Femtosecond Fiber Laser Applying for Cell Fusion Trang Dang Nguyen1, Yoshihiro Mizuta1 and Kozo Taguchi*1

Femtosecond Fiber Laser Applying for Cell Fusion Trang Dang Nguyen*1, Yoshihiro Mizuta*1 and Kozo Taguchi*1

APSAEM14 Journal of the Japan Society of Applied Electromagnetics and Mechanics Vol.23, No.3 (2015)

Regular Paper

Femtosecond Fiber Laser Applying for Cell Fusion Trang Dang Nguyen*1, Yoshihiro Mizuta*1 and Kozo Taguchi*1

We developed an actively mode-locked fiber laser that can generate 295 fs pulses at 9.188 MHz repetition rate. We built up a laser-induced cell fusion, in which the developed femtosecond laser was used as the laser source for both optical tweezers mode and laser scalpel mode, and thus improving cost-effectiveness. The cell fusion system also used a transparent dielectrophoresis chip as the specimen stage to create and manipulate the pearl chain of two or multiple cells for facilitating the cell fusion processes. We successfully developed the first optical tweezers using femtosecond fiber laser operating at 1530 nm, which can trap and transport cells effectively. With this developed system, we obtained the laser-induced fusion of red cabbage protoplasts. We also proposed a experimental cell fusion procedure which allows precisely selective cell fusion at the single-cell level. Therefore, the developed system would benefit basic research in biotechnology and biomedicine.

Keywords: fiber laser, active modelocking, femtosecond pulse generation, dielectrophoresis, laser-induced cell fusion. (Received: 24 July 2014, Revised: 7 May 2015)

1. Introduction peak power per a single pulse is lower compared with the case where only a single pulse is oscillated per a round- Cell fusion plays an important role in basic research trip. in biology, such as tissue regeneration, nuclear repro- Laser tweezers provide a non-invasive method for gramming, transplantation experiments etc. There are manipulating biological cells based on the optical gradi- three main methods of cell fusion. The first one uses ent forces. Recently, femtosecond laser has been theoreti- chemical solution (e.g. polyethylene glycol-PEG) to cally and experimentally demonstrated being able to use induce cell fusion [1]; however, this method produces for establishing femtosecond laser tweezers [10],[11]. very low fusion efficiency. The second method, so-called In this paper, we develop an actively mode-locked electrofusion, obtains cell fusion by applying pulses of mechanism that utilizes impulse modulation inside an intense electric field [2]; this method provides higher erbium-doped fiber ring cavity [12]. This mechanism can fusion efficiency. However, both mentioned methods generate very high-peak-power femtosecond laser pulses. cannot enable the fusion of individually selected cells Based on such femtosecond fiber laser, we propose a new present in clusters of other cells. laser-induced cell fusion system that combines a femto- By contrast, the third method utilizes focused laser second laser tweezers and a transparent dielectrophoresis beam of ultrashort pulses to create pores on the cell (DEP) chip. In addition to the femtosecond laser tweezers membrane and subsequently induces cell fusion, such function, the femtosecond laser also undertakes the processes are called laser-induced cell fusion [3]. The function of a laser scalpel to cut cell membranes and engineering approach of using laser tweezers to trap and induce cell fusion. transport cells, followed by fusion processes using ultrashort laser pulses, can enable selected cell fusion [4]- 2. Femtosecond fiber laser [6]. As regards the ultrashort laser pulse generation, Fig. 1 shows the schematic of the actively mode- mode-locking is the most common method. Actively locked fiber ring laser. The laser cavity consists of an mode-locked fiber lasers are stable and controllable erbium-doped fiber amplifier (EDFA), a fiber coupler, a because active elements are used inside the laser cavity. polarization controller (PC), and a dual-electrode Mach- Active mode-locking often generates relatively long pulse Zehnder intensity modulator. duration in the picosecond range [7],[8] due to the limited The commercial EDFA (FiberLabs AMP-FL8013- modulation speed of active modulators. To generate CB-13), which includes 8 m erbium-doped fiber (EDF) subpicosecond optical pulses, the active mode-locking and total 8 m single mode fiber (SMF) for the input and with conventional sinusoidal modulation needs to operate the output, provides the necessary gain for the cavity with at a high repetition rate of tens of GHz [9]. With such a a maximum output power of 13 dBm (20 mW). The high repetition rate, multiple pulses are simultaneously EDFA also have two optical isolators mounted at the oscillated inside the cavity per a round-trip; and thus the input and the output to guarantee unidirectional operation. Therefore, it is no need to use any external isolator in the ______cavity. Correspondence: K. Taguchi, Department of Electrical and The 10/90 fiber coupler extracts 10% power as laser Electronic Engineering, Ritsumeikan University, Kusatsu, output and provides 90% power feedback to the cavity. Shiga, Japan, 525-8577 The PC (Newport F-POL-APC) ensures optimal polar- email: [email protected] ized light enters the modulator. *1 Ritsumeikan University

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Finally, the 20 Gbit/s LiNbO3 modulator (Sumitomo optical field inside the ring cavity. This modulation is Osaka Cement T.DEH1.5-20-ADC) is used as the active called impulse modulation. element of the active mode-locking; it has 3 dB optical bandwidth of 18 GHz, insertion loss of 6 dB, on/off extinction ratio of 20 dB, polarization extinction ratio of 20 dB, and optical return loss of 30 dB. The modulator has a pigtailed polarization- maintaining (PM) fiber input and a single mode fiber output. The total fiber length of the modulator is about 3 m. The PM fiber input ensures that the modulator oper- ates stably. The two electrodes D1 and D2 of the modulator are driven by an electrical impulse generator (EIG), which generates a train of sharp electrical pulses. Phase difference between the two electrodes is controlled by a line stretcher connected with electrode D2. The total length of the laser cavity is about 20 m that is equivalent to the fundamental frequency of 9.188 MHz.

Fig. 2. (a) Measured results of two driving voltages fed into the two electrodes of the modulator. (b) Effective driving voltage. If the frequency of the impulse modulation is the same as the fundamental frequency of the laser cavity, Fig. 1. Experimental setup of the actively mode-locked then active mode-locking will take place, and just a single femtosecond fiber laser. optical pulse will oscillate in the cavity. The single pulse will contain almost entire energy of the cavity. In order to obtain a train of sharp driving pulses at the The cavity losses are modulated by the effective driv- fundamental frequency of the laser cavity, the EIG was designed and fabricated [13] instead of using a commer- ing voltage. As active mode-locking taken place, a cial pulse generator. perfectly timed optical pulse will be generated and passed Fig. 2 (a) displays the measured results of two driving by the modulator at the exact moments during every voltages on two electrodes D1 and D2 of the modulator. round-trip, where the losses are modulated at a minimum. The line stretcher made a delay of about 250 ps in the The pulse encounters higher attenuation in its wings, time domain between two driving voltages. compared with its peak. Therefore, the modulator tempo- For the sake of simplicity, let’s consider the operation rally shortens the pulse after every round-trip. However, characteristics of the dual-electrode Mach-Zehnder this shortening effect is not infinite because it is balanced intensity modulator in the time domain. Two driving by pulse-broadening effects (chromatic dispersion or the voltages applied to the two electrodes can be modeled as limited gain bandwidth). an effective driving voltage applied to the transmission If compared with the conventional sinusoidal modula- factor of the modulator. The effective driving voltage is tion operating at a high frequency (in the GHz range), the the difference of the two driving voltages applied to the impulse modulation though operating at the fundamental two electrodes as shown in Fig. 2 (b). That was an im- frequency (in the MHz range) could bring about similar pulse with 230 ps width and a very sharp peak. Such the shortening effect to the optical pulse, thanks to the sharp sharp peak would make fast and large modulation of the peak of the effective driving voltage.

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The top right inset of Fig. 4 shows the RF spectrum of the laser output, which was measured with a photodetec- tor (Focus 1454) and a 3-GHz RF spectrum analyzer (Advantest R3131A) with 199 kHz resolution. The RF spectrum shows a frequency comb of harmonics, which obviously corresponds to the repetition frequency of 9.188 MHz. There is no sideband observed, which sug- gests that stable mode-locked operation was obtained. The supermode suppression was about 32 dB. The bottom middle inset of Fig. 4 displays the same spectrum but unnormalized and presented in logarithmic scale. It shows a spectrum shape of pure mode-locking without any sideband of soliton. From the above values, the time-bandwidth product is 0.18, which is close to the expected transform-limited Fig. 3. Normalized autocorrelation trace (dotted line) and value of 0.142 for the Lorentzian pulse profile. corresponding Lorentzian fit (solid line). The inset shows the measured output pulse train. 3. DEP chip

The output optical pulses were measured by an auto- DEP chip is able to well manipulate dielectric parti- correlator (Alnair Labs HAC-150). Fig. 3 shows the cles (e.g. biological cells) and form pearl chain by using autocorrelation trace of the actively mode-locked femto- non-uniform electric field [2]. Fig.5 shows a proposed second fiber laser. It can be seen that the autocorrelation top-view design of a transparent DEP chip. The DEP chip trace of the output pulse had half-maximum width of 590 is fabricated on a thin glass. Two thin gold microelec- fs and Lorentzian-like shape. That means the output pulse trodes are coated on the glass by an ion coater and con- had pulsewidth of about 295 fs (assuming Lorentzian nected to an AC source that has adjustable voltage and pulse shape). The inset of Fig. 3 shows the output pulse frequency. Because viable and dead cells show different- train measured by a photodiode and an oscilloscope. It ly frequency-dependent characteristics in the DEP chip, shows the stable output pulse train with the repetition rate they can be easily distinguished by using positive and of 9.188 MHz, which is the same as the cavity round-trip negative DEP modes [14]. In the positive DEP mode, the frequency. Therefore, the active mode-locking has taken pearl chain of two or multiple cells is formed at the place inside the laser cavity. Average output power was region of strong electric field; and cell-cell contact about one mW and it could easily be increased up to 100 pressure can be effectively controlled by adjusting the mW level by using an external EDFA. applied voltage. These features facilitate the cell fusion processes.

Fig. 5. Illustration of the transparent DEP chip.

The fabricated DEP chip worked in the positive DEP mode at 2 V applied voltage and 1 MHz frequency. Protoplasts of red cabbage were used as biological cells Fig. 4. Normalized optical spectrum (solid line) and in our experiments with DEP chip. The size of protoplasts corresponding Lorentzian fit (dotted line). varies in 25-40 �m. Under the DEP force of the positive DEP mode, the pearl chain of two protoplasts were The optical spectrum was measured by an optical formed near the positive electrode, as shown in Fig. 6. spectrum analyzer (Advantest Q8384). The normalized The firm contact of cell-cell and the contact pressure optical spectrum (solid line) and its corresponding Lo- (controlled by adjusting the applied voltage) facilitate cell rentzian fit are displayed in Fig. 4. The 3 dB spectral fusion and consequently fusion efficiency is expected to bandwidth was 4.7 nm (0.61 THz). increase.

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also used to adjust the position of the specimen stage through a motorized stage. We calibrated the laser power after the collimator lens and after the objective lens. The output mean power after the collimator lens was in the range of 40 - 100 mW. After the objective lens, the output mean power was in the range of 18 – 45 mW, and thus the coupling efficiency of this mechanism at 1530 nm was about 45 %. Under this condition, the maximum peak power of a laser pulse after the objective lens was about 1.1×104 W. This maximum power level could puncture the membrane of cells at the focal volume of the beam.

4.2 Femtosecond optical tweezers Fig. 6. Pearl chain of cells formed by the fabricated DEP chip. One of the interesting points of our proposed system is that at the low output power, this system can also work as a femtosecond optical tweezers. This would be the first 4. Optical tweezers and laser-induced cell fusion femtosecond optical tweezers operating at 1530 nm wavelength band [12,13], to the best of our knowledge. 4.1 Experimental setup We carried out optical trapping experiment with both protoplasts and yeast cells. The average output power was set at the minimum of 18 mW to ensure no harm to the cells. A thin glass was used as the specimen stage for this experiment.

Fig. 8. Femtosecond laser tweezers experiment with protoplasts. Sequential video frames showing the trapping process with femtosecond laser pulse train. Fig. 7. Proposed setup of the laser-induced cell fusion. Protoplasts of red cabbage suspended in 0.5 M mannitol solution were inserted to the specimen stage. The The proposed setup of the laser-induced cell fusion is specimen stage was moved to scan a target protoplast into shown in Fig. 7. This system uses the developed femto- the laser focus axis. When the target protoplast was close second fiber laser to undertake both laser tweezers mode to the trap spot, it would be attracted to the center of the and laser scalpel mode. Average output power was trap spot, and thus it was trapped. Fig. 8 shows sequential adjustable by using an external EDFA (FiberLabs video frames during four continuous seconds. It can be FL8011-CB-22). The laser beam from the output of the seen that, at the beginning (0s) the trapped protoplast was external EDFA was collimated by a collimator lens attaching to the reference protoplast. During the next (Thorlabs LA1540-C), and then directed by a dichroic three seconds, by moving the specimen stage, the trapped mirror and focused by an objective lens (Edmund 100X protoplast was moved out of the attachment and went oil immersion NA = 1.25). We used a fast shutter (Suruga faraway the reference protoplast. The trapped protoplast Seiki F77-7) to control the laser exposure time. The could be manipulated in the trapping plane. However, the transparent DEP chip was used as the specimen stage for trapping efficiency of protoplasts suspended in mannitol cell manipulation and cell fusion. We used a CCD camera solution was quite low. This might be caused by the small to observe the fusion process through a computer that was

588 (154) 日本 AEM 学会誌 Vol. 23, No.3 (2015) difference in refractive indices between protoplast and bridges for connecting membranes of pair protoplasts, as mannitol, and thus lowering the optical trapping force. a result, fusion possibility was expected to increase. We are now in search for another solution for red cabbage protoplasts that can facilitate the trapping efficiency.

Fig. 10. Proposed cell fusion procedure. Photographs taken during the cell fusion process are shown in Fig. 11. A pair of protoplasts was selected. We started the process by shortly increasing the applied voltage on the two electrodes of the DEP chip to increase Fig. 9. Femtosecond laser trapping experiment with yeast cell-cell contact pressure. Then the femtosecond laser cells. Sequential video frames showing the trapping beam with about 1.1×104 W was controlled to shoot process with femtosecond laser pulse train. straight at the interface between the two contacted proto- We also did the same experiment but used yeast cells plasts by the shutter. The shot duration was about 0.2 s, as biological samples instead of protoplasts. The size of and we performed three shots per a pair. yeast cells varies in 4 - 8 µm. Yeast cells were suspended Fig. 11 (a) shows the pair of protoplasts just before in water solution. Due to higher difference in refractive femtosecond beam irradiation. After about 20 minutes, indices between yeast cell and water, trapping efficiency the cell fusion became visible. Fig. 11 (b) shows the was higher. The target yeast cell was well trapped and fusion process after 45 minutes. It was observed that, the manipulated as shown in Fig. 9. The trapping procedure fusion process was faster at the beginning than it was at in Fig. 9 is the same as that of Fig. 8 described above. the end (Fig. 11 (c) and (d)). After about 90 minutes, the two original protoplasts almost merged into one. At this 4.3 Laser-induced cell fusion stage, we conducted the same experimental conditions with three pairs of protoplasts, and we succeeded with To achieve precisely selective cell fusion at the single-cell level, we propose an experimental cell fusion two of them. procedure. The proposed cell fusion procedure performed by our system is shown in Fig. 10. First, the femtosecond optical tweezers will trap and transport individually selected cells close to the electrodes of the DEP chip (Fig. 10 (a)). The DEP chip will form the pearl chain of those selected cells and adjust contact pressure (Fig. 10 (b)). The shutter will shut the output laser beam, and the output power will be adjusted to a specific power that is enough for the laser scalpel mode. The focus point will be adjusted to exactly focus at the cell-cell membrane contact, and subsequently the shutter will enable a short series of high-power femtosecond pulses to puncture cells’ contact membranes (Fig. 10 (c)). Finally, two cells will start fusing (Fig. 10 (d)). Repeat the same processes for fusing multiple cells. By utilizing the proposed procedure, we carried out cell fusion experiment using red cabbage protoplasts. The room temperature was kept at about 22 oC by air condi- tioner. Protoplasts were suspended in mannitol solution. Fig. 11. Photographs of laser-induced cell fusion process After peal chains of protoplasts were formed by the DEP during 90 minutes. chip, polyethylene glycol (PEG) was added to the solution with the ratio of 20 %. PEG helps to provide Ca2+

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