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We propose the collinear balanced detection (CBD) technique for noise suppression in fiber laser (FL)-based stimulated Raman scattering (SRS) microscopy. This technique reduces the effect of laser intensity noise at a specific frequency by means of pulse splitting and recombination with a time delay difference. We experimentally confirm that CBD can suppress the intensity noise of second harmonic (SH) of Er-FL pulses by 13dB.The measured noise level including the thermal noise is higher by only ~1.4 dB than the shot noise limit. To demonstrate SRS imaging, we use 4-ps SH pulses and 3-ps Yb-FL pulses, which are synchronized subharmonically with a jitter of 227 fs. The effectiveness of the CBD technique is confirmed through SRS imaging of a cultured HeLa cell.
©2012 Optical Society of America
1. Introduction
Coherent Raman microscopy (CRM) [1–6] is an attractive technique for label-free biological imaging with high sensitivity. This technique provides chemical contrast based on intrinsic vibrational frequencies of sample molecules with subcellular spatial resolution.
In particular, coherent anti-Stokes Raman scattering (CARS) microscopy [1,2] has been extensively studied in the last decade. Compared with ordinary Raman microscopy, CARS microscopy offers much stronger signals by several orders of magnitude, and high speed imaging at up to the video rate [7–9]. However, quantitative interpretation is difficult because the CARS spectrum is different from its corresponding spontaneous Raman spectrum due to the nonresonant background, which distorts the spectral shape through the interference with the resonant signal. Furthermore, the nonresonant signal reduces image contrast because the solvent has broad nonresonant spectral response. Several techniques for suppression of the nonresonant background have been reported but at the expense of considerable complication of the experimental setup and/or loss of signal [10–14].
Recently, stimulated Raman scattering (SRS) microscopy has been reported as a technique for overcoming the nonresonant background limitation of CARS microscopy [3–5]. In SRS microscopy, we use two-color pulses, which are called pump and Stokes, at frequencies ωp and ωs, respectively. Then, one of the pulses is modulated temporally. The pulses are precisely overlapped in time, and collinearly and tightly focused on a sample. When the difference in frequency (Δω = ωp − ωs) corresponds to the intrinsic molecular vibrational frequency ΩR, the optical energy of the pump pulse is transferred to the Stokes pulse as a result of coherent excitation of molecular vibration through SRS process. Therefore, the intensity modulation is transferred to the other pulse, and this modulation transfer is measured by a lock-in amplifier. SRS offers several advantages such as quantitative contrast and accessibility to vibrational spectrum [15–18]. Furthermore, the video rate imaging is achievable [19] because the sensitivity of SRS microscopy at shot noise limit is comparable to that of CARS microscopy [4].
One of key technologies for realizing high-performance SRS microscopy is the generation of optical pulses. SRS imaging requires two-color synchronized trains of picosecond pulses with high-power and ultra-low noise. Furthermore, wavelength tunability over a wide spectral region is needed for hyperspectral SRS imaging [20, 21]. So far, mode-locked solid-state lasers and optical parametric oscillators have been most widely used for SRS microscopy because they almost satisfy the above requirements. Indeed, shot-noise limited sensitivity can be achieved by using these pulse sources [3, 22, 23]. In spite of that, it would be attractive to replace them by fiber lasers (FL’s) from a practical point of view, such as permanent optical alignment, good beam quality, and low cost. Indeed, FL-based optical pulse sources have been reported for CRM including CARS and SRS [24–29].
However, it is difficult to use FL’s in SRS microscopy because the intensity noise of FL’s is typically much higher than the shot noise limit due to the onset of excess noise through optical amplification and wavelength conversion process. The most common approach for reducing the effect of intensity noise is balanced detection [27]. In this technique, a pulse train is divided into signal and reference pulse trains whose intensities are adjusted to be the same for balanced photodetection. The reduction of intensity noise is accomplished by differencing between photocurrents originating from the signal and reference pulses electrically. In microscopy applications, however, it is difficult to maintain the balance of the intensities of detected pulses because the optical transmission of microscopes varies with focus position of sample. This may limit the performance of balanced detection.
In this paper, we propose the collinear balanced detection (CBD) technique for FL-based SRS microscopy. In this technique, the intensity balance between the signal and reference pulses is automatically maintained because both pulses are passed through a microscope collinearly. We experimentally confirm the effectiveness of CBD through the intensity noise measurement as well as label-free SRS imaging of a cultured cell.
2. Principle of collinear balanced detection technique
Figure 1(a) schematically illustrates the proposed CBD technique. An optical pulse train is divided into a signal pulse train and a reference pulse train. After a time delay difference of τ is given between these pulse trains by a delay-and-add line (DAL), they are combined collinearly. Then the signal and reference pulse trains are detected by a photodetector (PD). Here we denote the photocurrents contributed by the signal and reference pulse trains as isig(t) and iref(t), respectively. Since the reference pulse is a delayed replica of the signal,
Therefore, the total photocurrent I is given byIn the Fourier domain,where I(ω), Isig(ω) and Iref(ω) are Fourier transforms of i(t), isig(t) and iref(t), respectively. Equation (3) indicates that the photocurrents of signal and reference pulse trains add up destructively at ω = π/τ, because the delay of τ in the time domain corresponds to the linear spectral phase of ωτ. The RF spectral density of the photocurrent is proportional toIn this way, the intensity noise of the original pulse train can be eliminated at ω = π/τ. This is the principle of CBD.
Fig. 1 Schematic of the proposed CBD technique. (a) Principle of the CBD technique; DAL, delay and add line. (b) Arrangement of optical pulses in SRS microscopy with CBD; SRL, stimulated Raman loss.
Figure 1(b) explains a possible arrangement of pulse trains in SRS microscopy with CBD. Although CBD is applicable to SRS microscopy with any lock-in frequency in principle, we assume here that the lock-in frequency is the maximum, i.e. ωrep/2 [20, 22, 23], where ωrep is the repetition rate of the optical pulses. One can use a polarization beam splitter and a combiner to generate delayed reference pulses with a polarization orthogonal to the signal pulses. This allows us to reduce the optical loss of the DAL compared to an ordinary interferometer, where a beam combiner disposes a half of pulse energies. It is important that Stokes pulses are polarized in parallel to pump pulses so that we can detect ordinary Raman signal instead of Raman depolarization components.
From Eq. (4), one may think that optimum delay for suppressing noise at ωrep/2 may be τ = 2π/ωrep, which is equal to the pulse period. However, when the delay is precisely matched to the pulse delay, not only the signal pulses but also the reference pulses interact with Stokes pulses. In order to avoid such unwanted interaction, it is desirable to introduce additional small delayΔτ.
Through the stimulated Raman loss process, the intensity modulation of the Stokes pulses at ωrep/2 is transferred to the signal pulses. Then the signal pulses and the delayed reference pulses are detected by a photodetector. The intensity noise of signal pulses are cancelled out by CBD, whereas the transferred modulation can be detected by the lock-in technique to detect SRS signal.
Strictly speaking, the CBD technique has a slight drawback in terms of signal-to-noise ratio (SNR) because not only pump pulses but also reference pulses contribute to the shot noise and the total power. As a result, SNR achievable with CBD is 6 dB lower than that can be achieved under the same average power by quiet pulse trains with shot-noise-limited intensity noise. Therefore, it is redundant to incorporate CBD to SRS microscopy based on solid state lasers. Nevertheless, as will be demonstrated in the following sections, the CBD technique allows substantial improvement of SNR in FL-based SRS microscopy.
3. Experiments
3.1 Experimental setup
Figure 2 shows a schematic of the experimental setup of the FL-based SRS microscopy. A home-made figure-8 all polarization maintaining (PM) Er-FL [30], which was mode-locked by a nonlinear loop mirror, was used for generating pump pulses. The Er-FL produced 180-fs pulses at a repetition rate of f0 = 35.2 MHz. The center wavelength was 1560 nm and the bandwidth was14 nm. An electro-optic modulator (Photline Technologies, NIR-MPX-LN-10) was inserted in the Er-FL cavity as a mode-lock starter, and also used for high speed tuning of the repetition rate [31, 32]. The pulses were amplified by a PM-Er doped fiber amplifier (PM-EDFA). The amplified pulses were spectrally compressed by focusing in a 20-mm long periodically poled LiNbO3(PPLN) crystal (Covesion Ltd., MSHG1550-0.5-20) [26, 27]. The PPLN was equipped with multiple poled periods so as to generate narrowband (~0.3 nm) second harmonic (SH) pulses with an average power of up to 12 mW and a wavelength tunability from 760 to 810 nm. A Yb-FL generated sub-picosecond Stokes pulses at a repetition rate of 17.6 MHz. The Yb-FL was mode-locked by nonlinear polarization rotation. In the cavity of the Yb-FL, dispersion compensation was accomplished by a grating compressor which consisted of a grating (600 mm−1), a lens, and an end mirror. The center wavelength was 1026 nm and the bandwidth was 1.54 nm. For slow control of repetition rate over a wide range, a piezo actuator was inserted in the Yb-FL cavity. To equalize the pulse duration of the Stokes pulse to the SH pulse, Stokes pulses were introduced to a band-pass filter, which consisted of a lens pair, a grating (1200 mm−1), and a collimator. After the amplification by an Yb doped fiber amplifier, we obtained narrowband Stokes pulses with an average power of 15 mW and a bandwidth of 0.3 nm.
Fig. 2 Schematic of the FL-based SRS microscopy. LD, laser diode; PZT, piezo-transducer; HWP, half wave plate; QWP, quarter wave plate; YDF, Yb doped fiber; PBS, polarization beam splitter; BPF, band pass filter; G, reflection grating; YDFA, Yb doped fiber amplifier; PD, photo-detector; FL, fiber laser; DM, dichroic mirror; OL, objective lens; OF, optical filter; PM, polarization maintain; EDFA, Er doped fiber amplifier; EDF, Er doped fiber; PPLN, periodically poled LiNbO3; CGF, color glass filter; DAL, delay and add line; EOM, electric-optical modulator.
For subharmonic synchronization between two FL’s, we employed phase locked loop (PLL) technique with the two-photon absorption (TPA) detection scheme [22, 23, 33]. A part of pulses were tapped out by a polarization beam splitter’s (PBS’s), and combined on a dichroic mirror (DM) collinearly. The tapped pulses were introduced to a GaAsP-PD (Hamamatsu, G1115), which generated TPA photocurrent. Since this photocurrent reflects intensity cross correlation, it was used as an error signal. The photocurrent was fed back to the FL’s through a loop filter, which was equipped with an electronic amplifier and a proportional-integral controller. The in-loop jitter was measured by the same PD. The out-of-loop jitter was measured separately by using another GaAsP-PD at the input of the microscope.
Remaining pulses were used for SRS microscopy. The SH pulses were introduced to DAL for CBD. Intensity balance between the signal and reference pulses was adjusted by rotating a half wave plate in front of a PBS at DAL. The light path length of long and short delay lines were set to 8.6 m and 0.4 m, respectively for reducing the noise at f0/2. A lens pair was inserted in the long delay line of DAL to compensate for the divergence of the beam. After adjusting the timing between Stokes and twin SH pulses, these were combined on another DM collinearly and introduced to the objective lens ( × 100, NA 1.4, oil). The transmitted light was collected by another lens ( × 100, NA 1.4, oil). The sample position was scanned by a 3-axis piezo stage. Then, twin SH pulses was detected by Si-PD (Hamamatsu, S3399) after removing the Stokes pulses by an optical filter (OF). The photocurrent was filtered by band pass filters (BPF’s) and band elimination filters (BEF’s), and amplified before being measured by an RF spectrum analyzer (Agilent technologies, E4411B) or a lock-in amplifier (Stanford research systems, SR844). Reference signal at f0/2 for the lock-in detection was obtained through the photodetection of 0th diffraction from the grating compressor in the Yb-FL cavity.
3.2 Synchronization of two fiber lasers
Figures 3(a) and 3(b) show the error signal and its spectral density, respectively. The in-loop and out-of-loop jitter were measured to be 211 fs and 227 fs, respectively. The difference between these jitter values may be caused by intensity fluctuation of the lasers, which changes the DC bias of two-photon absorption photocurrent. Such a bias change is converted to timing shift by the PLL. Such a jitter cannot be monitored by in-loop measurement and can only be monitored by out-of-loop jitter measurement. Nevertheless, these jitter values were much smaller than the pulse widths, indicating that precise synchronization was achieved. The central peak in Fig. 3(b) indicates that PLL operated with a loop bandwidth of 1.6 kHz.
Fig. 3 Results of synchronization experiment. (a) In-loop (upper) and out-of-loop (lower) error signal in time domain. (b) Solid line, spectral densities of in-loop (grey) and out-of-loop (red) error signal; dashed line, integrated error signal.
3.3 Noise suppression by the CBD technique
We investigated the effectiveness of CBD for noise suppression at f0/2. The time delay of DAL was ~28 ns, which approximately corresponds to the period of the pump pulse train. The average power of the SH pulse train was 3.9 mW at the Si-PD.
Figure 4 shows the RF spectra of the measured photocurrents of the Si-PD. The noise spectrum had a broad peak reflecting the characteristics of BPF’s. The asymmetric spectral response is presumably due to the interference between the BEF and the BPF, which should be removed by further optimization of filter design. Green line shows the difference in RF spectra with and without CBD. When CBD was active, the noise spectrum was reduced by 13 dB at f0/2. At a frequency of f0/2, a measured noise level including the thermal noise was higher by only ~1.4 dB than the shot noise limit, which was estimated from a DC photocurrent of 2.5 mA.
Fig. 4 RF spectra of the photocurrent generated by the SH pulse train. Blue line, CBD is inactive; red line, CBD is active; grey line, circuit noise; dashed line, theoretical shot noise limit, difference in RF spectra; Green line. Average power of SH pulse, 3.9 mW; resolution bandwidth, 100 kHz.
We found that the intensity noise of the SH pulse train is very susceptible to pump levels of both the Er-FL and the EDFA, and that there is a trade-off between achievable optical power and intensity noise. The noise level without CBD presented in Fig. 4 was achieved through careful optimization of pump levels, and noise suppression of >20 dB was readily possible when the pump levels were not fully optimized. Additionally, we also confirmed that noise suppression at other frequency was accomplished by adjusting the delay length of DAL (not shown).
3.4 SRS imaging
We carried out SRS imaging with the developed system. The average power of the Stokes pulses was 4.5 mW at the focus. The Raman shift was set to 2850 cm−1 and 2950 cm−1,and the average power of the SH pulse were 5.4 mW and 8.7 mW at the focus, respectively. The pixel dwell time was 3 ms.
Figure 5 shows SRS images of a cultured HeLa cell obtained by CH2 stretching (Figs. 5(a) and 5(b)), and CH3 stretching (Figs. 5(c) and (d)) vibrations, respectively. Figures 5(e) and 5(f) are the histograms of pixel intensities in areas indicated by boxes in Figs. 5(a) and 5(c), respectively. Obviously the distribution became sharper by employing CBD. In order to evaluate the SNR improvement, root mean square of background noise in Fig. 5(e) was calculated to be 19 without CBD and 9.8 with CBD. Thus the SNR improvement was ~6 dB, which is in reasonable agreement with the RF noise suppression of 13 dB (Fig. 4) and 6-dB SNR drawback of CBD described in Section 2. In this way, we confirmed the effectiveness of the CBD technique.
Fig. 5 SRS images of a cultured HeLa cell. Pixel dwell time, 3 ms; number of pixels, 151 × 151; image size, 30 μm × 30 μm; scale bar, 5 μm. (a)(b) SRS images in 2850 cm−1, which show lipid rich region. (c)(d) SRS images in 2950 cm−1, which show protein. Upper row and lower show the cases where CBD is inactive and active, respectively. (e)(f) Histograms of square areas indicated in (a)(c) and same areas in (c)(d). Red line: with CBD. Black line: without CBD.
4. Conclusion
In conclusion, we have proposed and demonstrated the CBD technique for sensitivity enhancement of FL-based SRS microscopy. We confirmed the principle of CBD through the intensity noise measurement as well as label-free cell imaging with subharmonically synchronized FL’s. CBD will be especially useful for laser-scanning SRS microscopy because signal and reference pulses are passed through a microscope collinearly. Further improvement in imaging speed will be possible by optimizing the wavelength conversion process for generating intense pulses. In that case, the CBD technique would be more effective because the effect of classical noise will be more significant as the optical power increases. In addition, the experimental setup will be more compact by replacing DAL with optical fibers.
Acknowledgments
The authors would like to thank K. Goto of Osaka Univ. for providing samples.
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