Photostimulation of astrocytes with femtosecond laser pulses

doi:10.1364/OE.17.001291

« Show journal navigation

Photostimulation of astrocytes with femtosecond laser pulses

Yuan Zhao, Yuan Zhang, Xiuli Liu, Xiaohua Lv, Wei Zhou, Qingming Luo, and Shaoqun Zeng »View Author Affiliations

Optics Express, Vol. 17, Issue 3, pp. 1291-1298 (2009)
http://dx.doi.org/10.1364/OE.17.001291

View Full Text Article

Acrobat PDF (537 KB)

Journal Search
Article Lookup

Article Tools

Citations

Alert me when this article is cited
Export Citation/Save

Article Navigation

▸ Article Outline
– Abstract
1. Introduction
2. Materials and methods
3. Results
4. Discussions and conclusion
– References and links

Article Tools
Full Text
Article Info
References
Cited By
Figures
Multimedia
Metrics
Download PDF

Viewing Equations in the
HTML Article

Optimal Viewing Recommendations

Full Text
Article Info
References (40)
Cited By
Figures (6)
Multimedia (3)
Metrics

Abstract

The involvement of astrocytes in brain functions rather than support has been identified and widely concerned. However the lack of an effective stimulation of astrocytes hampers our understanding of their essential roles. Here, we employed 800-nm near infrared (NIR) femtosecond laser to induce Ca²⁺ wave in astrocytes. It was demonstrated that photostimulation of astrocytes with femtosecond laser pulses is efficient with the advantages of non-contact, non-disruptiveness, reproducibility, and high spatiotemporal precision. Photostimulation of astrocytes would facilitate investigations on information processing in neuronal circuits by providing effective way to excite astrocytes.

1. Introduction

Astrocytes are the major electrically non-excitable cells in the central nervous system, the role of which have long been constricted as supporting neurons. However, new functions of astrocytes have been identified in the past decades [1

A. Volterra and J. Meldolesi, “Astrocytes, from brain glue to communication elements: the revolution continues,” Nat. Rev. Neurosci. 6, 626–640 (2005). [CrossRef] [PubMed]

, 2

M. Nedergaard, B. Ransom, and S. A. Goldman, “New roles for astrocytes: redefining the functional architecture of the brain,” Trends Neurosci. 26, 523–530 (2003). [CrossRef] [PubMed]

], and become increasingly attractive to neuroscientists since early studies [3

M. Nedergaard, “Direct signaling from astrocytes to neurons in cultures of mammalian brain cells,” Science 263, 1768–1771 (1994). [CrossRef] [PubMed]

]. Astrocytes express various membrane receptors activated by neurotransmitters, in response of intracellular Ca²⁺ elevation and intercellular Ca²⁺ wave. In turn, they release chemical transmitters, regulating the synaptic transmission and strength, and influencing the cerebral microcirculation [4-6

P. G. Haydon and G. Carmignoto, “Astrocyte control of synaptic transmission and neurovascular coupling,” Physiol. Rev. 86, 1009–1031 (2006). [CrossRef] [PubMed]

]. In terms of the distinct bidirectional communications between astrocytes and neurons, it has been proposed that the astrocyte is the third functional element of the synapse [5

G. Carmignoto, “Reciprocal communication systems between astrocytes and neurones,” Prog. Neurobiol. 62, 561–581 (2000). [CrossRef] [PubMed]

]. Recently, precise consistency of tuned responses in astrocytes and neurons was proved, and astrocytes were proposed to contribute to functional brain imaging [7

J. Schummers, H. Yu, and M. Sur, “Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex,” Science 320, 1638–1643 (2008). [CrossRef] [PubMed]

], leading us to explore the delicate communications among these brain cells. While there are accumulative evidences that astrocytes are involved in brain functions through Ca²⁺ signaling, the essential roles have not been resolved. One of the key techniques is an effective stimulation.

Existing approaches developed for generating Ca²⁺ signaling in astrocytes, include mechanical stimulation [8

A. C. Charles, J. E. Merrill, E. R. Dirksen, and M. J. Sanderson, “Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate,” Neuron 6, 983–992 (1991). [CrossRef] [PubMed]

], electrical stimulation [3

M. Nedergaard, “Direct signaling from astrocytes to neurons in cultures of mammalian brain cells,” Science 263, 1768–1771 (1994). [CrossRef] [PubMed]

], uncaging [9

T. A. Fiacco and K. D. McCarthy, “Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons,” J. Neurosci. 24, 722–732 (2004). [CrossRef] [PubMed]

] and pharmacological applications [8

, 10

J. T. Porter and K. D. McCarthy, “Adenosine receptors modulate [Ca²⁺]_i in hippocampal astrocytes in situ,” J. Neurochem. 65, 1515–1523 (1995). [CrossRef] [PubMed]

]. However, despite of complexity and poor efficiency in performing, none of these allow noncontact and non-disruptive stimulation with selective and precise targeting, especially for in vivo studies. Hence new techniques are obliged to be introduced.

As a novel tool for nanoprocessing, femtosecond laser, not only for its multi-photon imaging, has been previously performed in various biological fields. Compared to ultraviolet light, high spatial resolution is achieved by two-photon excitation of femtosecond laser, for selectively labeling single organelles with photoconvertible proteins [11

W. Wantanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “ In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation,” J. Biomed. Opt. 13, 031213 (2008). [CrossRef]

, 12

W. Wantanabe, T. Shimada, S. Matsunaga, D. Kurihara, K. Fukui, S. Arimura, N. Tsutsumi, K. Isobe, and K. Itoh, “Single-organelle tracking by two-photon conversion,” Opt. Express 15, 2490–2498 (2007). [CrossRef]

]. Besides, and more interestingly, femtosecond laser has been widely adopted in nanosurgery. The laser beam is directed through an objective of high numerical aperture (NA), and focused onto subcellular structures, such as a chromosome [13

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

], single organelles [11

, 14-16

W. Wantanabe, 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, 4203–4213 (2004). [CrossRef]

], or even neuronal processes [17

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

, 18

L. Sacconi, R. P. O’Connor, A. Jasaitis, A. Masi, M. Buffelli, and F. S. Pavone, “ In vivo multiphoton nanosurgery on cortical neurons,” J. Biomed. Opt. 12, 050502 (2007). [CrossRef] [PubMed]

], resulting in ultra-precise disruption there. This makes it possible to inactivate subcellular functional regions, to examine the contributions and interactions of local structures. Besides, effects of femtosecond laser have also been studied at cell level [19-21

N. I. Smith, Y. Kumamoto, S. Iwanaga, J. Ando, K. Fujita, and S. Kawata, “A femtosecond laser pacemaker for heart muscle cells,” Opt. Express 16, 8604–8616 (2008). [CrossRef] [PubMed]

]. Laser targets can either be larger, like blood vessels [22

N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P. D. Lyden, and D. Kleinfeld, “Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke,” Nat. Methods 3(2006). [CrossRef] [PubMed]

], or highly localized on cell membrane [15

U. K. Tirlapur and K. König, “Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability,” The Plant Journal 31, 365–374 (2002). [CrossRef] [PubMed]

, 23

S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Nakamura, T. Takamatsu, and S. Kawata, “Location-dependent photogeneration of calcium waves in HeLa cells,” Cell Biochem. Biophys. 45, 167–176 (2006). [CrossRef] [PubMed]

, 24

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

], enabling tissue dissection with sub-micron precision, as called photoporation. Tightly focused femtosecond laser, with high peak intensity and low pulse energies, is proved to hardly compromise cell viability [11

, 14-21

, 23

, 24

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

]. Therefore, in addition with its high efficiency and precision, femtosecond laser has been confirmed to be a preferred tool compared to previous approaches in the fields above.

In this paper, we introduced 800-nm NIR femtosecond laser pulses focused on cell membrane, to evoke Ca²⁺ increase in the stimulated astrocyte and subsequent Ca²⁺ wave across other cells, which was demonstrated to be resulted from photoporation. Similar responses in astrocytes were observed by repetitive photostimulation with well-controlled laser intensity and target. Noncontact photostimulation employing femtosecond laser was proved to be non-disruptive, reproducible, and with high spatiotemporal precision. This method has potentials to be applied to investigations on detailed communications between astrocytes and other brains cells, especially in vivo or in live animals.

2. Materials and methods

All chemicals were obtained from Sigma (St Louis, MO, USA) unless indicated.

2.1 Cell cultures

Pure cortical astrocytes were prepared as described [25

Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.-s. Gu, and S. Duan, “Regulated ATP release from astrocytes through lysosome exocytosis,” Nat. Cell Biol. 9, 945–953 (2007). [CrossRef] [PubMed]

] with some modifications. In brief, cortex was obtained from postnatal 1- to 3-day Wistar rats, dissociated by trypsin (0.125%), and cultured with Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FCS) (HyClone, USA). As forming a confluent layer, astrocytes were ready for experiments. Use of animals followed the guideline of Huazhong University of Science and Technology for Biological Sciences Animal Research Advisory Committee.

2.2 Dyes and drugs

During experiments, cells were incubated in a Hepes-buffered solution (HBS, pH 7.2) containing (in mM): 145 NaCl, 3 KCl, 10 HEPES, 3 CaCl₂·2H₂O, 2 MgCl₂·6H₂O, 8 glucose. For Ca²⁺ imaging, cells were loaded with 4 μM Fluo-3-AM in HBS for 30 min at 37°C.

Propidium iodide (PI) was diluted in HBS at a final concentration of 50 μM. Ca²⁺-free solution contained 2 mM Ethylene glycol tetraacetic acid (EGTA) in HBS.

2.3 System for imaging and photostimulation

Experiments were carried out on a commercial confocal microscope system (FluoView1000, Olympus) equipped with multiphoton excitation settings, as well as a homebuilt random-access two-photon microscopy [26

X. Lv, C. Zhan, S. Zeng, W. R. Chen, and Q. Luo, “Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector,” Rev. Sci. Instrum. 77, 046101 (2006). [CrossRef]

]. The confocal system consisted of an inverted microscope (IX71) employing 20× 0.75 NA, and 60× 0.95 NA water-immersion objectives and a FV1000 confocal system (Olympus, Japan). Fluorescence of Fluo-3 was excited by the 488 nm band of the argon ion laser, relayed through a dichroic mirror (DM) 405/488, and collected by photo-multiplier tubes (PMTs). For stimulating, mode-locked laser beam (800 nm, ~90 fs pulses at 80 MHz) produced from a Ti: Sapphire laser (Mai Tai, Spectra-Physics) was coupled to the confocal scan head of FV1000, and directed into IX71, with the maximum average power of 60 mW after the 20× objective. A custom–built prism pair was typically used as a pulse compressor to compensate the dispersion in an acousto-optic modulator (AOM, A-A, France), which is employed to regulate the laser power. Pulse width was eventually recovered to nearly 90 fs. Time sequences of imaging and stimulating were computer controlled by FluoView software (Olympus). Images with each frame of 512 × 512 pixels were acquired at 2 μs/pixel, that is ~1 s/frame, and then analyzed in Image-Pro Plus5.0 (Media Cybernetics, USA).

3. Results

3.1 Photogenerated Ca²⁺ wave in astrocytes

Femtosecond laser was focused onto the upper membrane of a selected cell. Intracellular Ca²⁺ concentration in astrocytes was monitored by the fluorescence of Fluo-3 before and immediately after laser irradiation with controlled energies and durations. As shown in Fig. 1, femtosecond laser pulses with average power of 30 mW and duration of 4 ms, were targeted onto one of the astrocytes “As” (white dash curve in Fig. 1(a)). Ca²⁺ elevations in the stimulated cell and neighboring cells were detected instantaneously (Fig. 1(b), “1 s”), followed by obvious propagation throughout the astrocyte network, called Ca²⁺ wave. In most cases, the wave was already initiated within 1 s after laser irradiation, spreading concentrically (red dashed curves in Fig. 1(b), and see also supplementary Media 1) as in other studies [27-29

Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, “Astrocytes generate Na⁺-mediated metabolic waves,” Proc. Natl. Acad. Sci. USA 101, 14937–14942 (2004). [CrossRef] [PubMed]

]. With average laser power of 30~50 mW and duration of 1~4 ms, Ca²⁺ wave was always photogenerated, covering a range of 166.5 ± 63.9 μm in radius and at the average velocity of 11.5 ± 2.5 μm/s.

Fig. 1. Photogeneration of Ca²⁺ wave in astrocytes. (a) DIC image of cultured astrocytes and corresponding fluorescence images before and after laser irradiation. Red arrowhead (and in b) points to femtosecond laser target on an astrocyte “As”, which is indicated by white dashed curve. (b) Image series of fluorescence change ΔF, representing only Ca²⁺ increases. The edges of the wave at corresponding time points are marked by red dashed curves. Scale bar, 100 μm

3.2 Photoporation

We next verified whether poration was formed on cell membrane by femtosecond laser pulses [15

, 23

, 24

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

], resulting in astrocytic Ca²⁺ signaling. Virtually, local transformation of cell membrane was observed around the laser target directly in DIC images, suggesting the disruption there caused by femtosecond laser irradiation (Fig. 2(a), and see also supplementary Media 2). Astrocytes were bathed in HBS containing FM1-43, a membrane-staining dye, and photostimulation was carried out (see Media 3). Fluorescent spot appeared afterwards and was highly localized at the irradiated point initially, indicating a tiny pore open for the dye to fuse with cell membrane and emit fluorescence. This pore seemed to be closed soon, as fluorescence spot no longer extended after a while, suggesting FM1-43 hardly diffused any more intimately.

To further confirm it, astrocytes preloading Fluo-3 were imaged in the bath of PI, a membrane-impermeable nuclear stain. Fluorescence images for the two dyes were acquired sequentially. After photostimulation, fluorescence of PI (ΔF/F) was observed in the irradiated cell (dashed curve in Fig. 2(b)) with a slight increase (45.4 ± 3.2%), whereas there were no changes in other cells at all (Fig. 2(b), “PI”). Comparatively, fluorescence of Fluo-3 increased remarkably in both the irradiated cell (264.7 ± 160.0%) and surrounding cells, indicating the Ca²⁺ wave (Fig. 2(b), “Fluo-3”). Therefore, extracellular PI entered only into the stimulated cell, to which femtosecond laser was pointed with consequent Ca²⁺ signaling in astrocytes. Moreover, chelating extracellular Ca²⁺ with EGTA, we discovered that photogenerated Ca²⁺ elevation in the stimulated astrocyte disappeared (Fig. 2(c), “control” and “EGTA”). After washout of EGTA-containing (Ca²⁺-free) HBS, the cells were put back into normal solution, and response of the same astrocyte upon photostimulation recovered (Fig. 2(c), “washout”). Notably, the amplitude of photogenerated Ca²⁺ elevation reduced after washout, probably due to EGTA, which entered into cytoplasm during the trial in Ca²⁺-free solution. Moreover, Ca²⁺ wave would diminish as well, without Ca²⁺ elevation in the stimulated cell (data not shown). Thus we proposed that localized transient photoporation was available on cell membrane caused by femtosecond laser irradiation, through which extracellular Ca²⁺ entry was necessary for Ca²⁺ elevation in the stimulated cell and subsequent propagation across others.

Fig. 2. Photoporation on astrocytes caused by femtosecond laser pulses. (a) Transformation of cell membrane. The right two images are enlargements of boxed area. Arrowhead points to the femtosecond laser target. (b) PI-test. (Left) ΔF Images show fluorescence changes of Fluo-3 and PI after photostimulation. The stimulated cell is marked by dashed curves. (Right) Comparison of ΔF/F between Fluo-3 and PI. (c) EGTA-test. Time courses of fluorescence change (ΔF/F) in the same stimulated astrocyte under different conditions are shown. Arrows indicate the onset of laser irradiation. Scale bar, 20 μm.

3.3 Reproducible photostimulation

In virtue of the facility of manipulating laser, an astrocyte was photostimulated for several times, as shown in Fig. 3. During the five trials, femtosecond laser was focused at a certain plane, onto the same point of cell membrane, with average power of 24 mW and duration of 2 ms. Time courses of Ca²⁺ elevation showed similar responses in the stimulated cell, suggesting the reproducibility of photostimulation.

Fig. 3. Responses to repetitive photostimulation. (a) ΔF images show fluorescence changes after laser irradiation. Arrowheads point to the femtosecond laser targets. (b) Corresponding responses (ΔF/F) are plotted. Arrows indicate the onset of laser irradiation. Scale bar, 50 μm.

4. Discussions and conclusion

As suggested in section 1, existing approaches have limits for defined targeting to effectively induce Ca²⁺ signaling in astrocytes. Electrical devices and micropipettes are necessary to achieve local selective stimulation by mechanical [27-29

Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, “Astrocytes generate Na⁺-mediated metabolic waves,” Proc. Natl. Acad. Sci. USA 101, 14937–14942 (2004). [CrossRef] [PubMed]

], electrical [3

M. Nedergaard, “Direct signaling from astrocytes to neurons in cultures of mammalian brain cells,” Science 263, 1768–1771 (1994). [CrossRef] [PubMed]

, 27

Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, “Astrocytes generate Na⁺-mediated metabolic waves,” Proc. Natl. Acad. Sci. USA 101, 14937–14942 (2004). [CrossRef] [PubMed]

, 28

B. Innocenti, V. Parpura, and P. G. Haydon, “Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes,” J. Neurosci. 20, 1800–1808 (2000). [PubMed]

] and pharmacological methods [28

B. Innocenti, V. Parpura, and P. G. Haydon, “Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes,” J. Neurosci. 20, 1800–1808 (2000). [PubMed]

, 30

E. A. Newman, “Propagation of intercellular calcium waves in retinal astrocytes and müller cells,” J. Neurosci. 21, 2215–2223 (2001). [PubMed]

], which make it complex and invasive. Uncaging of uncaged Ca²⁺ [31

L. Leybaert, K. Paemeleire, A. Strahonja, and M. J. Sanderson, “Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells,” Glia 24, 398–407 (1998). [CrossRef] [PubMed]

] or uncaged IP₃ [9

, 31

] using ultraviolet lights seems more favorable, though similarly to pharmacological way, additional agents are involved. Moreover, multiphoton photolysis, especially of caged Ca²⁺, have been examined [32

F. DelPrincipe, M. Egger, G. C. R. Ellis-Davies, and E. Niggli, “Two-photon and UV-laser flash photolysis of the Ca²⁺ cage,” Cell Calcium 25, 85–91 (1999). [CrossRef] [PubMed]

, 33

E. B. Brown, J. B. Shear, S. R. Adams, R. Y. Tsien, and W. W. Webb, “Photolysis of caged calcium in femtoliter volumes using two-photon excitation,” Biophys. J 76, 489–499 (1999). [CrossRef] [PubMed]

], which allows in vivo studies. However, the effectiveness of this kind of application in astrocytes is controversial due to poor Ca²⁺-specificity of the multiphoton uncaged chemical compounds [34

G. C. R. Ellis-Davies, “DM-nitrophen AM is caged magnesium,” Cell Calcium 39, 471-173 (2006). [CrossRef] [PubMed]

, 35

G. C. Faas, K. Karacs, J. L. Vergara, and I. Mody, “Kinetic properties of DM-nitrophen binding to calcium and magnesium,” Biophys. J 88, 4421–4433 (2005). [CrossRef] [PubMed]

]. And basically, uncaged compounds enter into all cells by bath loading, whereas for selectively delivering them into target cells, micropipette is required. In this paper, the idea of photostimulation was motivated by successful biological applications of femtosecond laser, even in neural cells [20

W. Zhou, X. Liu, X. Lv, J. Li, Q. Luo, and S. Zeng, “Monitor and control of neuronal activities with femtosecond pulse laser,” Chinese Sci. Bull. 53, 687–694 (2008). [CrossRef]

, 21

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca²⁺ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett. 3, 154–161 (2006). [CrossRef]

, 36

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

]. The advantages over conventional stimulating approaches are summarized as following. (1) Laser beam is focused at a sub-femtolitre volume, and the target location, laser power and duration, onset of irradiation can be finely controlled, enabling high spatiotemporal accuracy of stimulation. (2) Photostimulation is noncontact, noninvasive, without any chemical compounds and easy to perform. (3) The facility of manipulating laser makes it feasible to carry out repetitive stimulation.

Extracellular Ca²⁺ influx through photoporation on cell membrane was proved underlying Ca²⁺ elevation evoked by femtosecond laser. This process could be regarded as simulating physiological Ca²⁺ entry from voltage-gated or ligand-operated channels in plasma membrane [5

G. Carmignoto, “Reciprocal communication systems between astrocytes and neurones,” Prog. Neurobiol. 62, 561–581 (2000). [CrossRef] [PubMed]

]. Intracellular Ca²⁺ increase was then evoked probably due to the well-known Ca²⁺-induced Ca²⁺ release (CICR) from intracellular stores, leading to intercellular Ca²⁺ wave [37

J. W. Deitmer, A. Verkhratsky, and C. Lohr, “Calcium signalling in glial cells,” Cell Calcium 24, 405–416 (1998). [CrossRef]

, 38

A. Verkhratsky, R. K. Orkand, and H. Kettenmann, “Glial calcium: homeostasis and signaling function,” Physol. Rev. 78, 99–141 (1998).

]. The spreading velocity and covering range were comparable with previous studies [8

, 29

A. Charles, “Intercellular calcium waves in glia,” Glia 24, 39–49 (1998). [CrossRef] [PubMed]

] by mechanical stimulation, in which the latter two were 11.25 μm/s in average and 250 μm in maximum.

Cell viability upon laser irradiation is a vital consideration, which has been confirmed in previous studies (see section 1). Because of the high peak intensity of femtosecond laser pulses to reduce the energy threshold [39

A. Vogel, J. Noack, K. Nahen, D. Theisen, S. Busch, U. Parlitz, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and R. Birngruber, “Energy balance of optical breakdown in water at nanosecond to femtosecond time scales,” Appl. Phys. B 68, 271–280 (1999). [CrossRef]

, 40

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]

] , tissue dissection is achieved with low-energy source [13

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

, 16

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

, 17

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

, 24

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

]. Here, minimum laser energy (0.225~0.75 nJ/pulse in the sample) was adopted, relatively low compared to studies on other cultured cells [16

, 20

W. Zhou, X. Liu, X. Lv, J. Li, Q. Luo, and S. Zeng, “Monitor and control of neuronal activities with femtosecond pulse laser,” Chinese Sci. Bull. 53, 687–694 (2008). [CrossRef]

, 21

, 23

, 24

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

]. The focus plane of femtosecond laser was carefully identified to be at the upper cell membrane, leading to formation of photoporation there. This process has been demonstrated to be mediated by nonlinear effects based on multi-photon ionization and plasma formation [39

, 40

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]

]. Thus photo-chemistry including bond breaking is involved, hardly related to mechanical or thermal stresses due to the ultrashort pulses. Therefore, laser-membrane interaction was supposed to be highly localized in the focal volume, with little extended damage to circumstances or the entire cell. Actually, the tiny cut on biological structures by femtosecond laser has been observed and measured to be no more than hundreds of nanometers in width [13

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

, 15

]. In our study, transformation of cell membrane appeared after laser irradiation (Fig. 2(a)). Moreover, when laser power as high as 60 mW was applied, a cavity could, sometimes, be observed on cell membrane (supplementary Media 2). Restricted fluorescence spot of FM1-43 (supplementary Media 3), as well as slight increase in the fluorescence of PI (Fig. 2(b)), implied the localized transient poration. Further, the viability and function of the stimulated astrocyte were supported by observations: (1) restoration by membrane fluidity immediately after pore formation (supplementary Media 2); (2) no stain of PI in the nuclear 0.5~2 h after photostimulation (data not shown); (3) similar Ca²⁺ responses by repetitive laser irradiation (Fig. 3). Note that, laser intensity should not be too high (at average power of over 60 mW for several milliseconds), otherwise large holes on the membrane and cell blebbing would be generated, due to accumulative heating [40

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]

], resulting in cell death (data not shown).

In conclusion, noncontact photostimulation of astrocyte employing femtosecond laser is confirmed to be non-disruptive, reproducible, and with high spatiotemporal precision. It is efficient and feasible for the research on Ca²⁺ signaling in astrocytes and communications with other brain cells. Associated with other technologies such as uncaging and transgenics, this versatile optical tool is promising and helpful for resolving problems that traditional approaches are difficult to achieve, especially in studies looking into the brain in live animals.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Grant No. 30727002, 30700215) and National High-Tech Research and Development Program of China (863 Program: 2006AA02Z343).

Supplementary movies

Supplementary Media 1. Photogenerated Ca²⁺ wave in astrocytes by femtosecond laser. Laser target is marked by white arrow. Scale bar, 100 μm.

Supplementary Media 2. Photogenerated pore on cell membrane and subsequent restoration after femtosecond laser irradiation. Laser target is marked by white arrow. Scale bar, 5 μm.

Supplementary Media 3. Photoporation on cell membrane labeled by FM 1-43. Laser target is marked by white arrow. Scale bar, 5 μm.

References and links

1.	A. Volterra and J. Meldolesi, “Astrocytes, from brain glue to communication elements: the revolution continues,” Nat. Rev. Neurosci. 6, 626–640 (2005). [CrossRef] [PubMed]
2.	M. Nedergaard, B. Ransom, and S. A. Goldman, “New roles for astrocytes: redefining the functional architecture of the brain,” Trends Neurosci. 26, 523–530 (2003). [CrossRef] [PubMed]
3.	M. Nedergaard, “Direct signaling from astrocytes to neurons in cultures of mammalian brain cells,” Science 263, 1768–1771 (1994). [CrossRef] [PubMed]
4.	P. G. Haydon and G. Carmignoto, “Astrocyte control of synaptic transmission and neurovascular coupling,” Physiol. Rev. 86, 1009–1031 (2006). [CrossRef] [PubMed]
5.	G. Carmignoto, “Reciprocal communication systems between astrocytes and neurones,” Prog. Neurobiol. 62, 561–581 (2000). [CrossRef] [PubMed]
6.	M. Simard, G. Arcuino, T. Takano, Q. S. Liu, and M. Nedergaard, “Signaling at the gliovascular interface,” J. Neurosci. 23, 9254–9262 (2003). [PubMed]
7.	J. Schummers, H. Yu, and M. Sur, “Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex,” Science 320, 1638–1643 (2008). [CrossRef] [PubMed]
8.	A. C. Charles, J. E. Merrill, E. R. Dirksen, and M. J. Sanderson, “Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate,” Neuron 6, 983–992 (1991). [CrossRef] [PubMed]
9.	T. A. Fiacco and K. D. McCarthy, “Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons,” J. Neurosci. 24, 722–732 (2004). [CrossRef] [PubMed]
10.	J. T. Porter and K. D. McCarthy, “Adenosine receptors modulate [Ca²⁺]_i in hippocampal astrocytes in situ,” J. Neurochem. 65, 1515–1523 (1995). [CrossRef] [PubMed]
11.	W. Wantanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “ In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation,” J. Biomed. Opt. 13, 031213 (2008). [CrossRef]
12.	W. Wantanabe, T. Shimada, S. Matsunaga, D. Kurihara, K. Fukui, S. Arimura, N. Tsutsumi, K. Isobe, and K. Itoh, “Single-organelle tracking by two-photon conversion,” Opt. Express 15, 2490–2498 (2007). [CrossRef]
13.	K. König, I. Riemann, and W. Fritzsche, “Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,” Opt. Lett. 26, 819–821 (2001). [CrossRef]
14.	W. Wantanabe, 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, 4203–4213 (2004). [CrossRef]
15.	U. K. Tirlapur and K. König, “Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability,” The Plant Journal 31, 365–374 (2002). [CrossRef] [PubMed]
16.	N. I. Smith, K. Fujita, T. Kaneko, K. Katoh, O. Nakamura, S. Kawata, and T. Takamatsu, “Generation of calcium waves in living cells by pulsed-laser-induced photodisruption,” Appl. Phys. Lett. 79, 1208–1210 (2001). [CrossRef]
17.	M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, “Functional regeneration after laser axotomy,” Nature 432, 822–822 (2004). [CrossRef] [PubMed]
18.	L. Sacconi, R. P. O’Connor, A. Jasaitis, A. Masi, M. Buffelli, and F. S. Pavone, “ In vivo multiphoton nanosurgery on cortical neurons,” J. Biomed. Opt. 12, 050502 (2007). [CrossRef] [PubMed]
19.	N. I. Smith, Y. Kumamoto, S. Iwanaga, J. Ando, K. Fujita, and S. Kawata, “A femtosecond laser pacemaker for heart muscle cells,” Opt. Express 16, 8604–8616 (2008). [CrossRef] [PubMed]
20.	W. Zhou, X. Liu, X. Lv, J. Li, Q. Luo, and S. Zeng, “Monitor and control of neuronal activities with femtosecond pulse laser,” Chinese Sci. Bull. 53, 687–694 (2008). [CrossRef]
21.	N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca²⁺ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett. 3, 154–161 (2006). [CrossRef]
22.	N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P. D. Lyden, and D. Kleinfeld, “Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke,” Nat. Methods 3(2006). [CrossRef] [PubMed]
23.	S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Nakamura, T. Takamatsu, and S. Kawata, “Location-dependent photogeneration of calcium waves in HeLa cells,” Cell Biochem. Biophys. 45, 167–176 (2006). [CrossRef] [PubMed]
24.	U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]
25.	Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.-s. Gu, and S. Duan, “Regulated ATP release from astrocytes through lysosome exocytosis,” Nat. Cell Biol. 9, 945–953 (2007). [CrossRef] [PubMed]
26.	X. Lv, C. Zhan, S. Zeng, W. R. Chen, and Q. Luo, “Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector,” Rev. Sci. Instrum. 77, 046101 (2006). [CrossRef]
27.	Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, “Astrocytes generate Na⁺-mediated metabolic waves,” Proc. Natl. Acad. Sci. USA 101, 14937–14942 (2004). [CrossRef] [PubMed]
28.	B. Innocenti, V. Parpura, and P. G. Haydon, “Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes,” J. Neurosci. 20, 1800–1808 (2000). [PubMed]
29.	A. Charles, “Intercellular calcium waves in glia,” Glia 24, 39–49 (1998). [CrossRef] [PubMed]
30.	E. A. Newman, “Propagation of intercellular calcium waves in retinal astrocytes and müller cells,” J. Neurosci. 21, 2215–2223 (2001). [PubMed]
31.	L. Leybaert, K. Paemeleire, A. Strahonja, and M. J. Sanderson, “Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells,” Glia 24, 398–407 (1998). [CrossRef] [PubMed]
32.	F. DelPrincipe, M. Egger, G. C. R. Ellis-Davies, and E. Niggli, “Two-photon and UV-laser flash photolysis of the Ca²⁺ cage,” Cell Calcium 25, 85–91 (1999). [CrossRef] [PubMed]
33.	E. B. Brown, J. B. Shear, S. R. Adams, R. Y. Tsien, and W. W. Webb, “Photolysis of caged calcium in femtoliter volumes using two-photon excitation,” Biophys. J 76, 489–499 (1999). [CrossRef] [PubMed]
34.	G. C. R. Ellis-Davies, “DM-nitrophen AM is caged magnesium,” Cell Calcium 39, 471-173 (2006). [CrossRef] [PubMed]
35.	G. C. Faas, K. Karacs, J. L. Vergara, and I. Mody, “Kinetic properties of DM-nitrophen binding to calcium and magnesium,” Biophys. J 88, 4421–4433 (2005). [CrossRef] [PubMed]
36.	H. Hirase, V. Nikolenko, J. H. Goldberg, and R. Yuste, “Multiphoton stimulation of neurons,” J Neurobiol. 51, 237–247 (2002). [CrossRef] [PubMed]
37.	J. W. Deitmer, A. Verkhratsky, and C. Lohr, “Calcium signalling in glial cells,” Cell Calcium 24, 405–416 (1998). [CrossRef]
38.	A. Verkhratsky, R. K. Orkand, and H. Kettenmann, “Glial calcium: homeostasis and signaling function,” Physol. Rev. 78, 99–141 (1998).
39.	A. Vogel, J. Noack, K. Nahen, D. Theisen, S. Busch, U. Parlitz, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and R. Birngruber, “Energy balance of optical breakdown in water at nanosecond to femtosecond time scales,” Appl. Phys. B 68, 271–280 (1999). [CrossRef]
40.	A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005). [CrossRef]

OCIS Codes
(000.1430) General : Biology and medicine
(170.1530) Medical optics and biotechnology : Cell analysis
(170.2655) Medical optics and biotechnology : Functional monitoring and imaging

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: November 6, 2008
Revised Manuscript: December 12, 2008
Manuscript Accepted: January 12, 2009
Published: January 21, 2009

Virtual Issues
Vol. 4, Iss. 4 Virtual Journal for Biomedical Optics

Citation
Yuan Zhao, Yuan Zhang, Xiuli Liu, Xiaohua Lv, Wei Zhou, Qingming Luo, and Shaoqun Zeng, "Photostimulation of astrocytes with femtosecond laser pulses," Opt. Express 17, 1291-1298 (2009)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-17-3-1291

Sort: Author | Year | Journal | Reset

References

A. Volterra and J. Meldolesi, "Astrocytes, from brain glue to communication elements: the revolution continues," Nat. Rev. Neurosci. 6, 626-640 (2005). [CrossRef] [PubMed]
M. Nedergaard, B. Ransom, and S. A. Goldman, "New roles for astrocytes: redefining the functional architecture of the brain," Trends Neurosci. 26, 523-530 (2003). [CrossRef] [PubMed]
M. Nedergaard, "Direct signaling from astrocytes to neurons in cultures of mammalian brain cells," Science 263, 1768-1771 (1994). [CrossRef] [PubMed]
P. G. Haydon and G. Carmignoto, "Astrocyte control of synaptic transmission and neurovascular coupling," Physiol. Rev. 86, 1009-1031 (2006). [CrossRef] [PubMed]
G. Carmignoto, "Reciprocal communication systems between astrocytes and neurones," Prog. Neurobiol. 62, 561-581 (2000). [CrossRef] [PubMed]
M. Simard, G. Arcuino, T. Takano, Q. S. Liu, and M. Nedergaard, "Signaling at the gliovascular interface," J. Neurosci. 23, 9254-9262 (2003). [PubMed]
J. Schummers, H. Yu, and M. Sur, "Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex," Science 320, 1638-1643 (2008). [CrossRef] [PubMed]
A. C. Charles, J. E. Merrill, E. R. Dirksen, and M. J. Sanderson, "Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate," Neuron 6, 983-992 (1991). [CrossRef] [PubMed]
T. A. Fiacco and K. D. McCarthy, "Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons," J. Neurosci. 24, 722-732 (2004). [CrossRef] [PubMed]
J. T. Porter and K. D. McCarthy, "Adenosine receptors modulate [Ca2+]i in hippocampal astrocytes in situ," J. Neurochem. 65, 1515-1523 (1995). [CrossRef] [PubMed]
W. Wantanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, "In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation," J. Biomed. Opt. 13, 031213 (2008). [CrossRef]
W. Wantanabe, T. Shimada, S. Matsunaga, D. Kurihara, K. Fukui, S. Arimura, N. Tsutsumi, K. Isobe, and K. Itoh, "Single-organelle tracking by two-photon conversion," Opt. Express 15, 2490-2498 (2007). [CrossRef]
K. König, I. Riemann, and W. Fritzsche, "Nanodissection of human chromosomes with near-infrared femtosecond laser pulses," Opt. Lett. 26, 819-821 (2001). [CrossRef]
W. Wantanabe, 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, 4203-4213 (2004). [CrossRef]
U. K. Tirlapur and K. König, "Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability," The Plant Journal 31, 365-374 (2002). [CrossRef] [PubMed]
N. I. Smith, K. Fujita, T. Kaneko, K. Katoh, O. Nakamura, S. Kawata, and T. Takamatsu, "Generation of calcium waves in living cells by pulsed-laser-induced photodisruption," Appl. Phys. Lett. 79, 1208-1210 (2001). [CrossRef]
M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, "Functional regeneration after laser axotomy," Nature 432, 822-822 (2004). [CrossRef] [PubMed]
L. Sacconi, R. P. O'Connor, A. Jasaitis, A. Masi, M. Buffelli, and F. S. Pavone, "In vivo multiphoton nanosurgery on cortical neurons," J. Biomed. Opt. 12, 050502 (2007). [CrossRef] [PubMed]
N. I. Smith, Y. Kumamoto, S. Iwanaga, J. Ando, K. Fujita, and S. Kawata, "A femtosecond laser pacemaker for heart muscle cells," Opt. Express 16, 8604-8616 (2008). [CrossRef] [PubMed]
W. Zhou, X. Liu, X. Lv, J. Li, Q. Luo, and S. Zeng, "Monitor and control of neuronal activities with femtosecond pulse laser," Chinese Sci. Bull. 53, 687-694 (2008). [CrossRef]
N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, "Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation," Laser Phys. Lett. 3, 154-161 (2006). [CrossRef]
N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P. D. Lyden, and D. Kleinfeld, "Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke," Nat. Methods 3 (2006). [CrossRef] [PubMed]
S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Nakamura, T. Takamatsu, and S. Kawata, "Location-dependent photogeneration of calcium waves in HeLa cells," Cell Biochem. Biophys. 45, 167-176 (2006). [CrossRef] [PubMed]
U. K. Tirlapur and K. König, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002). [CrossRef] [PubMed]
Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.-s. Gu, and S. Duan, "Regulated ATP release from astrocytes through lysosome exocytosis," Nat. Cell Biol. 9, 945-953 (2007). [CrossRef] [PubMed]
X. Lv, C. Zhan, S. Zeng, W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Instrum. 77, 046101 (2006). [CrossRef]
Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, "Astrocytes generate Na+-mediated metabolic waves," Proc. Natl. Acad. Sci. USA 101, 14937-14942 (2004). [CrossRef] [PubMed]
B. Innocenti, V. Parpura, and P. G. Haydon, "Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes," J. Neurosci. 20, 1800-1808 (2000). [PubMed]
A. Charles, "Intercellular calcium waves in glia," Glia 24, 39-49 (1998). [CrossRef] [PubMed]
E. A. Newman, "Propagation of intercellular calcium waves in retinal astrocytes and müller cells," J. Neurosci. 21, 2215-2223 (2001). [PubMed]
L. Leybaert, K. Paemeleire, A. Strahonja, and M. J. Sanderson, "Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells," Glia 24, 398-407 (1998). [CrossRef] [PubMed]
F. DelPrincipe, M. Egger, G. C. R. Ellis-Davies, and E. Niggli, "Two-photon and UV-laser flash photolysis of the Ca2+ cage," Cell Calcium 25, 85-91 (1999). [CrossRef] [PubMed]
E. B. Brown, J. B. Shear, S. R. Adams, R. Y. Tsien, and W. W. Webb, "Photolysis of caged calcium in femtoliter volumes using two-photon excitation," Biophys. J 76, 489-499 (1999). [CrossRef] [PubMed]
G. C. R. Ellis-Davies, "DM-nitrophen AM is caged magnesium," Cell Calcium 39, 471-473 (2006). [CrossRef] [PubMed]
G. C. Faas, K. Karacs, J. L. Vergara, and I. Mody, "Kinetic properties of DM-nitrophen binding to calcium and magnesium," Biophys. J 88, 4421-4433 (2005). [CrossRef] [PubMed]
H. Hirase, V. Nikolenko, J. H. Goldberg, and R. Yuste, "Multiphoton stimulation of neurons," J Neurobiol. 51, 237-247 (2002). [CrossRef] [PubMed]
J. W. Deitmer, A. Verkhratsky, and C. Lohr, "Calcium signalling in glial cells," Cell Calcium 24, 405-416 (1998). [CrossRef]
A. Verkhratsky, R. K. Orkand, and H. Kettenmann, "Glial calcium: homeostasis and signaling function," Physol. Rev. 78, 99-141 (1998).
A. Vogel, J. Noack, K. Nahen, D. Theisen, S. Busch, U. Parlitz, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and R. Birngruber, "Energy balance of optical breakdown in water at nanosecond to femtosecond time scales," Appl. Phys. B 68, 271-280 (1999). [CrossRef]
A. Vogel, J. Noack, G. Huttman, and G. Paltauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005). [CrossRef]

Appl. Phys. B

A. Vogel, J. Noack, K. Nahen, D. Theisen, S. Busch, U. Parlitz, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and R. Birngruber, "Energy balance of optical breakdown in water at nanosecond to femtosecond time scales," Appl. Phys. B 68, 271-280 (1999). [CrossRef]
A. Vogel, J. Noack, G. Huttman, and G. Paltauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005). [CrossRef]

Appl. Phys. Lett.

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

Biophys. J

E. B. Brown, J. B. Shear, S. R. Adams, R. Y. Tsien, and W. W. Webb, "Photolysis of caged calcium in femtoliter volumes using two-photon excitation," Biophys. J 76, 489-499 (1999). [CrossRef] [PubMed]
G. C. Faas, K. Karacs, J. L. Vergara, and I. Mody, "Kinetic properties of DM-nitrophen binding to calcium and magnesium," Biophys. J 88, 4421-4433 (2005). [CrossRef] [PubMed]

Cell Biochem. Biophys.

S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Nakamura, T. Takamatsu, and S. Kawata, "Location-dependent photogeneration of calcium waves in HeLa cells," Cell Biochem. Biophys. 45, 167-176 (2006). [CrossRef] [PubMed]

Cell Calcium

G. C. R. Ellis-Davies, "DM-nitrophen AM is caged magnesium," Cell Calcium 39, 471-473 (2006). [CrossRef] [PubMed]
F. DelPrincipe, M. Egger, G. C. R. Ellis-Davies, and E. Niggli, "Two-photon and UV-laser flash photolysis of the Ca2+ cage," Cell Calcium 25, 85-91 (1999). [CrossRef] [PubMed]
J. W. Deitmer, A. Verkhratsky, and C. Lohr, "Calcium signalling in glial cells," Cell Calcium 24, 405-416 (1998). [CrossRef]

Chinese Sci. Bull.

W. Zhou, X. Liu, X. Lv, J. Li, Q. Luo, and S. Zeng, "Monitor and control of neuronal activities with femtosecond pulse laser," Chinese Sci. Bull. 53, 687-694 (2008). [CrossRef]

Glia

L. Leybaert, K. Paemeleire, A. Strahonja, and M. J. Sanderson, "Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells," Glia 24, 398-407 (1998). [CrossRef] [PubMed]
A. Charles, "Intercellular calcium waves in glia," Glia 24, 39-49 (1998). [CrossRef] [PubMed]

J Neurobiol.

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

J. Biomed. Opt.

L. Sacconi, R. P. O'Connor, A. Jasaitis, A. Masi, M. Buffelli, and F. S. Pavone, "In vivo multiphoton nanosurgery on cortical neurons," J. Biomed. Opt. 12, 050502 (2007). [CrossRef] [PubMed]
W. Wantanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, "In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation," J. Biomed. Opt. 13, 031213 (2008). [CrossRef]

J. Neurochem.

J. T. Porter and K. D. McCarthy, "Adenosine receptors modulate [Ca2+]i in hippocampal astrocytes in situ," J. Neurochem. 65, 1515-1523 (1995). [CrossRef] [PubMed]

J. Neurosci.

T. A. Fiacco and K. D. McCarthy, "Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons," J. Neurosci. 24, 722-732 (2004). [CrossRef] [PubMed]
M. Simard, G. Arcuino, T. Takano, Q. S. Liu, and M. Nedergaard, "Signaling at the gliovascular interface," J. Neurosci. 23, 9254-9262 (2003). [PubMed]
E. A. Newman, "Propagation of intercellular calcium waves in retinal astrocytes and müller cells," J. Neurosci. 21, 2215-2223 (2001). [PubMed]
B. Innocenti, V. Parpura, and P. G. Haydon, "Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes," J. Neurosci. 20, 1800-1808 (2000). [PubMed]

Laser Phys. Lett.

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, "Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation," Laser Phys. Lett. 3, 154-161 (2006). [CrossRef]

Nat. Cell Biol.

Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.-s. Gu, and S. Duan, "Regulated ATP release from astrocytes through lysosome exocytosis," Nat. Cell Biol. 9, 945-953 (2007). [CrossRef] [PubMed]

Nat. Methods

N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P. D. Lyden, and D. Kleinfeld, "Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke," Nat. Methods 3 (2006). [CrossRef] [PubMed]

Nat. Rev. Neurosci.

A. Volterra and J. Meldolesi, "Astrocytes, from brain glue to communication elements: the revolution continues," Nat. Rev. Neurosci. 6, 626-640 (2005). [CrossRef] [PubMed]

Nature

M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, "Functional regeneration after laser axotomy," Nature 432, 822-822 (2004). [CrossRef] [PubMed]
U. K. Tirlapur and K. König, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002). [CrossRef] [PubMed]

Neuron

A. C. Charles, J. E. Merrill, E. R. Dirksen, and M. J. Sanderson, "Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate," Neuron 6, 983-992 (1991). [CrossRef] [PubMed]

Opt. Express

Opt. Lett.

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. Rev.

P. G. Haydon and G. Carmignoto, "Astrocyte control of synaptic transmission and neurovascular coupling," Physiol. Rev. 86, 1009-1031 (2006). [CrossRef] [PubMed]

Physol. Rev.

A. Verkhratsky, R. K. Orkand, and H. Kettenmann, "Glial calcium: homeostasis and signaling function," Physol. Rev. 78, 99-141 (1998).

Proc. Natl. Acad. Sci. USA

Y. Bernardinelli, P. J. Magistretti, and J. Y. Chatton, "Astrocytes generate Na+-mediated metabolic waves," Proc. Natl. Acad. Sci. USA 101, 14937-14942 (2004). [CrossRef] [PubMed]

Prog. Neurobiol.

G. Carmignoto, "Reciprocal communication systems between astrocytes and neurones," Prog. Neurobiol. 62, 561-581 (2000). [CrossRef] [PubMed]

Rev. Sci. Instrum.

X. Lv, C. Zhan, S. Zeng, W. R. Chen, and Q. Luo, "Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector," Rev. Sci. Instrum. 77, 046101 (2006). [CrossRef]

Science

M. Nedergaard, "Direct signaling from astrocytes to neurons in cultures of mammalian brain cells," Science 263, 1768-1771 (1994). [CrossRef] [PubMed]
J. Schummers, H. Yu, and M. Sur, "Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex," Science 320, 1638-1643 (2008). [CrossRef] [PubMed]

The Plant Journal

U. K. Tirlapur and K. König, "Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability," The Plant Journal 31, 365-374 (2002). [CrossRef] [PubMed]

Trends Neurosci.

M. Nedergaard, B. Ransom, and S. A. Goldman, "New roles for astrocytes: redefining the functional architecture of the brain," Trends Neurosci. 26, 523-530 (2003). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Click here to see a list of articles that cite this paper

Figures


Fig. 1.	Fig. 2.	Fig. 3.


Fig. fig04	Fig. fig05	Fig. fig06

Multimedia

Multimedia Files	Recommended Software
» Media 1: AVI (1219 KB)	QuickTime
» Media 2: AVI (456 KB)	QuickTime
» Media 3: AVI (210 KB)	QuickTime

« Previous Article | Next Article »

OSA is a member of CrossRef.

Select a Conference
Session or Paper #
Year

OpticsInfoBase

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

Photostimulation of astrocytes with femtosecond laser pulses

Article Tools

Article Navigation

Abstract

1. Introduction

2. Materials and methods

2.1 Cell cultures

2.2 Dyes and drugs

2.3 System for imaging and photostimulation

3. Results

3.1 Photogenerated Ca2+ wave in astrocytes

3.2 Photoporation

3.3 Reproducible photostimulation

4. Discussions and conclusion

Acknowledgment

Supplementary movies

References and links

References

Appl. Phys. B

Appl. Phys. Lett.

Biophys. J

Cell Biochem. Biophys.

Cell Calcium

Chinese Sci. Bull.

Glia

J Neurobiol.

J. Biomed. Opt.

J. Neurochem.

J. Neurosci.

Laser Phys. Lett.

Nat. Cell Biol.

Nat. Methods

Nat. Rev. Neurosci.

Nature

Neuron

Opt. Express

Opt. Lett.

Physiol. Rev.

Physol. Rev.

Proc. Natl. Acad. Sci. USA

Prog. Neurobiol.

Rev. Sci. Instrum.

Science

The Plant Journal

Trends Neurosci.

Cited By

Figures

Multimedia

3.1 Photogenerated Ca²⁺ wave in astrocytes