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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 3, Iss. 12 — Dec. 1, 2008
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Nonlinear-microscopy optical-pulse sources based on mode-locked semiconductor lasers

H. Yokoyama, A. Sato, H. -C. Guo, K. Sato, M. Mure, and H. Tsubokawa  »View Author Affiliations


Optics Express, Vol. 16, Issue 22, pp. 17752-17758 (2008)
http://dx.doi.org/10.1364/OE.16.017752


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Abstract

We developed picosecond optical-pulse sources suitable for multiphoton microscopy based on mode-locked semiconductor lasers. Using external-cavity geometry, stable hybrid mode locking was achieved at a repetition rate of 500 MHz. Semiconductor optical amplifiers driven by synchronized electric pulses reached subharmonic optical-pulse repetition rates of 1–100 MHz. Two-stage Yb-doped fiber amplifiers produced optical pulses of 2 ps duration, with a peak power of a few kilowatts at a repetition rate of 10 MHz. These were employed successfully for nonlinear-optic bio-imaging using two-photon fluorescence, second-harmonic generation, and sum-frequency generation of synchronized two-color pulses.

© 2008 Optical Society of America

1. Introduction

Since the first successful demonstration of two-photon excitation fluorescence imaging of bio-tissues [1

1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]

], a variety of novel nonlinear-optic multiphoton imaging (MPI) technologies have been developed [2

2. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003). [CrossRef] [PubMed]

]. Many of these technologies need a high-peak-power (over a kilowatt) ultrashort optical-pulse source at the heart of an imaging system to efficiently induce nonlinear-optic effects in bio-specimens. At present, most conventional optical-pulse sources are mode-locked solid-state lasers. However, even the current mode-locked solid-state lasers are large, expensive, and not maintenance-free. This makes it difficult for MPI to become a common technology. To render nonlinear-optic imaging technologies reliable and widely useful, we must be able to provide very stable, compact, and inexpensive optical-pulse sources. Semiconductor laser diodes (LDs) have had great success as reliable, low cost, and high-performance light sources in information technology (IT) applications. However, LDs can also be highly functional light sources in scientific and technological measurement systems. In this paper, we discuss potential applications of mode-locked LDs (MLLDs) in nonlinear bio-imaging.

2. Optical pulse source configuration

In the present optical-pulse sources, MLLDs are the core devices used to generate stable optical pulses of a few picoseconds in duration. Subsequent optical amplification by low-noise pre-amplifiers and low-nonlinear-effects main-amplifiers increases the peak power of the optical pulse to over a kilowatt, and the optical pulses can thus be used for nonlinear MPI purposes. We previously demonstrated the operation of a kilowatt-peak-power optical-pulse source of several picoseconds duration using a gain-switching method on the basis of a combination of a high-speed-response (>15 GHz bandwidth) 1.55 µm LD and a short-pulse electric-pulse generator. The second-harmonic 0.77 µm optical pulses were used successfully for two-photon excitation fluorescence imaging (TPI) of bio-specimens [3

3. H. Yokoyama, H. -C. Guo, T. Yoda, K. Takashima, K. Sato, H. Taniguchi, and H. Ito, “Two-photon bioimaging with picosecond optical pulses from a semiconductor laser,” Opt. Express 14, 3467–3471 (2006). [CrossRef] [PubMed]

].

Fig. 1. Schematic configuration of the high-peak-power 980-nm optical-pulse source. The MLLD-laser oscillator is in the area enclosed by the dashed line. BPF: tunable band-pass optical filter; BS-LD: bisection-laser diode; SOA: semiconductor optical amplifier; YDFA: Yb-doped fiber amplifier; LPF: long-wavelength-pass optical filter. The inset shows a SHG autocorrelation waveform for the optical pulses from the MLLD indicating a 3-ps pulse duration assuming a sech2 shape.

Starting from a 500 MHz MLLD, the optical-pulse repetition rate is decreased to a subharmonic frequency by using a semiconductor optical amplifier (SOA) as the optical gating device. Subsequently, optical-pulse amplification to kilowatt-peak-power level becomes possible by employing optical-fiber amplifiers, as described for the gain-switching LD light source [3

3. H. Yokoyama, H. -C. Guo, T. Yoda, K. Takashima, K. Sato, H. Taniguchi, and H. Ito, “Two-photon bioimaging with picosecond optical pulses from a semiconductor laser,” Opt. Express 14, 3467–3471 (2006). [CrossRef] [PubMed]

]. We have adopted this configuration for MLLDs operating at 980 and 1030 nm. Figure 1 illustrates the configuration for a high-peak-power 980 nm source. It consists of a MLLD for short-pulse generation, an SOA for subharmonic optical gating and pre-amplification, and a two-stage Yb-doped fiber amplifier (YDFA) for main amplification. The 980-nm LD chip used has a separate confinement heterostructure with a GaAs/InGaAs strained-layer double-quantum-well active layer. Characterized by a ridge waveguide structure, the bisection LD has a gain-section length of approximately 500 µm and a saturable-absorber (SA) section length of 30–50 µm. A bilayer antireflection (AR) coating was deposited on the cleaved facet of the gain-section side, resulting in a residual reflectivity of less than 10-3.

The external-cavity length was set to 300 mm for an optical-pulse-repetition frequency of 500 MHz. For hybrid mode-locking operation, the SA section was biased at −0.2 V for proper SA adjustment. The gain section was driven by a forward direct current of 14 mA, modulated by a 500 MHz microwave signal with an amplitude of 2 V at the 50 Ω termination. An optical isolator was inserted after the MLLD to eliminate any backward-reflected light. Under hybrid mode-locking conditions, the oscillation wavelength was tunable in the range of 965–990 nm with a 2-nm-bandwidth tunable band-pass optical filter (BPF) inside the cavity. The pulse width generated was mostly 2–3 ps, the average output power was about 0.6 mW (0.4–0.6 W peak power), and the timing-jitter was less than 1 ps. An example for the second-harmonic-generation (SHG) intensity autocorrelation waveform is shown in the inset of Fig. 1.

3. Sub-harmonic optical gating and high-peak-power amplification

Optical pulses with a low repetition rate in the megahertz range and a high peak power were generated by subharmonic gating of the SOA inside the fiber amplifier. Our SOA chip had the same 900-µm ridge-waveguide structure as the MLLD chip, but without the SA section. The SOA was driven synchronously by electronic pulses; these pulses were of 1.2 ns duration and had an amplitude of 4.4 V (at the 50 Ω termination) at a frequency of 1–100 MHz. Under these conditions, with an appropriately chosen incident optical-pulse polarization, we confirmed that the leaked (not gated) optical-pulse height was suppressed to less than 10-3. This high on/off extinction ratio is very important for concentrating the energy stored in the fiber amplifier, to produce the gated optical pulses by saturation amplification and to enable the kilowatt-peak-power optical pulses. The SOA also acted as a 10-dB net-gain preamplifier for the gated optical pulses. Because of losses in the fiber coupling and the optical isolator, the average optical power fed into the first Yb-doped fiber amplifier (YDFA) was approximately 40 µW.

Using the two-stage YDFA that we designed and built, the pulses were amplified after optical gating at the subharmonic repetition rate. With the exception of the Yb-doped fiber (YDF), we used free-space optics rather than fiber optics to reduce nonlinear optical effects due to amplification in the main YDFA. For the first YDFA, the 1.5-m-long YDF was excited by a forward-pumping LD (at a wavelength of 940 nm and a maximum power of 180 mW). For the second YDFA, the same type of YDF was co-excited by two orthogonally polarized LDs (at a wavelength of 915 nm and a maximum power of 180 mW) in a forward-pumping geometry. The Yb3+-weight concentration in the YDFs was 900 ppm. To eliminate optical feedback and unintentional laser oscillation, optical isolators were placed in front of the input ports of the first and second YDFAs, and the output end of the second YDFA was angle-cleaved. A 2-nm-wide BPF was inserted between the two YDFAs to reduce amplified spontaneous emission (ASE). To remove the residual pumping-laser light after the second YDFA, we used a long-wavelength-pass (>950 nm) optical filter (LPF). This resulted in a maximum output power of approximately 50 mW for a pulse-repetition rate of 10 MHz. The amplified optical-pulse power ratio was estimated at approximately 75%, with the remainder being due to ASE components. A further decrease in the pulse-repetition rate resulted in an increase in ASE, and thus we used a repetition rate of 10 MHz in the MPI experiment. Although the optical-pulse spectrum was broadened by a factor of 5 due to self-phase modulation inside the main YDFA, the pulse duration was not notably broadened because of a short YDF length, and thus the maximum peak power was kept at above 1 kW.

Except for the design of the MLLD chip and the optical components used, the 1030-nm optical-pulse source was developed using the same concept. However, the wavelength tuning range of the 1030-nm optical-pulse source was approximately 1020–1045 nm, while the 980-nm source was tuned only in the 977–982 nm range, determined by the gain width of the YDF used.

4. Two-photon fluorescence imaging

We examined the feasibility of the 980 and 1030 nm optical-pulse sources for TPI. Although the kilowatt-peak-power optical pulses obtained were successfully applicable to TPI of bio-specimens stained with various fluorescent dyes, very clear TPI for the specimens expressing GFP was not obtained with 1030-nm optical pulses. Therefore, we mainly used 980-nm optical pulses for TPI with GFP fluorescence. Figure 2 shows a TPI example of mouse brain neurons expressing GFP compared to a conventional single-photon confocal microscope image taken with a 488-nm continuous-wave laser. In this experiment, we attempted to image living brain neurons expressing GFP in a mouse (Acc. No. CDB0430T). In these neurons, GFP is fused to protein kinase C (PKC), an enzyme important in the regulation of several neuronal functions [8

8. K. Svoboda and W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385, 161 (1997).

,9

9. M. Matsuzaki, N. Honkura, G. C. R. Ellis-Davies, and H. Kasai, “Structural basis of long-term potentiation in single dendritic spines,” Nature 439, 761 (2004). [CrossRef]

]. The optical transmission efficiency from the output port of the main YDFA to the cover glass of the focus objective (60X, 1.2 NA, 280 µm WD) was 17% at a wavelength of 980 nm. As shown in Fig. 2 (b), single-photon images were sometimes blurred due to the scattering of fluorescence originating from the other branches close to the focal plane. The high spatial resolution of the TPI allowed us to investigate the intracellular three-dimensional structure of the neurons [8

8. K. Svoboda and W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385, 161 (1997).

,9

9. M. Matsuzaki, N. Honkura, G. C. R. Ellis-Davies, and H. Kasai, “Structural basis of long-term potentiation in single dendritic spines,” Nature 439, 761 (2004). [CrossRef]

]. Although nonlinear optical-pulse compression to the femtosecond temporal region is quite possible, this is not necessary for TPI. Our present results clearly indicate that picosecond optical pulses are suitable for practical use without pulse-broadening problems caused by optical dispersion in a microscope.

Fig. 2. (a) Two-photon and (b) single-photon excitation fluorescence imaging of the same branch in mouse brain neurons expressing GFP. The two-photon image was taken with 0.4 kW peak-power (at the surface of the specimen) optical pulses at a 10-MHz repetition rate, and was processed using a Kalman image filter. The single-photon image was taken with a 488-nm argon-ion laser.

5. Second-harmonic-generation imaging

We subsequently adopted these optical-pulse sources for nonfluorescent MPI. Among various nonfluorescent MPI methods, SHG imaging (SHI) would be most convenient for bio-specimens without using any fluorescent dyes or fluorescence proteins. A result of SHI is shown in Fig. 3 (a) for a specimen of unstained mouse adipose tissue; an auto-fluorescence TPI result is also displayed in Fig. 3 (b) for comparison. The 980-nm optical-pulse source was again used to take these MPI photos. The result shows clear differences between the performance of SHI compared to TPI. The differences can be attributed to the collagen structures inside the cells, which can efficiently produce SHG signals.

Fig. 3. (a) SHG imaging and (b) auto-fluorescent TPI of the same region in mouse adipose tissue. Both images were taken with 250-W peak-power (at the surface of the specimen) optical pulses at a 16-MHz repetition rate, and were processed using a Kalman image filter.

6. Sum-frequency-generation with two synchronized optical pulse sources

We also demonstrated all-electrically-controlled timing synchronization of the 980 and 1030-nm optical pulses. The synchronization was confirmed through sum-frequency generation (SFG) in a BiB3O6 crystal, as shown in Fig. 4, and was subsequently applied to SFG microscopy. Figure 5 indicates a demonstration of SFG imaging. We used a periodically-poled stoichiometric LiTaO3 (PPSLT) crystal instead of a bio-specimen as the imaging object to exclude unintentional two-photon fluorescence, and employed a narrow-band optical filter (in actual, this is a combination of a pair of optical filters, Semrock FF01-504/12 and Semrock FF01-500/15, giving 9nm transmission bandwidth) to assure SFG signal detection excluding SHG signals. Although SFG imaging may not provide very different information from SHI, the low-jitter synchronized operation of two (and more) different optical-pulse sources can be suitably extended to other nonlinear multiwavelength-excitation imaging technologies, including coherent anti-Stokes Raman scattering (CARS) [10

10. E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering micrsocopy with two tightly synchronized picosecond lasers,” Opt. Lett. 27, 1168 (2002). [CrossRef]

].

Fig. 4. Example of all-electrically-controlled timing synchronization of the 980- and 1030-nm optical pulses; prism-resolved photo of SFG and SHG pulses generated in a BiB3O6 crystal.
Fig. 5. SFG imaging for PPSLT crystal surface. The periodic SFG intensity contrast corresponds to the poling periodicity.

7. Summary

In summary, we have developed all-electrical timing-controllable optical-pulse sources using external-cavity MLLDs operating at wavelengths around 980 and 1030 nm. For each wavelength, with a combination of a SOA as the optical gating device and a two-stage YDFA as the main amplifier, we obtained kilowatt-peak-power optical pulses at a repetition rate of 10 MHz. Using these optical pulses, we obtained clear TPI for bio-specimens, especially for mouse brain neurons expressing GFP. The feasibility of SHG- and SFG-imaging applications was also demonstrated. Note, however, that if we develop high-power SOAs at the relevant wavelengths, the present optical-pulse source in which fiber amplifiers are used as the main amplifier will be modified to an all-semiconductor laser configuration [11

11. M. Kuramoto, N. Kitajima, H. -C. Guo, Y. Furushima, M. Ikeda, and H. Yokoyama, “Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source,” Opt. Lett. 32, 2726–2728 (2007). [CrossRef] [PubMed]

], and thus optical pulses at a variety of wavelengths can be obtained using many sets of MLLDs and SOAs. Therefore, we expect optical-pulse sources using MLLDs to see widespread adoption in practical nonlinear multiphoton microscopy.

Acknowledgments

The authors are grateful to K. Takashima and Y. Iseki for their technical supports and discussions. This work was supported by the Japan Science and Technology Agency (JST). H. C. Guo is supported by a JSPS Postdoctoral Fellowship.

References and links

1.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990). [CrossRef] [PubMed]

2.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003). [CrossRef] [PubMed]

3.

H. Yokoyama, H. -C. Guo, T. Yoda, K. Takashima, K. Sato, H. Taniguchi, and H. Ito, “Two-photon bioimaging with picosecond optical pulses from a semiconductor laser,” Opt. Express 14, 3467–3471 (2006). [CrossRef] [PubMed]

4.

N. Sakai, H. Tsubokawa, M. Matsuzaki, T. Kajimoto, E. Takahashi, Y. Ren, S. Ohmori, Y. Shirai, H. Matsubayashi, J. S. Chen, R. S. Duman, H. Kasai, and N. Saito, “Propagation of γPKC translocation along the dendrites of Purkinje cell in γPKC-GFP transgenic mice,” Genes to Cells 9, 945 (2004). [CrossRef] [PubMed]

5.

E. Spiess, F. Bestvater, A. Heckel-Pompey, K. Toth, M. Hacker, G. Stobrawa, T. Feurer, C. Wotzlaw, U. Berchner-Pfannschmidt, T. Porwol, and H. Acker, “Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP,” J. Microsc. 217, 200 (2004). [CrossRef]

6.

H. Yokoyama, H. Tsubokawa, H. -C. Guo, J. Shikata, K. Sato, K. Takashima, K. Kashiwagi, N. Saito, H. Taniguchi, and H. Ito, “Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source,” J. Biomed. Opt. 12, 054019 (2007). [CrossRef] [PubMed]

7.

H. Yokoyama, “Highly reliable mode-locked semiconductor lasers,” IEICE Trans. Electron. E85-C, 27–36 (2002).

8.

K. Svoboda and W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385, 161 (1997).

9.

M. Matsuzaki, N. Honkura, G. C. R. Ellis-Davies, and H. Kasai, “Structural basis of long-term potentiation in single dendritic spines,” Nature 439, 761 (2004). [CrossRef]

10.

E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering micrsocopy with two tightly synchronized picosecond lasers,” Opt. Lett. 27, 1168 (2002). [CrossRef]

11.

M. Kuramoto, N. Kitajima, H. -C. Guo, Y. Furushima, M. Ikeda, and H. Yokoyama, “Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source,” Opt. Lett. 32, 2726–2728 (2007). [CrossRef] [PubMed]

OCIS Codes
(140.4480) Lasers and laser optics : Optical amplifiers
(140.5960) Lasers and laser optics : Semiconductor lasers
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4180) Nonlinear optics : Multiphoton processes
(320.7090) Ultrafast optics : Ultrafast lasers

ToC Category:
Lasers and Laser Optics

History
Original Manuscript: August 7, 2008
Revised Manuscript: September 18, 2008
Manuscript Accepted: September 20, 2008
Published: October 17, 2008

Virtual Issues
Vol. 3, Iss. 12 Virtual Journal for Biomedical Optics

Citation
H. Yokoyama, A. Sato, H. -C. Guo, K. Sato, M. Mure, and H. Tsubokawa, "Nonlinear-microscopy optical-pulse sources based on mode-locked semiconductor lasers," Opt. Express 16, 17752-17758 (2008)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-16-22-17752


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003). [CrossRef] [PubMed]
  3. H. Yokoyama, H. -C. Guo, T. Yoda, K. Takashima, K. Sato, H. Taniguchi, and H. Ito, "Two-photon bioimaging with picosecond optical pulses from a semiconductor laser," Opt. Express 14, 3467-3471 (2006). [CrossRef] [PubMed]
  4. N. Sakai, H. Tsubokawa, M. Matsuzaki, T. Kajimoto, E. Takahashi, Y. Ren, S. Ohmori, Y. Shirai, H. Matsubayashi, J. S. Chen, R. S. Duman, H. Kasai, and N. Saito, "Propagation of γPKC translocation along the dendrites of Purkinje cell in γPKC-GFP transgenic mice," Genes to Cells 9, 945 (2004). [CrossRef] [PubMed]
  5. E. Spiess, F. Bestvater, A. Heckel-Pompey, K. Toth, M. Hacker, G. Stobrawa, T. Feurer, C. Wotzlaw, U. Berchner-Pfannschmidt, T. Porwol, and H. Acker, "Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP," J. Microsc. 217, 200 (2004). [CrossRef]
  6. H. Yokoyama, H. Tsubokawa, H. -C. Guo, J. Shikata, K. Sato, K. Takashima, K. Kashiwagi, N. Saito, H. Taniguchi, and H. Ito, "Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source," J. Biomed. Opt. 12, 054019 (2007). [CrossRef] [PubMed]
  7. H. Yokoyama, "Highly reliable mode-locked semiconductor lasers," IEICE Trans. Electron.E 85-C,27-36 (2002).
  8. K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, "In vivo dendritic calcium dynamics in neocortical pyramidal neurons," Nature 385, 161 (1997).
  9. M. Matsuzaki, N. Honkura, G. C. R. Ellis-Davies, and H. Kasai, "Structural basis of long-term potentiation in single dendritic spines," Nature 439, 761 (2004). [CrossRef]
  10. E. O. Potma, D. J. Jones, J. X. Cheng, X. S. Xie, and J. Ye, "High-sensitivity coherent anti-Stokes Raman scattering micrsocopy with two tightly synchronized picosecond lasers," Opt. Lett. 27, 1168 (2002). [CrossRef]
  11. M. Kuramoto, N. Kitajima, H. -C. Guo, Y. Furushima, M. Ikeda, and H. Yokoyama, "Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source," Opt. Lett. 32, 2726-2728 (2007). [CrossRef] [PubMed]

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