Widely tunable fiber optical parametric oscillator synchronized with a Ti:sapphire laser for stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a powerful vibrational imaging technique with high chemical specificity. However, the insufficient tuning range or speed of light sources limits the spectral range of SRS imaging and, hence, the ability to identify molecular species. Here, we present a widely tunable fiber optical parametric oscillator with a tuning range of 1470 cm−1, which can be synchronized with a Ti:sapphire laser. By using the synchronized light sources, we develop an SRS imaging system that covers the fingerprint and C–H stretching regions, without balanced detection. We validate its broadband imaging capability by visualizing a mixed polymer sample in multiple vibrational modes. We also demonstrate SRS imaging of HeLa cells, showing the applicability of our SRS microscope to biological samples.


Introduction
Vibrational imaging techniques based on stimulated Raman scattering (SRS) provide high chemical specificity and fast imaging speed [1][2][3][4].In SRS microscopy, two synchronized ultrashort pulses, referred to as pump and Stokes, coherently excite a molecular vibration that matches their frequency difference.Wide and fast tuning of this frequency difference is necessary to visualize distinct molecular species in diverse biomedical applications, such as cancer detection [5], monitoring drug delivery/interaction [6,7], and imaging cell metabolism [8][9][10][11][12][13].Ideally, the tunability from 500 to 3100 cm −1 is desired to cover the entire chemically informative molecular vibrational region [14,15].The high sensitivity of an imaging system is also crucial for acquiring clear images in a short time.The signal level is proportional to the power of pump and Stokes pulses, and the noise level is determined by the intensity noise of pump or Stokes pulses used for the lock-in detection [16,17].
One of the major challenges of current SRS imaging systems is the limited tuning range or speed of light sources.The most commonly used tunable light source for SRS is a synchronously pumped optical parametric oscillator (OPO) [18,19].While OPOs offer a wide tuning range over 4000 cm −1 , covering the entire Raman spectra, their tuning process can extend beyond one minute, especially when involving substantial wavelength changes.This lengthy time is attributed to the time-consuming temperature adjustments of the nonlinear crystal within the OPO.Recently, the spectral focusing method using a femtosecond OPO is becoming popular [20][21][22].This method uses two-color linearly chirped pulses, and tuning of the excitation wavenumber is realized by changing their temporal delay.Typically, the tuning range is limited to ∼300 cm −1 by the spectral widths of femtosecond pulses.Another commonly used tunable laser is a picosecond Yb-or Er-doped fiber laser that can tune the wavelength in the millisecond order or less [23][24][25].However, the tuning range of rare-earth-doped fiber lasers is, again, limited to ∼300 cm −1 by the gain bandwidth of the doped materials.
To overcome these limitations, tunable light sources using optical nonlinearities such as supercontinuum generation and four-wave mixing (FWM) have been proposed [26][27][28][29][30].Among them, a picosecond fiber optical parametric oscillator (FOPO) pumped by a tunable fiber laser is promising because of not only its remarkable tuning capability but also its high power spectral density [31][32][33].Wide and fast tuning with a FOPO was realized based on dispersion filtering and changing the repetition rate and the wavelength of a seed oscillator [31].Adjusting the repetition rate is advantageous over a previous tuning method that involved changing the cavity length by moving a delay stage, which hinders rapid wavelength tuning [34][35][36].For the application to SRS microscopy, however, the substantial noise associated with a FOPO and a fiber laser, which is a straightforward pump-Stokes combination, is a critical issue [32,37,38].Balanced detection can suppress the large excess noise, while it sacrifices at least 3 dB of signal-to-noise ratio (SNR) compared to shot-noise-limited detection with a single photodiode (PD).
In this work, we present a widely tunable picosecond FOPO with a tuning range from 819 to 931 nm, which corresponds to 1470 cm −1 .By synchronizing this FOPO and a tunable Yb fiber laser with a Ti:sapphire laser, we develop an SRS imaging system covering the fingerprint and C-H stretching regions.The wavelength of the FOPO can be tuned using an intracavity grating-based spectral filter while the pulse repetition rate is kept constant.This tuning method enables the FOPO to be synchronized with the external Ti:sapphire laser for the low-noise detection of SRS signals.To validate its broadband imaging capability, we demonstrate SRS imaging of a mixed polymer sample at several vibrational modes.We also demonstrate SRS imaging of HeLa cells to confirm the applicability of our SRS microscope to biomedical imaging.

Yb fiber laser for pumping the FOPO
Figure 1 shows the schematic of the SRS imaging system including the widely tunable FOPO and the Yb fiber laser for pumping it.Note that the Yb fiber laser produces pump pulses for the FOPO and Stokes pulses for SRS.The Yb fiber laser consists of an oscillator, a spectral broadening and filtering part, and amplifiers.The Yb fiber oscillator in a figure-nine configuration [14,39,40] generates seed pulses with a center wavelength of 1030 nm and a repetition rate of 38 MHz.The seed pulses are spectrally broadened via self-phase modulation (SPM) and spectrally filtered by a tunable filter [14,24], which consists of a diffraction grating and a galvanometer scanner.The spectral width of the filtered pulses is approximately 0.2 nm with a tuning range of more than 30 nm.The filtered seed pulses are amplified by cascaded two Yb-doped fiber amplifiers (YDFAs) and are divided into two branches.One branch is directed toward a FOPO port, and the other is toward a Stokes port for SRS.In the first branch, the pulses are further amplified by a double-clad YDFA to excite the following FOPO.The double-clad gain fiber (Yb1200-20/125DC-PM, Liekki) has a core diameter of 20 µm and is coiled for single-mode operation [41].This fiber has a low nonlinear coefficient and a large gain per length, both of which mitigate SPM-induced spectral broadening in the main amplifier.High power spectral density in the pump pulses is important for effectively pumping the FOPO.
The spectrum and intensity autocorrelation trace of the pump pulses generated from the Yb fiber laser are shown in Fig. 2. The spectral full-width at half-maximum (FWHM) is 0.48 nm when the average power is 870 mW at 1036 nm (Fig. 2(a)).Although the spectrum is broader than that before the power amplification, it does not exhibit the large SPM-induced spectral distortion, leading to the high power spectral density of the pump pulses compared with our previous work [33].The average power can exceed 2 W by increasing the pump power of the double-clad

FOPO
The setup of the FOPO presented here is based on that in our previous work [33].The pump pulses produced by the Yb fiber laser pass through an isolator and are focused into a 48 cm photonic crystal fiber (PCF, SUP-5-125-PM, Photonics Bretagne).The isolator prevents the pump pulses reflected on a PCF facet from returning to the fiber amplifier.The pulse power at the lens entrance is 840 mW, and the coupling efficiency to the PCF is approximately 75%.
Via FWM in the PCF, the pump pulses generate frequency up-shifted signal and down-shifted idler pulses, whose frequencies are determined by the phase-matching condition.The residual pump pulses and the idler pulses are blocked by a short-pass filter.This short-pass filter plays an important role in preventing pump pulses from entering the microscope or causing unwanted scattering within the free-space optics.At a polarizing beam splitter, most of the power of the YDFA.At such a high power, a spectral dip due to SPM appeared.The pulse duration at the same wavelength and at 870 mW is 7.2 ps under the assumption of a Gaussian waveform (Fig. 2(b)).The absence of oscillation in the spectrum and pedestal in the intensity autocorrelation trace indicates the high quality of the pulses.

FOPO
The setup of the FOPO presented here is based on that in our previous work [33].The pump pulses produced by the Yb fiber laser pass through an isolator and are focused into a 48 cm photonic crystal fiber (PCF, SUP-5-125-PM, Photonics Bretagne).The isolator prevents the pump pulses reflected on a PCF facet from returning to the fiber amplifier.The pulse power at the lens entrance is 840 mW, and the coupling efficiency to the PCF is approximately 75%.
Via FWM in the PCF, the pump pulses generate frequency up-shifted signal and down-shifted idler pulses, whose frequencies are determined by the phase-matching condition.The residual pump pulses and the idler pulses are blocked by a short-pass filter.This short-pass filter plays an important role in preventing pump pulses from entering the microscope or causing unwanted scattering within the free-space optics.At a polarizing beam splitter, most of the power of the

FOPO
The setup of the FOPO presented here is based on that in our previous work [33].The pump pulses produced by the Yb fiber laser pass through an isolator and are focused into a 48 cm photonic crystal fiber (PCF, SUP-5-125-PM, Photonics Bretagne).The isolator prevents the pump pulses reflected on a PCF facet from returning to the fiber amplifier.The pulse power at the lens entrance is 840 mW, and the coupling efficiency to the PCF is approximately 75%.
Via FWM in the PCF, the pump pulses generate frequency up-shifted signal and down-shifted idler pulses, whose frequencies are determined by the phase-matching condition.The residual pump pulses and the idler pulses are blocked by a short-pass filter.This short-pass filter plays an important role in preventing pump pulses from entering the microscope or causing unwanted scattering within the free-space optics.At a polarizing beam splitter, most of the power of the signal pulses is coupled out for the Stokes pulses of SRS, and the small remaining power is sent into the cavity.The output coupling ratio is adjusted by a half-wave plate (HWP).The signal pulses in the cavity pass through an automatic delay stage adjusting the cavity length, which is dependent on the wavelength due to the group velocity dispersion (GVD) of the PCF.They also pass through an intracavity spectral filter that consists of a galvanometer scanner, a 4f optical system, and a diffraction grating.The use of the spectral filter is essential to stably generate picosecond pulses; without it, the spectral width could exceed 10 nm.The passband of the spectral filter is controlled by changing the angle of the galvanometer scanner.After the delay stage and spectral filter, the signal pulses meet with the next pump pulses at a dichroic mirror.These pulses overlap in time and space, and the signal pulses are amplified via FWM in the PCF.Tuning of the pump wavelength shifts the resonant signal wavelength according to the phase-matching condition, leading to wide wavelength tuning of the FOPO.Depending on the signal wavelength, the delay stage and the spectral filter are adjusted.In contrast to dispersion tuning, the use of the intracavity filter allows us to keep the repetition rates unchanged during wavelength tuning.
To characterize the FOPO, we measured the spectrum, power, and intensity autocorrelation when tuning the wavelength.We controlled the automatic delay stage and two spectral filters in the Yb fiber laser and the FOPO.The HWP was also adjusted to maximize the output power.There was no need for alignment of other components.signal pulses is coupled out for the Stokes pulses of SRS, and the small remaining power is sent into the cavity.The output coupling ratio is adjusted by a half-wave plate (HWP).The signal pulses in the cavity pass through an automatic delay stage adjusting the cavity length, which is dependent on the wavelength due to the group velocity dispersion (GVD) of the PCF.
They also pass through an intracavity spectral filter that consists of a galvanometer scanner, a 4f optical system, and a diffraction grating.The use of the spectral filter is essential to stably generate picosecond pulses; without it, the spectral width could exceed 10 nm.The passband of the spectral filter is controlled by changing the angle of the galvanometer scanner.After the delay stage and spectral filter, the signal pulses meet with the next pump pulses at a dichroic mirror.These pulses overlap in time and space, and the signal pulses are amplified via FWM in the PCF.Tuning of the pump wavelength shifts the resonant signal wavelength according to the phase-matching condition, leading to wide wavelength tuning of the FOPO.Depending on the The measured spectrum and power are shown in Figs.3(a) and 3(b), respectively.The tuning range defined by the FWHM of powers is from 819 to 931 nm, which corresponds to 1470 cm −1 .This wide tuning range spans across the entire fingerprint region or multiple Raman regions.The FWHM of the spectrum is between 0.8 and 1.6 nm (10-19 cm −1 ).This spectral width is comparable to a typical Raman linewidth (<20 cm −1 ) and is narrow enough to distinguish Raman peaks [42].The output power reaches up to 81 mW.Considering the transmittance of our imaging system in the FOPO's tuning range, we can achieve an average power of tens of mW on the sample plane, which is sufficient for SRS. Figure 3(c) shows the intensity autocorrelation trace when the center wavelength is 885 nm.The pulse duration is 2.7 ps under the assumption of a Gaussian waveform, and it differs from 1.4 to 3.4 ps depending on the center wavelength.

Imaging system
The light sources of the SRS imaging system are the Ti:sapphire laser (Mira900D, Coherent) for the pump and the Yb fiber laser and the FOPO for the Stokes, as shown in Fig. 1.The pump pulses have a repetition rate of 76 MHz, a pulse duration of 3.5 ps, and a fixed wavelength of 789 nm.The Stokes pulses have a repetition rate of 38 MHz, which is exactly half of the pump pulse repetition rate.The Yb fiber laser is subharmonically synchronized with the Ti:sapphire laser by active feedback control.Specifically, this synchronization mechanism relies on adjusting the cavity length of the Yb fiber oscillator using an intracavity electro-optic phase modulator and piezo stage, based on the time delay between pump and Stokes pulses detected by a two-photon absorption PD [14].The Yb fiber laser and the FOPO are passively synchronized through synchronous pumping.These synchronizations result in the synchronization of the Ti:sapphire laser and the FOPO.The tuning range of the Yb fiber laser is from 1014 to 1047 nm (310 cm −1 ) [14,23], and that of the FOPO is from 819 to 931 nm (1470 cm −1 ).Considering the pump wavelength of 789 nm, the spectral region that can be accessed is 460-1930 cm −1 and 2810-3120 cm −1 , covering the fingerprint and C-H regions.
Two Stokes beams from the fiber laser and the FOPO are switched by a mirror.The pump beam and the selected Stokes beam are combined by a short-pass dichroic mirror (DMSP805R, Thorlabs), and a 2D galvanometer scanner scans the combined beam.The scanner plane is imaged to the pupil of a water immersion objective (60×, NA1.2, UPLSAPO60XW, Olympus) to focus the beam on the sample plane.The outgoing light transmitted through the sample is collected using another objective (UPLSAPO60XW, Olympus).The remaining Stokes beam is completely blocked by a short-pass filter (FESH0800, Thorlabs).After beam size reduction through a 4f optical system, only the pump beam is incident to a PD.The SRS signal is demodulated using a custom-made 38 MHz filter circuit and a homemade lock-in amplifier and is detected by a data acquisition system (USB-6363, National Instruments).All SRS images were acquired with an image size of 80 × 80 µm 2 , 500 × 500 pixels, 4 µs pixel dwell time, and no averaging.
HeLa cells used in this study were cultured in Dulbecco's modified Eagle's medium (12320-032, Gibco) supplemented with fetal bovine serum (SH30079.01,HyClone, GE Healthcare) and penicillin-streptomycin (15140148, Invitrogen) in an environment maintained at 37 • C with 5% CO 2 .The HeLa cells were seeded at a density of 4 × 10 4 cells in 500 µL of the medium onto a coverslip (C012001, Matsunami) in a 4-well dish (Thermo Scientific) and incubated for 3 days.The cell medium was replaced with 250 µL of fixation buffer (420801, BioLegend) and incubated for 20 minutes, followed by washing with phosphate-buffered saline (PBS), (166-23555, FUJIFILM Wako).For SRS imaging, the fixed cells were enclosed between two coverslips in PBS using an imaging spacer.

Results
To verify the broadband imaging capability of our SRS microscope, we first performed SRS imaging of a mixture of PMMA and PS beads.In the C-H region, PMMA and PS have Raman peaks at ∼2950 and ∼3050 cm −1 , respectively.They also have several vibrational modes in the fingerprint region [43].For example, the ring breathing mode of PS provides a strong Raman signal at ∼1000 cm −1 [44].We acquired a total of seven SRS images at 600, 813, 1000, 1452, and 1600 cm −1 in the fingerprint region as well as 2950 and 3050 cm −1 in the C-H region.SRS signals at 1000, 1600, and 3050 cm −1 targeted PS, while those at the other wavenumbers targeted PMMA.It took about one minute to tune the wavelength and to adjust the time delay between pump and Stokes pulses.The pump power was set to 5 mW on the sample plane to prevent signal saturation and minimize damage to the sample.The Stokes powers were 31-53 mW depending on the wavenumber.
Figure 4 shows the obtained SRS images of the mixed polymer sample.The images are arranged in two rows based on whether the targeted vibrational modes belong to PMMA or PS.Ring-like artifacts observed at 1600 cm −1 are attributed to the Stokes pulses that are generated by cascaded FWM and transmitted through the short-pass filter in front of the PD.These parasitic pulses can be blocked by placing a long-pass filter on the FOPO Stokes path.We can differentiate PMMA and PS beads from their signal levels in each SRS image.The SNR of an SRS image is defined by SNR = µ(S)/σ(N), where µ(S) is the signal mean measured in the area with the SRS signal, and σ(N) is the noise standard deviation measured in the background.The SNR of each SRS image is from 9 to 113.The difference comes mainly from the Raman cross section as well as from other factors such as pulse power and duration.The SRS image at 1000 cm −1 exhibits the highest SNR of all images owing to the strong PS signal.The relatively small difference between PMMA and PS at 813 and 1452 cm −1 is due to spectral overlap.Around these wavenumbers, while PS has very weak Raman signals compared to its own strong peaks, such as at ∼1000 cm −1 , Raman signal levels of PMMA and PS are not far apart [43].Next, we performed SRS imaging of fixed HeLa cells to demonstrate the applicability of our SRS microscope to biological imaging.Proteins and lipids are abundant and provide strong Raman signals in the C-H region.In the fingerprint region, a Raman peak at ∼1655 cm −1 is attributed to the amide I band of proteins and acyl C=C band of lipids [13,45].To visualize the distributions of proteins and lipids, we acquired SRS images at 1653, 2850, and 2940 cm −1 .The pump power was 62 mW on the sample plane.The Stokes powers were 36, 48, and 54 mW at 1653, 2850, and 2940 cm −1 , respectively.SRS images of HeLa cells are shown in Fig. 5.In contrast to the SRS image at 2850 cm −1 (Fig. 5(b)), which is associated with the CH 2 bond of lipids, the SRS image at 2940 cm −1 (Fig. 5(c)) exhibits the signal in nuclei, especially in nucleoli, whose signals are attributed to the CH 3 bond of proteins.Compared to the images acquired in the C-H regions, the SRS image at 1653 cm −1 (Fig. 5(a)) has a relatively low SNR because of the small Raman cross section in the fingerprint region.Nevertheless, we can see similar distributions of the SRS signal coming from proteins and lipids in Figs.5(a) and 5(c).These results validate the effectiveness of our SRS imaging system in biomedical applications.

Discussion
The present SRS imaging system has various advantages over previous ones.The FOPO has the potential of faster tuning capability than solid-state OPOs which require temperature adjustment of the nonlinear crystal [18,19].Compared with dispersion-tuning-based FOPOs [31,46], the present FOPO can tune the wavelength without changing the repetition rate, making it easier to synchronize the FOPO with the Ti:sapphire laser.Taking advantage of the low-noise property of the Ti:sapphire laser, SRS imaging can be accomplished without balanced detection, which is required to eliminate excess noise when noisy pulse sources are employed, at the expense of a 3 dB sensitivity drawback.As a result, SRS imaging of a polymer sample and HeLa cells was realized in a moderate pixel dwell time even in the fingerprint region, where the Raman cross section is small.Another notable advantage of our light source is its ability to access low vibrational wavenumbers such as 600 cm −1 or less, due to the independence of pump and Stokes wavelengths.
To improve this SRS imaging system, fast wavelength tuning can be realized by implementing automatic control of the spectral filters and delay stages.Its tuning rate is from a tenth of a second to a second depending on how far the wavelength is changed.It is possible to further reduce the tuning time to several milliseconds by passively compensating the GVD of the PCF to eliminate

Discussion
The present SRS imaging system has various advantages over previous ones.The FOPO has the potential of faster tuning capability than solid-state OPOs which require temperature adjustment of the nonlinear crystal [18,19].Compared with dispersion-tuning-based FOPOs [31,46], the present FOPO can tune the wavelength without changing the repetition rate, making it easier to synchronize the FOPO with the Ti:sapphire laser.Taking advantage of the low-noise property of the Ti:sapphire laser, SRS imaging can be accomplished without balanced detection, which is required to eliminate excess noise when noisy pulse sources are employed, at the expense of a 3 dB sensitivity drawback.As a result, SRS imaging of a polymer sample and HeLa cells was realized in a moderate pixel dwell time even in the fingerprint region, where the Raman cross section is small.Another notable advantage of our light source is its ability to access low vibrational wavenumbers such as 600 cm −1 or less, due to the independence of pump and Stokes wavelengths.
To improve this SRS imaging system, fast wavelength tuning can be realized by implementing automatic control of the spectral filters and delay stages.Its tuning rate is from a tenth of a second to a second depending on how far the wavelength is changed.It is possible to further reduce the tuning time to several milliseconds by passively compensating the GVD of the PCF to eliminate the need for delay stage movement.The tuning range of the FOPO can be widened by optimizing the Yb fiber laser pumping the FOPO so that the laser power is kept high and relatively constant across the tuning range of the fiber laser.The output power of the FOPO can also be improved in two ways.First, suppressing the SPM of FOPO pump pulses by shortening the double-clad gain fiber increases FWM energy conversion efficiency.Second, the output power becomes higher by increasing the pump power incident to the PCF.This power was set much lower than the maximum power (>2 W) in order to ensure a large margin for damage to a PCF facet.

Conclusion
We have developed the SRS imaging system using the widely tunable FOPO.The FOPO provided a maximum output power of 81 mW and a tuning range as broad as 1470 cm −1 .By synchronizing this FOPO and another tunable Yb fiber laser with the Ti:sapphire laser, we realized the SRS imaging system that covers the range from 460 to 1930 cm −1 in the fingerprint region and from 2810 to 3120 cm −1 in the C-H stretching region, without balanced detection.Its broadband imaging capability was verified by SRS imaging at multiple vibrational modes in the two regions.Furthermore, SRS imaging of HeLa cells shows the applicability of our SRS microscope to biological imaging.We expect that this imaging system with the FOPO will expand applications of SRS microscopy in various biomedical fields.

Fig. 1 .
Fig. 1.Schematic of the SRS imaging system.The light sources are a Ti:sapphire laser for the pump pulses and an Yb fiber laser and a FOPO for the Stokes pulses.The Stokes light sources can tune the wavelength across the C-H stretching and fingerprint regions, respectively.The three light sources are all synchronized through an active synchronization of the Yb fiber laser and the Ti:sapphire laser and a passive synchronization of the Yb fiber laser and the FOPO by synchronous pumping.TBPF: tunable bandpass filter, YDFA: Yb-doped fiber amplifier, DC: double clad, DM: dichroic mirror, PCF: photonic crystal fiber, SPF: short-pass filter, HWP: half-wave plate, PBS: polarizing beam splitter, OB: objective, PD: photodiode.

Fig. 2 .
Fig. 2. Characteristics of the Yb fiber laser in the FOPO port.(a) Spectrum at 1036 nm.The spectral width is 0.48 nm.(b) Corresponding intensity autocorrelation trace.The pulse duration is 7.2 ps under the assumption of a Gaussian waveform.

Fig. 3 .
Fig. 3. Characteristics of the FOPO when tuning the wavelength.(a) Spectrum and spectral FWHM, and (b) power.The tuning range is 819-931 nm, which corresponds to 1470 cm −1 .The power is up to 81 mW.(c) Intensity autocorrelation trace at 885 nm.The pulse duration is 2.7 ps under the assumption of a Gaussian waveform.

Fig. 5 .
Fig. 5. SRS imaging of fixed HeLa cells in the fingerprint and C-H regions.SRS images at (a) 1653 cm −1 in the amide I band of proteins and the acyl C=C band of lipids, (b) 2850 cm −1 in the CH 2 band of lipids, and (c) 2940 cm −1 in the CH 3 band of proteins and lipids.Scale bar: 10 µm.Pump power: 62 mW, Stokes power: 35-54 mW.Pixel dwell time: 4 µs.No averaging.