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We propose that a gain-switched laser diode (GS-LD) can be used as a picosecond laser source for stimulated Raman scattering (SRS) microscopy. We employed a 1.06-µm GS-LD to generate ~13-ps pulses at a repetition rate of 38 MHz and amplified them to >100 mW with Yb-doped fiber amplifiers. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF) so that the GS-LD pulses were synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We found the timing jitter of GS-LD pulses to be approximately 2.7 ps in a jitter bandwidth of 7 MHz. We also show that the delay of electrical pulses can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. We demonstrate the SRS imaging of polymer beads and of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.
© 2016 Optical Society of America
1. Introduction
Stimulated Raman scattering (SRS) microscopy is regarded as a powerful technique for high-speed label-free biomedical imaging based on the vibrational contrast of sample molecules [1–7]. Typical SRS microscopy uses pump and Stokes pulses at optical frequencies of ωp and ωs (ωp > ωs), respectively. After either pump or Stokes beam is intensity-modulated, these two-color pulses are simultaneously focused on a sample. When the vibrational frequency ωR of the molecules in the sample matches ωp – ωs, the pump pulses are attenuated and the Stokes pulses are amplified through SRS. As a result, the intensity modulation of one of the beams is transferred to the other beam. SRS signal is obtained by extracting the transferred modulation with the lock-in detection technique. Then, SRS images are taken by scanning the focal spot.
Compared with spontaneous Raman scattering microscopy, SRS microscopy is advantageous in terms of high signal intensity. This originates from the fact that the molecules are forced to oscillate by the intensity beat created by the two-color pulses. Consequently, SRS microscopy enables high-speed imaging at the video rate [4, 7]. It is true that coherent anti-Stokes Raman scattering (CARS) microscopy, which is another technique of label-free imaging, also allows for video-rate imaging [5]. However, CARS has suffered from the presence of unwanted background which originates from the nonresonant electronic response of sample molecules. This background reduces the image contrast and distorts the CARS spectrum, leading to the difficulty in interpreting CARS images. On the other hand, SRS signal straightforwardly reflects the same response as the spontaneous Raman scattering, and the signal background is almost suppressed. Because of these advantages, SRS microscopy has found a variety of applications in biology and medicine [8–39].
An important issue of SRS microscopy is that it requires a sophisticated light source which generates two-color and synchronized pulses. Therefore, many groups have used solid-state lasers and/or optical parametric oscillators (OPO) [1–3]. However, these sources are typically bulky, expensive, and susceptible to environmental perturbations. To promote the widespread use of SRS microscopy, it is important to develop a simple, low-cost and compact laser source.
So far, several groups have adopted fiber lasers (FLs) as alternative pulse sources for SRS microscopy [40–44] because FLs are compact and low-cost compared with solid-state lasers. Gambetta et al. and Freudiger et al. reported SRS microscopy using two-color pulses generated with a single fiber laser oscillator [40, 41]. In the application of FLs to SRS microscopy, it is important to consider the intensity noise of FLs, which severely deteriorates the signal-to-noise ratio (SNR) of SRS signal. The intensity noise of FLs is much larger than the shot noise because the pulse energy of FL pulses is much lower than those of solid state lasers. Therefore, optical amplification is necessary, which causes amplified spontaneous emission (ASE). To cope with this, several methods have been developed for suppressing the effect of ASE, leading to successful application of FLs to SRS imaging [41–44].
Apart from FLs, laser diodes (LDs) have been recently considered as laser sources for SRS microscopy because they are more compact and stable compared with solid-state lasers and FLs [45–47]. Wang et al. generated optical pulses from an LD under continuous wave (CW) operation using the time-lens method, where optical pulses are generated by applying strong phase modulation and group delay dispersion [45]. Steinle et al. used an LD, whose CW light is converted to laser pulses through optical parametric amplification with periodically poled lithium niobate (PPLN) crystals [46]. However, these techniques require several optics components to convert CW lights into pulse trains, leading to increased complexity of the laser system. Chun-Rui et al. used CW lasers as excitation sources for SRS microscopy [47]. Although the light sources are quite simplified therein, their method suffers from low SNR.
On the other hand, it is well known that optical pulses can be directly generated by gain-switched laser diodes (GS-LDs) [48–50], which are compact, low cost and long-term stable. A GS-LD driven by intense electrical pulses can produce optical pulses with pulse durations much shorter than the electrical pulses, taking advantage of fast carrier dynamics in the LD. Recently, GS-LDs at 1-µm wavelength region have been developed and applied to multiphoton microscopy [51, 52]. It would be attractive if a simple and stable pulse source such as a GS-LD could be applied to SRS microscopy. Indeed, we have recently reported SRS imaging of polymer beads [53] and live cells [54] with a 1.06-µm GS-LD that can directly generate picosecond pulses.
In this paper, we describe the experimental details on the SRS microscopy using the GS-LD. First, we characterize the GS-LD pulses. Then, we implement a pulse timing stabilization method of GS-LD pulses, which will be useful for SRS microscopy. Then, we experimentally confirm that the timing variation of GS-LD pulses can be suppressed by the proposed method. As a proof-of-principle experiment, we demonstrate SRS imaging of polymer beads and HeLa cells with a GS-LD and a Ti:sapphire laser (TSL).
2. Experiments and results
2.1 Experimental setup
Figure 1 shows a schematic of the experimental setup of SRS microscopy using a GS-LD and a TSL (Coherent, Mira 900D), which were used for generating Stokes pulses and pump pulses, respectively. The center wavelength of TSL pulses was manually controlled from 780 nm to 815 nm. The repetition rate, pulse width, and spectral width of the pump pulses were 76 MHz, 7 ps and 0.11 nm, respectively. A half-wave plate (HWP) and a polarizing beam splitter (PBS) were inserted to split TSL pulses into two. One is led to a Si-photodiode (PD, Kyosemi, KPID020D-H8) and the other is directed to an SRS microscope. A delay line stage was installed in the beam path of TSL pulses to control the relative delay of pump and Stokes pulses.
Fig. 1 A schematic of the experimental setup. HWP: half-wave plate, PD: photodetector, BPF: band-pass filter, YDFA: Yb-doped fiber amplifier, PBS: polarizing beam splitter, DM: dichroic mirror, T-FF: toggle flip-flop, EPG: electrical pulse generator, LIA: lock-in amplifier, SPF: short-pass filter, OL: objective lens.
In order to generate electrical pulses, the PD signal was input to a toggle flip-flop (T-FF, Hittite, HMC749LC3C). The output voltage (Vout) of the T-FF was switched every time when the input voltage (Vin) exceeded the threshold voltage (Vth). As a result, a square-wave voltage signal at a repetition rate of 38 MHz was generated by the T-FF. Then the signal was launched to an electrical pulse generator (EPG, Alnair Labs, EPG-200B-0100-S-P-T-A) so as to obtain 200-ps electrical pulses at a repetition rate of 38 MHz. These electrical pulses were used to drive the GS-LD. In this way, we generated 38-MHz GS-LD pulses and 76-MHz TSL pulses. Since this situation is equivalent to the intensity modulation of GS-LD pulses at 38 MHz, we can obtain SRS signal by the lock-in detection of the modulation transfer from GS-LD pulses to TSL pulses without using an intensity modulator [55].
Then, the optical pulses from the GS-LD were amplified by four concatenated polarization maintaining (PM) Yb-doped fiber amplifiers (YDFAs). The gain peak wavelength of the Yb-doped fiber (CorActive, Yb501-PM) used in the YDFAs was 1030 nm, which was shorter than the wavelength of Stokes pulses at 1060 nm. This is why 4-stage amplification was necessary in this experiment, whereas we expect that the number of amplification stages can be reduced by using YDF with a proper gain peak wavelength. In order to remove ASE caused by the YDFAs, we inserted two band-pass filters (BPFs), each of which was composed of a diffraction grating with a groove density of 1200 /mm (Thorlabs, GR25-1210) and two PM collimators with a beam diameter of 1.2 mm. To make sure that the pump pulses and Stokes pulses have the same polarization, we inserted a HWP and a PBS in the beam path of Stokes pulses. Then the pump and Stokes beams were combined by a dichroic mirror, and the combined beam was led to the SRS microscope, which was reported elsewhere [7]. Briefly, the laser beam was scanned by a resonant scanner at a scanning frequency of 8 kHz in the horizontal direction and a galvanometer scanner driven by a sawtooth wave at a frequency of 30 Hz in the vertical direction. The scanned laser beam was expanded by a lens pair and was focused onto a sample through a water-immersion objective lens (OL, 60 × , NA 1.2). The optical power of TSL and GS-LD beams at the back-aperture of the OL was measured to be 120 mW for each. Although the optical powers at the sample plane are not clear at the moment, we estimate that they were ~60 mW for each, considering the optical power of the transmitted beams of ~30 mW. The transmitted beams were collected by a condenser lens (NA 1.4). After the Stokes pulses were removed by a short-pass filter (SPF), the pump pulses were led to a Si-PD (Hamamatsu Photonics, S3399). Its photocurrent was input to a home-made lock-in amplifier (LIA) to obtain the SRS signal. The bandwidth of the lock-in amplifier output was approximately 7 MHz. As a reference signal of LIA, we used the other port of complementary outputs of the T-FF.
For the ease of operation, we stabilized the delay of the electrical pulses, which was found to be susceptible to the change of optical power of the TSL pulses. This is because the time at which Vin exceeds Vth is dependent on the amplitude of Vin as illustrated in Fig. 2(a). A common technique to cope with this problem is to use a constant fraction discriminator [56], which is composed of a delay line and a sophisticated digital circuit. Instead, we employed a simplified technique, where Vth was controlled in accordance to the optical power. Specifically, as shown in Fig. 1, the DC photocurrent from the PD was extracted by a low pass filter and amplified after an offset voltage was added properly. By doing so, the timing fluctuation of Vout was suppressed because Vth was changed in response to Vin as shown in Fig. 2(b).
Fig. 2 Illustrations of the input voltage (Vin) of T-FF for various optical powers of TSL pulses. When the threshold voltage (Vth) is fixed (a), the time at which Vin exceeds Vth is dependent on the optical power. By properly changing the threshold voltage (b), the time can be less sensitive to the change of the optical power.
2.2 Characterization of electric pulses and GS-LD pulses
Figure 3(a) shows the waveform of the electrical pulses generated by the EPG, measured with a sampling oscilloscope (Agilent Technologies, 86100C DCA-J). The duration and amplitude of the electrical pulses were measured to be 200 ps and 6.4 V, respectively. The timing jitter of the electrical pulses was measured to be less than 1 ps (not shown), which was limited by the trigger jitter of the sampling oscilloscope. We can see a zero-level signal in Fig. 3(a) because the repetition rate of the electrical pulses was 38 MHz while the pump pulses at 76 MHz were used for generating a trigger signal so as to confirm that the electrical pulses were synchronized to the pump pulses. Before amplification, the spectrum of the GS-LD pulses consists of a broad component centered at 1060.35 nm and a narrow peak at 1060.6 nm as shown with broken line in Fig. 3(b). The former is a result of GS operation, while the latter corresponds to a redundant tail part after the pulse is formed. Only the former was extracted by tuning the BPFs and amplified by the YDFAs. As a result, we obtained Stokes pulses with a spectral width of 0.20 nm as shown with solid line in Fig. 3(b).
Fig. 3 Generation and characterization of GS-LD pulses. (a) Sampling oscilloscope trace of the output signal from EPG. (b) Spectrum of the optical pulses of GS-LD before amplification (broken line) and after amplification by YDFA’s (solid line). (c) Cross-correlation trace of GS-LD pulses and TSL pulses (circles). Solid line: Gaussian fit. (d) Histogram of SRS signal intensity of water sample. We changed the delay of TSL pulses by moving the delay line stage to obtain the SRS signals at the three intensity levels, minimum (blue), half (red) and maximum (green).
2.3 Cross-correlation and jitter measurement of GS-LD pulses
GS-LDs are known for having timing jitter, which is much larger than mode-locked lasers. This is because the generation of each GS-LD pulse is initiated by spontaneous emission, which is a stochastic process. In order to investigate whether GS-LD pulses are applicable to SRS microscopy, we evaluated the timing jitter of the GS-LD by measuring the intensity cross-correlation between TSL and GS-LD pulses using SRS signal of water in the OH-stretching region. In this experiment, the wavelength of TSL pulses was set to 780 nm, while that of GS-LD was 1060.35 nm. Therefore, the Raman shift was 3390 cm−1. The pulse width of TSL pulses was set to be as small as ~3 ps. The TSL and GS-LD beams were loosely focused into a 10-mm cuvette filled with water (not shown in Fig. 1), while the delay of TSL pulses was manually scanned by the delay stage. As a result, we obtained the cross-correlation trace shown in Fig. 3(c), whose duration was found to be 14 ps.
To investigate the jitter characteristics, we acquired histograms of SRS signal intensity of the water sample, measured at three delay settings of pump pulses such that the SRS signal is minimized, at half maximum, and maximized. The result is shown in Fig. 3(d). Since the signal intensity was measured at the output of the lock-in amplifier with a bandwidth of 7 MHz, this measurement gives us a jitter in a bandwidth of 7 MHz. Then the standard deviations in these three settings were calculated to be σmin = 0.21, σhalf = 0.33, and σmax = 0.28, respectively. We can assume that σmin is dominated by the shot noise, which was confirmed by measuring the amount of noise as a function of the optical power, and that σhalf includes both the shot noise and jitter-induced signal fluctuation σjitter. Note that σjitter is approximately proportional to the slope of the intensity autocorrelation trace. Since the jitter and the shot noise can be considered to be independent of each other, we can estimate that σjitter = (σhalf2 – σmin2)1/2 = 0.25. We can see that σmax is larger than σmin because the timing jitter causes the signal fluctuation, as will be discussed later. From the waveform shown in Fig. 3(c), the slope at the half-maximum intensity was calculated to be 9.4 × 10−2 ps−1. Using this value, we found the jitter of GS-LD pulses to be 2.7 ps. Since this value is much smaller than the width of the cross-correlation trace, we conclude that the timing jitter is reasonably small for SRS microscopy. The actual duration of the GS-LD is estimated to be ~13 ps by taking the jitter and the TSL pulse duration into account. Note that σmin, σhalf, σmax, and the slope of the intensity autocorrelation were described incorrectly in [54].
2.4 Timing stabilization of electrical pulses
We investigated the effectiveness of the timing stabilization of electrical pulses, whose schematic was described in Section 2.1. First, we measured the delay of T-FF’s output signals with the sampling oscilloscope, while the optical power of pump pulses were changed by using a neutral density filter. The results are summarized in Fig. 4. When Vth was fixed, the variation of the time delay was as much as 12 ps for the variation in the input power from 2.1 mW to 3.0 mW as shown with red triangle. On the other hand, when Vth was controlled in response to the optical power, the variation was suppressed to 1.5 ps as shown with open blue circle. In this way, we confirmed that the time delay of T-FF’s output signals can be stabilized by the proposed technique. We expect that this technique can enhance the stability of the delay of Stokes pulses against the long-term intensity fluctuation of pump pulses and the intensity variation caused by wavelength tuning. Note that the above delay values are different from those in [54] because the range of the considered variation in the input power is also different.
Fig. 4 Experimentally measured time delay of the T-FF’s output signal as a function of the optical power of TSL pulses with variable Vth (open blue circle) and fixed Vth (red triangle).
2.5 SRS imaging with GS-LD and TSL
Figure 5 shows the SRS images of polystyrene (PS) and poly(methyl methacrylate) (PMMA) beads. The frame rate was 30 fps. We changed the wavelength of TSL pulses to change the Raman shift. In Figs. 5(a)-5(e), the Raman shift was set to 2850 cm−1, 2900 cm−1, 2950 cm−1, 3000 cm−1 and 3050 cm−1, respectively. We can clearly see the difference in SRS signal intensities depending on the wavenumber. Figure 5(f) shows SRS signals taken at the locations indicated by red and blue arrows in Fig. 5(a). We can confirm that the SRS spectra of PS and PMMA beads were successfully obtained.
Fig. 5 SRS images of polystyrene (PS) and poly(methyl methacrylate) (PMMA) beads. Number of pixels, 500 × 500; frame rate, 30 fps; image size, 80 µm × 80 µm; scale bar, 20 µm. SRS images at (a) 2850 cm−1, (b) 2900 cm−1, (c) 2950 cm−1, (d) 3000 cm−1, (e) 3050 cm−1. (f) SRS spectra of PS and PMMA beads. Blue line: PS. Red line: PMMA. Lines in Fig. 5(f) are for clarity.
We also acquired SRS images of HeLa cells as shown in Fig. 6. In Figs. 6(a) and 6(b), the Raman shift was set to 2850 cm−1 (CH2-stretching mode) and 2930 cm−1 (CH3-stretching mode), respectively. The acquisition time was 3.3 s. We can see lipid droplets and cytoplasm in Figs. 6(a) and 6(b) as well as nucleus and nucleoli in Fig. 6(b). The locations of lipid droplets are different because the lipid droplets were moving while the wavelength of TSL was changed manually in approximately 10 minutes. This means that the cells were living, and indicates the necessity of fast wavelength tuning for spectral imaging [7, 15, 19, 30]. Nevertheless, we have demonstrated SRS imaging of polymer beads and living cells using a GS-LD and a TSL. This result clearly proves that GS-LD is applicable to SRS microscopy as a Stokes pulse source.
Fig. 6 SRS images of HeLa cells at (a) 2850 cm−1, (b) 2930 cm−1. Number of pixels, 500 × 500; averaging time, 3.3 s; scale bar, 20 µm.
3. Discussion
In this section, we discuss the advantages and limitations of using GS-LD for SRS microscopy. First, the synchronization of GS-LD is quite easy and requires very small optical power for synchronization, compared with previous techniques using phase-locked loop (PLL) [7, 42, 43, 55] or wideband wavelength conversion [40, 41]. Second, we can omit intensity modulator for lock-in detection of SRS signal, leading to further simplification of the system. This is because we can use T-FF to easily generate subharmonically synchronized pulses that can be used for high-frequency lock-in detection [55]. Third, we can electrically control the timing of GS-LD pulses, as was demonstrated in the timing stabilization technique. This would allow us not only to stabilize the delay, but also to omit the free-space delay line in the TSL beam path.
On the other hand, there are remaining challenges in applying GS-LD to practical implementation of SRS microscopy at the moment. First, it is difficult to tune the wavelength of GS-LD pulses. Therefore, we have to employ a wavelength-tunable pulse source along with GS-LD as demonstrated in the previous section. Next, since the output power of our GS-LD is as small as several tens of µW, output pulses have to be amplified with a large optical gain. This also means that, when GS-LD is naively applied to the generation of pump pulses, the intensity noise due to ASE will reduce the SNR. To cope with this problem, the effect of the intensity noise should be reduced much more compared to other laser sources such as fiber lasers [41–44]. Moreover, the timing jitter is still a key issue although its effect is currently marginal.
Here, we estimate the effect of timing jitter on SRS signal. We denote the durations of pump pulses and Stokes pulses as τp and τs, respectively, and assume that both two-color pulses have Gaussian intensity waveforms given by
respectively, where Ep and Es represent the pulse energy of the pump pulse and the Stokes pulse, respectively. The SRS signal amplitude ISRS can be estimated by calculating the cross-correlation of two-color pulses given bywhere Δt is the delay of Stokes pulse relative to pump pulse. Equation (3) shows how SRS signal depends on the overlap of pulses, and how the timing jitter affects the signal. We can see that shorter pulse duration is favorable for enhancing the signal [57], but that the use of such short pulses may enhance the jitter-induced signal fluctuation. Considering that GS-LDs can be used for generating 7-ps pulses at 1.06 µm [51] and 5-ps pulses at 1.55 µm [50], we have a room to shorten the pulse duration by increasing the amplitude of the electric driving signal, while the effect of jitter should be investigated carefully.The fluctuation of SRS signal induced by the timing jitter can be estimated as follows. Assuming that , Eq. (3) can be approximated as
where . We denote the timing jitter as τ0 and assume that Δt obeys Gaussian probability density function with a standard deviation of τ0. Under this assumption, (Δt / τ0)2 is assumed to obey χ2-distribution with a degree of freedom of 1, whose standard deviation is known to be . In our experiment, the full widths at half maximum of TSL and GS-LD pulses were 7 ps and 13 ps, respectively. Thus, τp and τs are 4.2 ps and 7.8 ps, respectively. The timing jitter τ0 of GS-LD pulses is measured to be 2.7 ps in the video bandwidth Bv = 7 MHz. Therefore, in the Nyquist bandwidth BN = 19 MHz (i.e. half the repetition rate of GS-LD pulses), the timing jitter is estimated to be τ0(BN / Bv)1/2 = 4.4 ps. Using these values and Eq. (4), the standard deviation of ISRS / I0 in the bandwidth of Bv is given byThis value is in good agreement with the experimental result in Section 2.3, where the additional fluctuation of SRS signal is (σmax2 – σmin2)1/2 = 0.19. Thus, the current level of timing jitter leads to the signal fluctuation of on the order of 20%. The effect of jitter is less significant when the SNR of SRS signal is low because, in such a case, SRS signal should be averaged over a long time to enhance the SNR and hence jitter-induced fluctuation can also be averaged out. On the other hand, in order to apply GS-LD to high-speed imaging, we should reduce the timing jitter of GS-LD pulses. To this end, external injection seeding [53, 58] will be useful for reducing the timing jitter of GS-LD pulses and thereby enhancing the signal-to-noise ratio.4. Conclusion
In conclusion, we have demonstrated that a 1.06-µm GS-LD can be used as a Stokes pulse source for SRS microscopy. We generated ~13-ps GS-LD pulses, which were subharmonically synchronized to TSL, and measured the jitter to be 2.7 ps within a bandwidth of 7 MHz. We also implemented a technique of pulse timing stabilization of GS-LD pulses, and demonstrated that the timing variation of T-FF’s output signal was suppressed by varying the threshold voltage of triggering circuit. Furthermore, we successfully demonstrated SRS imaging of polymer beads and living cells with a GS-LD and a TSL, proving that GS-LD is readily applicable to SRS microscopy. The presented result is an important step toward realizing SRS microscopy with further simplified light sources such as a wavelength-tunable FL [59] and a GS-LD or a pair of GS-LDs, which would make SRS microscopy much more practical.
Acknowledgment
This work was supported by JSPS KAKENHI Grant Numbers 25702026 and 25600116, Advanced Photon Science Alliance, and Inamori Foundation. We would like to thank Prof. Akimitsu Okamoto, Prof. Satoshi Yamaguchi, Risa Takagi, and Koya Kobayashi of Univ. of Tokyo for preparing the live cell samples.
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