SUPPLEMENTARY INFORMATION

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1 Cell viability rate Exposure duration (s) Supplementary Figure 1. Femtosecond laser could disrupt and turn off GFP without photons at 473 nm and keep cells alive. a. GFP was turned off by the fs laser exposure without 473-nm laser scanning. b. GFP fluorescence was bleached by the fs laser treatment in 1000 s and remained dark 3 hours later. c. Viability of cells with fs laser exposure for different durations three hours later tested by trypan blue (n=20 for each point). Exposure for 0.2 s could be considered safe to cells with viability rate of more than 90%. Cells with 1-s exposure still had a relatively high viability (more than 50%). d. Disruption of GFP by fs laser at the focus spot (Red dotted circle). Left: GFP in HEK293T cells. Right: GFP fused with mitochondria by p33 in HeLa cells. The bleaching time in this case was 460±27.5 s (n=10). Bar: 10 μm. NATURE PHOTONICS 1

2 Ionomycin FCCP Supplementary Figure 2. GFP could be bleached by only H 2 O 2, ionomycin, and FCCP respectively. H 2 O 2 (10 mm) could supply direct oxidative stress to bleach GFP and such bleaching was also achieved at lower concentration (50 μm~ 1 mm). Cells in 2 mm Ca 2+ buffer could be bleached by adding ionomycin (1 μm) to induce large calcium influx. Besides, carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP, a mitochondrial protonophore on mitochondrial membrane for depolarization) from Sigma (1 μm) could also bleach cells by depolarizing mitochondria to leak ROS. Bar: 10 μm. 2 NATURE PHOTONICS

3 SUPPLEMENTARY INFORMATION Rhod-3 H 2 DCFDA H 2 DCFDA (Control) Supplementary Figure 3. Cellular Ca 2+ was labeled by Rhod-3 AM (Invitrogen) while ROS by 5-(and-6)-carboxy-2,7 - dichlorodihydrofluorescein diacetate (carboxy-h 2 DCFDA, Invitrogen). Left: cellular Ca 2+ was released at the exposure of fs laser and ROS increased after the exposure. Right: the highlighted DCFDA fluorescence at the focus spot of the fs laser by two-photon excitation (Red dashed circle). If the cellular Ca 2+ was removed, there would be only a modest ROS increase of the cell (the fluorescence of DCFDA by direct two-photon excitation of the fs laser could still exist during the exposure). This confirms that Ca 2+ release was necessary to the cellular ROS rise by fs laser. Bar: 10 μm. NATURE PHOTONICS 3

4 3 2 Fluo-4 CellROX CellROX Control Supplementary Figure 4. Cellular ROS was labeled with CellROX Deep Red Reagent while cellular Ca 2+ with Fluo-4. The ROS dynamics was consist with that probed by DHE and DCFDA. Bar: 10 μm. 4 NATURE PHOTONICS

5 SUPPLEMENTARY INFORMATION Supplementary Figure 5. GFP extracted from HEK293T cells and coated onto a glass slip was bleached by H 2 O 2. a. Pure GFP was bleached with continuous intense scanning at 473 nm (17 mw) in the presence of 50 μm H 2 O 2 or 30 μm cytochrome b. Pure GFP was bleached by 50 μm H 2 O 2 without any laser scanning. c. Pure GFP could not be bleached by fs laser. Bar: 10 μm. NATURE PHOTONICS 5

6 Supplementary Figure 6. HeLa cells with GFP were fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min. After being washed with PBS buffer for 3 times, cells treated with H 2 O 2 at different concentrations were subjected for confocal scanning. The GFP fluorescence was still bleached whose speed was proportional to the concentration of H 2 O 2 (0.05~5 mm). Hence the bleaching of GFP was owing to the oxidization of the chromophore. 6 NATURE PHOTONICS

7 SUPPLEMENTARY INFORMATION Red Fluorescence of GFP (a.u.) 350 HEK293T Cells with 5 mm H 2 O 2 HEK293T Cells with 0.2 mm H 2 O HEK293T Cells Number of 437-nm Laser Scans Supplementary Figure 7. Red fluorescent conversion of GFP in HEK293T by intense scanning of CW laser at 473 nm. Green fluorescence was bleached while red fluorescence appeared. Photos excited by the 543-nm laser were acquired after each time of 473-nm-laser scanning. Upper: Red fluorescence of GFP in HEK293T cells after 12 and 24 scans. Lower: Red fluorescence of GFP in HEK293T cells incubated with H 2 O 2 at 0.2 mm after 5 and 10 scans. The increase of red fluorescence here was faster, but soon bleached. Right: intensity of red fluorescence of GFP in HEK293T cells under different treatment. Bar: 20 μm. NATURE PHOTONICS 7

8 Subcellular area Whole cell Fluorescence (a.u) Supplementary Figure 8. a. HEK293T cells expressing GFP could be bleached by fs laser treatment (0.2 s) with (lower) or without (upper) any scanning. b. The lag period of GFP bleaching after the fs laser exposure in a subcellular area (red dotted circle) far away from the fs-laser focus (red arrow) can be observed. Insert: detailed bleaching curve in the orange box. It can be found the bleaching of GFP in the subcellular area had a lag period of around 3 s. Bar: 10 μm. 8 NATURE PHOTONICS

9 SUPPLEMENTARY INFORMATION Supplementary Figure 9. Mitochondria transition pore (MTP) in the inner mitochondrial membrane was detected by Image-IT LIVE Mitochondrial Transition Pore Assay Kit (Invitrogen). The green fluorescence came from calcein in mitochondria. MTP activation results from mitochondrial Ca 2+ overload, oxidation of mitochondrial glutathione, and increased levels of ROS in mitochondria. The calcein fluorescence will then decrease. Cytochrome c release from mitochondria and loss of mitochondrial membrane potential are observed subsequent to continuous pore activation. Bar: 10 μm. NATURE PHOTONICS 9

10 Supplementary Figure 10. Continuous intense scanning at 473 nm induced GFP bleaching and Ca 2+ rise. a. HeLa (upper) and HEK293T (lower) cells with GFP could be bleached by continuous intense scanning at 473 nm at 17 mw. b. The intense scanning by the 473 nm laser could induce Ca 2+ rise after cellular damage accumulation. The fluorescence would decrease at first due to the photobleaching of Fluo-4 by high-power excitation. Bar: 10 μm. 10 NATURE PHOTONICS

11 SUPPLEMENTARY INFORMATION Fluo-4 DHE 1.5 Fluo-4 TMRM Supplementary Figure 11. Cellular dynamics during removing calcium from cells. Ionomycin was slowly added into the Ca 2+ -free buffer. There was a modest Ca 2+ rise after adding ionomycin which would decrease in several seconds. ROS had a little increase (a) and MMP remained the same (b). This means there was no influence to cells in a short time after this calcium depletion. Cells were bathed in solutions as indicated: the number after Ca and Ionomucin reflects the Ca 2+ and ionomycin concentration in μm NATURE PHOTONICS 11

12 TMRM 60 DHE Fluorescence decrease (%) Normal VC treatment Fluorescence increase (%) Normal VC treatment Supplementary Figure 12. Vitamin C can not prevent MMP from depolarization but can suppress ROS increase. a. MMP decreased at 47.7±3.8% (n=10) 10 s after the fs laser exposure with vitamin C (10 mm), a little less than the group without vitamin C (62.7±2.9% ). The depolarization was still significant, which means vitamin C had little effect on MMP. b. ROS increased by only 6.5±0.7% in the presence of vitamin C (10 mm) compared with the control group 47.7±3.8% 10 s after the laser exposure. Vitamin C significantly scavenged ROS and thus decreased the ROS-induced mitochondria depolarization. 12 NATURE PHOTONICS

13 SUPPLEMENTARY INFORMATION Supplementary Methods Materials Fluo-4/AM, TMRM, DHE, Image-IT LIVE Mitochondrial Transition Pore Assay Kit (MTP Assay), 5-(and-6)-carboxy-2,7 -dichlorodihydrofluorescein diacetate (carboxy-h 2 DCFDA), Rhod-3, and CellROX Deep Red Reagent were all purchased from Invitrogen. Fluorescence labeling was performed following their corresponding protocols. In short-time experiments (< 5 min), Ca 2+ -free buffer was phosphate buffered saline (PBS) with 1 μm ethylene glycol tetraacetic acid (EGTA). Otherwise, the buffer with (concentration in mm) 1 EGTA, 140 NaCl, 5 KCl, 1 MgCl 2, 10 glucose, and 10 Na + -N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) (final ph 7.2) was used. Cytochrome C (from equine heart) were from Sigma and used at 0.3 mm. Vitamin C was used at 0.5 mm and incubated with cells for 10 minutes before experiments. NATURE PHOTONICS 13

14 Supplementary Discussion It has been reported that the scanning of Ti: Sapphire femtosecond (fs) laser could induce harm to mitochondria, involved with oscillations of the membrane potential and release of ROS in two-photon laser scanning microscopy 1,2. The fs laser could induce Ca 2+ release directly by plasma and thermal effect with little harm to the whole cell 3. Those effects are finely confined in the focal volume due to the ultra-short pulse duration and ultra-high peak power, enabling high spatial and temporal accuracy. Higher elevation and longer duration of Ca 2+ elevation could be induced by higher laser power 4, due to larger damage to the cell. Similarly, at the same power, longer exposure time of the fs laser also induce larger amount of Ca 2+ release. This will result in more release of reactive oxygen species (ROS) and thus larger oxidative stress. In this way, the bleaching time T b will decrease when the exposure duration of the fs laser increases as the data in Fig. 1(d). When Ca 2+ was released from the laser focus, it can be amplified by the Ca 2+ -induced Ca 2+ release (CICR) effect 5. For example, the original released Ca 2+, if enough, can induce the activation of inositol 1,4,5-trisphosphate (IP 3 ) receptors 6 and depolarize some Ca 2+ stores like mitochondria 7 to release more Ca 2+. This effect is very important for local cellular communication especially between cytoplasm and nucleus. The Ca 2+ rise in cytoplasm can trigger Ca 2+ in nucleus and so can the inverse process, modulated by mitochondria, IP 3 receptors, and some cellular structures 8. This is the reason why a global Ca 2+ rise can be triggered by exposure of the fs laser exposed in one spot of the cell. It can be found in Supplementary Fig. 11, small volume and duration of Ca 2+ rise induced by ionomycin in Ca 2+ -free buffer can not lead to mitochondrial membrane potential (MMP) 14 NATURE PHOTONICS

15 SUPPLEMENTARY INFORMATION depolarization. The reason is the release of Ca 2+ is too little to be amplified by CICR against the Ca 2+ efflux to the buffer and the uptake by mitochondria and other organelles. Reactive oxygen species (ROS) exist naturally in cells due to the electron leakage from the electron transport chain (ETC) in the process of respiration in mitochondria, in the forms like H 2 O 2, -O 2 -. The normal natural level of cellular ROS is very low. However, when the MMP was depolarized, the leakage of ROS was highly enhanced, involved with the opening of mitochondria permeability transition pore for the ROS release from mitochondria 9. Only sufficient Ca 2+ concentration in cells can induce MMP depolarization and ROS release. In this case, the released ROS can be also amplified by ROS-induced ROS release (RIRR) effect 10. The mechanism is similar with CICR. Large amount of ROS will harm more mitochondria and release more ROS. Such ROS supply high oxidative stress to cells and GFP. Released ROS can be cleared slowly by the ROS scavenger in cells 11. However, if the ROS volume is too large, apoptosis of cells can be initiated 12. Hence long-time fs laser exposure (> 1 s) will cause low cell viability. The integration of cells with fs laser irradiation was tested with propidium iodide (PI). Cells were at first incubated with PI (1.5 μg/ml) for 1 hour. The cells with fs laser irradiation showed a good integration in 3 hours. The rate of cells with PI fluorescence with the 0.2-s laser irradiation, indicating those with pores in the membrane, was 12.5±1.71% (n=50 cells for each independent experiment, and 4 independent experiments). References 1 Aon, M. A. Cortassa, S. Marba n, E. & O Rourke, B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. The Journal Of Biological Chemistry 278, (2003). NATURE PHOTONICS 15

16 2 Cortassa, S. Aon, M.A. Winslow, R.L. & O Rourke B. A mitochondrial oscillator dependent on reactive oxygen species. Biophysical Journal 87, (2004). 3 Zhao, Y. et al. Photostimulation of astrocytes with femtosecond laser pulses. Optics Express 17, (2009). 4 Zhao Y. et al. Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser. Journal of Biomedical Optics 15, (2010). 5 Wakui, M. Osipchuk, Y. V. & Petersen O. H. Receptor-activated cytoplasmic Ca 2+ spiking mediated by inositol trisphosphate is due to Ca 2+ -induced Ca 2+ release. Cell 63, (1990). 6 Bootman, M. D. Fearnley, C. Smyrnias, I. MacDonald, F. & Roderick, H. L. An update on nuclear calcium signaling. Journal of Cell Science 122, (2009). 7 Alonso, M. T. Villalobos, C. Chamero, P. Alvarez, J. & García-Sancho, J. Calcium microdomains in mitochondria and nucleus. Cell Calcium 40, (2006). 8 Echevarría, W. Leite, M. F. Guerra, M. T. Zipfel W. R. & Nathanson, M. H. Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum. Nature Cell Biology 5, (2003). 9 Zorov, D. B. Filburnb, C. R. Klotzb, L. Zweierc, J. L. & Sollott, S. J. Reactive Oxygen Species (Ros-Induced) Ros Release A New Phenomenon Accompanying Induction of the Mitochondrial Permeability Transition in Cardiac Myocytes. The Journal of Experimental Medicine 192, (2000). 10 Zorov, D. B. Juhaszova, M. Sollott, S. J. Mitochondrial ROS-induced ROS release: An update and review. Biochimica et Biophysica Acta 1757, (2006). 11 Baumgart, J. et al. Repetition rate dependency of reactive oxygen species formation during femtosecond laser based cell surgery. Journal of Biomedical Optics 14, (2009). 12 Ferri K. F. & Kroemer, G. Organelle-specific initiation of cell death pathways. Nature Cell Biology 3, E255-E263 (2001). 16 NATURE PHOTONICS