Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells
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We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca2+ concentration and membrane potential revealed coincident photogeneration of Ca2+ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca2+ which acts on Ca2+ activated K+ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.
KeywordsFemtosecond laser Membrane potential Laser-cell interaction Hyperpolarization Depolarization
Intracellular calcium ion concentration
- GHK voltage equation
The use of the near-infrared femtosecond laser has become popular for biomedical experiments, due to the long penetration depth, low scattering, and localized nonlinear absorption (Denk et al. 1990; Dombeck et al. 2005; Smith et al. 2001; König et al. 1999; Hirase et al. 2002). These advantages have been used to carry out novel experiments in cells such as generation of intracellular Ca2+ waves (Smith et al. 2001), dissection of intracellular chromosomes (König et al. 1999), as well as inducing action potentials in neurons (Hirase et al. 2002). While similar effects have been produced using different laser sources (Uzdensky and Savransky 1997; Wells et al. 2007), the merits of femtosecond laser irradiations are being seen in the emergence of new applications which exploit the particular properties of ultrashort pulsewidths and near-infrared wavelengths. Although such applications are now emerging, the use of femtosecond laser illumination to provoke biological reactions in cells is in part limited by the incomplete understanding of the cellular response to high intensity light. There is therefore significant interest in finding new ways to understand and measure the cell response to high intensity light, and particularly the response to femtosecond laser irradiation.
In this report, we use patch-clamp measurement to monitor cellular membrane potential during femtosecond laser irradiation. Patch-clamp recording allows quantitative evaluation of the laser effect on the cell membrane condition. HeLa cells were chosen specifically as targets for their lack of action potentials and slow electrophysiological response, allowing us to determine the laser effect on the cell electrophysiology. Changes in membrane potential following laser irradiation were monitored, and clear differences in membrane potential response were observed depending on the location of the laser focus.
Results and discussion
To consider the nature of the photoinduced membrane potential change, we also monitored the membrane potential of HeLa cells under the influence of continuous-wave 780 nm irradiation, and no response in membrane potential or Ca2+ concentration was observed even with 60 mW of laser power, while femtosecond pulsed 780 nm laser with 60 mW of laser power causes apparent membrane potential hyperpolarization. This result is similar to results reported by Sacconi et al., where continuous wave near-infrared laser irradiation was not able to ablate microtubules, while femtosecond pulsed laser of the same power could cause ablation (Sacconi et al. 2005). The requirement of the ultrashort pulsed mode indicates that the nature of the photostimulation of the membrane potential hyperpolarization is clearly nonlinear, and is likely related to multiphoton-based ionization, in agreement with previous work (Iwanaga et al. 2006; Vogel and Venugopalan 2003). The requirement for the femtosecond pulsed mode occurs due to the low linear absorption of photons at 780 nm, and a less stringent requirement for pulsed irradiation would be expected if the wavelength was reduced. However, 780 nm is ideal for irradiation of living biological targets due to the high penetration and low absorption in areas outside the focal region (Denk et al. 1990). Previous work using the same cell type showed that, depending on the pulse width and repetition rate, the interaction between near-infrared ultrashort pulsed irradiation and the resulting cell response can even become independent of the exposure time, showing the highly nonlinear nature of the interaction (Iwanage et al. 2005). The observation of such Ca2+ responses in living cells from even a single pulse of low pulse energy further supports the premise that the interaction is based on the nonlinear interactions resulting from the ultrashort pulsewidth rather than single photon, thermal, or other mediating processes in the laser-cell interaction.
We then lowered the extracellular potassium concentration from 5.4 mM to 0.4 mM and repeated the experiment. In this case, the average depth of membrane potential hyperpolarization changed to −111.5 mV (n = 5). This value again corresponds well with the prediction of −114.9 mV, as calculated by the GHK equation with the appropriate modification for the lowered K+ concentration. This indicates that the femtosecond laser induced membrane potential hyperpolarization was caused by rapid elevation of the K+ channels open probability, which was in turn triggered by laser-induced [Ca2+] i elevation.
The cytoplasm irradiation experiments caused hyperpolarization that was consistent with known cellular properties. We also performed similar experiments, instead targeting the outer cell membrane, where we expected to see significantly different responses in membrane potential due to the disruption of the membrane. Evidence of membrane disruption after membrane irradiation by femtosecond laser has been previously seen, where influx of Ca2+ or other extracellular molecules could occur and affect the intracellular Ca2+ response (Iwanaga et al. 2006), as well as by other experiments where influx of extracellular molecules was intentionally induced in order to modify the cell (Tirlapur and König 2002). It should be noted, however, that the laser disruption of the outer cell membrane precludes the use of the GHK equations to calculate the membrane potential, as was done in the case of cytosol irradiation, since the ion-specific permeability after irradiation is unknown and the ion flow across the membrane cannot be approximated as steady-state.
In addition, the results in this manuscript also clarify the Ca2+ dye influence on laser induced intracellular Ca2+ dynamics. In 2006, Iwanaga et al. reported on the mechanism of near-infrared femtosecond laser induced Ca2+ release which triggers Ca2+ waves in cells (Iwanaga et al. 2006). The presence of the dye is necessary to measure the Ca2+ dynamics, but does have the potential to affect the laser-cell interaction. In Fig. 2d, by comparing the hyperpolarization power dependence (using no fluorescent dye) and the Ca2+ wave generation probability (with Fluo-4), we can conclude that the presence of the dye does not significantly affect the photogeneration of Ca2+ waves in living cells. The femtosecond laser is already showing promise as a tool for minimally invasive perturbation of biosystems with 3D resolution, and has recently been applied as a pacemaker of heart muscle cell contraction (Smith et al. 2008). The independence of the dye from the laser-cell interaction is quite relevant since it implies that the dye is not necessary for the stimulation effect, but also that the dye can be used to monitor Ca2+ levels without concern for overly affecting the laser-cell physics.
In summary, we have demonstrated photogeneration of membrane potential hyperpolarization and depolarization in living non-excitable cells induced by femtosecond laser illumination on cellular cytoplasm and cellular plasma membrane. This report quantitatively reveals cellular biological responses under exposure to high intensity femtosecond laser on living cells. Similarly, Sacconi et al. recently determined the optimum laser power for second-harmonic imaging of action potentials by analysis of femtosecond laser-induced photodamage and membrane potential change in living neurons (Sacconi et al. 2006). The quantitative analysis of femtosecond laser causing biological response reported here will contribute to the large field of biological research which requires high intensity of femtosecond laser. It also demonstrates that for applications involving femtosecond laser nanosurgery, gene transfection or manipulation of living cells, both the laser power and focus position must be taken into account in research where ion concentrations and membrane potential are under observation.
The authors thank Dr. Hideo Tanaka at the Kyoto Prefectural University of Medicine for technical advice in electrophysiological measurement, and Mr. Tomoya Uchiyama for help with the patch-clamp experimental setup.
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