Abstract

We demonstrated stimulation of Ca2+ in living cells by near-infrared laser pulses operated at sub-MHz repetition rates. HeLa cells were exposed to focused 780 nm femtosecond pulses, generated by a titanium-sapphire laser and adjusted by an electro-optical modulator. We found that the laser-induced Ca2+ waves could be generated over three orders of magnitude in repetition rates, with required laser pulse energy varying by less than one order of magnitude. Ca2+ wave speed and gradients were reduced with repetition rate, which allows the technique to be used to modulate the strength and speed of laser-induced effects. By lowering the repetition rate, we found that the laser-induced Ca2+ release is partially mediated by reactive oxygen species (ROS). Inhibition of ROS was successful only at low repetition rates, with the implication that ROS scavengers may in general be depleted in experiments using high repetition rate laser irradiation.

© 2006 Optical Society of America

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References

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Am. J. Physiol. Cell Physiol. (1)

J. I. Kourie, "Interaction of reactive oxygen species with ion transport mechanisms," Am. J. Physiol. Cell Physiol. 275, C1-C24 (1998).

Appl. Phys. Lett. (1)

N. I. Smith, K. Fujita, T. Kaneko, K. Kato, O. Nakamura, T. Takamatsu, and S. Kawata, "Generation of calcium waves in living cells by pulsed-laser-induced photodisruption," Appl. Phys. Lett. 79, 1208-1210 (2001).
[CrossRef]

Biochem. Pharmacol. (1)

T. T. Rohn, T. R. Hinds, and F. F. Vincenzi, "Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-DI-1-pyrrolidinyl-7H-pyrrolo[2,3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger," Biochem. Pharmacol. 51, 471-476 (1996).
[CrossRef] [PubMed]

Bull. Korean Chem. Soc (1)

S. H. Kang, and Q. Chae, "Ultraviolet Light-induced Lipid Peroxidation of Cultured Skin Fibroblast Membrane," Bull. Korean Chem. Soc., 14, 371-374 (1993).

Cell Mol. Biol. (1)

K. König, I. Riemann, P. Fischer, and K.J. Halbhuber, "Intracellular nanosurgery with near infrared femtosecond laser pulses," Cell Mol. Biol. 45, 195-201 (1999).
[PubMed]

Chem. Rev. (1)

A. Vogel and V. Venugopalan, "Mechanisms of pulsed laser ablation of biological tissues," Chem. Rev. 103, 577-644 (2003).
[CrossRef] [PubMed]

Circ. Res. (1)

T. Kaneko, H. Tanaka, M. Oyamada, S. Kawata, and T. Takamatsu, "Three distinct types of Ca2+ waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy," Circ. Res. 86, 1093-1099 (2000).
[PubMed]

Circulation (1)

Q. Hu, S. Corda, J. L. Zweier, M. C. Capogrossi, and R. C. Ziegelstein, "Hydrogen peroxide induces intracellular calcium oscillations in human aortic endothelial cells," Circulation. 97, 268-275 (1998).
[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, 88-97 (2001).
[CrossRef] [PubMed]

Free Radic. Res. (1)

A. Mahns, I. Melchheier, C. V. Suschek, H. Sies, and Lars-Oliver Klotz, "Irradiation of cells with ultraviolet-A (320-400nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide," Free Radic. Res. 37, 391-397 (2003).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

M. A. J. Rodgers, and P. T. Snowden, "Lifetime of O2 (1Δg) in liquid water as determined by time-resolved infrared luminescence measurements," J. Am. Chem. Soc. 104, 5541-5543 (1982).
[CrossRef]

J. Cell Mol. Med. (1)

C. Batandier, E. Fontaine, C. Kériel, X. M. Leverve, "Determination of mitochondrial reactive oxygen species: methodological aspects," J. Cell Mol. Med. 6, 175-187 (2002).
[CrossRef] [PubMed]

J. Neurobiol. (1)

H. Hirase, V. Nikolenko, J.H. Goldberg, and R. Yuste, "Multiphoton stimulation of neurons," J. Neurobiol. 51, 237-247 (2002).
[CrossRef] [PubMed]

J. Photochem. Photobiol. B (1)

M. S. Patterson, "Experimental tests of the feasibility of singlet oxygen luminescence monitoring in vivo during photodynamic therapy," J. Photochem. Photobiol. B 5, 69-84 (1990).
[CrossRef] [PubMed]

J. Photochem. Photobiol. B Biology (1)

A.B. Uzdensky, and V.V. Savransky, "Single neuron response to pulse-periodic laser microirradiation. Action spectra and two-photon effect," J. Photochem. Photobiol. B Biology 39, 224-228 (1997).
[CrossRef]

J. Physiol. (1)

M. J. Berridge "Elementary and global aspects of calcium signalling," J. Physiol. 499, 291-306 (1997).
[PubMed]

Nature (3)

M. J. Berridge, P. Lipp, and M. D. Bootman, "Calcium - a life and death signal," Nature 395, 645-648 (1998).
[CrossRef] [PubMed]

M. Endo, M. Tanaka, and Y. Ogawa, "Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres," Nature 228, 34-36 (1970).
[CrossRef] [PubMed]

M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, "Functional regeneration after laser axotomy," Nature 432, 822 (2004).
[CrossRef] [PubMed]

Opt. Lett. (1)

K. König, I. Riemann, and W. Fritzsche, "Nanodissection of human chromosomes with near-infrared femtosecond laser pulses," Opt. Lett. 26, 819-821 (2001).
[CrossRef]

Physiol. Chem. Phys. & Med. NMR (1)

D. N. Wheatley, A. Redfern, and R. P. C. Johnson, "Heat-induced disturbances of intracellular movement and the consistency of the aqueous cytoplasm in HeLa S-3 cells: A laser-doppler and proton NMR study," Physiol. Chem. Phys. & Med. NMR 23, 199-216 (1991)
[PubMed]

Proc. Natl. Acad. Sci. USA (1)

P. E. Hockberger, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, "Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells," Proc. Natl. Acad. Sci. USA 96, 6255-6260 (1999).
[CrossRef] [PubMed]

Proc. SPIE (1)

S. Iwanaga, N. Smith, K. Fujita, T. Kaneko, O. Nakamura, S. Kawata, M. Oyamada, and T. Takamatsu, "Stimulation of living cells by femtosecond near-infrared laser pulses," in Commercial and Biomedical Applications of Ultrafast Lasers 3, J. Neev, A. Ostendorf, and C. B. Schaffer, eds., Proc. SPIE 4978, 122-128 (2003).
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Sov. Phys. JETP (1)

L. V. Keldysh, "Ionization in the field of a strong electromagnetic wave," Sov. Phys. JETP 20, 1307-1314 (1965).

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Figures (7)

Fig. 1.
Fig. 1.

The effect of leakage pulses on average power at each repetition rate. Dots are measured laser powers. The solid line is the curve fit to experimental data shown by dots (1.52 nJ pulse energy, 0.0033 EOM extinction ratio). Dotted lines are calculated curves with constant pulse energy (1.52 nJ) and each extinction ratio (0, 0.1, 0.5, 1.0, 5.0, and 10%)

Fig. 2.
Fig. 2.

The probability of intracellular Ca2+ wave generation at each number of pulses. The laser (780 nm wavelength, (a) 10 kHz, 100 kHz, and 1 MHz repetition rate, 1.31 nJ pulse energy, 0.0024 EOM extinction ratio, (b) 100 kHz, 400 kHz, and 1 MHz repetition rate, 0.99 nJ, 0.0036 EOM extinction ratio) was focused into the cytoplasm of the HeLa cell. n=30.

Fig. 3.
Fig. 3.

The probability of intracellular Ca2+ wave generation with or without Trolox. The laser (780 nm wavelength, 100 kHz repetition rate, 520 μW average power, 10 s exposure time) was focused into the cytoplasm of the HeLa cell. Error bar is calculated from standard deviation in binominal distribution. (n=40)

Fig. 4.
Fig. 4.

The probability of intracellular Ca2+ wave generation at each average power with or without Trolox. The laser (780 nm wavelength) was focused into the cytoplasm of the HeLa cell. Error bar is calculated from standard deviation in binominal distribution. (n=20) (a) 100 kHz repetition rate, 10 s exposure time (b) 400 kHz repetition rate, 10 s exposure time (c) 82 MHz repetition rate, 13 ms exposure time

Fig. 5.
Fig. 5.

Simulation of diffusion-based recovery of an ROS scavenger into a depleted region inside the focal zone. The graph is based on complete depletion of Trolox by a single laser pulse in a sphere with a 0.5 μm radius. After complete depletion, 50% recovery is achieved within 100 μs, corresponding to a laser repetition rate of 10 kHz.

Fig. 6.
Fig. 6.

(a) Fluorescence images of 100 kHz laser-induced intracellular Ca2+ wave (false color). Arrow indicates the position of laser irradiation. The time shown below each figure is counted from the start of laser irradiation. The pulse energy was 1.36 nJ and exposure time was 10 s. White scale bar = 25 μm. (b) Time course of fluorescence intensity averaged in a whole laser-irradiated cell. The fluorescence intensity corresponds to Ca2+ concentration becomes maximum in 30.7 s after laser irradiation. Black bars above each figure show the laser irradiation.

Fig. 7.
Fig. 7.

Time course of 100 kHz (a,b) or 82 MHz (c,d) laser-induced intracellular Ca2+ wave (false color). A straight yellow line represents the axis through which the video sequence was resliced, to produce the images shown in (b) and (d). The pulse energy was 1.36 nJ and exposure time was 10 s. Scale bar = 2 μm (a, c) or 1s (b, d).

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P ave = { R R × E p + E p × E r × ( 82000 R R ) } 1000
U t = κ ( 2 r U t + 2 U r 2 )

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