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Abstract
An ultrafast, thulium-doped fiber laser system is developed for three-photon microscopy. The system generates 150 fs pulses at the center wavelength of 1.82 µm with a pulse energy of 1.1 µJ at the repetition rate of 1 MHz. The generated pulses are applied to a three-photon fluorescence microscope, with which biological samples expressing red fluorescent proteins are observed through three-photon excitation processes.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Multi-photon microscopy is widely used for the imaging of biological samples. Two-photon microscopy [1] enables the observation of more in-depth sites of tissues, organs, and living animals, compared to conventional fluorescence microscopy, thanks to the low scattering of longer-wavelength excitation light [2,3]. Recently, Horton et al. demonstrated that the penetration depth could be further improved by using three-photon excitation using a 1675 nm laser [4]. The three-photon excitation has two significant advantages. One is the improved signal-to-noise ratio because the background signal caused by out-of-focus fluorescence is significantly reduced compared to two-photon excitation. The other is the better penetration property because the attenuation of excitation laser with the wavelengths around 1.7–1.8 µm is less than that with the wavelength of 0.8–1.0 µm used in typical two-photon microscopes. However, light sources operating around 1.7 µm are not readily available, and all the previous work started from pulses at 1.5 µm and then shifted its wavelengths through soliton self-frequency shift through intrapulse stimulated Raman scattering processes. While this technique enables fine-tuning of the wavelength, it is coupled to the initial power of the seed pulses. Therefore, the laser parameters, such as the output power, are not very flexible.
An alternative approach is to use thulium-doped fiber lasers to generate ultrashort pulses directly around 1.8 µm instead of relying on frequency shifting. Since thulium has a very broad emission spectrum extending from 1.6 µm to 2.2 µm, this approach can work in principle. However, it is rather difficult to operate thulium-doped fiber lasers in the relatively short wavelength region below 1.8 µm because the absorption band of thulium is located around this wavelength region and thus cancels the gain. In fact, most of the high-power thulium-doped fiber lasers in previous work operate with wavelengths longer than 1.9 µm [5–11]. This problem can be alleviated by increasing the fraction of the population inversion [12]. An amplifier based on thulium-doped fibers operating around 1.7 µm is demonstrated in the CW regime with the output power of $\sim$100 mW [13]. Li et al. demonstrated amplification of ultrashort pulses centered at 1785 nm, where pulses with 445 fs duration are obtained with the pulse energy of 5.7 nJ [14].
Here, we demonstrate a thulium-doped fiber laser system generating ultrashort pulses around 1.8 µm with a duration of 150 fs and the pulse energy of 1.1 µJ. The generated pulses are intense enough to be used as the excitation source for three-photon fluorescence microscopes, which is confirmed by observing biological tissues expressing red fluorescent proteins through three-photon-excited fluorescence.
2. Laser setup
The laser system consists of an oscillator and two amplifier stages as shown in Fig. 1.
The oscillator is developed using fibers made of fluoride glass called ZBLAN (ZrF$_4$-BaF$_2$-LaF$_3$-AlF$_3$-NaF) [15]. Here, we decided to use ZBLAN fibers because ZBLAN glass is known for its high transmission and low dispersion in the mid-infrared region, which makes them a suitable choice for developing an ultrafast fiber-based oscillator in 2 µm region. It is also reported that a ZBLAN fiber has a higher efficiency than a silica fiber when used as an active fiber [16], which is most probably because of its lower phonon energy compared to that of a silica fiber [17,18]. In silica fibers, the higher phonon energy makes multiphonon relaxation processes quite efficient. This means that high pump power is required to keep a sufficient population inversion ratio. It also means that amplification becomes efficient only when the seed power is very high so that the stimulated emission rate is much higher compared to the multiphonon relaxation rate. In contrast, the lower phonon energy of ZBLAN glass leads to significant suppression of multiphonon relaxation such that it is almost negligible, and thus the stimulated emission process becomes significantly more efficient. This property makes ZBLAN fibers advantageous especially when the pump power or the seed power is relatively low, e.g., for developing oscillators and moderate-power amplifiers. All the ZBLAN fibers used for the experiments are custom fibers produced by Fiberlabs, Inc. by specifying the parameters.
While the detail of the oscillator is presented in Ref. [15], the parameters are slightly updated by optimizing the alignment. The developed oscillator generates output pulses with a very broad spectrum extending from 1.7 µm to 2.1 µm at 67.5 MHz repetition rate. The pulses can be compressed to the duration of 41 fs using an external compressor. The output power of 36 mW is obtained at the total pump power of 380 mW.
The output pulses generated from the oscillator are amplified with the first amplifier stage. As the amplifier fiber, a 0.5-m-long, large-mode-area thulium-doped fiber made of ZBLAN is used because we found that its amplification efficiency is much higher compared to that of silica-based fibers at the power level of a few watts [18]. The setup of the first amplifier is basically the same as that presented in Ref. [19]. The core of the fiber has a diameter of 20 µm and a numerical aperture of 0.08, which supports up to two modes at the wavelength longer than 1.3 µm. A Raman fiber laser at the wavelength of 1.6 µm (RLR-30-1620, IPG Photonics) is used as the pump source and is coupled into the core of the amplifier fiber in the counter-propagating direction. The core-pumping scheme helps to shift the center of the gain spectrum to the short-wavelength region. The stretcher before the amplifier is used to adjust the pre-chirp of the input pulse. It should be noted that the stretcher before the amplifier is not intended for chirped-pulse amplification [20] but rather for slightly adjusting the pre-chirp. In fact, the pulse experiences strong nonlinear effects within the amplifier fiber so that the output spectrum is significantly broadened [19]. This spectral broadening compensates for the strong gain-narrowing during the amplification, and consequently, the width of the output spectrum is similar to that of the oscillator output. By adjusting the stretcher, the strength of the nonlinear effects within the amplifier fiber is controlled and the output spectral shape is optimized. The pulse right after the first amplifier fiber has a duration of less than 50 fs at the average output power of 3.3 W.
Although the amplified spectrum extends from 1.7 µm to 2.0 µm, the wavelength component above 1.85 µm needs to be removed if the pulses are to be applied for three-photon microscopes intended for biological observation. This is because radiation with the wavelength longer than 1.85 µm is strongly absorbed by substances within biological tissues such as water molecules and will lead to damages to the samples. To remove the long-wavelength component, we used a 4-$f$ system with a beam block inside. The bandwidth of the output spectrum can be tuned by adjusting the position of the beam block. When we position the beam block so that the intensity at 1.85 µm is $\sim$10 dB lower than that at the 1.82 µm peak, we obtain a filtered spectrum with the average power of 0.9 W. Since the 4-$f$ setup is similar to a common grating-based stretcher, we can add a large amount of positive dispersion by shifting the position of the folding mirror.
Although we could further amplify the pulses using another amplifier stage, the pulse repetition rate is reduced first because biological samples can be easily damaged if the average power is too high. Before the second amplifier stage, a Pockels cell (RTP-3-20, Lysop) and a polarizer are used to reduce the pulse repetition rate to 1 MHz. Here, we chose a Pockels cell as a pulse picker because of its high efficiency compared to acousto-optic modulators at this wavelength region. The polarization extinction ratio is $\sim$300:1 whereas the transmission is $\sim$80 % around 1.8 µm. The pulse train with the reduced repetition rate is sent into the second amplifier stage, which is based on a thulium-doped ZBLAN fiber as well. The parameters of the fiber are the same as those of the first amplifier except for the length, which is 0.2 m for the second amplifier. The second amplifier stage is pumped by the same type of Raman fiber laser used in the first stage. The output pulses are amplified to the average power of 2 W, which corresponds to the pulse energy of 2 µJ, with the slope efficiency of 37 %. The evolution of the optical spectrum measured with a spectrum analyzer (AQ6375, Yokogawa) is shown in Fig. 2. As the bottom panel of Fig. 2 shows, no sign of amplified spontaneous emission is observed after the second amplifier stage above the background level of −40 dB. The power stability of the output beam is $\sim$0.4 % over 1 h whereas the pulse-to-pulse stability is $\sim$2 %.
Fig. 2. Evolution of the optical spectrum throughout the setup. The spectra after the oscillator, the first amplifier, 4-$f$ setup, and the second amplifier are shown from top to bottom.
The output pulses are compressed with a compressor based on a pair of gratings with grooves of 560 mm−1. The compressed pulses are characterized by using a home-built second-harmonic generation frequency-resolved optical gating (SHG-FROG) device. The results of SHG-FROG measurements are shown in Fig. 3. Analyzing the measured FROG trace shows that the compressed pulses have a duration of 150 fs. The small side pulses observed in Fig. 3(b) is due to the remaining third-order dispersion, which can be observed in the phase curve shown in Fig. 3(c). The average power after the compressor is $\sim$1.1 W.
Fig. 3. Characteristics of the output pulses. (a) SHG-FROG trace measured after compression. (b) Pulse shape (filled curve) and its phase (dashed curve) retrieved from the SHG-FROG trace shown in (a). (c) Spectral shape (filled curve) and its phase (dashed curve) retrieved from the SHG-FROG trace shown in (a).
3. Three-photon microscope
The pulses were sent into a microscope system consisting of galvanometer scanners (Cambridge Technology), a scan lens (SL50-3P, Thorlabs), a trinocular tube (U-TR30IR, Olympus), and a water-immersion objective (LUMPLFLN40XW, Olympus), as shown in Fig. 4. The galvanometer scanners were controlled with the ScanImage software [21]. The total transmission of the optics within the microscope is approximately 10%. The major loss is due to the microscope objective, where the transmission is less than 30%, because there was no objective designed for 2 µm laser commercially available at the moment and we had to use an objective designed for another wavelength. A photomultiplier tube (R3896, Hamamatsu) was used to detect the fluorescence signal emitted from the samples in the backward direction.
To test whether our setup is capable of inducing three-photon excitation processes, we observed a red fluorescent dye called cresyl violet dissolved in dimethyl sulfoxide. Cresyl violet absorbs radiation around 600 nm and emits radiation around 620 nm. Therefore, if laser pulses with 1.8 µm wavelength are focused into the solution, fluorescence emission by three-photon excitation should be observed. In fact, we successfully observed the fluorescence signal under our setup. To confirm if the signal is due to a three-photon process, we observed the dependence of the signal strength as a function of the input power [Fig. 5(a)]. We should note that strong background signals are observed when the beam is focused at the surface of the dish holding the sample. Since this background signal is due to third-harmonic generation and has the same input-power dependency as the three-photon fluorescence, we inserted a bandpass filter (FF01-709/167, Semrock) in front of the photomultiplier tube to remove the third-harmonic wavelength. After placing the filter, the background signal disappeared even when the focus is placed at the surface of the dish. Nevertheless, we carefully adjusted the position of the focal point to make sure that it is not located at the surface of the dish. The compressor of the laser system is re-adjusted to maximize the signal. The data shown in Fig. 5(a) are obtained under this condition and clearly show cubic dependence of the signal to the input power. This result indicates that the microscope is capable of observing red fluorescent dye through three-photon-excited fluorescence generation.
Fig. 5. Results of three-photon excitation measurements using the microscope. (a) Fluorescence signal strength measured from cresyl violet dissolved in dimethyl sulfoxide. (b) A typical image obtained by observing red fluorescent beads dispersed in agarose gel.
To determine the spatial resolution of the microscope, we dispersed red fluorescent beads (0.2 µm, FluoSpheres, crimson fluorescent 625/645) into agarose gel and observed them. A typical image is shown in Fig. 5(b). Bright spots originating from the fluorescence from the beads were clearly observed. If we magnify into one of the spots, the spot can be resolved by more than ten pixels and can be fitted with a gaussian to determine the spot size. Since the spot is much larger than the size of the beads, the spot size is directly reflected from the point spread function of the microscope system. From the size of the point spread function, we could determine the resolution of the microscope as 0.8 µm in the transverse direction and 1.9 µm in the longitudinal direction.
Next, to test whether we can observe the fluorescence from living cells, we transfected a red fluorescent protein, TurboFP635 (a gift from Samantha Sampson, Addgene plasmid # 78314 [22]), in HeLa cells using Lipofectamine 3000 (Invitrogen). After 24 hours, we successfully observed the fluorescence from individual HeLa cells under our microscope [Fig. 6(a)]. The laser power used for the observation was 20 mW at the sample.
Fig. 6. Typical images of living cells expressing a red fluorescent protein, TurboFP635. (a) HeLa cells expressing TurboFP635. (b) Neurons expressing TurboFP635 in hippocampal slices of rat brain.
We further tested the microscope using living tissues from a rat brain. A hippocampus from a 6-day-old rat was sliced into 350 µm thickness, and the next day, the adeno-associated virus encoding TurboFP635 was introduced at a depth of approximately 50 µm from the surface of the slice. The slices were incubated at 35 °C in 5% CO$_2$ for 10 days so that sufficient level of TurboFP635 expression was obtained. For observation, the slices were taken out from the incubator and observed under the microscope within 1 hour so that the cells are still alive. Figure 6(b) shows one of the typical images taken with the laser power of 70 mW at the sample. The shapes of the neurons were clearly observed.
To observe deep within biological samples, it would be beneficial to increase the peak power while maintaining the average power at a similar level. The most favorable solution would be decreasing the pulse duration. One possibility is to improve the transmission within the microscope by replacing the optics. This way, we do not need very high output power from the laser, and the lower amplification factor would reduce the gain narrowing effect in the second amplifier stage. Unfortunately, optics designed for this wavelength region is not very common at the moment. Another possibility is to add a so-called nonlinear compression stage [23–25] to increase the bandwidth, which is a very powerful method but the setup would be rather complicated. It is also possible to further decrease the pulse repetition rate so that the energy of the single pulse would be higher, but that would eventually compromise the image acquisition rate. Nevertheless, there are several approaches to improve the current system, which would add a valuable tool for deep-tissue imaging.
4. Conclusion
We have developed an ultrashort laser system centered around 1.8 µm wavelength. The pulses with 150 fs duration at the center wavelength of 1.82 µm are obtained with an average power of 1.1 W at the repetition rate of 1 MHz, corresponding to the pulse energy of 1.1 µJ. Using these pulses, a three-photon fluorescence microscope has been developed. The microscope can be used to observe biological samples expressing red-fluorescent proteins through three-photon excitation processes. The developed system could be further improved to be used as an effective tool for deep-tissue imaging.
Funding
Japan Science and Technology Agency (JPMJPR168B), (JPMJCR17N5); Amada Foundation (AF-2015207).
Acknowledgments
The authors would like to thank Yoshinori Mimura and Kazuhiko Ogawa of FiberLabs Inc. for providing the ZBLAN fibers used for experiments and giving us many useful insights through fruitful discussion. Y. N. thanks Dr. Takayuki Yamashita for his help in conducting preliminary experiments using mouse brain slices at the initial stage of development.
Disclosures
The authors declare no conflicts of interest.
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