisted laser inactivation--towards a spatiotemporal-functional analysis of proteins, and the ablation of chromatin, organelle and cell function,” J. Cell Sci. 127(8), 1621–1629 (2014).
[Crossref] [PubMed]

2013 (1)

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics. 7(2), 93–101 (2013).

2012 (4)

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

L. A. Sena and N. S. Chandel, “Physiological Roles of Mitochondrial Reactive Oxygen Species,” Mol. Cell 48(2), 158–167 (2012).
[Crossref] [PubMed]

A. H. Schapira, “Mitochondrial diseases,” Lancet 379(9828), 1825–1834 (2012).
[Crossref] [PubMed]

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

2011 (1)

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

2010 (2)

A. Valle-Prieto and P. A. Conget, “Human Mesenchymal Stem Cells Efficiently Manage Oxidative Stress,” Stem Cells Dev. 19(12), 1885–1893 (2010).
[Crossref] [PubMed]

E. Sahin and R. A. Depinho, “Linking functional decline of telomeres, mitochondria and stem cells during ageing,” Nature 464(7288), 520–528 (2010).
[Crossref] [PubMed]

2009 (2)

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

S. H. Chung and E. Mazur, “Surgical applications of femtosecond lasers,” J. Biophotonics 2(10), 557–572 (2009).
[Crossref] [PubMed]

2006 (2)

D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial ROS-induced ROS release: an update and review,” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1757(5–6), 509–517 (2006).
[Crossref]

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

2005 (2)

2004 (1)

2001 (2)

W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
[PubMed]

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

Arakawa, N.

Baumgart, J.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Bintig, W.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Brugués, J.

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

Cao, Y.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Chandel, N. S.

L. A. Sena and N. S. Chandel, “Physiological Roles of Mitochondrial Reactive Oxygen Species,” Mol. Cell 48(2), 158–167 (2012).
[Crossref] [PubMed]

Chang, D. C.

W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
[PubMed]

Choi, C.

J. Yoon, S. W. Ryu, S. Lee, and C. Choi, “Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-Like Cell Death via Intrinsic Reactive Oxygen Cascades,” Sci. Rep. 5, 8231 (2015).
[Crossref] [PubMed]

Chung, S. H.

S. H. Chung and E. Mazur, “Surgical applications of femtosecond lasers,” J. Biophotonics 2(10), 557–572 (2009).
[Crossref] [PubMed]

Cleland, M. M.

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

Conget, P. A.

A. Valle-Prieto and P. A. Conget, “Human Mesenchymal Stem Cells Efficiently Manage Oxidative Stress,” Stem Cells Dev. 19(12), 1885–1893 (2010).
[Crossref] [PubMed]

Depinho, R. A.

E. Sahin and R. A. Depinho, “Linking functional decline of telomeres, mitochondria and stem cells during ageing,” Nature 464(7288), 520–528 (2010).
[Crossref] [PubMed]

Ertmer, W.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Fukui, K.

Gao, W.

W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
[PubMed]

Halbhuber, K.-J.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

He, H.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Heisterkamp, A.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13(10), 3690–3696 (2005).
[Crossref] [PubMed]

Higashi, T.

Hoover, E. E.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics. 7(2), 93–101 (2013).

Hu, M.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Ingber, D. E.

Ishii, H.

Isobe, K.

Itoh, K.

Jeong, S.

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

Juhaszova, M.

D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial ROS-induced ROS release: an update and review,” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1757(5–6), 509–517 (2006).
[Crossref]

Karbowski, M.

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

König, K.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

Krieg, R.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

Kuetemeyer, K.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Kumar, S.

Lee, S.

J. Yoon, S. W. Ryu, S. Lee, and C. Choi, “Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-Like Cell Death via Intrinsic Reactive Oxygen Cascades,” Sci. Rep. 5, 8231 (2015).
[Crossref] [PubMed]

Li, S.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Lubatschowski, H.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Luo, K. Q.

W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
[PubMed]

Matsunaga, S.

Maxwell, I. Z.

Mazur, E.

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

S. H. Chung and E. Mazur, “Surgical applications of femtosecond lasers,” J. Biophotonics 2(10), 557–572 (2009).
[Crossref] [PubMed]

A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13(10), 3690–3696 (2005).
[Crossref] [PubMed]

Needleman, D. J.

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

Ngezahayo, A.

J. Baumgart, K. Kuetemeyer, W. Bintig, A. Ngezahayo, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery,” J. Biomed. Opt. 14(5), 054040 (2009).
[Crossref] [PubMed]

Nickerson, J. A.

Norris, K. L.

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

Nuzzo, V.

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

Peuckert, C.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

Pu, Y.

W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang, “Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells,” J. Cell Sci. 114(Pt 15), 2855–2862 (2001).
[PubMed]

Ryu, S. W.

J. Yoon, S. W. Ryu, S. Lee, and C. Choi, “Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-Like Cell Death via Intrinsic Reactive Oxygen Cascades,” Sci. Rep. 5, 8231 (2015).
[Crossref] [PubMed]

Sahin, E.

E. Sahin and R. A. Depinho, “Linking functional decline of telomeres, mitochondria and stem cells during ageing,” Nature 464(7288), 520–528 (2010).
[Crossref] [PubMed]

Sano, Y.

Y. Sano, W. Watanabe, and S. Matsunaga, “Chromophore-assisted laser inactivation--towards a spatiotemporal-functional analysis of proteins, and the ablation of chromatin, organelle and cell function,” J. Cell Sci. 127(8), 1621–1629 (2014).
[Crossref] [PubMed]

Schapira, A. H.

A. H. Schapira, “Mitochondrial diseases,” Lancet 379(9828), 1825–1834 (2012).
[Crossref] [PubMed]

Sena, L. A.

L. A. Sena and N. S. Chandel, “Physiological Roles of Mitochondrial Reactive Oxygen Species,” Mol. Cell 48(2), 158–167 (2012).
[Crossref] [PubMed]

Shimada, T.

Sollott, S. J.

D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial ROS-induced ROS release: an update and review,” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1757(5–6), 509–517 (2006).
[Crossref]

Squier, J. A.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics. 7(2), 93–101 (2013).

Tirlapur, U. K.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

Underwood, J. M.

Valle-Prieto, A.

A. Valle-Prieto and P. A. Conget, “Human Mesenchymal Stem Cells Efficiently Manage Oxidative Stress,” Stem Cells Dev. 19(12), 1885–1893 (2010).
[Crossref] [PubMed]

Wang, C.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Wang, S.

H. He, S. Li, S. Wang, M. Hu, Y. Cao, and C. Wang, “Manipulation of cellular light from green fluorescent protein by a femtosecond laser,” Nat. Photonics 6(10), 651–656 (2012).
[Crossref]

Watanabe, W.

Wu, S.

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

Xing, D.

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

Yoon, J.

J. Yoon, S. W. Ryu, S. Lee, and C. Choi, “Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-Like Cell Death via Intrinsic Reactive Oxygen Cascades,” Sci. Rep. 5, 8231 (2015).
[Crossref] [PubMed]

Youle, R. J.

M. Karbowski, K. L. Norris, M. M. Cleland, S. Jeong, and R. J. Youle, “Role of Bax and Bak in mitochondrial morphogenesis,” Nature 443(7112), 658–662 (2006).

Zhang, Z.

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

Zhou, F.

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

Zorov, D. B.

D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial ROS-induced ROS release: an update and review,” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1757(5–6), 509–517 (2006).
[Crossref]

Biochimica et Biophysica Acta (BBA) - Bioenergetics (1)

D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial ROS-induced ROS release: an update and review,” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1757(5–6), 509–517 (2006).
[Crossref]

Cell (1)

J. Brugués, V. Nuzzo, E. Mazur, and D. J. Needleman, “Nucleation and Transport Organize Microtubules in Metaphase Spindles,” Cell 149(3), 554–564 (2012).
[Crossref] [PubMed]

Exp. Cell Res. (1)

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263(1), 88–97 (2001).
[Crossref] [PubMed]

FEBS J. (1)

S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278(6), 941–954 (2011).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

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Figures (5)

Fig. 1
Fig. 1 Optical setup based on a confocal system. One more laser at 543 nm was also used in this setup for red fluorescence excitation. A lens pair (Lens 1 and 2) was used for beam expansion, collimation, and divergence control of the femtosecond laser. DM1: reflection < 750 nm, transmition >750 nm. DM2: transmition at only 488/543/632 nm, and reflective at all other wavelengths. BS: beam splitter.
Fig. 2
Fig. 2 Mitochondrial fragmentation and swelling after photostimulation. Fragmented mitochondria would recover very soon (a) (Bar: 20 μm.) but swelling ones could not (b) (Bar: 10 μm). (c) More mitochondria would response to photostimulation with higher power (n = 14, 20, 16, 15, 24 respectively at each point).
Fig. 3
Fig. 3 The rates of fragmented and swelling mitochondria by photostimulation at 8 mW (a), 10 mW (b), 12 mW (c), 17 mW (d) for 100 ms, 300 ms, and 3 s respectively.
Fig. 4
Fig. 4 mROS and mPTP were involved in mitochondrial responses to photostimulation. (a) Left panel: The photostimulation (6mW for 0.1 s, performed at 3.33 s) initiated mROS increase and MMP depolarization. Right panel: mROS (indicated by the green fluorescence) was immediately generated at the photostimulation (4.4 s, arrow: position of the laser focus). Bar: 10 μm (b) Mitochondria were divided into 9 groups to be photostimulated with mROS scavenged or enhanced (by MitoTEMPO or TBHP respectively) at different laser powers (n = 30 mitochondria in each group). (c) mPTP opening could be observed by the calcein fluorescence quenching (n = 5 mitochondria). (d) Control experiments by using CsA inhibiting mPTP (n = 50 in control, n = 60 in CsA group).
Fig. 5
Fig. 5 Colocalization of the stimulated mitochondria (indicated by TMRM) and translocation of proapoptotic molecules (indicated by immunofluorescent microscopy). The localized concentration of Bax (a) and release of cytochrome C (b) could be found in the stimulated mitochontria.

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