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 al- so 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 sys- tem, 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 (151) 585 日本 AEM 学会誌 Vol. 23, No.3 (2015) 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 modula- tor 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. 586 (152) 日本 AEM 学会誌 Vol. 23, No.3 (2015) 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.